POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Patent Application Serial No.

62/902,300, filed on September 18, 2019, the entire disclosure of each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING STATEMENT

[0002] A sequence listing text (.txt) file is submitted herewith under 37 CFR.

1.821(c) and is hereby incorporated by reference in its entirely. The details of the file as required under 37 CFR. 1.52(e)(5) and 37 CFR 1.77(b)(5) are as follows: Name of file is DEMT_007_01WO_SeqList_ST25.txt; date of creation is September 17, 2020; size is 697176 bytes. The content of the sequence listing information recorded in computer readable form is identical to the written sequence listing (if any) and identical to the sequence information provided with the original filed application and contains no new matter. The information recorded in electronic form (if any) submitted under Rule 13ter with this application is identical to the sequence listing as contained in the application as filed.

BACKGROUND

[0003] Plants from the genus Cannabis have been used by humans for their medicinal properties for thousands of years. In modern times, the bioactive effects of Cannabis are attributed to a class of compounds termed “cannabinoids,” of which there are hundreds of structural analogs including tetrahydrocannabinol (THC) and cannabidiol (CBD). These molecules and preparations of Cannabis material have recently found application as therapeutics for chronic pain, multiple sclerosis, cancer-associated nausea and vomiting, weight loss, appetite loss, spasticity, seizures, and other conditions.

Cannabidiol / CBD Tetrahydrocannabinol / THC / Dronabinol / Marinol

[0004] The physiological effects of certain cannabinoids are thought to be mediated by their interaction with two cellular receptors found in humans and other animals. Cannabinoid receptor type 1 (CB1) is common in the brain, the reproductive system, and the eye. Cannabinoid receptor type 2 (CB2) is common in the immune system and mediates therapeutic effects related to inflammation in animal models. The discovery of cannabinoid receptors and their interactions with plant-derived cannabinoids predated the identification of endogenous ligands.

[0005] Besides THC and CBD, hundreds of other cannabinoids have been identified in Cannabis. However, many of these compounds exist at low levels and alongside more abundant cannabinoids, making it difficult to obtain pure samples from plants to study their therapeutic potential. Similarly, methods of chemically synthesizing these types of products have been cumbersome and costly, and tend to produce insufficient yield. Accordingly, additional methods of making pure cannabinoids or cannabinoid derivatives are needed. [0006] One possible method is production via fermentation of engineered microbes, such as yeast. By engineering production of the relevant plant enzymes in microbes, it may be possible to achieve conversion of various feedstocks into a range of cannabinoids, potentially at much lower cost and with much higher purity than what is available from the plant. A key challenge to this effort is the difficulty of expressing plant enzymes in the microbe, particularly secreted enzymes such as the cannabinoid synthases, which must successfully traverse the microbe’s secretory pathway to fold and function properly. Engineered variants of cannabinoid synthases, modified host cells, and new methods are needed to address these challenges.

Summary

[0007] The present disclosure provides engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing cannabinoids or cannabinoid derivatives, methods of producing cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide. The engineered variants of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids). The modified host cells of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids) and/or for expressing engineered variants of the disclosure. The disclosure also provides for modified host cells for expressing the engineered variants of the disclosure. Additionally, the disclosure provides for preparation of engineered variants of the disclosure.

[0008] An aspect of the disclosure relates to an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions. In some embodiments, the engineered variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:44. In some embodiments, the engineered variant comprises at least one amino acid substitution in a signal polypeptide, a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing. In some embodiments, the engineered variant comprises substitution of at least one surface exposed amino acid.

[0009] In some embodiments, the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of R31, P43, P49, K50, L51, Q55, H56, L59, M61, M61, S62, L71, S100, V103, T109, Q124, V125, L132, S137, H143, V149, W161, K165, N168, E167, S170, F171, P172, Y175, G180, N196, H208,

G235, A250, 1257, K261, L269, G311, F317, L327, K390, T379, S429, N467, Y500, N528, P539, P542, H543, H544, and H545. In some embodiments, the engineered variant comprises at least one amino acid substitution selected from the group consisting of R31Q, P43E, P49E, P49K, P49Q, K50T, L51I, Q55E, Q55P, H56E, L59E, M61W, M61H, M61S, S62Q, L71A, S100A, V103F, T109V, Q124D, Q124E, Q124N, V125E, V125Q, L132M, S137G, H143D, W161R, W161Y, W161K, K165A, N168S, E167P, Y175F, G180A,

N196Q, N196V, H208T, A250T, I257V, K261C, G311A, F317Y, L327I, K390E, T379S, Y500M, Y500V, N528E, P542E, P542V, H543 V, H544A, H545D, and H545E [0010] In some embodiments, the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, and SEQ ID NO: 186.

[0011] In some embodiments, the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, and SEQ ID NO: 186. [0012] In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.

[0013] In some embodiments, the engineered variant comprises at least one immutable amino acid in a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing. In some embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 immutable amino acids in the FAD binding domain. In some embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 immutable amino acids in the BBE domain.

[0014] In some embodiments, the engineered variant comprises at least one immutable amino acid selected from the group consisting of A28, F34, L35, C37, L64, N70, P87, 193, C99, R108, R110, G112, E117, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, A173, G174, C176, P177, T178,

V179, G182, G183, H184, F185, G187, G188, G189, Y190, G191, P192, L193, R195,

A201, D202, 1205, D206, V210, G214, G223, D225, L226, F227, W228, R231, G234, S237, F238, G239, K245, 1246, L248, V251, V259, Q276, F312, S313, L323, C341, F352, S354, F380, K381, 1382, K383, D385, Y386, 1391, M412, L415, G419, M422, 1425, 1430, P431, P433, H434, R435, G437, Y440, W443, Y444, 1445, 1464, Y465, M468, T469, Y471, V472, P476, R484, N498, A502, N513, F514, K521, N528, F529, E533, Q534, and S535. In some embodiments, the engineered variant comprises at least one immutable amino acid selected from the group consisting of C37, N70, 193, C99, El 17, S120, F127, D131, G156, E157, Y159, G174, C176, G182, G183, F185, G187, G188, G189, Y190, G191, P192, R195,

D202, D206, G214, W228, G234, F238, and L248.

[0015] In some embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 immutable amino acids. [0016] In some embodiments, the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase (THCAS) polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. In some embodiments, the engineered variant produces THCA from CBGA in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0017] In some embodiments, the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. In some embodiments, the engineered variant produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500: 1.

[0018] In some embodiments, the engineered variant comprises a truncation at an N- terminus, at a C-terminus, or at both the N- and C-termini. In some embodiments, the truncated engineered variant comprises a signal polypeptide or a membrane anchor. In some embodiments, the engineered variant lacks a native signal polypeptide. In some embodiments, the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the C-terminus. In some embodiments, the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus.

[0019] Another aspect of the disclosure relates to a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID N0:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID N0:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID N0:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID

NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID

NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID

NO:145, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID

NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID

NO:175, SEQ ID NO: 177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO: 183, and SEQ ID NO: 185. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID

NO: 131, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID

NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:151, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID

NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID

NO: 181, SEQ ID NO: 183, and SEQ ID NO: 185. In some embodiments of the nucleic acids of the disclosure, the nucleotide sequence is codon-optimized.

[0020] An aspect of the disclosure relates to a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure into a host cell. [0021] Another aspect of the disclosure relates to a vector comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.

[0022] An aspect of the disclosure relates to a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more vectors comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure into a host cell.

[0023] Another aspect of the disclosure relates to a modified host cell for producing a cannabinoid or a cannabinoid derivative, wherein the modified host cell comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.

[0024] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide. In certain such embodiments, the GOT polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 17. In some embodiments, the modified host cell comprises two or more heterologous nucleic acids comprising the nucleotide sequence encoding the GOT polypeptide.

[0025] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide. In certain such embodiments, the NphB polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 188.

[0026] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tetraketide synthase (TKS) polypeptide and one or more heterologous nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase (OAC) polypeptide. In certain such embodiments, the TKS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 19. In some embodiments, the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide. In some embodiments, the OAC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:21 or SEQ ID NO:48. In some embodiments, the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide. [0027] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acyl activating enzyme (AAE) polypeptide. In certain such embodiments, the AAE polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:23. In some embodiments, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide.

[0028] In some embodiments of the disclosure, the modified host cell comprises one or more of the following: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMG-CoA synthase (HMGS) polypeptide; b) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a truncated 3- hydroxy-3-methyl-glutaryl-CoA reductase (tHMGR) polypeptide; c) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate kinase (MK) polypeptide; d) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a phosphomevalonate kinase (PMK) polypeptide; e) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate pyrophosphate decarboxylase (MVD1) polypeptide; or f) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a isopentenyl diphosphate isomerase (IDIl) polypeptide. In some embodiments, the IDI1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:25. In some embodiments, the tHMGR polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:27. In some embodiments, the HMGS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:29. In some embodiments, the MK polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:39. In some embodiments, the PMK polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:37. In some embodiments, the MVD1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:33.

[0029] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide. In certain such embodiments, the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:31. [0030] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a pyruvate decarboxylase (PDC) polypeptide. In certain such embodiments, the PDC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:35. [0031] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate synthetase (GPPS) polypeptide. In certain such embodiments, the GPPS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:41.

[0032] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. In certain such embodiments, the KAR2 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:5. In some embodiments, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[0033] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDI1 polypeptide. In certain such embodiments, the PDI1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:9.

[0034] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IRE1 polypeptide. In certain such embodiments, the IRE1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 190.

[0035] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide. In certain such embodiments, the EROl polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:7.

[0036] In some embodiments of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a FAD1 polypeptide. In certain such embodiments, the FAD1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 192.

[0037] In some embodiments of the disclosure, the modified host cell comprises a deletion or downregulation of one or more genes encoding a PEP4 polypeptide. In certain such embodiments, the PEP4 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15.

[0038] In some embodiments of the disclosure, the modified host cell comprises a deletion or downregulation of one or more genes encoding a ROT2 polypeptide. In certain such embodiments, the ROT2 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13.

[0039] In some embodiments of the disclosure, the modified host cell is a eukaryotic cell. In certain such embodiments, the eukaryotic cell is a yeast cell. In certain such embodiments, the yeast cell is Saccharomyces cerevisiae. In certain such embodiments, the Saccharomyces cerevisiae is a protease-deficient strain of Saccharomyces cerevisiae.

[0040] In some embodiments of the disclosure, at least one of the one or more nucleic acids are integrated into the chromosome of the modified host cell. In some embodiments of the disclosure, at least one of the one or more nucleic acids are maintained extrachromosomally. In some embodiments of the disclosure, at least one of the one or more nucleic acids are operably-linked to an inducible promoter. In some embodiments of the disclosure, at least one of the one or more nucleic acids are operably-linked to a constitutive promoter.

[0041] In some embodiments of the disclosure, the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under similar culture conditions for the same length of time.

[0042] In some embodiments of the disclosure, the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under similar culture conditions for the same length of time.

[0043] In some embodiments of the disclosure, the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under similar culture conditions for the same length of time.

[0044] In some embodiments of the disclosure, the modified host cell has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under similar culture conditions for the same length of time.

[0045] In some embodiments of the disclosure, the modified host cell produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under similar culture conditions for the same length of time.

[0046] In some embodiments of the disclosure, the modified host cell produces

THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

[0047] Another aspect of the disclosure relates to a method of producing a cannabinoid or a cannabinoid derivative, the method comprising: a) culturing a modified host cell of the disclosure in a culture medium. In certain such embodiments, the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative. In some embodiments, the culture medium comprises a carboxylic acid. In certain such embodiments, the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid.

In certain such embodiments, the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid. In some embodiments, the culture medium comprises olivetolic acid or an olivetolic acid derivative. In some embodiments, the cannabinoid is cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin. In some embodiments, the culture medium comprises a fermentable sugar. In some embodiments, the culture medium comprises a pretreated cellulosic feedstock. In some embodiments, the culture medium comprises a non-fermentable carbon source. In certain such embodiments, the non-fermentable carbon source comprises ethanol. In some embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium.

[0048] In some embodiments of the methods of the disclosure, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, are cultured under similar culture conditions for the same length of time.

[0049] In some embodiments of the methods of the disclosure, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, are cultured under similar culture conditions for the same length of time.

[0050] In some embodiments of the methods of the disclosure, the cannabinoid is tetrahydrocannabinolic acid (THCA), and wherein the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under similar culture conditions for the same length of time. [0051] An aspect of the disclosure relates to a method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of the disclosure. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In some embodiments of the methods of the disclosure, the cannabinoid is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin.

[0052] In some embodiments of the methods of the disclosure, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure, wherein the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time.

[0053] In some embodiments of the methods of the disclosure, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure, wherein the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time. [0054] In some embodiments of the methods of the disclosure, the cannabinoid is tetrahydrocannabinolic acid (THCA), and wherein the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA) compared to that produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instread of the engineered variant of the disclosure, wherein the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time.

[0055] In some embodiments of the methods of the disclosure, the method produces

THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

[0056] Another aspect of the disclosure relates to a method of screening an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, the method comprising: a) dividing a population of host cells into a control population and a test population; b) co-expressing in the control population a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 and a comparison cannabinoid synthase polypeptide, wherein the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 can convert cannabigerolic acid (CBGA) to a first cannabinoid, tetrahydrocannabinolic acid (THCA), and the comparison cannabinoid synthase polypeptide can convert the same CBGA to a different second cannabinoid; c) co-expressing in the test population the engineered variant and the comparison tetrahydrocannabinolic acid synthase polypeptide, wherein the engineered variant may convert CBGA to the same first cannabinoid, tetrahydrocannabinolic acid (THCA), as the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44, and wherein the comparison cannabinoid synthase polypeptide can convert the same CBGA to the second cannabinoid and is expressed at similar levels in the test population and in the control population; d) measuring a ratio of the first cannabinoid, tetrahydrocannabinolic acid (THCA), over the second cannabinoid produced by both the test population and the control population; and e) measuring an amount, in mg/L or mM, of the first cannabinoid produced by both the test population and the control population. In certain such embodiments, the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase (THCAS) polypeptide having an amino acid sequence of SEQ ID NO:44, wherein improved in vivo performance is demonstrated by an increase in the ratio of the first cannabinoid over the second cannabinoid produced by the test population compared to that produced by the control population under similar culture conditions for the same length of time. In some embodiments of the method of screening the engineered variant of a THCAS polypeptide, the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO: 3 by producing the first cannabinoid in a greater amount, as measured in mg/L or mM, by the test population compared to the amount produced by the control population under similar culture conditions for the same length of time.

[0057] In some embodiments of the method of screening the engineered variant of a

THCAS polypeptide, the cannabinoid synthase polypeptide is a cannabidiolic acid synthase polypeptide. In certain such embodiments, the cannabidiolic acid synthase (CBDAS) polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:3. In some embodiments of the method of screening the engineered variant of a THCAS polypeptide, the second cannabinoid is cannabidiolic acid (CBDA).

[0058] In some embodiments of the method of screening the engineered variant of a

THCAS polypeptide, the engineered variant is an engineered variant of the disclosure.

Brief Description Of The Drawings

[0059] FIGS. 1 A, IB, and 1C depict expression constructs used in the production of the S29 strain. The expression constructs depicted in FIGS. 1 A, IB, and 1C were also used in the production of the following strains: S61, S122, S171, S181, S220, S241, S270, S487, S951, S1000-S1059, S1072-1079, and S1081. Throughout the figures, in addition to the specified coding sequences from Table 1, construct maps depict regulatory, non-coding and genomic cassette sequences described in Table 5. Construct maps also depict genes denoted with a preceding “m” (e.g. mERG13), which specify open reading frames from Table 1 with 200-250 base pairs (bp) of downstream regulatory (terminator) sequence. Arrows in construct maps indicate the directionality of certain DNA parts. The “!” preceding a part name is an output of the DNA design software used, is redundant with the arrow directionality, and can be ignored.

[0060] FIG. 2 depicts an expression construct used in the production of the S181 strain. The expression construct depicted in FIG. 2 was also used in the production of following strains: S220, S241, S270, S487, S951, S1000-S1059, SI 072- 1079, and SI 081. [0061] FIG. 3 depicts an expression construct used in the production of the S220 strain. The expression construct depicted in FIG. 3 was also used in the production of following strains: S241, S270, S487, S951, S1000-S1059, SI 072- 1079, and SI 081.

[0062] FIG. 4 depicts expression constructs used in the production of the S241 strain.

The expression constructs depicted in FIG. 4 were also used in the production of following strains: S270, S487, S951, S1000-S1059, S1072-1079, and S1081.

[0063] FIG. 5 depicts a landing pad construct used in the production of the S61 strain. The construct depicted in FIG. 5 was also used in the production of the following strains: S122, S171, S181, S220, S241, S270, S487, S951, S1000-S1059, S1072-1079, and S 1081.

[0064] FIG. 6 depicts expression constructs used in the production of the S122 strain.

The expression constructs depicted in FIG. 6 were also used in the production of the following strains: S 171, SI 81, S220, S241, S270, S487, S951, S1000-S1059, SI 072- 1079, and S 1081.

[0065] FIG. 7 depicts an expression construct used in the production of the S 171 strain. The expression construct depicted in FIG. 7 was also used in the production of the following strains: S181, S220, S241, S270, S487, S951, S1000-S1059, SI 072- 1079, and S 1081.

[0066] FIG. 8 depicts expression constructs used in the production of the S270 strain.

The expression constructs depicted in FIG. 8 were also used in the production of the following strains: S487, S951, S1000-S1059, SI 072- 1079, and SI 081.

[0067] FIGS. 9A and 9B depict an expression construct used in the production of the

S487 strain. The expression constructs depicted in FIGS. 9A and 9B were also used in the production of the following strains: S951 S1000-S1059, S1072-1079, and S1081.

[0068] FIG. 10 depicts an expression construct used in the production of the SI 042 strain.

[0069] FIG. 11 depicts an expression construct used in the production of the following strains: S951, S1000-S1041, S1043-S1059, S1072-1079, and S1081. Detailed Description

[0070] Synthetic biology allows for the engineering of industrial host organisms — e.g., microbes — to convert simple sugar feedstocks into medicines. This approach includes identifying genes that produce the target molecules and optimizing their activities in the industrial host. Microbial production can be significantly cost-advantaged over agriculture and chemical synthesis, less variable, and allow tailoring of the target molecule. However, reconstituting or creating a pathway to produce a target molecule in an industrial host organism can require significant engineering of both the pathway genes and the host. The present disclosure provides engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing cannabinoids or cannabinoid derivatives, methods of producing cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the THCAS polypeptide. The engineered variants of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids). The modified host cells of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids) and/or for expressing engineered variants of the disclosure. The disclosure also provides for modified host cells for expressing the engineered variants of the disclosure. Additionally, the disclosure provides for preparation of engineered variants of the disclosure.

[0071] Cannabinoid synthase polypeptides, such as tetrahydrocannabinolic acid synthase, cannabichromenic acid synthase, or cannabidiolic acid synthase polypeptides, play an important role in the biosynthesis of cannabinoids. However, reconstituting their activity in a modified host cell has proven challenging, hampering progress in the production of cannabinoids or cannabinoid derivatives. Cannabinoid synthases must successfully traverse the secretory pathway to fold and function properly. These secreted plant enzymes have not evolved to be expressed in a yeast cell, and as a result have poor activity, with limited conversion of their substrate cannabigerolic acid (CBGA) into cannabidiolic acid (CBDA) or tetrahydrocannabinolic acid (THCA). A simple method to increase activity of an enzyme is to increase its copy number (expression). However, expression of the CBDAS and tetrahydrocannabinolic acid synthase (THCAS) genes in yeast is toxic (likely owing to misfolding of the protein), frustrating straightforward attempts to boost activity by integrating multiple copies of the genes. Product profile presents another problem. While the primary product of the natural CBDAS enzyme is CBDA, the enzyme also makes significant amounts of THCA, an undesired byproduct, which would require expensive additional downstream purification steps to separate in an industrial process.

[0072] For these reasons, the natural CBDAS or THCAS enzymes are not optimal for industrial purposes, and improved enzymes are required. Parameters of interest include catalytic activity, product profile, enzyme stability, and pH and temperature optima. Enzyme improvement is typically accomplished by coupling the generation of diversity (a library of engineered variants) to a screen or selection for the properties of interest. DNA libraries encoding engineered variants can be generated in a variety of ways. For example, libraries can be generated using error prone PCR using the wild type gene sequence as a template.

The resulting library can be quite large, consisting of genes with variable numbers of mutations at random positions. Error prone PCR is inexpensive and convenient but has several drawbacks. First, instead of a precise number of mutations per construct, a distribution is obtained. This presents an unfortunate trade-off. A distribution centered around a low number of mutations will include a significant amount of zero-mutation wild- type constructs that waste screening capacity. A distribution centered around a higher number of mutations is likely to generate constructs that have accumulated loss of function mutations that would prevent identification of the desired gain of function mutations.

Second, error prone PCR introduces mutational bias (an intrinsic property of the low fidelity polymerases used) which means that the library underrepresents certain types of mutation. A powerful alternative to error prone PCR is saturation mutagenesis, which involves synthesis of a library containing every possible amino acid at every position in the protein. Recent advances in DNA synthesis technologies have improved the quality of these libraries significantly.

[0073] Once a library encoding engineered variants is generated, it is necessary to select or screen for engineered variants with the properties of interest. This can be accomplished by using a protein production host to express and purify the engineered variants, followed by testing in vitro. Such an approach allows careful measurement of the engineered variants’ kinetic parameters and assessment of performance under carefully controlled conditions. However, for application in an engineered microbial strain, in vitro data can be highly misleading as no in vitro system can represent the cellular milieu accurately. In this case, the best option is to test the engineered variants in the exact context they must eventually perform — inside an engineered production strain. In the case of the cannabinoid synthases, such a production strain would be engineered to produce the substrate CBGA in excess. One challenge with this in vivo system is that variability is higher. When testing a large library, this variability can make it difficult to distinguish clones with more subtle improvements over the wild type enzyme activity. By calculating the ratio of the library enzyme product titer and the invariant competition enzyme titer, it is possible to reduce the variability in data significantly. This is because biological variables tend to affect both of the enzymes in the same way, allowing normalization of the effect. Unlike a kinetic parameter, the competition ratio reports on both changes in both enzyme catalytic parameters such as K _m and K _cat as well as changes in the steady state levels of functional engineered variant (expression and stability).

[0074] Through use of the above methods, the present disclosure provides engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide. Herein, various engineered variants were screened. THCA titers were improved (outside standard deviation of wild type) in 58 distinct variants. These engineered variants of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids). The engineered variants of the disclosure may produce tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. Additionally, the engineered variants of the disclosure may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. Similar conditions may include the same temperature, pH, buffer, and/or fermentation conditions and in the same culture medium and/or reaction solvent.

[0075] The methods of the disclosure may include using engineered microorganisms

(e.g., modified host cells) or engineered variants of a THCAS polypeptide of the disclosure to produce naturally-occurring and non-naturally occurring cannabinoids. Naturally- occurring cannabinoids and non-naturally occurring cannabinoids (e.g., cannabinoid derivatives) are challenging to produce using chemical synthesis due to their complex structures. The methods of the disclosure enable the construction of metabolic pathways inside living cells to produce bespoke cannabinoids or cannabinoid derivatives from simple precursors such as sugars and carboxylic acids. One or more nucleic acids (e.g., heterologous nucleic acids) disclosed herein comprising nucleotide sequences encoding one or more polypeptides or engineered variants disclosed herein can be introduced into host microorganisms allowing for the stepwise conversion of inexpensive feedstocks, e.g., sugar, into final products: cannabinoids or cannabinoid derivatives. These products can be specified by the choice and construction of expression constructs or vectors comprising one or more nucleic acids (e.g., heterologous nucleic acids) disclosed herein, allowing for the efficient bioproduction of chosen cannabinoids, such as THC and THCA and less common cannabinoid species found at low levels in Cannabis ; or cannabinoid derivatives. Bioproduction also enables synthesis of cannabinoids or cannabinoid derivatives with defined stereochemistries, which is challenging to do using chemical synthesis. To produce cannabinoids or cannabinoid derivatives and create biosynthetic pathways within modified host cells, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of a THCAS polypeptide of the disclosure may express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, the nucleotide sequences encoding the polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis are codon- optimized.

[0076] The disclosure also provides for modification of the secretory pathway of a host cell modified with one or more nucleic acids (e.g., heterologous nucleic acids) comprising a nucleotide sequence encoding an engineered variant of a THCAS polypeptide of the disclosure. In some embodiments, the nucleotide sequence encoding the engineered variant of a THCAS polypeptide is codon-optimized. Modification of the secretory pathway in the host cell may improve expression and solubilization of the engineered variants of the disclosure, as these variants are processed through the secretory pathway. Reconstituting the activity of polypeptides processed through the secretory pathway, such as the engineered variants of the disclosure, in a modified host cell, such as a modified yeast cell, can be challenging and unreliable. Often the expressed engineered variants may be misfolded or mislocalized, resulting in low expression, expressed engineered variants lacking activity, engineered variant aggregation, reduced host cell viability, and/or cell death. Additionally, a backlog of misfolded or mislocalized expressed engineered variants can induce metabolic stress within the modified host cell, harming the modified host cell. The expressed engineered variants may lack necessary posttranslational modifications for folding and activity, such as disulfide bonds, glycosylation and trimming, and cofactors, affording inactive polypeptides or polypeptides with reduced enzymatic activity.

[0077] The modified host cell of the disclosure may be a modified yeast cell. Yeast cells may be cultured using known conditions, grow rapidly, and are generally regarded as safe. Yeast cells contain the secretory pathway common to all eukaryotes. As disclosed herein, manipulation of that secretory pathway in yeast host cells modified with one or more nucleic acids (e.g., heterologous nucleic acids) comprising a nucleotide sequence encoding an engineered variant of a THCAS polypeptide of the disclosure may improve expression, folding, and enzymatic activity of the engineered variant as well as viability of the modified yeast host cell, such as modified Saccharomyces cerevisiae. Further, use of codon- optimized nucleotide sequences encoding engineered variants of the disclosure, may improve expression and activity of the engineered variant and viability of modified yeast host cells, such as modified Saccharomyces cerevisiae.

[0078] Besides allowing for the production of desired cannabinoids or cannabinoid derivatives, the present disclosure provides a more reliable and economical process than agriculture-based production. Microbial fermentations can be completed in days versus the months necessary for an agricultural crop, are not affected by climate variation or soil contamination (e.g., by heavy metals), and can produce pure products at high titer.

[0079] The present disclosure also provides a platform for the economical production of high-value cannabinoids, including THC, as well as derivatives thereof. It also provides for the production of different cannabinoids or cannabinoid derivatives for which no viable method of production exists. Using the engineered variants, methods, and modified host cells disclosed herein, cannabinoids and cannabinoid derivatives may be produced in an amount of over 100 mg per liter of culture medium, over 1 g per liter of culture medium, over 10 g per liter of culture medium, over 100 g per liter of culture medium.

[0080] Additionally, the disclosure provides engineered variants of a THCAS polypeptide, methods, modified host cells, and nucleic acids to produce cannabinoids or cannabinoid derivatives in vivo or in vitro from simple precursors. Nucleic acids (e.g., heterologous nucleic acids) disclosed herein can be introduced into microorganisms (e.g., modified host cells), resulting in expression or overexpression of one or more polypeptides, such as the engineered variants of the disclosure, which can then be utilized in vitro or in vivo for the production of cannabinoids or cannabinoid derivatives. In some embodiments, the in vitro methods are cell-free.

Cannabinoid Biosynthesis

[0081] In addition to one or more nucleic acids (e.g., heterologous nucleic acids) encoding an engineered variant of a THCAS polypeptide, one or more nucleic acids (e.g., heterologous nucleic acids) encoding one or more polypeptides having at least one activity of a polypeptide present in the cannabinoid or cannabinoid precursor biosynthetic pathway may be useful in the methods and modified host cells for the synthesis of cannabinoids or cannabinoid derivatives. Cannabinoid precursors may include, for example, geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA.

[0082] In Cannabis , cannabinoids are produced from the common metabolite precursors geranylpyrophosphate (GPP) and hexanoyl-CoA by the action of three polypeptides. Hexanoyl-CoA and malonyl-CoA are combined to afford a 12-carbon tetraketide intermediate by a tetraketide synthase (TKS) polypeptide. This tetraketide intermediate is then cyclized by an olivetolic acid cyclase (OAC) polypeptide to produce olivetolic acid. Olivetolic acid is then prenylated with the common isoprenoid precursor GPP by a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide (e.g., a CsPT4 polypeptide) to produce CBGA, the cannabinoid also known as the “mother cannabinoid.” The engineered variants of a THCAS polypeptide of the disclosure then convert CBGA into other cannabinoids, e.g., THCA, etc. In the presence of heat or light, the acidic cannabinoids can undergo decarboxylation, e.g., THCA producing THC.

[0083] GPP and hexanoyl-CoA can be generated through several pathways. One or more nucleic acids (e.g., heterologous nucleic acids) encoding one or more polypeptides having at least one activity of a polypeptide present in these pathways can be useful in the methods and modified host cells for the synthesis of cannabinoids or cannabinoid derivatives.

[0084] Polypeptides that generate GPP or are part of a biosynthetic pathway that generates GPP may be one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway (e.g., one or more MEV pathway polypeptides). The term “mevalonate pathway” or “MEV pathway,” as used herein, may refer to the biosynthetic pathway that converts acetyl-CoA to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The mevalonate pathway comprises polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoAto generate acetoacetyl-CoA(e.g., by action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoAto form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by action of a HMG-CoA synthase (HMGS) polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by action of a HMG-CoA reductase (HMGR) polypeptide); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of a mevalonate kinase (MK) polypeptide); (e) converting mevalonate 5-phosphate to mevalonate 5- pyrophosphate (e.g., by action of a phosphomevalonate kinase (PMK) polypeptide); (f) converting mevalonate 5 -pyrophosphate to isopentenyl pyrophosphate (e.g., by action of a mevalonate pyrophosphate decarboxylase (MVD1) polypeptide); and (g) converting isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP) (e.g., by action of an isopentenyl pyrophosphate isomerase (IDI1) polypeptide). A geranyl pyrophosphate synthetase (GPPS) polypeptide then acts on IPP and/or DMAPP to generate GPP.

[0085] Polypeptides that generate hexanoyl-CoA may include polypeptides that generate acyl-CoA compounds or acyl-CoA compound derivatives (e.g., an acyl-activating enzyme polypeptide, a fatty acyl-CoA synthetase polypeptide, or a fatty acyl-CoA ligase polypeptide). Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such polypeptides.

[0086] GPP and hexanoyl-CoA may also be generated through pathways comprising polypeptides that condense two molecules of acetyl-CoA to generate acetoacetyl-CoA and pyruvate decarboxylase polypeptides that generate acetyl-CoA from pyruvate via acetaldehyde. Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such pathways.

General Information

[0087] In certain aspects, the practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature: “ Molecular Cloning: A Laboratory Manual ,” second edition (Sambrook et al., 1989); “ Oligonucleotide Synthesis ” (M. J. Gait, ed., 1984); “ Animal Cell Culture ” (R. I. Freshney, ed., 1987);

“ Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology ” (F. M. Ausubel et al., eds., 1987, and periodic updates); “ PCR : The Polymerase Chain Reaction ,” (Mullis et al., eds., 1994). Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0088] “Cannabinoid” or “cannabinoid compound” as used herein may refer to a member of a class of unique meroterpenoids found until now only in Cannabis sativa. Cannabinoids may include, but are not limited to, cannabichromene (CBC) type (e.g. cannabichromenic acid), cannabigerol (CBG) type (e.g. cannabigerolic acid), cannabidiol (CBD) type (e.g. cannabidiolic acid), A ⁹ -trans-tetrahydrocannabinol (D ⁹ -THC) type (e.g. D ⁹ - tetrahydrocannabinolic acid), A ⁸ -trans-tetrahydrocannabinol (D ⁸ -THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, cannabigerolic acid (CBGA), cannabigerolic acid monomethylether (CBGAM), cannabigerol (CBG), cannabigerol monomethylether (CBGM), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-Ci), D ⁹ -tetrahydrocannabinolic acid A (THCA-A), D ⁹ -tetrahydrocannabinolic acid B (THCA-B), D ⁹ -tetrahydrocannabinol (THC), D ⁹ -tetrahydrocannabinolic acid-C4 (THCA-C4), D ⁹ -tetrahydrocannabinol-C4 (THC-C4), D ⁹ -tetrahydrocannabivarinic acid (THCVA), D ⁹ -tetrahydrocannabivarin (THCV), D ⁹ - tetrahydrocannabiorcolic acid (THCA-Ci), D ⁹ -tetrahydrocannabiorcol (THC-Ci), D ⁷ -cis- iso-tetrahydrocannabivarin, D ⁸ -tetrahydrocannabinolic acid (D ⁸ -THCA), D ⁸ - tetrahydrocannabinol (D ⁸ -THC), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovarin (CBLV), cannabielsoic acid A (CBEA-A), cannabielsoic acid B (CBEA- B), cannabielsoin (CBE), cannabielsoinic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methylether (CBNM), cannabinol-C4, (CBN-C4), cannabivarin (CBV), cannabinol-C2 (CNB-C2), cannabiorcol (CBN-Ci), cannabinodiol (CBND), cannabinodivarin (CBVD), cannabitriol (CBT), 10-ethyoxy-9-hydroxy-delta-6a- tetrahydrocannabinol, 8,9-dihydroxyl-delta-6a-tetrahydrocannabinol, cannabitriolvarin (CBTVE), dehydrocannabifuran (DCBF), cannabifuran (CBF), cannabichromanon (CBCN), cannabicitran (CBT), 10-oxo-delta-6a-tetrahydrocannabinol (OTHC), delta-9-cis- tetrahydrocannabinol (cis-THC), 3,4,5,6-tetrahydro-7-hydroxy-alpha-alpha-2-trimethyl-9-n- propyl-2,6-methano-2H-l-benzoxocin-5-methanol (OH-iso-HHCV), cannabiripsol (CBR), and trihydroxy-delta-9-tetrahydrocannabinol (triOH-THC).

[0089] An acyl-CoA compound as detailed herein may include compounds with the following structure: wherein R may be an unsubstituted fatty acid side chain or a fatty acid side chain substituted with or comprising one or more functional and/or reactive groups as disclosed herein (i.e., an acyl-CoA compound derivative).

[0090] As used herein, a hexanoyl CoA derivative, an acyl-CoA compound derivative, a cannabinoid derivative, or an olivetolic acid derivative may refer to hexanoyl CoA, an acyl-CoA compound, a cannabinoid, or olivetolic acid substituted with or comprising one or more functional and/or reactive groups. Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl (including branched and straight chain alkyl groups), alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, heterocyclyl, spirocyclyl, heterospirocyclyl, thioalkyl (or alkylthio), arylthio, heteroarylthio, sulfone, sulfonyl, sulfoxide, amido, alkylamino, dialkylamino, arylamino, alkylarylamino, diarylamino, N-oxide, imide, enamine, imine, oxime, hydrazone, nitrile, aralkyl, cycloalkylalkyl, haloalkyl, heterocyclylalkyl, heteroarylalkyl, nitro, thioxo, and the like. Suitable reactive groups may include, but are not necessarily limited to, azide, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), halide, ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, and the like. A reactive group may facilitate covalent attachment of a molecule of interest. Suitable molecules of interest may include, but are not limited to, a detectable label; imaging agents; a toxin (including cytotoxins); a linker; a peptide; a drug (e.g., small molecule drugs); a member of a specific binding pair; an epitope tag; ligands for binding by a target receptor; tags to aid in purification; molecules that increase solubility; molecules that enhance bioavailability; molecules that increase in vivo half-life; molecules that target to a particular cell type; molecules that target to a particular tissue; molecules that provide for crossing the blood- brain barrier; molecules to facilitate selective attachment to a surface; and the like. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.

[0091] A cannabinoid derivative or olivetolic acid derivative may also refer to a compound lacking one or more chemical moieties found in naturally-occurring cannabinoids or olivetolic acid. Such chemical moieties may include, but are not limited to, methyl, alkyl, alkenyl, methoxy, alkoxy, acetyl, carboxyl, carbonyl, oxo, ester, hydroxyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkenylalkyl, cycloalkenylalkenyl, heterocyclylalkenyl, heteroarylalkenyl, arylalkenyl, heterocyclyl, aralkyl, cycloalkylalkyl, heterocyclylalkyl, heteroarylalkyl, and the like. In some embodiments, a cannabinoid derivative or olivetolic acid derivative may also comprise one or more of any of the functional and/or reactive groups described herein. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.

[0092] The term “nucleic acid” used herein, may refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term may include, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, genes, synthetic DNA or RNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other naturally-occurring, chemically or biochemically modified, non- naturally-occurring, or derivatized nucleotide bases.

[0093] The terms “peptide,” “polypeptide,” and “protein” may be used interchangeably herein, and may refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The polypeptides disclosed herein may include full- length polypeptides, fragments of polypeptides, truncated polypeptides, fusion polypeptides, or polypeptides having modified peptide backbones. The polypeptides disclosed herein may also be variants differing from a specifically recited “reference” polypeptide (e.g., a wild- type polypeptide) by amino acid insertions, deletions, mutations, and/or substitutions.

[0094] An “engineered variant of a tetrahydrocannabinolic acid synthase polypeptide” or “engineered variant of the disclosure” may indicate a non-wild type polypeptide having tetrahydrocannabinolic acid synthase activity. One skilled in the art can measure the tetrahydrocannabinolic acid synthase activity of the engineered variants using known methods. For example, by GC-MS or LC-MS or as described in the examples provided herein. Engineered variants may have amino acid substitutions compared to a wild type tetrahydrocannabinolic acid synthase sequence, such as the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44. In addition to substitutions, engineered variants may comprise truncations, additions, and/or deletions, and/or other mutations compared to a wild type tetrahydrocannabinolic acid synthase sequence, such as the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44. Engineered variants may have substitutions compared a non-wild type tetrahydrocannabinolic acid synthase sequence. In addition to substitutions, engineered variants may comprise truncations, additions, and/or deletions and/or other mutations compared to a non-wild type tetrahydrocannabinolic acid synthase sequence. The engineered variants described herein contain at least one amino acid residue substitution from a parent tetrahydrocannabinolic acid synthase polypeptide. In some embodiments, the parent tetrahydrocannabinolic acid synthase polypeptide is a wild type sequence. In some embodiments, the parent tetrahydrocannabinolic acid synthase polypeptide is a non-wild type sequence.

[0095] As used herein, the term “heterologous” may refer to what is not normally found in nature. As such, a heterologous nucleotide sequence may be: (a) foreign to its host cell (i.e., is “exogenous” to the cell); (b) naturally found in the host cell (i.e., “endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); (c) be naturally found in the host cell but positioned outside of its natural locus; or (d) be naturally found in the host cell, but with introns removed or added. The term “heterologous nucleotide sequence” or the term “heterologous nucleic acid” may refer to a nucleic acid or nucleotide sequence not normally found in a given cell in nature. A codon-optimized nucleotide sequence may be an example of a heterologous nucleotide sequence. The term “heterologous enzyme” or “heterologous polypeptide” may refer to an enzyme or polypeptide that is not normally found in a given cell in nature. The term encompasses an enzyme or polypeptide that is: (a) exogenous to a given cell (i.e., encoded by a nucleic acid that is not naturally present in the host cell or not naturally present in a given context in the host cell); or (b) naturally found in the host cell (e.g., the enzyme or polypeptide is encoded by a nucleic acid that is endogenous to the cell) but that is produced in an unnatural amount (e.g., greater or lesser than that naturally found) in the host cell. For example, a heterologous polypeptide may include a mutated version of a polypeptide naturally occurring in a host cell. A heterologous nucleic acid may be: (a) foreign to its host cell (i.e., is “exogenous” to the cell); (b) naturally found in the host cell (i.e., “endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus. In some embodiments, a heterologous nucleic acid may comprise a codon- optimized nucleotide sequence.

[0096] As used herein, the term “one or more heterologous nucleic acids” or “one or more heterologous nucleotide sequences” may refer to heterologous nucleic acids comprising one or more nucleotide sequences encoding one or more polypeptides. In some embodiments, these one or more heterologous nucleic acids may comprise a nucleotide sequence encoding one polypeptide. In other embodiments, these one or more heterologous nucleic acids may comprise nucleotide sequences encoding more than one polypeptide. In some embodiments, these one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of the same polypeptide. In some embodiments, these one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of different polypeptides.

[0097] As used herein, “increased ratio” may refer to an increase in the molar ratio, an increase in the mass (or weight) ratio, an increase in the molarity ratio, or an increase in the mass concentration (e.g., mg/L or mg/mL) ratio between two products produced by a polypeptide, engineered variant, method, and/or modified host cell disclosed herein compared to the molar ratio, mass (or weight) ratio, molarity ratio, or mass concentration ratio between the same two products produced by another polypeptide, engineered variant, method, and/or modified host cell disclosed herein (e.g., a comparative polypeptide, engineered variant, method, and/or modified host cell disclosed herein). For example, a 100: 1 ratio of THCA over CBCA produced by an engineered variant disclosed herein would be an increased ratio of THCA over CBCA compared to an 11:1 ratio of THCA over CBCA produced by a different engineered variant disclosed herein.

[0098] As used herein, a ratio of products produced by a polypeptide, engineered variant, method, and/or modified host cell disclosed herein, such as the ratio of THCA over CBCA, may refer to a molar ratio, a mass (or weight) ratio, molarity ratio, or a mass concentration (e.g., mg/L or mg/mL) ratio. For example, if a modified host cell disclosed herein produced 4 mM THCA and 1 mM CBCA, the ratio of THCA over CBCA would be 4:1.

[0099] “Operably linked” may refer to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0100] “Isolated” may refer to polypeptides or nucleic acids that are substantially or essentially free from components that normally accompany them in their natural state.

An isolated polypeptide or nucleic acid may be other than in the form or setting in which it is found in nature. Isolated polypeptides and nucleic acids therefore may be distinguished from the polypeptides and nucleic acids as they exist in natural cells. An isolated nucleic acid or polypeptide may be purified from one or more other components in a mixture with the isolated nucleic acid or polypeptide, if such components are present.

[0101] A “modified host cell” (also may be referred to as a “recombinant host cell”) may refer to a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector or construct. For example, a modified eukaryotic host cell may be produced through introduction into a suitable eukaryotic host cell of a heterologous nucleic acid.

[0102] As used herein, a “cell-free system” may refer to a cell lysate, cell extract or other preparation in which substantially all of the cells in the preparation have been disrupted or otherwise processed so that all or selected cellular components, e.g., organelles, proteins, nucleic acids, the cell membrane itself (or fragments or components thereof), or the like, are released from the cell or resuspended into an appropriate medium and/or purified from the cellular milieu. Cell-free systems can include reaction mixtures prepared from purified and/or isolated polypeptides and suitable reagents and buffers.

[0103] In some embodiments, conservative substitutions may be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions may be accomplished by the skilled artisan by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Additionally, by comparing aligned sequences of homologous proteins from different species, conservative substitutions may be identified by locating amino acid residues that have been mutated between species without altering the basic functions of the encoded proteins. The term “conservative amino acid substitution” may refer to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains may consist of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains may consist of serine and threonine; a group of amino acids having amide containing side chains may consist of asparagine and glutamine; a group of amino acids having aromatic side chains may consist of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains may consist of lysine, arginine, and histidine; a group of amino acids having acidic side chains may consist of glutamate and aspartate; and a group of amino acids having sulfur containing side chains may consist of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0104] A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/ebi.ac.uk/ Tools/msa/muscle/mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.

[0105] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0106] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0107] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0108] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cannabinoid compound” or “cannabinoid” may include a plurality of such compounds and reference to “the modified host cell” may include reference to one or more modified host cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. [0109] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

Engineered Variants of the Tetrahydrocannabinolic Acid Synthase (THCAS) Polypeptide

[0110] Disclosed herein are engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions. The inventors have identified amino acid locations of the THCAS polypeptide comprising an amino acid sequence of SEQ ID NO:44 that when substituted, may result in one or more improved properties of the engineered variant. In one aspect of the disclosure, the substitution is at a location corresponding to the position in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa. The THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa comprises the following domains:

1. Signal polypeptide: amino acids 1-28.

2. FAD binding domain: amino acids 77-251.

3. BBE domain: amino acids 480-538.

[0111] The THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa also comprises the following domains surface exposed amino acids: 28-33, 35, 36, 39-45, 47-50,

52, 55-59, 61, 62, 65, 66, 69, 71-77, 79, 80, 82, 88, 89, 90, 94, 98, 101, 102, 104, 109, 114, 115, 124, 125, 126, 133, 134, 136-139, 141-145, 148, 150, 161, 164-168, 176, 183, 197, 202,

205, 208, 213, 215-221, 223, 224, 225, 231, 236, 245, 247, 250, 252, 253, 259, 261, 262-

268, 271, 274, 275, 278, 279, 281, 282, 284, 285, 286, 292, 294, 296-306, 312, 318, 321,

322, 323, 326, 327, 329, 330, 331, 333, 334, 336, 338-341, 343, 344, 349, 356, 358-368,

371-374, 377, 378, 389, 390, 391, 393, 394, 395, 399, 402, 403, 405, 406, 408, 409, 410, 413, 422, 424-430, 437, 438, 444, 446, 448, 450, 451-454, 456, 457, 460, 463, 464, 467, 468, 470, 471, 472, 475-478, 483, 484, 487, 488, 491, 493-502, 504, 505, 508, 509, 513,

516, 517, 520, 524, 525, 527, 528, 530, 532, and 540-545.

[0112] Residue positions in the engineered variants discussed herein are identified with respect to a reference amino acid sequence, the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa (shown herein in Table 1; UniProtKB/Swiss-Prot: Q8GTB6). Accordingly, a reference to “F317” identifies an amino acid that, in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa , is the 317 ^th amino acid from the N-terminus, wherein the methionine is the first amino acid. The 317 ^th amino acid is a phenylalanine (F) in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa. Those of skill in the art appreciate that the F317 amino acid may have a different position in the THCAS polypeptides from different species or in different isoforms. These engineered variants are intended to be encompassed by this disclosure.

[0113] The polypeptide sequence position at which a particular amino acid or amino acid change (“residue difference”) is present is sometimes described herein as “Xn”, or “position n”, where n refers to the amino acid position with respect to the reference sequence. Accordingly, a reference to “X317” identifies an amino acid that, in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa , is the 317 ^th amino acid from the N- terminus.

[0114] A specific substitution mutation, which is a replacement of the specific amino acid in a reference sequence with a different specified residue may be denoted by the conventional notation “X (number) Y”, where X is the single letter identifier of the amino in the reference sequence, “number” is the amino acid position in the reference sequence, and Y is the single letter identifier of the amino acid substitution in the engineered sequence. Accordingly, a reference to “F317Y” identifies a substitution that, in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa , is the 317 ^th amino acid from the N- terminus, phenylalanine, being replaced by tyrosine.

[0115] Cannabinoid synthase polypeptides, secreted polypeptides, have structural features that may hinder expression in modified host cells, such as modified yeast cells. Cannabinoid synthase polypeptides comprise disulfide bonds, numerous glycosylation sites, including A -glycosylation sites, and a bicovalently attached flavin adenine dinucleotide (FAD) cofactor moiety. Accordingly, reconstituting the activity of or expressing cannabinoid synthase polypeptides in a modified host cell, such as a modified yeast cell, can be challenging and unreliable. Often these secreted polypeptides are misfolded or mislocalized, resulting in low expression, polypeptides lacking activity, reduced host cell viability, and/or cell death. As disclosed herein, engineered variants may have improved expression, folding, and enzymatic activity compared to the THCAS polypeptide comprising an amino acid sequence of SEQ ID NO:44. Additionally, expression of the engineered variants of the disclosure may enhance viability of the modified host cells disclosed herein compared to modified host cells expressing a THCAS polypeptide comprising an amino acid sequence of SEQ ID NO:44.

[0116] The disclosure provides for an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions. In certain such embodiments, the engineered variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:44 . In some embodiments, the engineered variant comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:44.

[0117] The disclosure provides for an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, wherein the engineered variant comprises at least one amino acid substitution in a signal polypeptide, a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing. In some embodiments, at least one amino acid substitution is present in the signal polypeptide. In certain such embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions in the signal polypeptide. In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in the signal polypeptide. In some embodiments, at least one amino acid substitution is present in the FAD binding domain. In certain such embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions in the FAD binding domain. In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in the FAD binding domain. In some embodiments, wherein at least one amino acid substitution is present in the FAD domain, the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of X100, X103, X109, X124, X125, X132, X137, X143, X149, X161,

X165, X167, X168, X170, X171, X172, X175, X180, X196, X208, X235, and X250. In some embodiments, wherein at least one amino acid substitution is present in the FAD domain, the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of S100, V103, T109, Q124, V125, L132, S137, H143, V149, W161, K165, E167, N168, S170, F171, P172, Y175, G180, N196, H208,

G235, and A250. In some embodiments, wherein at least one amino acid substitution is present in the FAD domain, the engineered variant comprises at least one amino acid substitution selected from the group consisting of L132M, S170T, F 1711, N196T, N196Q, and N196V. In some embodiments, at least one amino acid substitution is present in the BBE domain. In certain such embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions in the BBE domain. In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, or 15 amino acid substitutions in the BBE domain. In some embodiments, wherein at least one amino acid substitution is present in the BBE domain, the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of X500 and X528. BBE domain, the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of Y500 and N528. In some embodiments, wherein at least one amino acid substitution is present in the BBE domain, the engineered variant comprises at least one amino acid substitution selected from the group consisting of Y500M, Y500V, and N528E.

[0118] The disclosure provides for an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, wherein the engineered variant comprises substitution of at least one surface exposed amino acid. In certain such embodiments, at least one hydrophobic surface exposed amino acid is substituted with a hydrophilic amino acid. In some embodiments, at least one hydrophilic surface exposed amino acid is substituted with a hydrophobic amino acid. In some embodiments, the engineered variant comprises substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 surface exposed amino acids. In some embodiments, the engineered variant comprises substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 surface exposed amino acids. In some embodiments, wherein the engineered variant comprises substitution of at least one surface exposed amino acid, the engineered variant comprises at least one amino acid substitution selected from the group consisting of X132, X170, X171, X196, X261, X269, X317, and X539. In some embodiments, wherein the engineered variant comprises substitution of at least one surface exposed amino acid, the engineered variant comprises at least one amino acid substitution selected from the group consisting of L132, S170, F171, N196, K261,

L269, F317, and P539. In some embodiments, wherein the engineered variant comprises substitution of at least one surface exposed amino acid, the engineered variant comprises at least one amino acid substitution selected from the group consisting of L132M, S170T,

F 1711, N196T, N196Q, N196V, K261C, L269I, F317Y, and P539T. Substitution of hydrophobic surface exposed amino acids with hydrophilic amino acids may increase the hydrophilicity of solvent-exposed amino acids, which may improve solubility of the engineered variants of the disclosure in an aqueous (non-trichome) environment.

[0119] The disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of X31, X43, X49, X50, X 51, X55, X56, X59, X61, X62, X71, X100, X103, X109, X124, X125, X132, X137, X143, X149, X161, X165, X168, X167, X170,

X171, X172, X175, X180, X196, X208, X235, X250, X257, X261, X269, X311, X317,

X327, X390, X379, X429, X467, X500, X528, X539, X542, X543, X544, and X545. Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. In some embodiments, such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. [0120] The disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of R31, P43, P49, K50, L51, Q55, H56, L59, M61, S62, L71, S100, V103, T109, Q124, V125, L132, S137, H143, W161, K165, N168, E167, Y175, G180, N196, H208, A250, 1257, K261, G311, F317, L327, K390, T379, D429, Y500, N528, P542, H543, H544, and H545. Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. In some embodiments, such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0121] The disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution selected from the group consisting of R31Q, P43E, P49E, P49K, P49Q, K50T, L51I, Q55E, Q55P, H56E, L59E, M61W, M61H, M61S, S62Q, L71A, S100A, V103F,T109V,Q124D, Q124E, Q124N, V125E, V125Q, L132M, S137G, H143D, W161R, W161Y, W161K, K165A, N168S, E167P, Y175F, G180A, N196Q, N196V, H208T, A250T, I257V, K261C, G311A, F317Y, L327I, K390E, T3797S, Y500M, Y500V, N528E, P542E, P542V, H543V, H544A, H545D, and H545E. Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. In some embodiments, such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0122] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO: 88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID

NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID

NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID

NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID

NO: 134, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, and SEQ ID NO:186. Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. In some embodiments, such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g. CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO: 44 under similar conditions for the same length of time.

[0123] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions. The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. Combinations of the amino acid substitutions described herein can be made and the resulting engineered variants screened for improved tetrahydrocannabinolic acid synthase (THCAS) properties. Engineered variants comprising combinations of all of the substitutions described herein are intended to be encompassed by this disclosure. In some embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the amino acid substitutions described herein (e.g., 1-30 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the amino acid substitutions described herein (e.g., 1-15 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acid substitutions described herein (e.g., 1-10 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, 3, 4, or 5 of the amino acid substitutions described herein (e.g., 1-5 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, 3, or 4 of the amino acid substitutions described herein (e.g., 1-4 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, or 3 of the amino acid substitutions described herein (e.g., 1-3 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1 or 2 of the amino acid substitutions described herein (e.g., 1-2 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 2 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 3 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 4 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 5 of the amino acid substitutions described herein. [0124] The disclosure provides for an engineered variant, wherein the engineered variant comprises at least one immutable amino acid. The disclosure provides for an engineered variant, wherein the engineered variant comprises at least one immutable amino acid in a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing.

[0125] In some embodiments, the engineered variant comprises at least one immutable amino acid in the FAD binding domain. In certain such embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 immutable amino acids in the FAD binding domain. In some embodiments, the engineered variant comprises at least one immutable amino acid in the FAD binding domain, wherein the at least one immutable amino acid is selected from the group consisting of X87, X93, X99, X108, X110, X112, X117, X118, X120, X126, X127, X131, X141, X148, X152, X153, X155, X156, X157, X159, X160, X163, X170, X171, X172, X173, X174, X176,

X177, X178, X179, X182, X183, X184, X185, X187, X188, X189, X190, X191, X192,

X193, X195, X201, X202, X205, X206, X210, X214, X223, X225, X226, X227, X228,

X231, X234, X237, X238, X239, X245, X246, X248, and X251. In some embodiments, mutation of one or more of these immutable amino acids reduces titer of one or more cannabinoids. In some embodiments, mutation of one or more of amino acids XI 70, X171, and/or XI 72 reduces titer of one or more cannabinoids. In some embodiments, wherein the engineered variant comprises at least one immutable amino acid in the FAD binding domain, the at least one immutable amino acid is selected from the group consisting of P87, 193, C99, R108, R110, G112, E117, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, S170, F171, P172, G173, G174, C176, P177, T178, V179, G182, G183, H184, F185, G187, G188, G189, Y190, G191, A192, L193, R195, A201,

D202, 1205, D206, V210, G214, G223, D225, L226, F227, W228, R231, G234, N237, F238, G239, K245, 1246, L248, and V251. In some embodiments, mutation of one or more of amino acids SI 70, F171, and/or PI 72 reduces titer of one or more cannabinoids.

[0126] In some embodiments, the engineered variant comprises at least one immutable amino acid in the BBE domain. In certain such embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 immutable amino acids in the BBE domain. In some embodiments, wherein the engineered variant comprises at least one immutable amino acid in the BBE domain, the at least one immutable amino acid is selected from the group consisting of X485, X499, X503, X514, X515, X522, X529, X530, X534, X535, and X536. In some embodiments, wherein the engineered variant comprises at least one immutable amino acid in the BBE domain, the at least one immutable amino acid is selected from the group consisting of R485, N499, A503, N514, F515, K522, N529, F530, E534, Q535, and S536.

[0127] The disclosure provides for an engineered variant, wherein the engineered variant comprises at least one immutable amino acid selected from the group consisting of X28, X34, X35, X37, X64, X70, X87, X93, X99, X108, XI 10, XI 12, XI 17, XI 18, X120, X126, X127, X131, X141, X148, X152, X153, X155, X156, X157, X159, X160, X163,

X173, X174, X176, X177, X178, X179, X182, X183, X184, X185, X187, X188, X189,

X190, X191, X192, X193, X195, X201, X202, X205, X206, X210, X214, X223, X225,

X226, X227, X228, X231, X234, X237, X238, X239, X245, X246, X248, X251, X260,

X277, X313, X314, X324, X342, X353, X355, X381, X382, X383, X384, X386, X387,

X392, X413, X416, X420, X423, X426, X431, X432, X434, X435, X436, X438, X441,

X444, X445, X446, X465, X466, X469, X470, X472, X473, X477, X485, X499, X503,

X514, X515, X522, X529, X530, X534, X535, and X536. In certain such embodiments, the engineered variant comprises at least one immutable amino acid selected from the group consisting ofX37, X70, X93, X99, XI 17, X120, X127, X131, X156, X157, X159, X174, X176, X182, X183, X185, X187, X188, X189, X190, X191, X192, X195, X202, X206,

X214, X228, X234, X238, X248, X277, X314, X324, X355, X382, X384, X386, X420,

X423, X436, X441, X444, X445, X472, X477, X514, X515, X529, and X535. In some embodiments, the engineered variant comprises at least one immutable amino acid selected from the group consisting of A28, F34, L35, C37, L64, N70, P87, 193, C99, R108, R110, G112, El 17, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, A173, G174, C176, P177, T178, V179, G182, G183, H184, F185,

G187, G188, G189, Y190, G191, P192, L193, R195, A201, D202, 1205, D206, V210, G214, G223, D225, L226, F227, W228, R231, G234, S237, F238, G239, K245, 1246, L248, V251, V260, Q277, F313, S314, L324, C342, F353, S355, F381, K382, 1383, K384, D386, Y387, 1392, M413, L416, G420, M423, 1426, 1431, P432, P434, H435, R436, G438, Y441, W444, Y445, 1446, 1465, Y466, M469, T470, Y472, V473, P477, R485, N499, A503, N514, F515, K522, N529, F530, E534, Q535, and S536. In certain such embodiments, the engineered variant comprises at least one immutable amino acid selected from the group consisting of C37, N70, 193, C99, E117, S120, F127, D131, G156, E157, Y159, G174, C176, G182,

G183, F185, G187, G188, G189, Y190, G191, P192, R195, D202, D206, G214, W228,

G234, F238, L248, Q277, S314, L324, S355, K382, K384, D386, G420, M423, R436, Y441, W444, Y445, Y472, P477, N514, F515, N529, and Q535.

[0128] The disclosure provides for an engineered variant, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 immutable amino acids, provided that the engineered variant has at least one amino acid substitution compared to SEQ ID NO:44. Engineered variants with combinations of the immutable amino acids and substitiutions described herein can be made and the resulting engineered variants screened for improved tetrahydrocannabinolic acid synthase (THCAS) properties. Engineered variants comprising combinations of all of the substitutions and immutable amino acids described herein are intended to be encompassed by this disclosure.

[0129] The disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution at the C-terminus. In certain such embodiments, a hydrophilic amino acid is replaced with a hydrophobic amino acid. In some embodiments, wherein the engineered variant comprises at least one amino acid substitution at the C-terminus, a hydrophobic amino acid is replaced with a hydrophilic amino acid. Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0130] The disclosure provides for an engineered variant, wherein the engineered variant comprises a truncation at the N-terminus, at the C-terminus, or at both the N- and C- termini. In some embodiments, the engineered variant comprises a truncation at the N- terminus. In some embodiments, the engineered variant comprises a truncation at the C- terminus. In some embodiments, the engineered variant comprises a truncation at both the Isl and C-termini. In some embodiments, the engineered variant lacks a native signal polypeptide (i.e., amino acids 1-28 of SEQ ID NO:44).

[0131] In some embodiments, the engineered variant comprises a truncation at the N- terminus, at the C-terminus, or at both the N- and C-termini, and comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:44. In some embodiments, the engineered variant comprises a truncation at the N-terminus, at the C- terminus, or at both the N- and C-termini, and comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:44.

[0132] In some embodiments, the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the C-terminus. In some embodiments, the engineered variant comprises a truncation of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the C-terminus. In some embodiments, the engineered variant comprises a truncation of at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids at the C-terminus. In some embodiments, the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus (e.g., 1-10 amino acids at the C-terminus). In some embodiments, the engineered variant comprises a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the C-terminus (e.g., 11- 20 amino acids at the C-terminus). In some embodiments, the engineered variant comprises a truncation of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the C-terminus (e.g., 21-30 amino acids at the C-terminus).

[0133] In some embodiments, the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the N-terminus. In some embodiments, the engineered variant comprises a truncation of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus. In some embodiments, the engineered variant comprises a truncation of at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids at the N-terminus. In some embodiments, the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus (e.g., 1-10 amino acids at the N-terminus). In some embodiments, the engineered variant comprises a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminus (e.g., 11- 20 amino acids at the N-terminus). In some embodiments, the engineered variant comprises a truncation of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the N-terminus (e.g., 21-30 amino acids at the N-terminus).

[0134] In some embodiments, a truncated engineered variant of the disclosure may comprise a signal polypeptide. In certain such embodiments, the truncated engineered variant lacks a native signal polypeptide. In some embodiments, the signal polypeptide is a secretory signal polypeptide. In some embodiments, the secretory signal polypeptide is a native secretory signal polypeptide. In some embodiments, the secretory signal polypeptide is a synthetic secretory signal polypeptide. In some embodiments, the secretory signal polypeptide is an endoplasmic reticulum retention signal polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal polypeptide is a mitochondrial targeting signal polypeptide. In some embodiments, the secretory signal polypeptide is a Golgi targeting signal polypeptide. In some embodiments, the secretory signal polypeptide is a vacuolar localization signal polypeptide. In certain such embodiments, the vacuolar localization signal polypeptide is a PEP4t polypeptide or a PRC It polypeptide. In certain such embodiments, the vacuolar localization signal polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal polypeptide is a plasma membrane localization signal polypeptide. In some embodiments, the secretory signal polypeptide is a peroxisome targeting signal polypeptide. In some embodiments, the peroxisome targeting signal polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal polypeptide is a mating factor secretory signal polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal polypeptide is linked to the N-terminus of the engineered variant.

[0135] In some embodiments, a truncated engineered variant of the disclosure may comprise a membrane anchor. A membrane anchor may be a sequence that inserts into a membrane in the cell and anchor an attached polypeptide there. A membrane anchor may be present in a membrane external to the cell (e.g., GPI polypeptides) or internal to the cell (e.g., tail anchors, ER anchoring). Examples of membrane anchors include, but are not limited to, glycosylphosphatidylinositol membrane anchors (GPI polypeptides, e.g. AGA1), CAAX box polypeptides (get prenylated, e.g. RASl), or tail anchored polypeptides with a hydrophobic C-terminus (e.g. phosphatidylinositol 4,5-bisphosphate 5-phosphatase (INP54) has a hydrophobic tail anchor in ER membrane or synaptobrevin 2 (VAMP2) has a hydrophobic poly-I tail anchor in vesicle membranes). [0136] The disclosure provides for an engineered variant, wherein the engineered variant comprises an addition and/or deletion of one or more amino acids.

[0137] Engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, production of THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. Additionally, engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, production of THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time. Similar conditions may refer to reaction conditions at the same temperature, pH, buffer, and/or fermentation conditions and in the same culture medium and/or reaction solvent.

[0138] In some embodiments of the disclosure, the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0139] In some embodiments of the disclosure, the engineered variant produces

THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

[0140] These improved properties may be assessed by the conversion of CBGA to

THCA, or alternatively the conversion of another starting material to a desired cannabinoid or cannabinoid derivative, in vitro with isolated and/or purified engineered variants of the disclosure or in vivo in the context of a modified host cell expressing the engineered variant. In some embodiments, the modified host cell expresses polypeptides involved in the MEV pathway and/or polypeptides involved in cannabinoid biosynthesis and/or comprises modifications to the secretory pathway. It is contemplated that engineered variants of the disclosure having various degrees of stability, solubility, activity, and/or expression level in one or more of the test conditions will find use in the present disclosure for the production of cannabinoids or cannabinoid derivatives in a diversity of host cells.

[0141] Additionally, engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, production of cannabinoids or cannabinoid derivatives by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.

[0142] Additionally, engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant have a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. Additionally, engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. Similar culture conditions may refer to host cells grown in the same culture medium at the same temperature, pH, and/or fermentation conditions.

[0143] Moreover, engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant do not have significantly decreased growth or viability compared to modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. Additionally, engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant do not have significantly decreased growth or viability compared to an unmodified host cell.

Nucleic Acids Comprising Nucleotide Sequences Encoding Engineered Variants of the Tetrahydrocannabinolic Acid Synthase (THCAS) Polypeptide and Expression Vectors and Constructs

[0144] The disclosure provides for nucleic acids comprising nucleotide sequences encoding engineered variants of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein and expression vectors and constructs comprising said nucleic acids.

[0145] The disclosure provides nucleic acids comprising nucleotide sequences encoding engineered variants of the disclosure. Some embodiments of the disclosure relate to a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprising an amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:86, SEQ ID NO: 88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:132, SEQ ID NO: 134, SEQ ID NO:136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, and SEQ ID NO: 186. In some embodiments, the nucleotide sequence is codon-optimized.

[0146] Some embodiments of the disclosure relate to a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprising an amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, or SEQ ID NO: 186. In some embodiments, the nucleotide sequence is codon-optimized.

[0147] The disclosure also provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:145, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, or SEQ ID NO: 185. In some embodiments, the nucleotide sequence is codon- optimized.

[0148] The disclosure provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:145, SEQ ID NO: 147, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO: 159, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, or SEQ ID NO: 185, or a codon degenerate sequence of any of the foregoing. In some embodiments, the nucleotide sequence is codon-optimized.

[0149] The disclosure provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO: 177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, or SEQ ID NO: 185, or a codon degenerate sequence of any of the foregoing. In some embodiments, the nucleotide sequence is codon-optimized.

[0150] The disclosure provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:145, SEQ ID NO: 147, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO: 159, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, or SEQ ID NO: 185. In some embodiments, the nucleotide sequence is codon- optimized.

[0151] Further included are nucleic acids that hybridize to the nucleic acids disclosed herein. Hybridization conditions may be stringent in that hybridization will occur if there is at least a 90%, at least a 95%, or at least a 97% sequence identity with the nucleotide sequence present in the nucleic acid encoding the polypeptides disclosed herein. The stringent conditions may include those used for known Southern hybridizations such as, for example, incubation overnight at 42 °C in a solution having 50% formamide, 5><SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt’s solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, following by washing the hybridization support in O.lxSSC at about 65 °C. Other known hybridization conditions are well known and are described in Sambrook et ah, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2001). [0152] The length of the nucleic acids disclosed herein may depend on the intended use. For example, if the intended use is as a primer or probe, for example for PCR amplification or for screening a library, the length of the nucleic acid will be less than the full length sequence, for example, 15-50 nucleotides. In certain such embodiments, the primers or probes may be substantially identical to a highly conserved region of the nucleotide sequence or may be substantially identical to either the 5’ or 3’ end of the nucleotide sequence. In some cases, these primers or probes may use universal bases in some positions so as to be “substantially identical” but still provide flexibility in sequence recognition. It is of note that suitable primer and probe hybridization conditions are well known in the art.

[0153] Some embodiments of the disclosure relate to a vector comprising one or more nucleic acids disclosed herein. Some embodiments of the disclosure relate to an expression construct comprising one or more nucleic acids disclosed herein. Some embodiments of the disclosure relate to nucleic acids comprising codon-optimized nucleotide sequences encoding the engineered variants of the disclosure. In some embodiments, the nucleic acids disclosed herein are heterologous.

Methods of Screening Engineered Variants of the Tetrahydrocannabinolic Acid Synthase (THCAS) Polypeptide

[0154] The disclosure provides a method of screening an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions. In certain such embodiments, the method involves a competition assay wherein the engineered variant of the disclosure is expressed in a modified host cells alongside a related enzyme.

[0155] Some embodiments of the disclosure relate to a method of screening an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, the method comprising: a) dividing a population of host cells into a control population and a test population; b) co-expressing in the control population a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 and a comparison cannabinoid synthase polypeptide, wherein the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 can convert CBGA to a first cannabinoid, THCA, and the comparison cannabinoid synthase polypeptide can convert the same CBGA to a different second cannabinoid; c) co-expressing in the test population the engineered variant and the comparison cannabinoid synthase polypeptide, wherein the engineered variant may convert CBGA to the same first cannabinoid, THCA, as the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44, and wherein the comparison cannabinoid synthase polypeptide can convert the same CBGA to the second cannabinoid and is expressed at similar levels in the test population and in the control population; d) measuring a ratio of the first cannabinoid, THCA, over the second cannabinoid produced by both the test population and the control population; and e) measuring an amount, in mg/L or mM, of the first cannabinoid produced by both the test population and the control population. In cetain such embodiments, the engineered variant is an engineered variant of the disclosure.

[0156] In some embodiments, the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 by producing the first cannabinoid in a greater amount, as measured in mg/L or mM, by the test population compared to the amount produced by the control population under similar culture conditions for the same length of time. In some embodiments, the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein improved in vivo performance is demonstrated by an increase in the ratio of the first cannabinoid over the second cannabinoid produced by the test population compared to that produced by the control population under similar culture conditions for the same length of time.

[0157] In some embodiments, the cannabinoid synthase polypeptide is a cannabidiolic acid synthase (CBDAS) polypeptide. In certain such embodiments, the CBDAS polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:3. In some embodiments, a nucleotide sequence encoding the CBDAS polypeptide is the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, a nucleotide sequence encoding the CBDAS polypeptide is the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, or a codon degenerate nucleotide sequence thereof. In some embodiments, a nucleotide sequence encoding the CBDAS polypeptide has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:l or SEQ ID NO:2. In some embodiments, the second cannabinoid is CBDA.

Modified Host Cells for Expressing Engineered Variants of the Tetrahydrocannabinolic Acid Synthase (THCAS) Polypeptide and for Producing Cannabinoids and Cannabinoid Derivatives

[0158] The present disclosure provides modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In certain such embodiments, the modified host cells of the disclosure are for expressing an engineered variant and/or for producing a cannabinoid or a cannabinoid derivative. In some embodiments, the nucleotide sequence encoding the engineered variant is codon-optimized.

[0159] The disclosure also provides nucleic acids (e.g., heterologous nucleic acids), which can be introduced into microorganisms (e.g., modified host cells), resulting in expression or overexpression of the engineered variants of the disclosure, which can then be utilized in vitro (e.g., cell-free) or in vivo for the production of cannabinoids or cannabinoid derivatives. In some embodiments, these nucleic acids comprise a codon-optimized nucleotide sequence encoding the engineered variant.

[0160] Cannabinoid synthase polypeptides, secreted polypeptides, such as the engineered variants of the disclosure, have structural features that may hinder expression in modified host cells, such as modified yeast cells. Cannabinoid synthase polypeptides, including the engineered variants of the disclosure, comprise disulfide bonds, numerous glycosylation sites, including A-glycosylation sites, and a bicovalently attached flavin adenine dinucleotide (FAD) cofactor moiety. Often these secreted polypeptides are misfolded or mislocalized, resulting in low expression, polypeptides lacking activity, reduced host cell viability, and/or cell death. As disclosed herein, manipulation of secretory pathway in host cells modified with one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure may improve expression, folding, and enzymatic activity of the engineered variant of the disclosure as well as viability of the modified host cell. In certain such embodiments, the nucleotide sequence encoding the engineered variant is codon-optimized.

[0161] To produce cannabinoids or cannabinoid derivatives and create biosynthetic pathways within modified host cells, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure may express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, the nucleotide sequences encoding the polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis are codon- optimized. In some embodiments, the modified host cells of the disclosure for producing cannabinoid or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprise one or more modifications to modulate the expression of one or more secretory pathway polypeptides. The one or more modifications to modulate the expression of one or more secretory pathway polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure for producing cannabinoids or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, resulting in expression or overexpression of the one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized. In some embodiments, the modified host cell for producing cannabinoids or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, reducing or eliminating the expression of the one or more secretory pathway polypeptides.

In certain such embodiments, the modified host cells comprise a deletion of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the modified host cells comprise a downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[0162] In some embodiments, culturing of a modified host cell for producing cannabinoids or cannabinoid derivatives in a culture medium provides for synthesis of the cannabinoid or the cannabinoid derivative.

[0163] To express an engineered variant of the disclosure, the modified host cells may express or overexpress one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant. In some embodiments, the nucleotide sequences encoding the engineered variants are codon-optimized. In some embodiments, the modified host cells of the disclosure for expressing an engineered variant of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant comprise one or more modifications to modulate the expression of one or more secretory pathway polypeptides. The one or more modifications to modulate the expression of one or more secretory pathway polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure for expressing an engineered variant of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, resulting in expression or overexpression of the one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, reducing or eliminating the expression of the one or more secretory pathway polypeptides. In certain such embodiments, the modified host cells comprise a deletion of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the modified host cells comprise a downregulation of one or more genes encoding one or more secretory pathway polypeptides. Secretory Pathway Modifications

[0164] Secretory pathway polypeptides with modulated expression in the modified host cells of the disclosure may include, but are not limited to: a KAR2 polypeptide, a ROT2 polypeptide, aPDIl polypeptide, an EROl polypeptide, FAD1 polypeptide, aPEP4 polypeptide, and an IREl polypeptide. Expression of secretory pathway polypeptides may be modulated by introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.

[0165] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more of the following genes: a ROT2 gene or a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of one or more of the following genes: a ROT2 gene or a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of one or more of the following genes: a ROT2 gene or a PEP4 gene.

[0166] The secretory pathway polypeptides and heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides may be derived from any suitable source, for example, bacteria, yeast, fungi, algae, human, plant, or mouse. In some embodiments, the secretory pathway polypeptides and heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides may be derived from Pichia pastoris (now known as Komagataella phaffii ), Pichia fmlandica , Pichia trehalophila , Pichia koclamae , Pichia memhranaefaciens , Pichia opuntiae , Pichia thermotolerans , Pichia salictaria , Pichia guercuum , Pichia pijperi , Pichia stiptis , Pichia methanolica , Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha (now known as Pichia angusta ), Yarrowia lipolytica ,

Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pomhe , Scheffer somyces stipites, Dekkera hruxellensis , Blastobotrys adeninivorans (formerly Arxula adeninivorans ), Candida albicans , Aspergillus nidulans , Aspergillus niger , Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense , Fusarium sp., Fusarium gramineum , Fusarium venenatum , Neurospora crassa, and the like. In some embodiments, the disclosure also encompasses orthologous genes encoding the secretory pathway polypeptides disclosed herein. Exemplary secretory pathway polypeptides disclosed herein may also include a full-length secretory pathway polypeptide, a fragment of a secretory pathway polypeptide, a variant of a secretory pathway polypeptide, a truncated secretory pathway polypeptide, or a fusion polypeptide that has at least one activity of a secretory pathway polypeptide.

[0167] Exemplary KAR2 polypeptides disclosed herein may include a full-length

KAR2 polypeptide, a fragment of a KAR2 polypeptide, a variant of a KAR2 polypeptide, a truncated KAR2 polypeptide, or a fusion polypeptide that has at least one activity of a KAR2 polypeptide.

[0168] Exemplary ROT2 polypeptides disclosed herein may include a full-length

ROT2 polypeptide, a fragment of a ROT2 polypeptide, a variant of a ROT2 polypeptide, a truncated ROT2 polypeptide, or a fusion polypeptide that has at least one activity of a ROT2 polypeptide.

[0169] Exemplary PDI1 polypeptides disclosed herein may include a full-length

PDI1 polypeptide, a fragment of a PDI1 polypeptide, a variant of a PDI1 polypeptide, a truncated PDI1 polypeptide, or a fusion polypeptide that has at least one activity of a PDI1 polypeptide.

[0170] Exemplary EROl polypeptides disclosed herein may include a full-length

EROl polypeptide, a fragment of an EROl polypeptide, a variant of an EROl polypeptide, a truncated EROl polypeptide, or a fusion polypeptide that has at least one activity of an EROl polypeptide.

[0171] Exemplary FAD1 polypeptides disclosed herein may include a full-length

FAD1 polypeptide, a fragment of a FAD1 polypeptide, a variant of a FAD1 polypeptide, a truncated FAD1 polypeptide, or a fusion polypeptide that has at least one activity of a FAD1 polypeptide.

[0172] Exemplary PEP4 polypeptides disclosed herein may include a full-length

PEP4 polypeptide, a fragment of a PEP4 polypeptide, a variant of a PEP4 polypeptide, a truncated PEP1 polypeptide, or a fusion polypeptide that has at least one activity of a PEP4 polypeptide.

[0173] Exemplary IREl polypeptides disclosed herein may include a full-length

IREl polypeptide, a fragment of an IREl polypeptide (e.g., missing the first 7 amino acids), a variant of an IREl polypeptide, a truncated IREl polypeptide, or a fusion polypeptide that has at least one activity of an IREl polypeptide. [0174] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more of a KAR2 polypeptide, a ROT2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, a PEP4 polypeptide, or an IREl polypeptide. The one or more modifications to modulate the expression of one or more of a KAR2 polypeptide, a ROT2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, a PEP4 polypeptide, or an IREl polypeptide may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of the KAR2 polypeptide, the PDI1 polypeptide, the EROl polypeptide, the FAD1 polypeptide, or the IREl polypeptide and/or deletion or downregulation of one or more genes encoding one or more of the ROT2 polypeptide or the PEP4 polypeptide in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide resulting in expression or overexpression of the KAR2 polypeptide, the PDI1 polypeptide, the EROl polypeptide, the FAD1 polypeptide, or the IREl polypeptide. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, reducing or eliminating the expression of the ROT2 polypeptide or PEP4 polypeptide.

[0175] In some embodiments, the one or more modifications to modulate the expression of one or more secretory pathway polypeptides may improve modified host cell viability. Improving modified host cell viability may improve the industrial fermentation process. The EROl polypeptide may serve as a partner to the PDI1 polypeptide, a protein disulfide isomerase polypeptide. Modulating the expression of an IREl polypeptide may prevent degradation of expressed engineered variants of the disclosure.

[0176] In some embodiments, the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.

[0177] In some embodiments, the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprising the amino acid sequences set forth in SEQ ID NO:5 (a KAR2 polypeptide), SEQ ID NO:9 (a PDI1 polypeptide), SEQ ID NO:7 (an EROl polypeptide), SEQ ID NO: 192 (a FAD1 polypeptide), SEQ ID NO: 11 (an IRE1 polypeptide), or SEQ ID NO: 190 (a fragment IRE1 polypeptide).

[0178] In some embodiments, the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprising the amino acid sequences set forth in SEQ ID NO:5 (a KAR2 polypeptide), SEQ ID NO:9 (a PDI1 polypeptide), SEQ ID NO:7 (an EROl polypeptide), SEQ ID NO: 192 (a FAD1 polypeptide), SEQ ID NO: 11 (an IRE1 polypeptide), or SEQ ID NO: 190 (a fragment IRE1 polypeptide), or a conservatively substituted amino acid sequence of any of the foregoing.

[0179] In some embodiments, the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprising amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 5 (a KAR2 polypeptide), SEQ ID NO: 9 (a PDI1 polypeptide), SEQ ID NO: 7 (an EROl polypeptide), SEQ ID NO: 192 (a FAD1 polypeptide), SEQ ID NO: 11 (an IRE1 polypeptide), or SEQ ID NO: 190 (a fragment IREl polypeptide).

[0180] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.

[0181] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides comprising the amino acid sequences set forth in SEQ ID NO: 13 (a ROT2 polypeptide) or SEQ ID NO: 15 (a PEP4 polypeptide).

[0182] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IREl polypeptide. [0183] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding a FAD1 polypeptide. In some embodiments, the nucleotide sequences encoding the one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide are codon-optimized.

[0184] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of genes encoding a ROT2 polypeptide and a PEP4 polypeptide. [0185] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a secretory pathway polypeptide, such as, a full-length secretory pathway polypeptide, a fragment of a secretory pathway polypeptide, a variant of a secretory pathway polypeptide, a truncated secretory pathway polypeptide, or a fusion polypeptide that has at least one activity of a secretory pathway polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[0186] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a KAR2 polypeptide, such as, a full- length KAR2 polypeptide, a fragment of a KAR2 polypeptide, a variant of a KAR2 polypeptide, a truncated KAR2 polypeptide, or a fusion polypeptide that has at least one activity of a KAR2 polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0187] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a ROT2 polypeptide, such as, a full- length ROT2 polypeptide, a fragment of a ROT2 polypeptide, a variant of a ROT2 polypeptide, a truncated ROT2 polypeptide, or a fusion polypeptide that has at least one activity of a ROT2 polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0188] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a PDI1 polypeptide, such as, a full- length PDI1 polypeptide, a fragment of a PDI1 polypeptide, a variant of a PDI1 polypeptide, a truncated PDI1 polypeptide, or a fusion polypeptide that has at least one activity of a PDI1 polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[0189] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an EROl polypeptide, such as, a full- length EROl polypeptide, a fragment of an EROl polypeptide, a variant of an EROl polypeptide, a truncated EROl polypeptide, or a fusion polypeptide that has at least one activity of an EROl polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0190] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a FAD1 polypeptide, such as, a full- length FAD1 polypeptide, a fragment of a FAD1 polypeptide, a variant of a FAD1 polypeptide, a truncated FAD1 polypeptide, or a fusion polypeptide that has at least one activity of a FAD1 polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0191] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a PEP4 polypeptide, such as, a full- length PEP4 polypeptide, a fragment of a PEP4 polypeptide, a variant of a PEP4 polypeptide, a truncated PEP1 polypeptide, or a fusion polypeptide that has at least one activity of a PEP4 polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0192] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an IRE1 polypeptide, such as, a full- length IRE1 polypeptide, a fragment of an IRE1 polypeptide (e.g., missing the first 7 amino acids), a variant of an IRE1 polypeptide, a truncated IRE1 polypeptide, or a fusion polypeptide that has at least one activity of an IRE1 polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[0193] In some embodiments, one or more secretory pathway polypeptides, such as a

KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding one or more secretory pathway polypeptides, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, a FAD1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.

[0194] In some embodiments, the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides selected from the group consisting of nucleotide sequences set forth in SEQ ID NO:4 (encodes a KAR2 polypeptide), SEQ ID NO:8 (encodes a PDI1 polypeptide), SEQ ID NO:6 (encodes an EROl polypeptide), SEQ ID NO: 191 (encodes a FADl polypeptide), SEQ ID NO: 10 (encodes an IREl polypeptide), and SEQ ID NO: 189 (encodes a fragment IREl polypeptide).

[0195] In some embodiments, the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides selected from the group consisting of nucleotide sequences set forth in SEQ ID NO:4 (encodes a KAR2 polypeptide), SEQ ID NO:8 (encodes a PDI1 polypeptide), SEQ ID NO:6 (encodes an EROl polypeptide), SEQ ID NO: 191 (encodes a FADl polypeptide), SEQ ID NO: 10 (encodes an IREl polypeptide), and SEQ ID NO: 189 (encodes a fragment IREl polypeptide), or a codon degenerate nucleotide sequence of any of the foregoing.

[0196] In some embodiments, the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides selected from the group consisting of nucleotide sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:4 (encodes a KAR2 polypeptide), SEQ ID NO: 8 (encodes a PDI1 polypeptide), SEQ ID NO:6 (encodes an EROl polypeptide), SEQ ID NO: 191 (encodes a FAD1 polypeptide), SEQ ID NO: 10 (encodes an IRE1 polypeptide), and SEQ ID NO: 189 (encodes a fragment IRE1 polypeptide).

[0197] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides encoded by nucleotide sequences selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 12 (encodes a ROT2 polypeptide) and SEQ ID NO: 14 (encodes a PEP4 polypeptide).

[0198] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a ROT2 gene.

[0199] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a PEP4 gene.

[0200] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a PEP4 gene and a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a PEP4 gene and a ROT2 gene.

In some embodiments, the modified host cells of the disclosure comprise a downregulation of a PEP4 gene and a ROT2 gene.

Cannabinoid and Cannabinoid Precursor Biosynthetic Pathway Modifications [0201] A modified host cell of the present disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure may also comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In addition to engineered variants of the disclosure, such polypeptides may include, but are not limited to: a geranyl pyrophosphate: olivetolic acid geranyltransferase (GOT) polypeptide, a tetraketide synthase (TKS) polypeptide, an olivetolic acid cyclase (OAC) polypeptide, one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway (e.g., one or more MEV pathway polypeptides), an acyl-activating enzyme (AAE) polypeptide, a polypeptide that generates GPP (e.g., a geranyl pyrophosphate synthetase (GPPS) polypeptide), a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., an acetoacetyl-CoA thiolase polypeptide), and a pyruvate decarboxylase polypeptide. In some embodiments, the nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis are codon-optimized.

[0202] The polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis and heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis may be derived from any suitable source, for example, bacteria, yeast, fungi, algae, human, plant (e.g., Cannabis), or mouse. In some embodiments, the disclosure also encompasses orthologous genes encoding the polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis disclosed herein.

Engineered Variants of the Tetrahydrocannabinolic Acid Synthase (THCAS) Polypeptide [0203] A modified host cell of the present disclosure may comprise one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein. In certain such embodiments, the tetrahydrocannabinolic acid synthase polypeptide has an amino acid sequence of SEQ ID NO:44.

[0204] In some embodiments a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, wherein the engineered variant comprises the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, or SEQ ID NO: 186. In some embodiments, the nucleotide sequence is codon- optimized.

[0205] In some embodiments, a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, wherein the engineered variant comprises the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, or SEQ ID NO: 186. In some embodiments, the nucleotide sequence is codon-optimized.

[0206] In some embodiments, the engineered variant of the disclosure is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the engineered variant of the disclosure to a strong promoter. In some embodiments, the modified host cell has one copy of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has two copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has three copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has four copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has five copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has six copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has seven copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has eight copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has eight or more copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure.

[0207] In some embodiments, a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,

SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID

NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID

NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID

NO: 149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID

NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID

NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, or SEQ ID NO: 185. In some embodiments, the nucleotide sequence is codon-optimized. [0208] In some embodiments, a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,

SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:181, SEQ ID NO:183, or SEQ ID NO:185, or a codon degenerate nucleotide sequence of any of the foregoing. In some embodiments, the nucleotide sequence is codon- optimized.

[0209] In some embodiments, a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,

SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO:141, SEQ ID NO: 143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:151, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, or SEQ ID NO: 185. In some embodiments, the nucleotide sequence is codon-optimized.

[0210] In some embodiments, a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105,

SEQ ID NO: 107, SEQ ID NO:109, SEQ ID NO:l l l, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:151, SEQ ID NO: 155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, or SEQ ID NO: 185 or a codon degenerate sequence of any of the foregoing. In some embodiments, the nucleotide sequence is codon-optimized.

[0211] In some embodiments, at least one of the one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure is operably linked to an inducible promoter. In some embodiments, at least one of the one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure is operably linked to a constitutive promoter.

Geranyl Pyrophosphate :Olivetolic Acid Geranyltransferase (GOT) Polypeptides [0212] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide.

[0213] Exemplary GOT polypeptides disclosed herein may include a full-length

GOT polypeptide, a fragment of a GOT polypeptide, a variant of a GOT polypeptide, a truncated GOT polypeptide, or a fusion polypeptide that has at least one activity of a GOT polypeptide. In some embodiments, the GOT polypeptide has aromatic prenyltransferase (PT) activity. In some embodiments, the GOT polypeptide modifies a cannabinoid precursor or a cannabinoid precursor derivative. In certain such embodiments, the GOT polypeptide modifies olivetolic acid or an olivetolic acid derivative.

[0214] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 17.

[0215] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 17. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 17. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 17. [0216] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a GOT polypeptide, such as, a full- length GOT polypeptide, a fragment of a GOT polypeptide, a variant of a GOT polypeptide, a truncated GOT polypeptide, or a fusion polypeptide that has at least one activity of a GOT polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[0217] In some embodiments, the GOT polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the GOT polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the GOT polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide.

[0218] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 16. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 16, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 16.

[0219] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 16. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 16.

[0220] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 16. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 16.

NphB Polypeptides

[0221] In some embodiments, a NphB polypeptide is used instead of a GOT polypeptide to generate cannabigerolic acid from GPP and olivetolic acid. A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide.

[0222] Exemplary NphB polypeptides disclosed herein may include a full-length

NphB polypeptide, a fragment of a NphB polypeptide, a variant of a NphB polypeptide, a truncated NphB polypeptide, or a fusion polypeptide that has at least one activity of a NphB polypeptide. In some embodiments, the NphB polypeptide has aromatic prenyltransferase (PT) activity. In some embodiments, the NphB polypeptide modifies a cannabinoid precursor or a cannabinoid precursor derivative. In certain such embodiments, the NphB polypeptide modifies olivetolic acid or an olivetolic acid derivative.

[0223] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 188, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises an amino acid sequence having at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 188. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 188. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 188.

[0224] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a NphB polypeptide, such as, a full- length NphB polypeptide, a fragment of a NphB polypeptide, a variant of a NphB polypeptide, a truncated NphB polypeptide, or a fusion polypeptide that has at least one activity of a NphB polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0225] In some embodiments, the NphB polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the NphB polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the NphB polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide.

[0226] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 187. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 187, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 187. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 187.

[0227] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 187. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 187. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 187. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 187.

Polypeptides that Generate Acyl-CoA Compounds or Acyl-CoA Compound Derivatives [0228] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates acyl-CoA compounds or acyl-CoA compound derivatives. Such polypeptides may include, but are not limited to, acyl-activating enzyme (AAE) polypeptides, fatty acyl-CoA synthetases (FAA) polypeptides, or fatty acyl-CoA ligase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide. [0229] AAE polypeptides, FAA polypeptides, and fatty acyl-CoA ligase polypeptides can convert carboxylic acids to their CoA forms and generate acyl-CoA compounds or acyl-CoA compound derivatives. Promiscuous acyl -activating enzyme polypeptides, such as CsAAEl and CsAAE3 polypeptides, FAA polypeptides, or fatty acyl- CoA ligase polypeptides, may permit generation of cannabinoid derivatives (e.g., cannabigerolic acid derivatives), as well as cannabinoids (e.g., cannabigerolic acid). In some embodiments, unsubstituted or substituted hexanoic acid or carboxylic acids other than unsubstituted or substituted hexanoic acid are fed to modified host cells expressing an AAE polypeptide, FAA polypeptide, or fatty acyl-CoA ligase polypeptide (e.g., are present in the culture medium in which the cells are grown) to generate hexanoyl-CoA, acyl-CoA compounds, derivatives of hexanoyl-CoA, or derivatives of acyl-CoA compounds. The hexanoyl-CoA, acyl-CoA compounds, derivatives of hexanoyl-CoA, or derivatives of acyl- CoA compounds can then be further utilized by a modified host cell to generate cannabinoids or cannabinoid derivatives. In certain such embodiments, the cell culture medium comprising the modified host cells comprises unsubstituted or substituted hexanoate. In some embodiments, the cell culture medium comprising the modified host cells comprises a carboxylic acid other than unsubstituted or substituted hexanoate.

[0230] Exemplary AAE, FAA, or fatty acyl-CoA ligase polypeptides disclosed herein may include a full-length AAE, FAA, or fatty acyl-CoA ligase polypeptide; a fragment of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a variant of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a truncated AAE, FAA, or fatty acyl-CoA ligase polypeptide; or a fusion polypeptide that has at least one activity of an AAE, FAA, or fatty acyl-CoA ligase polypeptide.

[0231] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:23. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:23. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:23.

[0232] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an AAE, FAA, or fatty acyl-CoA ligase polypeptide, such as, a full-length AAE, FAA, or fatty acyl-CoA ligase polypeptide; a fragment of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a variant of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a truncated AAE, FAA, or fatty acyl-CoA ligase polypeptide; or a fusion polypeptide that has at least one activity of an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0233] In some embodiments, one or more AAE, FAA, or fatty acyl-CoA ligase polypeptide are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the AAE, FAA, or fatty acyl-CoA ligase polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking a nucleotide sequence encoding the AAE, FAA, or fatty acyl-CoA ligase polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide.

[0234] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:22. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:22, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:22. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:22.

Polypeptides that Condense an Acyl-CoA Compound or an Acyl-CoA Compound Derivative with Malonyl-CoA to Generate Olivetolic Acid or Derivatives of Olivetolic Acid [0235] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more polypeptides that condense an acyl-CoA compound, such as hexanoyl-CoA, or an acyl-CoA compound derivative, such as a hexanoyl-CoA derivative, with malonyl-CoA to generate olivetolic acid, or a derivative of olivetolic acid. Polypeptides that react an acyl-CoA compound or an acyl-CoA compound derivative with malonyl-CoA to generate olivetolic acid, or a derivative of olivetolic acid, may include TKS and OAC polypeptides. TKS and OAC polypeptides have been found to have broad substrate specificity, enabling production of cannabinoid derivatives or cannabinoids. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide.

[0236] Exemplary TKS or OAC polypeptides disclosed herein may include a full- length TKS or OAC polypeptide, a fragment of a TKS or OAC polypeptide, a variant of a TKS or OAC polypeptide, a truncated TKS or OAC polypeptide, or a fusion polypeptide that has at least one activity of a TKS or OAC polypeptide.

[0237] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 19. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 19. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 19.

[0238] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:48. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:48, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:21 or SEQ ID NO:48. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:21 or SEQ ID NO:48. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:21 or SEQ ID NO:48.

[0239] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:21. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:21. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:21.

[0240] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:48, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:48. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:48. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:48.

[0241] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a TKS or OAC polypeptide, such as, a full-length TKS or OAC polypeptide, a fragment of a TKS or OAC polypeptide, a variant of a TKS or OAC polypeptide, a truncated TKS or OAC polypeptide, or a fusion polypeptide that has at least one activity of a TKS or OAC polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[0242] In some embodiments, the TKS polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the TKS polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the TKS polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has nine copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has ten copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has eleven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has twelve copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has twelve or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide.

[0243] In some embodiments, the OAC polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the OAC polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has nine copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has ten copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has eleven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has twelve copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has twelve or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide.

[0244] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 18. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 18, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 18. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 18.

[0245] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20 or SEQ ID NO:47. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20 or SEQ ID NO:47, or a codon degenerate nucleotide sequence of any of the foregoing. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:20 or SEQ ID NO:47. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:20 or SEQ ID NO:47.

[0246] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:20. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:20.

[0247] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:47. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:47, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:47. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:47.

Polypeptides that Generate Geranyl Pyrophosphate

[0248] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates GPP. In some embodiments, the polypeptide that generates GPP is a geranyl pyrophosphate synthetase (GPPS) polypeptide. In some embodiments, the GPPS polypeptide also has farnesyl diphosphate synthase (FPPS) polypeptide activity. In some embodiments, the GPPS polypeptide is modified such that it has reduced FPPS polypeptide activity (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than at least 90%, less FPPS polypeptide activity) than the corresponding wild-type or parental GPPS polypeptide from which the modified GPPS polypeptide is derived. In some embodiments, the GPPS polypeptide is modified such that it has substantially no FPPS polypeptide activity. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide.

[0249] Exemplary GPPS polypeptides disclosed herein may include a full-length

GPPS polypeptide, a fragment of a GPPS polypeptide, a variant of a GPPS polypeptide, a truncated GPPS polypeptide, or a fusion polypeptide that has at least one activity of a GPPS polypeptide.

[0250] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:41, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:41. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:41. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:41. The mutation in this amino acid sequence shifts the ratio of GPP to farnesyl diphosphate (FPP), increasing the production of the GPP required to produce CBDA or THCA.

[0251] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a GPPS polypeptide, such as, a full- length GPPS polypeptide, a fragment of a GPPS polypeptide, a variant of a GPPS polypeptide, a truncated GPPS polypeptide, or a fusion polypeptide that has at least one activity of a GPPS polypeptide. In some embodiments, the nucleotide sequence is codon- optimized.

[0252] In some embodiments, the GPPS polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the GPPS polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the GPPS polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide.

[0253] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:40. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:40, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:40. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:40.

Polypeptides that Generate Acetyl-CoA from Pyruvate

[0254] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates acetyl-CoA from pyruvate. Polypeptides that generate acetyl-CoA from pyruvate may include a pyruvate decarboxylase (PDC) polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide.

[0255] Exemplary PDC polypeptides disclosed herein may include a full-length PDC polypeptide, a fragment of a PDC polypeptide, a variant of a PDC polypeptide, a truncated PDC polypeptide, or a fusion polypeptide that has at least one activity of a PDC polypeptide. [0256] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:35, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:35. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:35. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises an amino acid sequence having at least

85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:35.

[0257] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a PDC polypeptide, such as, a full- length PDC polypeptide, a fragment of a PDC polypeptide, a variant of a PDC polypeptide, a truncated PDC polypeptide, or a fusion polypeptide that has at least one activity of a PDC polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[0258] In some embodiments, the PDC polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDC polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the PDC polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide.

[0259] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:34. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:34, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:34. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:34.

Polypeptides that Condense Two Molecules of Acetyl-CoA to Generate Acetoacetyl-Co A [0260] A modified host cell of the disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA. In some embodiments, the polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA is an acetoacetyl-CoA thiolase polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide.

[0261] Exemplary acetoacetyl-CoA thiolase polypeptides disclosed herein may include a full-length acetoacetyl-CoA thiolase polypeptide, a fragment of an acetoacetyl- CoA thiolase polypeptide, a variant of an acetoacetyl-CoA thiolase polypeptide, a truncated acetoacetyl-CoA thiolase polypeptide, or a fusion polypeptide that has at least one activity of an acetoacetyl-CoA thiolase polypeptide.

[0262] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl- CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least

50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:31. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:31. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:31.

[0263] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an acetoacetyl-CoA thiolase polypeptide, such as, a full-length acetoacetyl-CoA thiolase polypeptide, a fragment of an acetoacetyl-CoA thiolase polypeptide, a variant of an acetoacetyl-CoA thiolase polypeptide, a truncated acetoacetyl-CoA thiolase polypeptide, or a fusion polypeptide that has at least one activity of an acetoacetyl-CoA thiolase polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[0264] In some embodiments, the acetoacetyl-CoA thiolase polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the acetoacetyl- CoA thiolase polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide.

[0265] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl- CoA thiolase polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:30. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:30, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:30. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:30.

Mevalonate Pathway Polypeptides

[0266] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway. In certain such embodiments, the one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway comprise one or more MEV pathway polypeptides.

[0267] In some embodiments, the one or more polypeptides that are part of a biosynthetic pathway that generates GPP are one or more polypeptides having at least one activity of a polypeptide present in the mevalonate pathway. The mevalonate pathway may comprise polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoAto generate acetoacetyl-CoA (e.g., by action of an acetoacetyl-CoAthiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoAto form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by action of a HMGS polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by action of an HMGR polypeptide); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of a MK polypeptide); (e) converting mevalonate 5-phosphate to mevalonate 5 -pyrophosphate (e.g., by action of a PMK polypeptide); (f) converting mevalonate 5 -pyrophosphate to isopentenyl pyrophosphate (e.g., by action of a mevalonate pyrophosphate decarboxylase (MPD or MVD1) polypeptide); and (g) converting isopentenyl pyrophosphate to dimethylallyl pyrophosphate (e.g., by action of an isopentenyl pyrophosphate isomerase (IDI1) polypeptide).

[0268] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding a MEV pathway polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than one MEV pathway polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than two MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than three MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than four MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than five MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than six MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding all MEV pathway polypeptides.

[0269] Exemplary MEV pathway polypeptides disclosed herein may include a full- length MEV pathway polypeptide, a fragment of a MEV pathway polypeptide, a variant of a MEV pathway polypeptide, a truncated MEV pathway polypeptide, or a fusion polypeptide that has at least one activity of a MEV pathway polypeptide. In some embodiments, the one or more MEV pathway polypeptides are selected from the group consisting of an acetoacetyl-CoA thiolase polypeptide, a HMGS polypeptide, a HMGR polypeptide, an MK polypeptide, a PMK polypeptide, an MVD1 polypeptide, and an IDI1 polypeptide.

[0270] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:29. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:29. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:29.

[0271] In some embodiments, the HMGR polypeptide is a truncated HMGR

(tHMGR) polypeptide. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:27. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:27. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:27.

[0272] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:39. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:39, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:39. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:39. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:39.

[0273] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:37. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:37. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:37.

[0274] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:33, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:33. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:33. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:33.

[0275] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDI1 polypeptide, wherein the IDI1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the IDIl polypeptide comprises the amino acid sequence set forth in SEQ ID NO:25, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the IDIl polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:25. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the IDIl polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:25. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDI1 polypeptide, wherein the IDI1 polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:25.

[0276] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a MEV pathway polypeptide, such as, a full-length MEV pathway polypeptide, a fragment of a MEV pathway polypeptide, a variant of a MEV pathway polypeptide, a truncated MEV pathway polypeptide, or a fusion polypeptide that has at least one activity of a polypeptide that is part of the MEV pathway.

In some embodiments, the nucleotide sequence is codon-optimized.

[0277] In some embodiments, one or more MEV pathway polypeptides are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding a MEV pathway polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding a MEV pathway polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide.

[0278] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:28. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:28, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:28. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:28.

[0279] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:26. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:26, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:26. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:26.

[0280] In some embodiments, a modified host cell of the present disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence that encodes a tHMGR polypeptide. In some embodiments, a modified host cell of the present disclosure comprises two heterologous nucleic acids comprising a nucleotide sequence that encodes a tHMGR polypeptide.

[0281] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:38. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:38, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:38. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:38.

[0282] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:36. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:36, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:36. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:36.

[0283] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:32. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:32, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:32. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:32.

[0284] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:24. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:24, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:24. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:24.

Modified Host Cells to Produce Cannabinoids or Cannabinoid Derivatives and/or Express Engineered Variants of the Disclosure

[0285] The present disclosure provides modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. The modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure may be for producing cannabinoids or cannabinoid derivatives and/or for expressing an engineered variant of the disclosure.

[0286] The disclosure provides for modified host cells for producing cannabinoids or cannabinoid derivatives. For producing cannabinoids or cannabinoid derivatives, modified host cells disclosed herein may be modified to express or overexpress one or more nucleic acids disclosed herein comprising nucleotide sequences encoding an engineered variant of the disclosure, one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, and/or one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. A modified host cell for producing cannabinoids or cannabinoid derivatives may comprise a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In certain such embodiments, the modified host cell for producing cannabinoids or cannabinoid derivatives may comprise a deletion of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the modified host cell for producing cannabinoids or cannabinoid derivatives may comprise a downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a codon- optimized nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments, the nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, and/or one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis are codon-optimized.

[0287] The disclosure also provides modified host cells modified to express or overexpress one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments of the modified host cell for expressing an engineered variant of the disclosure, the modified host cell comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure and one or more heterologous nucleic acids disclosed herein comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, aFADl polypeptide, or an IRE1 polypeptide. In some embodiments of the modified host cell for expressing an engineered variant of the disclosure, the modified host cell comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In certain such embodiments, the modified host cell may comprise a deletion of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the modified host cell may comprise a downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments of the modified host cell for expressing an engineered variant of the disclosure, the nucleotide sequence encoding the engineered variant of the disclosure is a codon-optimized nucleotide sequence. In some embodiments, the nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide are codon-optimized.

[0288] To produce cannabinoids or cannabinoid derivatives, expression or overexpression of one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure in a modified host cell may be done in combination with expression or overexpression by the modified host cell of one or more heterologous nucleic acids disclosed herein (e.g., one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide) and/or with deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the nucleotide sequence encoding the engineered variant of the disclosure is a codon-optimized nucleotide sequence. [0289] To express or overexpress an engineered variant of the disclosure, expression or overexpression of one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant in a modified host cell may be done in combination with expression or overexpression by the modified host cell of one or more heterologous nucleic acids disclosed herein (e.g., one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide) and/or with deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the nucleotide sequence encoding the engineered variant is a codon-optimized nucleotide sequence.

[0290] In some embodiments, a modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell for producing cannabinoids or cannabinoid derivatives produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.

[0291] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0292] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FADl polypeptide, or an IRE1 polypeptide produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, a FAD1 polypeptide, an EROl polypeptide, or an IREl polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0293] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0294] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0295] In some embodiments, the modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield and an increased titer of THCA compared to a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.

[0296] In some embodiments, the modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.

[0297] In some embodiments, the modified host cell of the disclosure for expressing an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell of the disclosure expressing an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield and an increased titer of THCA compared to a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.

[0298] In some embodiments, the modified host cell of the disclosure for expressing an engineered variant of the disclosure has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell of the disclosure for expressing an engineered variant of the disclosure has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. [0299] In some embodiments, the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield and an increased titer of THCA compared to a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.

[0300] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0301] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FADl polypeptide, or an IREl polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FADl polypeptide, or an IREl polypeptide has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, the modified host cells comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0302] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0303] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0304] In some embodiments, a modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell for producing cannabinoids or cannabinoid derivatives produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

[0305] In some embodiments, a modified host cell of the disclosure for expressing an engineered variant of the disclosure produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

[0306] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200: 1, about 500: 1, or greater than about 500: 1. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0307] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FADl polypeptide, or an IREl polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FADl polypeptide, or an IREl polypeptide produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FADl polypeptide, or an IRE1 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0308] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0309] In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces THCA from CBGA in an increased ratio of THCA over another a cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500: 1. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0310] In some embodiments, the growth and/or viability of modified host cells of the disclosure for producing cannabinoids or cannabinoid derivatives is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure for producing cannabinoids or cannabinoid derivatives has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions.

[0311] In some embodiments, the growth and/or viability of modified host cells of the disclosure for expressing an engineered variant of the disclosure is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure for expressing an engineered variant of the disclosure has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions.

[0312] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0313] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide is not significantly decreased compared to the growth and/or viability of an unmodified host cell.

In some embodiments, a culture of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. [0314] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide is not significantly decreased compared to the growth and/or viability of an unmodified host cell.

In some embodiments, a culture of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

[0315] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments of the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.

Suitable Host Cells

[0316] Parent host cells that are suitable for use in generating a modified host cell of the present disclosure may include eukaryotic cells. In some embodiments, the eukaryotic cells are yeast cells.

[0317] Host cells (including parent host cells and modified host cells) are in some embodiments unicellular organisms, or are grown in culture as single cells. In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells may include, but are not limited to, yeast cells and fungal cells. Suitable eukaryotic host cells may include, but are not limited to, Pichia pastoris (now known as Komagataella phaffii ), Pichia fmlandica , Pichia trehalophila , Pichia koclamae , Pichia membranaefaciens , Pichia opuntiae , Pichia thermotolerans , Pichia salictaria , Pichia guercuum , Pichia pijperi , Pichia stiptis , Pichia methanolica , Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha (now known as Pichia angusta ), Yarrowia lipolytica , Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pombe , Scheffer somyces stipites, Dekkera bruxellensis , Blastobotrys adeninivorans (formerly Arxula adeninivorans ), Candida albicans , Aspergillus nidulans , Aspergillus niger , Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense , Fusarium sp., Fusarium gramineum , Fusarium venenatum , Neurospora crassa, and the like. In some embodiments, the modified host cell disclosed herein is cultured in vitro.

[0318] In some embodiments, the host cell of the disclosure is a yeast cell. In some embodiments, the host cell is a protease-deficient strain of Saccharomyces cerevisiae. Protease-deficient yeast strains may be effective in reducing the degradation of expressed heterologous proteins. Examples of proteases deleted in such strains may include one or more of the following: PEP 4 , PRBf and KEX1.

[0319] In some embodiments, the host cell is Saccharomyces cerevisiae. In some embodiments, the host cell for use in generating a modified host cell of the present disclosure may be selected because of ease of culture; rapid growth; availability of tools for modification, such as promoters and vectors; and the host cell’s safety profile. In some embodiments, the host cell for use in generating a modified host cell of the present disclosure may be selected because of its ability or inability to introduce certain posttranslational modifications onto expressed polypeptides, such as engineered variants of the disclosure. For instance, modified Komagataella phaffli host cells may hyperglycosylate engineered variants of the disclosure and hyperglycosylation may alter the activity of the resultant expressed polypeptide.

Genetic Modification of Host Cells and Exemplary Modified Host Cells of the Disclosure [0320] The present disclosure provides for modified host cells and methods of making modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments, the method of making a modified host cell of the disclosure comprises introducing into a host cell one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments, the modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments, the nucleic acids comprise codon-optimized nucleotide sequences.

[0321] The present disclosure provides for modified host cells and methods of making modified host cells for producing a cannabinoid or a cannabinoid derivative, comprising introducing into a host cell one or more nucleic acids (e.g., heterologous) disclosed herein. In some embodiments, the nucleic acids comprise codon-optimized nucleotide sequences.

[0322] The disclosure provides a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising a) introducing into a host cell one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In certain such embodiments, the method comprises b) introducing into the host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide. In some embodiments, the method comprises b) introducing into the host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide. In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a codon-optimized nucleotide sequence encoding an engineered variant of the disclosure.

[0323] In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide. In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. [0324] In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide or a FAD1 polypeptide. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide. In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. [0325] In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of ROT2 polypeptide or a PEP4 polypeptide. In certain such embodiments, the modified host cell comprises a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide. The disclosure provides a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing into a host cell one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.

[0326] In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide and a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide. In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[0327] The disclosure provides a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and c) a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.

[0328] The disclosure provides a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.

[0329] In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative may comprise one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, the methods of making a modified host cell for producing a cannabinoid or a cannabinoid derivative comprise introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis.

[0330] In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IRE1 polypeptide and a deletion or downregulation of the genes encoding the ROT2 polypeptide and the PEP4 polypeptide. In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[0331] In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide. In some embodiments, the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. [0332] The present disclosure provides for a method of making a modified host cell for expressing an engineered variant of the disclosure, the method comprising introducing into a host cell one or more nucleic acids disclosed herein. The disclosure provides a method of making a modified host cell for expressing an engineered variant of the disclosure, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide. The disclosure provides a method of making a modified host cell for expressing an engineered variant of the disclosure, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a codon-optimized nucleotide sequence encoding the engineered variant of the disclosure.

[0333] In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure and comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. [0334] In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure and comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. [0335] In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In certain such embodiments, the modified host cell comprises a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide. The disclosure provides a method of making a modified host cell for expressing an engineered variant of the disclosure, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and b) a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.

[0336] In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide and a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. [0337] The disclosure provides a method of making a modified host cell for expressing an engineered variant of the disclosure, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and c) a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.

[0338] The disclosure provides a method of making a modified host cell for expressing an engineered variant of the disclosure, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.

[0339] In some embodiments, the modified host cell for expressing an engineered variant of the disclosure may comprise one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, the methods of making a modified host cell for expressing an engineered variant of the disclosure comprise introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis.

[0340] In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IRE1 polypeptide and a deletion or downregulation of the genes encoding the ROT2 polypeptide and the PEP4 polypeptide. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[0341] In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide. In some embodiments, the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. [0342] To modify a parent host cell to produce a modified host cell of the present disclosure, one or more nucleic acids (e.g., heterologous) disclosed herein may be introduced stably or transiently into a host cell, using established techniques. Such techniques may include, but are not limited to, electroporation, calcium phosphate precipitation, DEAE- dextran mediated transfection, liposome-mediated transfection, the lithium acetate method, and the like. See Gietz, R.D. and R. A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. For stable transformation, a nucleic acid (e.g., heterologous) will generally include a selectable marker, e.g., any of several well- known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like. In some embodiments, a parent host cell is modified to produce a modified host cell of the present disclosure using a CRISPR/Cas9 system to modify a parent host cell with one or more nucleic acids (e.g., heterologous) disclosed herein.

[0343] In some embodiments, varying engineered variant expression level and/or the production of cannabinoids or cannabinoid derivatives in a modified host cell may be done by changing the gene copy number, promoter strength, and/or promoter regulation.

[0344] One or more nucleic acids (e.g., heterologous) disclosed herein can be present in an expression vector or construct. Suitable expression vectors may include, but are not limited to, plasmids, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as yeast). Thus, for example, one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding a mevalonate pathway gene product(s) is included in any one of a variety of expression vectors for expressing the mevalonate pathway gene product(s). Such vectors may include chromosomal, non- chromosomal, and synthetic DNA sequences.

[0345] The present disclosure provides for a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing into a host cell one or more vectors disclosed herein. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising a nucleotide sequence encoding an engineered variant of the disclosure. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In some embodiments, the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequences encoding one or more secretory pathway polypeptides are codon- optimized. In some embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis. In some embodiments, the nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis are codon- optimized. [0346] The present disclosure provides for a method of making a modified host cell for expressing a cannabinoid synthase polypeptide, the method comprising introducing into a host cell one or more vectors disclosed herein. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising a nucleotide sequence encoding an engineered variant of the disclosure. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequences encoding one or more secretory pathway polypeptides are codon- optimized. In some embodiments, the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[0347] Numerous additional suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for yeast, the low copy CEN ARS and high copy 2 micron plasmids. However, any other plasmid or other vector may be used so long as it is compatible with the host cell. [0348] In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, two or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, three or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, four or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, five or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, six or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, seven or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector.

[0349] In some embodiments, two or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, three or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, four or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, five or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, six or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, seven or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, eight or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, nine or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, ten or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors.

[0350] In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct. In some embodiments, two or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct. In some embodiments, three or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct. In some embodiments, four or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct. In some embodiments, five or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct. In some embodiments, six or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct. In some embodiments, seven or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.

[0351] In some embodiments, two or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, three or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, four or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, five or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, six or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, seven or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, eight or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, nine or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs. In some embodiments, ten or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs.

[0352] In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a high copy number plasmid, e.g., a plasmid that exists in about 10-50 copies per cell, or more than 50 copies per cell. In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a low copy number plasmid. In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a medium copy number plasmid. The copy number of the plasmid may be selected to reduce expression of one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression by limiting the copy number of the plasmid may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability. [0353] In some embodiments, the modified host cell has one copy of a nucleic acid

(e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has two copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has three copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has four copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has five copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has six copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has seven copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has eight copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has nine copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has ten copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has eleven copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has twelve copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has twelve or more copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. [0354] Depending on the host/vector or host/construct system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector or construct (see e.g., Bitter et al. (1987) Methods in Emymology , 153:516-544).

[0355] In some embodiments, the nucleic acids (e.g., heterologous) disclosed herein are operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is functional in a eukaryotic cell. In some embodiments, the promoter can be a strong driver of expression. In some embodiments, the promoter can be a weak driver of expression. In some embodiments, the promoter can be a medium driver of expression. The promoter may be selected to reduce expression of one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression through promoter selection may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability. Examples of strong constitutive promoters include, but are not limited to: pTDH3 and pFBAl. Examples of medium constitutive promoters include, but are not limited to: pACTl and pCYCl. An example of a weak constitutive promoter includes, but is not limited to: pSLNl. Examples of strong inducible promoters include, but are not limited to: pGALl and pGALlO. An example of a medium inducible promoter includes, but is not limited to: pGAL7. An example of a weak inducible promoter includes, but is not limited to: pGAL3. [0356] Non-limiting examples of suitable eukaryotic promoters may include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector, construct, and promoter is well within the level of ordinary skill in the art. The expression vector or construct may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector or construct may also include appropriate sequences for amplifying expression.

[0357] In yeast, a number of vectors or constructs containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathem et al., Cold Spring Harbor Press, Vols. I and II. A constitutive yeast promoter such as pADH, pTDH3, pFBAl, pACTl, pCYCl, and pSLNl or an inducible promoter such as pGALl, pGALlO, pGAL7, and pGAL3 may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.

[0358] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the S. cerevisiae TRPl gene or a gene cassette encoding resistance to an antibiotic, etc.; and a promoter derived from a highly-expressed gene to direct transcription of the coding sequence. Such promoters can be derived from genetic sequences encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.

[0359] Inducible promoters are well known in the art. Suitable inducible promoters may include, but are not limited to, a tetracycline-inducible promoter; an estradiol inducible promoter, a sugar inducible promoter, e.g, pGall or pSUC2, an amino acid inducible promoter, e.g. pMet25; a metal inducible promoter, e.g. pCupl, a methanol-inducible promoter, e.g. pAOXl, and the like.

[0360] In addition, the expression vectors or constructs will in many embodiments contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture.

[0361] In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into the genome of the modified host cell disclosed herein. In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into a chromosome of the modified host cell disclosed herein. In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein remains episomal (i.e., is not integrated into the genome or a chromosome of the modified host cell). In some embodiments, at least one of the one or more nucleic acids (e.g., heterologous) disclosed herein is maintained extrachromosomally. The gene copy number of one or more genes encoding one or more polypeptides disclosed herein, such as an engineered variant of the disclosure, may be selected to reduce expression of the one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression by limiting the gene copy number may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability.

[0362] As will be appreciated by the skilled artisan, slight changes in nucleotide sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. It will be appreciated by persons skilled in the art that changes in the identities of nucleotides in a specific gene sequence that change the amino acid sequence of the encoded polypeptide may result in reduced or enhanced effectiveness of the genes and that, in some applications (e.g., anti-sense, co-suppression, or RNAi), partial sequences often work as effectively as full length versions. The ways in which the nucleotide sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of the altered genes. In certain embodiments, effectiveness may easily be tested by, for example, conventional gas chromatography. All such variations of the genes are therefore included as part of the present disclosure.

[0363] Genomic deletion of the open reading frame encoding the protein may abolish all expression of a gene. Downregulation of a gene can be accomplished in several ways at the DNA, RNA, or protein level, with the result being a reduction in the amount of active protein in the cell. Truncations of the open reading frame or the introduction of mutations that destabilize the protein or reduce catalytic activity achieve a similar goal, as does fusing a “degron” polypeptide that destabilizes the protein. Engineering of the regulatory regions of the gene can also be used to change gene expression. Alteration of the promoter sequence or replacement with a different promoter is one method. Truncation of the terminator, known as decreased abundance of mRNA perturbation (DAmP) is also known to reduce gene expression. Other methods that reduce the stability of the mRNA include the use of cis- or trans-acting ribozymes, e.g., self-cleaving ribozymes, or RNA elements that recruite an exonuclease, or antisense DNA. RNAi may be used to silence genes in budding yeast strains via import of the required protein factors from other species, e.g., Drosha or Dice (Drinnenberg et al 2009). Gene expression may also be silenced in S. cerevisiae via recruitment of native or heterologous silencing factors or repressors, which may be accomplished at arbitrary loci using the D-Cas9 CRISPR system (Qi et al 2013). Protein level can also be reduced by engineering the amino acid sequence of the target protein. A variety of degron sequences may be used to target the protein for rapid degradation, including, but not limited to, ubiquitin fusions and N-end rule residues at the amino terminus. These methods may be implemented in a constitutive or conditional fashion.

Induction Systems

[0364] To adapt to a constantly changing environment, microbes such as yeast have evolved a wide range of natural inducible promoter systems. Any promoter that is regulated by a small molecule or change in environment (temperature, pH, oxygen level, osmolarity, oxidative damage) can in principle be converted into an inducible system for the expression of heterologous genes. The best known system in S. cerevisiae is the galactose regulon, which is strongly repressed by glucose and activated by galactose. Heterologous genetic pathways under the control of galactose-inducible promoters are regulated in the same way, and thus an engineered strain can be grown in glucose media to build biomass, and then switched to galactose to induce pathway expression. A range of expression levels can be achieved, from very strong pGALl to relatively weak pGAL3. However, galactose may be expensive and a poor carbon source for S. cerevisiae. Therefore, for industrial applications, it may be advantageous to re-engineer the regulon such that the cells can be induced in a non galactose media. The galactose regulon can be modified for this purpose in many ways, including:

Overexpressing the negative regulator of GAL80, GAIA, from an inducible promoter, e.g. pSUC2-GAL3, such that switching from glucose to sucrose relieves GAL80 expression and activates the pathway.

Deleting the repressor GAL80 and replacing the native GAL4 cassette with a version under the control of a sucrose inducible promoter, e.g. pSUC2-GAL4, such that expression is induced by a switch from glucose to sucrose. Replacing the native GAL80 gene with an inducible version, e.g. pSUC2- GAL80, such that expression is induced by a switch from sucrose to glucose. [0365] These strategies often require fine-tuning of the activator and repressor levels to achieve the proper dynamics (very low or no expression in the off state, and desired expression level in the on state). There are a variety of ways to fine tune protein expression, including use of protein stabilization or degradation tags (e.g. degrons) or use of temperature sensitive mutants of the activators or regulators. In the examples above, the pSUC2 promoter is used to induce the galactose regulon in sucrose media. However, any inducible promoter can be used for this purpose, or for control of individual genes outside of the context of the galactose regulon. The list below provides some examples:

• Phosphate regulated promoters, e.g. pPH05

• Carbon source regulated promoters, e.g. pADH2

• Amino acid regulated promoters, e.g. pMET25

• Metal ion induced promoters, e.g. pCUPl

• Temperature regulated promoters, e.g. pHSP12, pHSP26

• pH regulated promoters, e.g. pHSP12, pHSP26

• Oxygen level regulated promoters, e.g. pDANl

• Oxidative stress regulated promoters, e.g. promoters from AHPl, TRR1, TRX2, TSAI, GPX2, GSH1, GSH2, GLR1, SOD1, or SOD2 genes.

• ER stress regulated promoters, e.g. unfolded protein response element promoters.

[0366] In addition to these natural examples, there are a variety of synthetic inducible promoter systems. These are generally based on re-arrangement of native or foreign transcriptional elements into a basal promoter scaffold and/or fusions of activator domains and DNA binding domains to create novel transcription factors. Two examples are provided below:

• Estradiol-inducible systems involving fusion of the estradiol receptor to DNA-binding and transcriptional activation domain, paired with synthetic or native promoters with binding sites.

• tet Trans Activator (tTA) or reverse tet Trans Activator (rtTA) systems paired with tetO-containing promoters.

[0367] In some embodiments, one of the above inducible promoter systems is used in a modified host cell of the disclosure. In some embodiments, the inducible promoter system is a natural inducible promoter system. In some embodiments, the inducible promoter system is a synthetic inducible promoter system. In some embodiments, a suitable media for culturing modified host cells of the disclosure comprises one or more of the inducers disclosed herein. Possible inducers include:

• Phosphate regulated promoters, e.g. pPH05 o KH2PO4

• Carbon source regulated promoters, e.g. pADH2 o Galactose (e.g. pGALl) o Glucose (e.g. pADH2) o Sucrose (e.g. pSUC2, pGPHl, pMAL12) o Maltose (e.g. pMAL12, pMAL32)

• Amino acid regulated promoters, e.g. pMET25 o Methionine (e.g. pMET25) o Lysine (e.g. pLYS9) o Other amino acids

• Metal ion induced promoters, e.g. pCUPl o CuS0 ₄

• Temperature regulated promoters, e.g. pHSP12, pHSP26 o Change in temperature, e.g. 30 °C to 37 °C

• pH regulated promoters, e.g. pHSP12, pHSP26 o Change in pH, e.g. pH 6 to pH 4

• Oxygen level regulated promoters, e.g. pDANl o Change in oxygen level, e.g. 20% to 1% dissolved oxygen levels

• Oxidative stress regulated promoters, e.g. pSODl o Addition of hydrogen peroxide or superoxide-generating drug menadione

• ER stress regulated promoters, e.g. unfolded protein response element promoters. o Tunicamycin, or expression of proteins prone to misfolding (e.g., cannabinoid synthases)

• Estradiol-inducible systems involving fusion of the estradiol receptor to DNA- binding and transcriptional activation domain, paired with synthetic or native promoters with binding sites. o estradiol

• tet Trans Activator (tTA) or reverse tet Trans Activator (rtTA) systems paired with tetO-containing promoters. o doxycyclin Codon Usage

[0368] As is well known to those of skill in the art, it is possible to improve the expression of a heterologous nucleic acid in a host organism by replacing the nucleotide sequences coding for a particular amino acid (i.e., a codon) with another codon which is better expressed in the host organism (i.e., codon-optimization). One reason that this effect arises is due to the fact that different organisms show preferences for different codons. In some embodiments, a nucleic acid disclosed herein is modified or optimized such that the nucleotide sequence reflects the codon preference for the particular host cell. For example, the nucleotide sequence will in some embodiments be modified or optimized for yeast codon preference. In some embodiments, a nucleotide sequence disclosed herein is codon- optimized. See, e.g., Bennetzen and Hall (1982) J Biol. Chem. 257(6): 3026-3031.

[0369] Statistical methods have been generated to analyze codon usage bias in various organisms and many computer algorithms have been developed to implement these statistical analyses in the design of codon optimized gene sequences (Lithwick G, Margalit H (2003) Hierarchy of sequence-dependent features associated with prokaryotic translation. Genome Research 13: 2665-73). Other modifications in codon usage to increase protein expression that are not dependent on codon bias have also been described (Welch et al. (2009).

[0370] In some embodiments, the codon usage of a nucleotide sequence is modified or optimized such that the level of translation of the encoded mRNA is decreased. In some embodiments, a codon-optimized nucleotide sequence may be optimized such that the level of translation of the encoded mRNA is decreased. Reducing the level of translation of an mRNA by modifying codon usage may be achieved by modifying the nucleotide sequence to include codons that are rare or not commonly used by the host cell. Codon usage tables for many organisms are available that summarize the percentage of time a specific organism uses a specific codon to encode for an amino acid. Certain codons are used more often than other, “rare” codons. The use of “rare” codons in a nucleotide sequence generally decreases its rate of translation. Thus, e.g., the nucleotide sequence is modified by introducing one or more rare codons, which affect the rate of translation, but not the amino acid sequence of the polypeptide translated. For example, there are six codons that encode for arginine: CGT, CGC, CGA, CGG, AGA, and AGG. In E. coli the codons CGT and CGC are used far more often (encoding approximately 40% of the arginines in E. coli each) than the codon AGG (encoding approximately 2% of the arginines in E. coli). Modifying a CGT codon within the sequence of a gene to an AGG codon would not change the sequence of the polypeptide, but would likely decrease the gene’s rate of translation.

[0371] In some embodiments, a codon-optimized nucleotide sequence may be optimized for expression in a yeast cell. In certain such embodiments, the yeast cell is Saccharomyces cerevisiae.

[0372] Further, it will be appreciated that this disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art and illustrated in the following table.

Codon Degeneracies

Methods of Producing a Cannabinoid or a Cannabinoid Derivative or of Expressing and/or Preparing Engineered Variants of the Tetrahydrocannabinolic Acid Synthase (THCAS) Polypeptide

[0373] The disclosure provides methods for expressing an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide of the disclosure. In certain such embodiments, the methods comprise culturing a modified host cell of the disclosure in a culture medium. The disclosure also provides methods for preparing an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide of the disclosure. The disclosure also provides methods of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of the disclosure.

[0374] The present disclosure also provides methods of producing a cannabinoid or a cannabinoid derivative. The methods of the present disclosure may involve production of cannabinoids or cannabinoid derivatives using an engineered variant disclosed herein. The methods may involve culturing a modified host cell of the present disclosure in a culture medium and recovering the produced cannabinoid or cannabinoid derivative. The methods may also involve cell-free production of cannabinoids or cannabinoid derivatives using one or more polypeptides disclosed herein, such as an engineered variant of the disclosure, expressed or overexpressed by a modified host cell of the disclosure. The methods may also involve cell-free production of cannabinoids or cannabinoid derivatives using an engineered variant disclosed herein.

[0375] Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may include, but are not limited to, cannabichromene (CBC) type (e.g., cannabichromenic acid), cannabidiol (CBD) type (e.g. cannabidiolic acid), A ⁹ -trans-tetrahydrocannabinol (D ⁹ -THC) type (e.g. A ⁹ -tetrahydrocannabinolic acid), A ⁸ -trans-tetrahydrocannabinol (D ⁸ -THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, derivatives of any of the foregoing, and others as listed in Elsohly M.A. and Slade D., Life Sci. 2005 Dec 22;78(5):539-48. Epub 2005 Sep 30. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0376] Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), CBD A, cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-Ci), D ⁹ - tetrahydrocannabinolic acid A (THCA-A), D ⁹ -tetrahydrocannabinolic acid B (THCA-B), D ⁹ -tetrahydrocannabinol (THC), D ⁹ -tetrahydrocannabinolic acid-C4 (THCA-C4), D ⁹ - tetrahydrocannabinol-C4 (THC-C4), D ⁹ -tetrahydrocannabivarinic acid (THCVA), D ⁹ - tetrahydrocannabivarin (THCV), D ⁹ -tetrahydrocannabiorcolic acid (THCA-Ci), D ⁹ - tetrahydrocannabiorcol (THC-Ci), D ⁷ -cis-iso-tetrahydrocannabivarin, D ⁸ - tetrahydrocannabinolic acid (D ⁸ -THCA), D ⁸ -tetrahydrocannabinol (D ⁸ -THC), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovarin (CBLV), cannabielsoic acid A (CBEA-A), cannabielsoic acid B (CBEA-B), cannabielsoin (CBE), cannabielsoinic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methylether (CBNM), cannabinol-C4, (CBN-C4), cannabivarin (CBV), cannabinol-C2 (CNB-C2), cannabiorcol (CBN-Ci), cannabinodiol (CBND), cannabinodivarin (CBVD), cannabitriol (CBT), 10-ethyoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9- dihydroxyl-delta-6a-tetrahydrocannabinol, cannabitriolvarin (CBTVE), dehydrocannabifuran (DCBF), cannabifuran (CBF), cannabichromanon (CBCN), cannabicitran (CBT), 10-oxo- delta-6a-tetrahydrocannabinol (OTHC), delta-9-cis-tetrahydrocannabinol (cis-THC), 3,4,5,6- tetrahydro-7-hydroxy-alpha-alpha-2-trimethyl-9-n-propyl-2,6- methano-2H-l-benzoxocin-5- methanol (OH-iso-HHCV), cannabiripsol (CBR), trihydroxy-delta-9-tetrahydrocannabinol (triOH-THC), CBGA-hydrocinnamic acid (3-[(2E)-3,7-dimethylocta-2,6-dien-l-yl]-2,4- dihydroxy-6-(2-phenylethyl)benzoic acid), CBG-hydrocinnamic acid (2-[(2E)-3,7- dimethylocta-2,6-dien-l-yl]-5-(2-phenylethyl)benzene-l,3-dio l), CBDA-hydrocinnamic acid (2,4-dihydroxy-3-[3-methyl-6-(prop-l-en-2-yl)cyclohex-2-en-l -yl]-6-(2- phenylethyl)benzoic acid), CBD-hydrocinnamic acid (2-[3-methyl-6-(prop-l-en-2- yl)cyclohex-2-en-l-yl]-5-(2-phenylethyl)benzene-l,3-diol), THCA-hydrocinnamic acid (1- hydroxy-6,6,9-trimethyl-3-(2-phenylethyl)-6H,6aH,7H,8H,10aH- benzo[c]isochromene-2- carboxylic acid), THC-hydrocinnamic acid (6,6,9-trimethyl-3-(2-phenylethyl)- 6H,6aH,7H,8H,10aH-benzo[c]isochromen-l-ol, perrottetinene), and derivatives of any of the foregoing. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0377] In some embodiments, the cannabinoid produced with the engineered variants, methods, or modified host cells of the present disclosure is D ⁹ - tetrahydrocannabinolic acid, A ⁹ -tetrahydrocannabinol, A ⁸ -tetrahydrocannabinolic acid, D ⁸ - tetrahydrocannabinol, cannabidiolic acid, cannabidiol, cannabichromenic acid, cannabichromene, cannabinolic acid, cannabinol, cannabidivarinic acid, cannabidivarin, tetrahydrocannabivarinic acid, tetrahydrocannabivarin, cannabichromevarinic acid, cannabichromevarin, cannabigerovarinic acid, cannabigerovarin, cannabicyclolic acid, cannabicyclol, cannabielsoinic acid, cannabielsoin, cannabicitranic acid, or cannabicitran. In some embodiments, the cannabinoid is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid is produced in an amount of more than 50 mg/L culture medium.

[00100] In some embodiments, the cannabinoid produced with the engineered variants, methods, or modified host cells of the present disclosure is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin. In some embodiments, the cannabinoid is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid is produced in an amount of more than 50 mg/L culture medium.

[0378] Additional cannabinoids and cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, CBDA, CBD, CBGA, THC, THCA, THCVA, CBDVA, (6aR,10aR)-l-hydroxy-6,6,9-trimethyl-3-butyl-6a,7,8,10a-tetr ahydro-6H-dibenzo[b,d]pyran-

2-carboxylic acid, (6aR,10aR)-l-hydroxy-6,6,9-trimethyl-3-(3-methylpentyl)-6a,7 ,8,10a- tetrahydro-6H-dibenzo[b,d]pyran-2-carboxylic acid, (6aR, 10aR)-l -hydroxy-6, 6, 9-trimethyl-

3-(4-pentenyl)-6a,7,8,10a-tetrahydro-6H-dibenzo[b,d]pyran -2-carboxylic acid, (6aR,10aR)- l-hydroxy-6,6,9-trimethyl-3-hexyl-6a,7,8,10a-tetrahydro-6H-d ibenzo[b,d]pyran-2- carboxylic acid, (6aR,10aR)-l-hydroxy-6,6,9-trimethyl-3-(5-hexynyl)-6a,7,8,10 a-tetrahydro- 6H-dibenzo[b,d]pyran-2-carboxylic acid, and others as listed in Bow, E. W. and Rimoldi, J. M., “The Structure-Function Relationships of Classical Cannabinoids: CB1/CB2 Modulation,” Perspectives in Medicinal Chemistry 2016:8 17-39 doi:

10.4137/PMC. S32171, incorporated by reference herein. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.

[0379] Additional cannabinoids and cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, (l'R,2'R)-4-(hexan-2-yl)-5'-methyl-2'-(prop-l-en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, (l'R,2'R)-4-hexyl-5'-methyl-2'-(prop- 1 -en-2- yl)-l',2',3',4'-tetrahydro-[l,r-biphenyl]-2,6-diol, (rR,2'R)-5'-methyl-4-(3-methylpentyl)-2'- (prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, (l'R,2'R)-4-(4-chlorobutyl)-5'- methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, (1 'R,2'R)-5'-methyl- 4-(4-methylpentyl)-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol,

( 1 'R,2'R)-5'-methyl-4-(4-(methylthio)butyl)-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1,1'- biphenyl]-2,6-diol, ( 1 'R,2'R)-5'-methyl-4-((E)-pent- 1 -en- 1 -yl)-2'-(prop- 1 -en-2-yl)- l',2',3 ',4'- tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-5'-methyl-4-((E)-pent-3 -en- 1 -yl)-2'-(prop- 1 -en- 2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-5'-methyl-4-((E)-pent-2-en- 1 - yl)-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-4-(but-3 -yn- 1 - yl)-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, (l'R,2'R)-4- ((E)-but- 1 -en- 1 -yl)-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6- diol, (l'R,2'R)-5'-methyl-4-(pent-4-yn- 1 -yl)-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1,1'- biphenyl]-2,6-diol, (l'R,2'R)-5'-methyl-2'-(prop-l-en-2-yl)-4-undecyl-r,2',3',4' -tetrahydro- [l,l'-biphenyl]-2,6-diol, (l'R,2'R)-4-(hex-5-yn-l-yl)-5'-methyl-2'-(prop-l-en-2-yl)-r, 2',3',4'- tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-4-((E)-hept- 1 -en- 1 -yl)-5'-methyl-2'-(prop- 1 -en- 2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-5'-methyl-4-octyl-2'-(prop- 1 -en- 2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-5'-methyl-4-((E)-oct- 1 -en- 1 -yl)- 2'-(prop-l-en-2-yl)-l',2',3',4'-tetrahydro-[l,r-biphenyl]-2, 6-diol, (l'R,2'R)-5'-methyl-4- nonyl-2'-(prop-l-en-2-yl)-l',2',3',4'-tetrahydro-[l,r-biphen yl]-2,6-diol, (l'R,2'R)-5'-methyl- 4-(3 -phenylpropyl)-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol,

( 1 'R,2'R)-5'-methyl-4-(4-phenylbutyl)-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , G- biphenyl]-2,6-diol, (l'R,2'R)-5'-methyl-4-(5-phenylpentyl)-2'-(prop-l-en-2-yl)-r ,2',3',4'- tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-5'-methyl-4-(6-phenylhexyl)-2'-(prop- 1 -en-2- yl)-l',2',3',4'-tetrahydro-[l,r-biphenyl]-2,6-diol, (l'R,2'R)-5'-methyl-4-(2-methylpentyl)-2'- (prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, (l'R,2'R)-4-isopropyl-5'- methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, ( 1 'R,2'R)-4-decyl- 5'-methyl-2'-(prop-l-en-2-yl)-l',2',3',4'-tetrahydro-[l,r-bi phenyl]-2,6-diol, (l'R,2'R)-5'- methyl-2'-(prop- 1 -en-2-yl)-4-tridecyl- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, (E)-3 - ((l'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]- 4-yl)acrylic acid, (Z)-3-((l'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-l-en-2-yl) -r,2',3',4'- tetrahydro-[l,l'-biphenyl]-4-yl)acrylic acid, 7-((l'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- l-en-2-yl)-l',2',3',4'-tetrahydro-[l,r-biphenyl]-4-yl)heptan oic acid, 8-((l'R,2'R)-2,6- dihydroxy-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-4-yl)octanoic acid, 9-((l'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1,1'- biphenyl]-4-yl)nonanoic acid, 1 l-((l'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-l-en-2-yl)- ,2',3',4'-tetrahydro-[l,l'-biphenyl]-4-yl)undecanoic acid, (l"R,2"R)-3',5'-dihydroxy-5"- methyl-2"-(prop- 1 -en-2-yl)- 1 ",2",3 ",4"-tetrahydro-[ 1 , 1 ' :4', 1 "-terphenyl]-2-carboxylic acid, (l"R,2"R)-3',5'-dihydroxy-5"-methyl-2"-(prop-l-en-2-yl)-l",2 ",3",4"-tetrahydro-[l,r:4',l"- terphenyl]-3-carboxylic acid, (l"R,2"R)-3',5'-dihydroxy-5"-methyl-2"-(prop-l-en-2-yl)- l",2",3",4"-tetrahydro-[l,r:4',l"-terphenyl]-4-carboxylic acid, (l"R,2"R)-3',5'-dihydroxy-5"- methyl-2"-(prop- 1 -en-2-yl)- 1 ",2",3 ",4"-tetrahydro-[ 1 , G :4', 1 "-terphenyl]-3 , 5-dicarboxylic acid, ( 1 'R,2'R)-4-(4-hydroxybutyl)-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1,1'- biphenyl]-2,6-diol, (rR,2'R)-4-(4-aminobutyl)-5'-methyl-2'-(prop-l-en-2-yl)-r,2' ,3',4'- tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, 5-(( 1 'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1 -en-2- yl)-r,2',3',4'-tetrahydro-[l,r-biphenyl]-4-yl)pentanenitrile , (rR,2'R)-5'-methyl-4-(3- methylhexan-2-yl)-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol,

( 1 'R,2'R)-5'-methyl-2'-(prop- 1 -en-2-yl)-4-propyl- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6- diol, ( 1 'R,2'R)-4-butyl-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]- 2,6-diol, (rR,2'R)-5'-methyl-4-pentyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , G- biphenyl]-2,6-diol, (rR,2'R)-4-heptyl-5'-methyl-2'-(prop-l-en-2-yl)-r,2',3',4'-t etrahydro- [1,1 '-biphenyl]-2,6-diol, (rR,2'R)-5'-methyl-4-(pent-4-en- 1 -yl)-2'-(prop- 1 -en-2-yl)- 1',2',3 ',4'- tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, 3 -(( 1 'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1 -en-2- yl)-r,2',3',4'-tetrahydro-[l,r-biphenyl]-4-yl)propanoic acid, (rR,2'R)-4,5'-dimethyl-2'- (prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, 2-((l'R,2'R)-2,6-dihydroxy-5'- methyl-2'-(prop-l-en-2-yl)-r,2',3',4'-tetrahydro-[l,r-biphen yl]-4-yl)acetic acid, 4- ((rR,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]- 4-yl)butanoic acid, ( 1 'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1 -en-2-yl)- 1',2',3 ',4'- tetrahydro-[l,l'-biphenyl]-4-carboxylic acid, 5-((rR,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- l-en-2-yl)-r,2',3',4'-tetrahydro-[l,r-biphenyl]-4-yl)pentano ic acid, and 6-((l'R,2'R)-2,6- dihydroxy-5'-methyl-2'-(prop- 1 -en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-4-yl)hexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0380] A cannabinoid derivative may lack one or more chemical moieties found in a naturally-occurring cannabinoid. Such chemical moieties may include, but are not limited to, methyl, alkyl, alkenyl, methoxy, alkoxy, acetyl, carboxyl, carbonyl, oxo, ester, hydroxyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkenylalkyl, cycloalkenylalkenyl, heterocyclylalkenyl, heteroarylalkenyl, arylalkenyl, heterocyclyl, aralkyl, cycloalkylalkyl, heterocyclylalkyl, heteroarylalkyl, and the like. In some embodiments, a cannabinoid derivative lacking one or more chemical moieties found in a naturally-occurring cannabinoid may also comprise one or more of any of the functional and/or reactive groups described herein. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.

[0381] A cannabinoid derivative may be a cannabinoid substituted with or comprising one or more functional and/or reactive groups. Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl, alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, spirocyclyl, heterospirocyclyl, heterocyclyl, thioalkyl (or alkylthio), arylthio, heteroarylthio, sulfone, sulfonyl, sulfoxide, amido, alkylamino, dialkylamino, arylamino, alkylarylamino, diarylamino, N-oxide, imide, enamine, imine, oxime, hydrazone, nitrile, aralkyl, cycloalkylalkyl, haloalkyl, heterocyclylalkyl, heteroarylalkyl, nitro, thioxo, and the like. Suitable reactive groups may include, but are not necessarily limited to, azide, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), halide, ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, acetyl, and the like. In some embodiments, the reactive group is selected from a carboxyl, a carbonyl, an amine, an ester, a thioester, a thioether, a sulfonyl halide, an alcohol, a thiol, an alkyne, alkene, an azide, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide, and a hydrazine. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.

[0382] “Alkyl” may refer to a straight or branched chain saturated hydrocarbon. For example, Ci-C6alkyl groups contain 1 to 6 carbon atoms. Examples of a Ci-C6alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec- butyl and /e/V-butyl, isopentyl, and neopentyl.

[0383] “Alkenyl” may include an unbranched (i.e., straight) or branched hydrocarbon chain containing 2-12 carbon atoms. The “alkenyl” group contains at least one double bond. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Examples of alkenyl groups may include, but are not limited to, ethylenyl, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2- ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl and the like.

[0384] Compounds disclosed herein, such as cannabinoids and cannabinoid derivatives, may be substituted with one or more substituents, such as those illustrated generally herein, or as exemplified by particular classes, subclasses, and species of the present disclosure. In general, the term “substituted” refers to the replacement of a hydrogen atom in a given structure with a specified substituent. Combinations of substituents envisioned by the present disclosure are typically those that result in the formation of stable or chemically feasible compounds.

[0385] As used herein, the term “unsubstituted” may mean that the specified group bears no substituents beyond the moiety recited (e.g., where valency satisfied by hydrogen). [0386] A reactive group may facilitate covalent attachment of a molecule of interest.

Suitable molecules of interest may include, but are not limited to, a detectable label; imaging agents; a toxin (including cytotoxins); a linker; a peptide; a drug (e.g., small molecule drugs); a member of a specific binding pair; an epitope tag; ligands for binding by a target receptor; tags to aid in purification; molecules that increase solubility; and the like. A linker may be a peptide linker or a non-peptide linker.

[0387] In some embodiments, a cannabinoid derivative substituted with an azide may be reacted with a compound comprising an alkyne group via “click chemistry” to generate a product comprising a heterocycle, also known as an azide-alkyne cycloaddition. In some embodiments, a cannabinoid derivative substituted with an alkyne may be reacted with a compound comprising an azide group via click chemistry to generate a product comprising a heterocycle.

[0388] Additional molecules of interest that may be desirable for attachment to a cannabinoid derivative may include, but are not necessarily limited to, detectable labels (e.g., spin labels, fluorescence resonance energy transfer (FRET)-type dyes, e.g., for studying structure of biomolecules in vivo ); small molecule drugs; cytotoxic molecules (e.g., drugs); imaging agents; ligands for binding by a target receptor; tags to aid in purification by, for example, affinity chromatography (e.g., attachment of a FLAG epitope); molecules that increase solubility (e.g., polyethylene glycol); molecules that enhance bioavailability; molecules that increase in vivo half-life; molecules that target to a particular cell type (e.g., an antibody specific for an epitope on a target cell); molecules that target to a particular tissue; molecules that provide for crossing the blood-brain barrier; and molecules to facilitate selective attachment to a surface, and the like.

[0389] In some embodiments, a molecule of interest comprises an imaging agent.

Suitable imaging agents may include positive contrast agents and negative contrast agents. Suitable positive contrast agents may include, but are not limited to, gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA); gadolinium- diethylenetriaminepentaacetic acid (Gd-DTPA); gadolinium-1, 4, 7-tris(carbonylmethyl)-10- (2'-hydroxypropyl)- 1 ,4,7, 10-tetraazacyclododecane (Gd-HP-D03 A); Manganese(II)- dipyridoxal diphosphate (Mn-DPDP); Gd-diethylenetriaminepentaacetate-bis(methylamide) (Gd-DTPA-BMA); and the like. Suitable negative contrast agents may include, but are not limited to, a superparamagnetic iron oxide (SPIO) imaging agent; and a perfluorocarbon, where suitable perfluorocarbons may include, but are not limited to, fluoroheptanes, fluorocycloheptanes, fluoromethylcycloheptanes, fluorohexanes, fluorocyclohexanes, fluoropentanes, fluorocyclopentanes, fluoromethylcyclopentanes, fluorodimethylcyclopentanes, fluoromethylcyclobutanes, fluorodimethylcyclobutanes, fluorotrimethylcyclobutanes, fluorobutanes, fluorocyclobutanse, fluoropropanes, fluoroethers, fluoropolyethers, fluorotriethylamines, perfluorohexanes, perfluoropentanes, perfluorobutanes, perfluoropropanes, sulfur hexafluoride, and the like.

[0390] Additional cannabinoid derivatives that can be produced with an engineered variant, method, or modified host cell of the present disclosure may include derivatives that have been modified via organic synthesis or an enzymatic route to modify drug metabolism and pharmacokinetics (e.g. solubility, bioavailability, absorption, distribution, plasma half- life and metabolic clearance). Modification examples may include, but are not limited to, halogenation, acetylation, and methylation.

[0391] The cannabinoids or cannabinoid derivatives described herein further include all pharmaceutically acceptable isotopically labeled cannabinoids or cannabinoid derivatives. An “isotopically-” or “radio-labeled” compound is a compound where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). For example, in some embodiments, in the cannabinoids or cannabinoid derivatives described herein, hydrogen atoms are replaced or substituted by one or more deuterium or tritium. Certain isotopically labeled cannabinoids or cannabinoid derivatives of this disclosure, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e., ³ H, and carbon 14, i.e., ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e., ² H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Suitable isotopes that may be incorporated in cannabinoids or cannabinoid derivatives described herein include but are not limited to ² H (also written as D for deuterium), ³ H (also written as T for tritium), ^U C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ 0, ¹⁷ 0, ¹⁸ 0, ¹⁸ F, ³⁵ S, ³⁶ C1 , ⁸² Br, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, and ¹³¹ I. Substitution with positron emitting isotopes, such as ^U C, ¹⁸ F, ¹⁵ 0, and ¹³ N, can be useful in Positron Emission Topography (PET) studies.

[0392] The methods of bioproduction, modified host cells, and engineered variants disclosed herein enable synthesis of cannabinoids or cannabinoid derivatives with defined stereochemistries, which is challenging to do using chemical synthesis. Cannabinoids or cannabinoid derivatives disclosed herein may be enantiomers or disastereomers. The term “enantiomers” may refer to a pair of stereoisomers which are non-superimposable mirror images of one another. In some embodiments the cannabinoids or cannabinoid derivatives may be the fV)-enantiomer In some embodiments the cannabinoids or cannabinoid derivatives may be the (//(-enantiomer In some embodiments, the cannabinoids or cannabinoid derivatives may be the (+) or (-) enantiomers. The term “diastereomers” may refer to the set of stereoisomers which cannot be made superimposable by rotation around single bonds. For example, cis- and trans- double bonds, endo- and exo- substitution on bicyclic ring systems, and compounds containing multiple stereogenic centers with different relative configurations may be considered to be diastereomers. The term “diastereomer” may refer to any member of this set of compounds. Cannabinoids or cannabinoid derivatives disclosed herein may include a double bond or a fused ring. In certain such embodiments, the double bond or fused ring may be cis or trans, unless the configuration is specifically defined. If the cannabinoid or cannabinoid derivative contains a double bond, the substituent may be in the E or Z configuration, unless the configuration is specifically defined.

[0393] In some embodiments when the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; from the cell lysate, the modified host cell, and the culture medium; or from a cell- free reaction mixture comprising one or more polypeptides and/or engineered variants disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.

[0394] The disclosure includes pharmaceutically acceptable salts of the cannabinoids or cannabinoid derivatives described herein. “Pharmaceutically acceptable salts” may refer to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable. Representative pharmaceutically acceptable salts include, but are not limited to, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,· 4-diaminostilbene-2, 2-di sulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, sethionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (l,l-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p- toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.

[0395] “Pharmaceutically acceptable salt” also includes both acid and base addition salts. “Pharmaceutically acceptable acid addition salt” may refer to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo- glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5 -di sulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4- aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p- toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0396] “Pharmaceutically acceptable base addition salt” may refer to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. For example, inorganic salts include, but are not limited to, ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.

[0397] The disclosure provides a method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of of the disclosure. In certain such embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure. In certain such embodiments, the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time. In some embodiments of the methods of producing a cannabinoid or a cannabinoid derivative of the disclosure, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure. In certain such embodiments, the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time. [0398] In some embodiments of the methods of producing a cannabinoid or a cannabinoid derivative of the disclosure, the cannabinoid is THCA and the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure. In certain such embodiments, the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time. In some embodiments of the methods of producing a cannabinoid or a cannabinoid derivative of the disclosure, the cannabinoid is THCA and the method produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

Methods of Using Host Cells to Generate Cannabinoids or Cannabinoid Derivatives [0399] The disclosure provides methods of producing a cannabinoid or a cannabinoid derivative, such as those described herein, the method comprising: culturing a modified host cell of the disclosure in a culture medium. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In certain such embodiments, the produced cannabinoid or cannabinoid derivative is then purified as disclosed herein.

[0400] In some embodiments, culturing of the modified host cells of the disclosure in a culture medium provides for synthesis of a cannabinoid or a cannabinoid derivative, such as those described herein, in an increased amount compared to an unmodified host cell cultured under similar conditions.

[0401] The disclosure provides methods of producing a cannabinoid or a cannabinoid derivative, such as those described herein, the method comprising: culturing a modified host cell of the disclosure in a culture medium comprising a carboxylic acid. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In certain such embodiments, the produced cannabinoid or cannabinoid derivative is then purified as disclosed herein.

[0402] In some embodiments, the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; from the cell lysate, the modified host cell, and the culture medium. In certain such embodiments, the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. In some embodiments when the cannabinoid or cannabinoid derivative is recovered from the cell lysate; from the culture medium; from the modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; from the cell lysate, the modified host cell, and the culture medium; or from the cell-free reaction mixture comprising one or more polypeptides disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0403] In some embodiments, the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid. In some embodiments, the carboxylic acid may be substituted with or comprise one or more functional and/or reactive groups. Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl, alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, spirocyclyl, heterospirocyclyl, heterocyclyl, thioalkyl (or alkylthio), arylthio, heteroarylthio, sulfone, sulfonyl, sulfoxide, amido, alkylamino, dialkylamino, arylamino, alkylarylamino, diarylamino, N-oxide, imide, enamine, imine, oxime, hydrazone, nitrile, aralkyl, cycloalkylalkyl, haloalkyl, heterocyclylalkyl, heteroarylalkyl, nitro, thioxo, and the like. Reactive groups may include, but are not necessarily limited to, azide, halogen, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, and the like. In some embodiments, the reactive group is selected from a carboxyl, a carbonyl, an amine, an ester, thioester, thioether, a sulfonyl halide, an alcohol, a thiol, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide, an azide, an alkyne, an alkene, and a hydrazine. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.

[0404] In some embodiments, the carboxylic acid is isotopically- or radio-labeled. In some embodiments, the carboxylic acid may be an enantiomer or disastereomer. In some embodiments the carboxylic acid may be the fV)-enantiomer In some embodiments the carboxylic acid may be the (//(-enantiomer In some embodiments, the carboxylic acid may be the (+) or (-) enantiomer. In some embodiments, the carboxylic acid may include a double bond or a fused ring. In certain such embodiments, the double bond or fused ring may be cis or trans, unless the configuration is specifically defined. If the carboxylic acid contains a double bond, the substituent may be in the E or Z configuration, unless the configuration is specifically defined.

[0405] In some embodiments, the carboxylic acid comprises a C=C group. In some embodiments, the carboxylic acid comprises an alkyne group. In some embodiments, the carboxylic acid comprises an N3 group. In some embodiments, the carboxylic acid comprises a halogen. In some embodiments, the carboxylic acid comprises a CN group. In some embodiments, the carboxylic acid comprises iodo. In some embodiments, the carboxylic acid comprises bromo. In some embodiments, the carboxylic acid comprises chloro. In some embodiments, the carboxylic acid comprises fluoro. In some embodiments, the carboxylic acid comprises a carbonyl. In some embodiments, the carboxylic acid comprises an acetyl.

In some embodiments, the carboxylic acid comprises an alkyl group. In some embodiments, the carboxylic acid comprises an aryl group. [0406] Carboxylic acids may include, but are not limited to, unsubstituted or substituted C3-C18 fatty acids, C3-C18 carboxylic acids, C1-C18 carboxylic acids, butyric acid, isobutyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, myristic acid, C15-C18 fatty acids, C15-C18 carboxylic acids, fumaric acid, itaconic acid, malic acid, succinic acid, maleic acid, malonic acid, glutaric acid, glucaric acid, oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, glutaconic acid, ortho-phthalic acid, isophthalic acid, terephthalic acid, citric acid, isocitric acid, aconitic acid, tricarballylic acid, and trimesic acid. Carboxylic acids may include unsubstituted or substituted C1-C18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C ₃ -C18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C ₃ -C12 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C4-C1 ₀ carboxylic acids. In some embodiments, the carboxylic acid is an unsubstituted or substituted C4 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted Cs carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted G carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C7 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted G carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C9 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C10 carboxylic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted butyric acid. In some embodiments, carboxylic acid is unsubstituted or substituted valeric acid. In some embodiments, the carboxylic acid is unsubstituted or substituted hexanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted heptanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted octanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted nonanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted decanoic acid.

[0407] Carboxylic acids may include, but are not limited to, 2-methylhexanoic acid,

3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3- hexenoic acid, 4-hexenoic acid, 5-hexenoic acid, 5-chlorovaleric acid, 5-aminovaleric acid, 5-cyanovaleric acid, 5-(methylsulfanyl)valeric acid, 5-hydroxyvaleric acid, 5-phenylvaleric acid, 2,3-dimethylhexanoic acid, d ₃ -hexanoic acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, trans-2- nonenoic acid, 4-phenylbutyric acid, 6-phenylhexanoic acid, 7-phenylyheptanoic acid, and the like. In some embodiments, the carboxylic acid is 2-methylhexanoic acid. In some embodiments, the carboxylic acid is 3-methylhexanoic acid. In some embodiments, the carboxylic acid is 4-methylhexanoic acid. In some embodiments, the carboxylic acid is 5- methylhexanoic acid. In some embodiments, the carboxylic acid is 2-hexenoic acid. In some embodiments, the carboxylic acid is 3-hexenoic acid. In some embodiments, the carboxylic acid is 4-hexenoic acid. In some embodiments, the carboxylic acid is 5-hexenoic acid. In some embodiments, the carboxylic acid is 5-chlorovaleric acid. In some embodiments, the carboxylic acid is 5-aminovaleric acid. In some embodiments, the carboxylic acid is 5-cyanovaleric acid. In some embodiments, the carboxylic acid is 5- (methylsulfanyl)valeric acid. In some embodiments, the carboxylic acid is 5-hydroxyvaleric acid. In some embodiments, the carboxylic acid is 5 -phenyl valeric acid. In some embodiments, the carboxylic acid is 2,3-dimethylhexanoic acid. In some embodiments, the carboxylic acid is d3-hexanoic acid. In some embodiments, the carboxylic acid is 4- pentynoic acid. In some embodiments, the carboxylic acid is trans-2-pentenoic acid. In some embodiments, the carboxylic acid is 5-hexynoic acid. In some embodiments, the carboxylic acid is trans-2-hexenoic acid. In some embodiments, the carboxylic acid is 6-heptynoic acid. In some embodiments, the carboxylic acid is trans-2-octenoic acid. In some embodiments, the carboxylic acid is trans-2-nonenoic acid. In some embodiments, the carboxylic acid is 4- phenylbutyric acid. In some embodiments, the carboxylic acid is 6-phenylhexanoic acid. In some embodiments, the carboxylic acid is 7-phenylheptanoic acid.

[0408] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid. In certain such embodiments, the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.

[0409] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is butyric acid, valeric acid, hexanoic acid, octanoic acid, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4- hexenoic acid, 5-hexenoic acid, heptanoic acid, 5-chlorovaleric acid, 5- (methylsulfanyl)valeric acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, nonanoic acid, trans-2- nonenoic acid, decanoic acid, undecanoic acid, dodecanoic acid, myristic acid, 4- phenylbutyric acid, 5 -phenyl valeric acid, 6-phenylhexanoic acid, 7-phenylheptanoic acid, isobutyric acid, fumaric acid, itaconic acid, malic acid, succinic acid, maleic acid, malonic acid, glutaric acid, glucaric acid, oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecandioic acid, glutaconic acid, ortho-phthalic acid, isophthalic acid, terephthalic acid, citric acid, isocitric acid, aconitic acid, tricarballylic acid, trimesic acid, 5- aminovaleric acid, 5-cyanovaleric acid, 5-hydroxyvaleric acid, or 2,3-dimethylhexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0410] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is butyric acid, valeric acid, hexanoic acid, octanoic acid, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4- hexenoic acid, 5-hexenoic acid, heptanoic acid, 5-chlorovaleric acid, 5- (methylsulfanyl)valeric acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, nonanoic acid, trans-2- nonenoic acid, decanoic acid, undecanoic acid, dodecanoic acid, myristic acid, 4- phenylbutyric acid, 5 -phenyl valeric acid, 6-phenylhexanoic acid, 7-phenylheptanoic acid, isobutyric acid, fumaric acid, succinic acid, maleic acid, malonic acid, glutaric acid, oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecandioic acid, ortho-phthalic acid, isophthalic acid, terephthalic acid, trimesic acid, 5-aminovaleric acid, 5- cyanovaleric acid, 5-hydroxyvaleric acid, or 2,3-dimethylhexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.

[0411] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is 2- methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4-hexenoic acid, heptanoic acid, 5-chlorovaleric acid, 5-(methylsulfanyl)valeric acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, nonanoic acid, trans-2- nonenoic acid, decanoic acid, undecanoic acid, dodecanoic acid, myristic acid, 4- phenylbutyric acid, 5 -phenyl valeric acid, 6-phenylhexanoic acid, 7-phenylheptanoic acid, isobutyric acid, fumaric acid, itaconic acid, malic acid, maleic acid, glucaric acid, suberic acid, azelaic acid, sebacic acid, dodecandioic acid, glutaconic acid, ortho-phthalic acid, isophthalic acid, terephthalic acid, citric acid, isocitric acid, aconitic acid, tricarballylic acid, trimesic acid, 5-aminovaleric acid, 5-cyanovaleric acid, 5-hydroxyvaleric acid, or 2,3- dimethylhexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.

[0412] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is 2- methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4-hexenoic acid, heptanoic acid, 5-chlorovaleric acid, 5-(methylsulfanyl)valeric acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, nonanoic acid, trans-2- nonenoic acid, decanoic acid, undecanoic acid, dodecanoic acid, myristic acid, 4- phenylbutyric acid, 5 -phenyl valeric acid, 6-phenylhexanoic acid, 7-phenylheptanoic acid, isobutyric acid, fumaric acid, maleic acid, suberic acid, azelaic acid, sebacic acid, dodecandioic acid, ortho-phthalic acid, isophthalic acid, terephthalic acid, trimesic acid, 5- aminovaleric acid, 5-cyanovaleric acid, 5-hydroxyvaleric acid, or 2,3-dimethylhexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0413] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is 4- pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, nonanoic acid, trans-2-nonenoic acid, decanoic acid, undecanoic acid, dodecanoic acid, 4-phenylbutyric acid, 5-phenylvaleric acid, 6-phenylhexanoic acid, or 7-phenylheptanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.

[0414] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is 2- methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3- hexenoic acid, 4-hexenoic acid, heptanoic acid, 5 -chi or oval eric acid, or 5- (methylsulfanyl)valeric acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.

[0415] The disclosure also provides methods of producing a cannabinoid or a cannabinoid derivative, such as those described herein, the method comprising: culturing a modified host cell of the disclosure in a culture medium comprising olivetolic acid or an olivetolic acid derivative. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In certain such embodiments, the produced cannabinoid or cannabinoid derivative is then purified as disclosed herein.

[0416] Olivetolic acid derivatives used herein may be substituted with or comprise one or more reactive and/or functional groups as disclosed herein. In some embodiments, an olivetolic acid derivative may lack one or more chemical moieties found in olivetolic acid. In some embodiments when the culture medium comprises an olivetolic acid derivative, the olivetolic acid derivative is orsellinic acid. In some embodiments when the culture medium comprises an olivetolic acid derivative, the olivetolic acid derivative is divarinic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.

[0417] The disclosure provides methods of using a modified host cell of the disclosure for producing a cannabinoid or cannabinoid derivative. In some embodiments of the methods of using a modified host cell of the disclosure for producing a cannabinoid or cannabinoid derivative, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method instead comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant. In certain such embodiments, the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, are cultured under similar culture conditions for the same length of time.

[0418] In some embodiments of the methods of using a modified host cell of the disclosure for producing a cannabinoid or cannabinoid derivative, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method instead comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant. In certain such embodiments, the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, are cultured under similar culture conditions for the same length of time.

[0419] In some embodiments of the methods of using a modified host cell of the disclosure for producing a cannabinoid or cannabinoid derivative, the cannabinoid is THCA and the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced in a method instead comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments of the methods of using a modified host cell of the disclosure for producing a cannabinoid or cannabinoid derivative, the cannabinoid is THCA and the method produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80: 1, about 90: 1, about 100: 1, about 150: 1, about 200: 1, about 500: 1, or greater than about 500:1.

Exemplary Cell Culture Conditions

[0420] Suitable media for culturing modified host cells of the disclosure may include standard culture media (e.g., Luria-Bertani broth, optionally supplemented with one or more additional agents, such as an inducer (e.g., where nucleic acids disclosed herein are under the control of an inducible promoter, etc.); standard yeast culture media; and the like). In some embodiments, the culture medium can be supplemented with a fermentable sugar (e.g., a hexose sugar, e.g., glucose, xylose, and the like). Sugars fermentable by yeast may include, but are not limited to, sucrose, dextrose, glucose, fructose, mannose, galactose, and maltose. [0421] In some embodiments, the culture medium can be supplemented with unsubstituted or substituted hexanoate, carboxylic acids other than unsubstituted or substituted hexanoate, olivetolic acid, or olivetolic acid derivatives. In some embodiments, the culture medium can be supplemented with pretreated cellulosic feedstock (e.g., wheat grass, wheat straw, barley straw, sorghum, rice grass, sugarcane straw, bagasse, switchgrass, corn stover, com fiber, grains, or any combination thereof). In some embodiments, the culture medium can be supplemented with oleic acid. In some embodiments, the culture medium comprises a non-fermentable carbon source. In certain such embodiments, the non- fermentable carbon source comprises ethanol. In some embodiments, the suitable media comprises an inducer. In certain such embodiments, the inducer comprises galactose. In some embodiments, the inducer comprises KH2PO4, galactose, glucose, sucrose, maltose, an amino acid (e.g., methionine, lysine), CuSCri, a change in temperature (e.g., 30 °C to 37 °C), a change in pH (e.g., pH 6 to pH 4), a change in oxygen level (e.g., 20% to 1% dissolved oxygen levels), addition of hydrogen peroxide or superoxide-generating drug menadione, tunicamycin, expression of proteins prone to misfolding (e.g. cannabinoid synthases), estradiol, or doxycycline. Additional induction systems are detailed herein. [0422] The carbon source in the suitable media can vary significantly, from simple sugars like glucose to more complex hydrolysates of other biomass, such as yeast extract. The addition of salts generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize polypeptides and nucleic acids. The suitable media can also be supplemented with selective agents, such as antibiotics, to select for the maintenance of certain plasmids and the like. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent cells lacking the resistance from growing. The suitable media can be supplemented with other compounds as necessary to select for desired physiological or biochemical characteristics, such as particular amino acids and the like. [0423] In some embodiments, modified host cells disclosed herein are grown in minimal medium. As used herein, the terms “minimal medium” may refer to growth medium containing the minimum nutrients possible for cell growth, generally, but not always, without the presence of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids). Minimal medium typically contains: (1) a carbon source for cellular (e.g. bacterial or yeast) growth; (2) various salts, which can vary among cellular (e.g. bacterial or yeast) species and growing conditions; and (3) water.

[0424] In some embodiments, modified host cells disclosed herein are grown in rich medium. In certain such embodiments, the rich medium or rich media comprises yeast extract peptone dextrose (YPD) media comprising water, 10 g/L yeast extract, 20 g/L Bacto peptone, and 20 g/L dextrose (glucose). In some embodiments, the rich medium or rich media comprises YP + 20 g/L galactose and 1 g/L glucose. In some embodiments, the rich medium or rich media comprises a carboxylic acid (e.g., 1 mM olivetolic acid, 1 mM olivetolic acid derivative, 2 mM unsubstituted or substituted hexanoic acid, or 2 mM of a carboxylic acid other than unsubstituted or substituted hexanoic acid). In some embodiments, rich medium or rich media affords more rapid cell growth compared to minimal media or minimal medium.

[0425] Materials and methods suitable for the maintenance and growth of the recombinant cells of the disclosure are described herein, e.g., in the Examples section. Other materials and methods suitable for the maintenance and growth of cell (e.g. bacterial or yeast) cultures are well known in the art. Exemplary techniques can be found in International Publication No. W02009/076676, U.S. Patent Application No. 12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. No. 2010/0048964, WO 2009/132220, US Publ. No. 2010/0003716, Manual of Methods for General Bacteriology Gerhardt et al, eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA.

[0426] Standard cell culture conditions can be used to culture the modified host cells disclosed herein (see, for example, WO 2004/033646 and references cited therein). In some embodiments, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20 °C to about 37 °C, at about 0.04% to about 84% CO2, at about 0% to about 100% dissolved oxygen, and at a pH between about 2 to about 9). In some embodiments, modified host cells disclosed herein are grown at about 34 °C in a suitable cell culture medium. In some embodiments, modified host cells disclosed herein are grown at about 20 °C to about 37 °C in a suitable cell culture medium. While the growth optimum for S. cerevisiae is about 30 °C, culturing cells at a higher temperature, e.g. 34 °C may be advantageous by reducing the costs to cool industrial fermentation tanks. In some embodiments, modified host cells disclosed herein are grown at about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, or about 37 °C in a suitable cell culture medium. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 9.0 (such as about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). In some embodiments, the pH ranges for fermentation are between about pH 4.5 to about pH 5.5. In some embodiments, the pH ranges for fermentation are between about pH 4.0 to about pH 6.0. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 6.0. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 5.5. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 5.0. In some embodiments, the dissolved oxygen is between about 0% to about 10%, about 0% to about 20%, about 0% to about 30%, about 0% to about 40%, about 0% to about 50%, about 0% to about 60%, about 0% to about 70%, about 0% to about 80%, about 0% to about 90%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40% or about 10% to about 50%. In some embodiments, the CO2 level is between about 0.04% to about 0.1% CO2, about 0.04% to about 1% CO2, about 0.04% to about 5% CO2, about 0.04% to about 10% CO2, about 0.04% to about 20% CO2, about 0.04% to about 30% CO2, about 0.04% to about 40% CO2, about 0.04% to about 50% CO2, about 0.04% to about 60% CO2, about 0.04% to about 70% CO2, about 0.1% to about 5% CO2, about 0.1% to about 10% CO2, about 0.1% to about 20% CO2, about 0.1% to about 30% CO2, about 0.1% to about 40% CO2, about 0.1% to about 50% CO2, about 1% to about 5% CO2, about 1% to about 10% CO2, about 1% to about 20% CO2, about 1% to about 30% CO2, about 1% to about 40% CO2, about 1% to about 50% CO2, about 5% to about 10%

CO2, about 10% to about 20% CO2, about 10% to about 30% CO2, about 10% to about 40%

CO2, about 10% to about 50% CO2, about 10% to about 60% CO2, about 10% to about 70%

CO2, about 10% to about 80% CO2, about 50% to about 60% CO2, about 50% to about 70%

CO2, or about 50% to about 80% CO2. Modified host cells disclosed herein disclosed herein can be grown under aerobic, anoxic, microaerobic, or anaerobic conditions based on the requirements of the cells.

[0427] Standard culture conditions and modes of fermentation, such as batch, fed- batch, or continuous fermentation that can be used are described in International Publication No. WO 2009/076676, U.S. Patent Application No. 12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. No. 2010/0048964, WO 2009/132220, US Publ. No. 2010/0003716, the contents of each of which are incorporated by reference herein in their entireties. Batch and Fed-Batch fermentations are common and well known in the art and examples can be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.

Production and Recovery of Produced Cannabinoids or Cannabinoid Derivatives [0428] The present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, by modified host cells of the disclosure in an amount of from about 1 mg/L culture medium to about 1 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 mg/L culture medium to about 500 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 mg/L culture medium to about 100 mg/L culture medium. For example, in some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 mg/L culture medium to about 5 mg/L culture medium, from about 5 mg/L culture medium to about 10 mg/L culture medium, from about 10 mg/L culture medium to about 25 mg/L culture medium, from about 25 mg/L culture medium to about 50 mg/L culture medium, from about 50 mg/L culture medium to about 75 mg/L culture medium, or from about 75 mg/L culture medium to about 100 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 100 mg/L culture medium to about 150 mg/L culture medium, from about 150 mg/L culture medium to about 200 mg/L culture medium, from about 200 mg/L culture medium to about 250 mg/L culture medium, from about 250 mg/L culture medium to about 500 mg/L culture medium, from about 500 mg/L culture medium to about 750 mg/L culture medium, or from about 750 mg/L culture medium to about 1 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about from about 50 mg/L culture medium to about 100 mg/L culture medium, 50 mg/L culture medium to about 150 mg/L culture medium, from about 50 mg/L culture medium to about 200 mg/L culture medium, from about 50 mg/L culture medium to about 250 mg/L culture medium, from about 50 mg/L culture medium to about 500 mg/L culture medium, or from about 50 mg/L culture medium to about 750 mg/L culture medium.

[0429] In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of from about 50 mg/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of from about 50 mg/L culture medium to about 100 mg/L culture medium, or more than 100 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of more than 50 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of more than 100 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 100 mg/L culture medium to about 500 mg/L culture medium, or more than 500 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 500 mg/L culture medium to about 1 g/L culture medium, or more than 1 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 10 g/L culture medium, or more than 10 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 20 g/L culture medium, or more than 20 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 30 g/L culture medium, or more than 30 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 40 g/L culture medium, or more than 40 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 50 g/L culture medium, or more than 50 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium.

In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 90 g/L culture medium, or more than 90 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 20 g/L culture medium, or more than 20 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 30 g/L culture medium, or more than 30 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 40 g/L culture medium, or more than 40 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 50 g/L culture medium, or more than 50 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 90 g/L culture medium, or more than 90 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 90 g/L culture medium, or more than 90 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 30 g/L culture medium, or more than 30 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 40 g/L culture medium, or more than 40 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 50 g/L culture medium, or more than 50 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 90 g/L culture medium, or more than 90 g/L culture medium.

[0430] In some embodiments, the modified host cell disclosed herein is cultured in a liquid medium comprising a carboxylic acid, olivetolic acid, or an olivetolic acid derivative. [0431] In some embodiments, a method of producing a cannabinoid or a cannabinoid derivative, such as those disclosed herein, may involve culturing a modified yeast cell of the present disclosure under conditions that favor production of a cannabinoid or a cannabinoid derivative; wherein the cannabinoid or the cannabinoid derivative is produced by the modified yeast cell and is present in the culture medium (e.g., a liquid culture medium) in which the modified yeast cell is cultured. In some embodiments, the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount of from 1 ng/L to 1 g/L (e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L to 500 ng/L, from 500 ng/L to 1 pg/L, from 1 pg/L to 50 pg/L, from 50 pg/L to 100 pg/L, from 100 pg/L to 500 pg/L, from 500 pg/L to 1 mg/L, from 1 mg/L to 50 mg/L, from 50 mg/L to 100 mg/L, from 100 mg/L to 500 mg/L, or from 500 mg/L to 1 g/L). In certain such embodiments, the modified yeast cell is a modified S. cerevisiae. In some embodiments, the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount from 50 mg/L to 100 mg/L. In certain such embodiments, the modified yeast cell is a modified S. cerevisiae. In some embodiments, the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount from 100 mg/L to 500 mg/L. In certain such embodiments, the modified yeast cell is a modified S. cerevisiae. In some embodiments, the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount from 500 mg/L to 1 g/L. In certain such embodiments, the modified yeast cell is a modified S. cerevisiae. In some embodiments, the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L. In certain such embodiments, the modified yeast cell is a modified S. cerevisiae.

[0432] In some embodiments, a method of producing a cannabinoid or a cannabinoid derivative, such as those disclosed herein, may involve culturing a modified yeast cell of the present disclosure under conditions that favor fermentation of a sugar, and under conditions that favor production of a cannabinoid or a cannabinoid derivative; wherein the cannabinoid or the cannabinoid derivative is produced by the modified yeast cell and is present in alcohol produced by the modified yeast cell. The present disclosure provides an alcoholic beverage produced by the modified yeast cell, where the alcoholic beverage comprises the cannabinoid or cannabinoid derivative produced by the modified yeast cell. Alcoholic beverages may include beer, wine, and distilled alcoholic beverages. In some embodiments, an alcoholic beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount of from 1 ng/L to 1 g/L (e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L to 500 ng/L, from 500 ng/L to 1 pg/L, from 1 pg/L to 50 pg/L, from 50 pg/L to 100 pg/L, from 100 pg/L to 500 pg/L, from 500 pg/L to 1 mg/L, from 1 mg/L to 50 mg/L, from 50 mg/L to 100 mg/L, from 100 mg/L to 500 mg/L, or from 500 mg/L to 1 g/L). In some embodiments, an alcoholic beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L.

[0433] The present disclosure provides a beverage produced by the modified yeast cell, where the beverage comprises the cannabinoid or cannabinoid derivative, such as those disclosed herein, produced by the modified yeast cell. In some embodiments, a beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount of from 1 ng/L to 1 g/L (e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L to 500 ng/L, from 500 ng/L to 1 pg/L, from 1 pg/L to 50 pg/L, from 50 pg/L to 100 pg/L, from 100 pg/L to 500 pg/L, from 500 pg/L to 1 mg/L, from 1 mg/L to 50 mg/L, from 50 mg/L to 100 mg/L, from 100 mg/L to 500 mg/L, or from 500 mg/L to 1 g/L). In some embodiments, a beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L. In some embodiments, a beverage of the present disclosure is non-alcoholic.

[0434] In some embodiments, a method of the present disclosure provides for increased production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein. In certain such embodiments, culturing of the modified host cell disclosed herein in a culture medium provides for synthesis of a cannabinoid or a cannabinoid derivative in an increased amount compared to an unmodified host cell cultured under similar conditions.

The production of a cannabinoid or a cannabinoid derivative by the modified host cells disclosed herein may be increased by about 5% to about 1,000,000 folds compared to an unmodified host cell cultured under similar conditions. The production of a cannabinoid or a cannabinoid derivative by the modified host cells disclosed herein may be increased by about 10% to about 1,000,000 folds (e.g., about 50% to about 1,000,000 folds, about 1 to about 500,000 folds, about 1 to about 50,000 folds, about 1 to about 5,000 folds, about 1 to about 1,000 folds, about 1 to about 500 folds, about 1 to about 100 folds, about 1 to about 50 folds, about 5 to about 100,000 folds, about 5 to about 10,000 folds, about 5 to about 1,000 folds, about 5 to about 500 folds, about 5 to about 100 folds, about 10 to about 50,000 folds, about 50 to about 10,000 folds, about 100 to about 5,000 folds, about 200 to about 1,000 folds, about 50 to about 500 folds, or about 50 to about 200 folds) compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions. The production of a cannabinoid or a cannabinoid derivative by modified host cells disclosed herein may also be increased by at least about any of 10%,

20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds, 10,000 folds, 20,000 folds, 50,000 folds, 100,000 folds, 200,000 folds, 500,000 folds, or 1,000,000 folds compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions.

[0435] In some embodiments, the production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, by modified host cells of the disclosure may also be increased by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions. In some embodiments, the production of a cannabinoid or a cannabinoid derivative by modified host cells disclosed herein may also be increased by at least about any of 1-20%, 2-20%, 5-20%, 10-20%, 15-20%, 1-15%, 1-10%, 2-15%, 2-10%, 5-15%, 10-15%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50%, 50-100%, 50- 60%, 50-70%, 50-80%, or 50-90% compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions.

[0436] In some embodiments, production of a cannabinoid or a cannabinoid derivative by modified host cells of the disclosure is determined by LC-MS analysis. In certain such embodiments, each cannabinoid or cannabinoid derivative is identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition.

[0437] In some embodiments, the modified host cell of the disclosure is a yeast cell.

In certain such embodiments, the modified host cell disclosed herein is cultured in a bioreactor. In some embodiments, the modified host cell is cultured in a culture medium supplemented with unsubstituted or substituted hexanoic acid, a carboxylic acid other than unsubstituted or substituted hexanoic acid, olivetolic acid, or an olivetolic acid derivative. In some embodiments, the modified yeast cell is a modified S. cerevisiae.

[0438] In some embodiments, the cannabinoid or cannabinoid derivative, such as those disclosed herein, is recovered from a cell lysate, e.g., by lysing the modified host cell disclosed herein and recovering the cannabinoid or cannabinoid derivative derivative from the lysate. In other cases, the cannabinoid or cannabinoid derivative is recovered from the culture medium in which the modified host cell disclosed herein is cultured. In other cases, the cannabinoid or cannabinoid derivative is recovered from both the cell lysate and the culture medium. In other cases, the cannabinoid or cannabinoid derivative is recovered from a modified host cell. In other cases, the cannabinoid or cannabinoid derivative is recovered from both the modified host cell and the culture medium. In other cases, the cannabinoid or cannabinoid derivative is recovered from the cell lysate, the modified host cell, and the culture medium. In some embodiments when the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; from the cell lysate, the modified host cell, and the culture medium; or from a cell- free reaction mixture comprising one or more polypeptides disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.

[0439] In some embodiments, the recovered cannabinoid or cannabinoid derivative, such as those disclosed herein, is then purified. In some embodiments, whole-cell broth from cultures comprising modified host cells of the disclosure may be extracted with a suitable organic solvent to afford cannabinoids or cannabinoid derivatives. Suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate. In some embodiments, the suitable organic solvent comprises hexane. In some embodiments, the suitable organic solvent may be added to the whole-cell broth from fermentations comprising modified host cells of the disclosure at a 10:1 ratio (10 parts whole-cell broth - 1 part organic solvent) and stirred for 30 minutes. In certain such embodiments, the organic fraction may be separated and extracted twice with an equal volume of acidic water (pH 2.5). The organic layer may then be separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid or cannabinoid derivative crystals. In certain such embodiments, the crude crystals may be heated or exposed to light to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystals may be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystalline product may be re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered to remove any insoluble material. In certain such embodiments, the solvent may then be removed e.g. by rotary evaporation, to produce pure crystalline product.

[0440] In some embodiments, the cannabinoid or cannabinoid derivative is pure, e.g., at least about 40% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98%, or more than 98% pure, where “pure” in the context of a cannabinoid or a cannabinoid derivative may refer to a cannabinoid or a cannabinoid derivative that is free from other cannabinoids or cannabinoid derivatives, macromolecules, contaminants, etc.

Methods of Preparing Engineered Variants of a Tetrahydrocannabinolic Acid Synthase ( THCAS ) Polypeptide

[0441] In an aspect, the present disclosure provides methods for preparing engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide. In certain such embodiments, the methods may comprise culturing a modified host cell of the disclosure in a culture medium. In some embodiments, the modified host cell of the disclosure is a Pichia sp. The method can comprise isolating and/or purifying the expressed engineered variants, as described herein.

[0442] In some embodiments, the method for preparing engineered variants comprises the step of isolating or purifying the engineered variants. The engineered variants of the disclosure can be expressed in modified host cells, as described herein, and isolated from the modified host cells and/or culture medium using any one or more of the well known techniques used for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Chromatographic techniques for isolation of the engineered variants of the disclosure may include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. In some embodiments, affinity chromatography is used.

[0443] In some embodiments, the engineered variants of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in various forms including but not limited to crude extracts (e.g., cell-free lysates), powders (e.g., shake-flask powders), lyophilizates, frozen stocks made with glycerol or another cryoprotectant, and substantially pure preparations (e.g., DSP powders).

[0444] In some embodiments, the engineered variants of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in purified form. Generally, conditions for purifying a particular engineered variant will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. Cell-Free Methods of Producing Cannabinoids or Cannabinoid Derivatives [0445] The methods of the disclosure may involve cell-free production of cannabinoids or cannabinoid derivatives, such as those disclosed herein, using engineered variants disclosed herein expressed or overexpressed by a modified host cell of the disclosure. In some embodiments, an engineered variant disclosed herein is used in a cell- free system for the production of cannabinoids or cannabinoid derivatives. In certain such embodiments, the engineered variant of the disclosure is isolated and/or purified. In some embodiments, appropriate starting materials for use in producing cannabinoids or cannabinoid derivatives may be mixed together with engineered variants disclosed herein in a suitable reaction vessel to effect the reaction. The engineered variants disclosed herein may be used in combination to effect a complete synthesis of a cannabinoid or cannabinoid derivative from the appropriate starting materials. In some embodiments, the cannabinoid or cannabinoid derivative is recovered from a cell-free reaction mixture comprising engineered disclosed herein.

[0446] In some embodiments, the recovered cannabinoids or cannabinoid derivatives, such as those disclosed herein, are then purified. In certain such embodiments, a cell-free reaction mixture comprising an engineered variant disclosed herein may be extracted with a suitable organic solvent to afford cannabinoids or cannabinoid derivatives. Suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate. In some embodiments, the suitable organic solvent comprises hexane. In some embodiments, the suitable organic solvent may be added to the cell-free reaction mixture comprising one or more of the polypeptides disclosed herein at a 10:1 ratio (10 parts reaction mixture - 1 part organic solvent) and stirred for 30 minutes. In certain such embodiments, the organic fraction may be separated and extracted twice with an equal volume of acidic water (pH 2.5). The organic layer may then be separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid or cannabinoid derivative crystals. In certain such embodiments, the crude crystals may be heated or exposed to light to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystals may be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystalline product may be re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered to remove any insoluble material. In certain such embodiments, the solvent may then be removed e.g. by rotary evaporation, to produce pure crystalline product.

[0447] In some embodiments when the cannabinoid or cannabinoid derivative is recovered from a cell-free reaction mixture comprising one or more engineered variants disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.

[0448] In some embodiments, cell-free production of a cannabinoid or a cannabinoid derivative by engineered variants disclosed herein is determined by LC-MS analysis. In certain such embodiments, each cannabinoid or cannabinoid derivative is identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition.

EXAMPLES OF NON-LIMITING EMBODIMENTS OF THE DISCLOSURE [0449] Embodiments of the present subject matter disclosed herein may be beneficial alone or in combination with one or more other embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure, numbered 1-1 to 1-121 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below.

[0450] Some embodiments of the disclosure are of Embodiment I:

[0451] Embodiment 1-1. An engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions.

[0452] Embodiment 1-2. The engineered variant of Embodiment 1-1, wherein the engineered variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44. [0453] Embodiment 1-3. The engineered variant of Embodiment I- 1 or 1-2, wherein the engineered variant comprises at least one amino acid substitution in a signal polypeptide, a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing.

[0454] Embodiment 1-4. The engineered variant of Embodiment 1-3, wherein the engineered variant comprises at least one amino acid substitution in the signal polypeptide.

[0455] Embodiment 1-5. The engineered variant of Embodiment 1-3 or 1-4, wherein the engineered variant comprises at least one amino acid substitution in the FAD binding domain.

[0456] Embodiment 1-6. The engineered variant of any one of Embodiments 1-3 to 1-5, wherein the engineered variant comprises at least one amino acid substitution in the BBE domain.

[0457] Embodiment 1-7. The engineered variant of any one of Embodiments 1-3 to 1-6, wherein the engineered variant comprises substitution of at least one surface exposed amino acid.

[0458] Embodiment 1-8. The engineered variant of Embodiment I- 1 or 1-2, wherein the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of R31, P43, P49, K50, L51, Q55, H56, L59, M61, S62, L71, S100, V103, T109, Q124, V125, L132, S137, H143, V149, W161, K165, N168, E167, S170, F171, P172, Y175, G180, N196, H208, G235, A250, 1257, K261, L269, G311, F317, L327, K390, T379, S429, N467, Y500, N528, P539, P542, H543, H544, and H545 [0459] Embodiment 1-9. The engineered variant of Embodiment 1-8, wherein the engineered variant comprises at least one amino acid substitution selected from the group consisting of R31Q, P43E, P49E, P49K, P49Q, K50T, L51I, Q55E, Q55P, H56E, L59E, M61W, M61H, M61S, S62Q, L71A, S100A, V103F,T109V,Q124D, Q124E, Q124N, V125E, V125Q, L132M, S137G, H143D, V149I, W161R, W161Y, W161K, K165A, N168S, E167P, S170T, F171I, P172V, Y175F, G180A, N196Q, N196V, H208T, G235P, A250T, 1257V, K261W, K261C, L269I, G311A, G311C, F317Y, L327I, K390E, T379S, S429L, N467D, Y500M, Y500V, N528E, P539T, P542E, P542V, H543V, H544A, H545D, and H545E.

[0460] Embodiment I- 10. The engineered variant of Embodiment I- 1 or 1-2, wherein the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID

NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID

NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID

NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID

NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID

NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID

NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID

NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID

NO: 182, SEQ ID NO: 184, and SEQ ID NO: 186.

[0461] Embodiment 1-11. The engineered variant of any one of Embodiments 1-1 to 1-9, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions.

[0462] Embodiment 1-12. The engineered variant of any one of Embodiments 1-1 to 1-9, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, or 30 amino acid substitutions.

[0463] Embodiment 1-13. The engineered variant of any one of Embodiments 1-1 to 1-12, wherein the engineered variant comprises at least one immutable amino acid in a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing.

[0464] Embodiment 1-14. The engineered variant of Embodiment 1-13, wherein the engineered variant comprises at least one immutable amino acid in the FAD binding domain.

[0465] Embodiment 1-15. The engineered variant of Embodiment 1-14, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 immutable amino acids in the FAD binding domain.

[0466] Embodiment 1-16. The engineered variant of any one of Embodiments I-

13 to 1-15, wherein the engineered variant comprises at least one immutable amino acid in the BBE domain.

[0467] Embodiment 1-17. The engineered variant of Embodiment 1-16, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 immutable amino acids in the BBE domain.

[0468] Embodiment 1-18. The engineered variant of any one of Embodiments 1-1 to 1-17, wherein the engineered variant comprises at least one immutable amino acid selected from the group consisting of A28, F34, L35, C37, L64, N70, P87, 193, C99, R108, R110, G112, El 17, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, A173, G174, C176, P177, T178, V179, G182, G183, H184, F185,

G187, G188, G189, Y190, G191, P192, L193, R195, A201, D202, 1205, D206, V210, G214, G223, D225, L226, F227, W228, R231, G234, S237, F238, G239, K245, 1246, L248, V251, V259, Q276, F312, S313, L323, C341, F352, S354, F380, K381, 1382, K383, D385, Y386, 1391, M412, L415, G419, M422, 1425, 1430, P431, P433, H434, R435, G437, Y440, W443, Y444, 1445, 1464, Y465, M468, T469, Y471, V472, P476, R484, N498, A502, N513, F514, K521, N528, F529, E533, Q534, and S535.

[0469] Embodiment 1-19. The engineered variant of Embodiment 1-18, wherein the engineered variant comprises at least one immutable amino acid selected from the group consisting of C37, N70, 193, C99, El 17, S120, F127, D131, G156, E157, Y159, G174,

C176, G182, G183, F185, G187, G188, G189, Y190, G191, P192, R195, D202, D206,

G214, W228, G234, F238, L248, Q277, S314, L324, S355, K382, K384, D386, G420,

M423, R436, Y441, W444, Y445, Y472, P477, N514, F515, N529, and Q535.

[0470] Embodiment 1-20. The engineered variant of any one of Embodiments 1-1 to 1-19, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 immutable amino acids.

[0471] Embodiment 1-21. The engineered variant of any one of Embodiments 1-1 to 1-20, wherein the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in a greater amount, as measured in mg/L or mM, than an amount of THC A produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0472] Embodiment 1-22 The engineered variant of any one of Embodiments 1-1 to 1-21, wherein the engineered variant produces tetrahydrocannabinolic acid (THC A) from cannabigerolic acid (CBGA) in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0473] Embodiment 1-23. The engineered variant of any one of Embodiments 1-1 to 1-22, wherein the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an increased ratio of THCA over another cannabinoid (e.g. cannabichromenic acid (CBCA)) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.

[0474] Embodiment 1-24. The engineered variant of any one of Embodiments 1-1 to 1-23, wherein the engineered variant produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500: 1, or greater than about 500: 1.

[0475] Embodiment 1-25. The engineered variant of any one of Embodiments 1-1 to 1-24, wherein the engineered variant comprises a truncation at an N-terminus, at a C- terminus, or at both the N- and C-termini.

[0476] Embodiment 1-26. The engineered variant of Embodiment 1-25, wherein the truncated engineered variant comprises a signal polypeptide or a membrane anchor. [0477] Embodiment 1-27. The engineered variant of Embodiment 1-25 or 1-26, wherein the engineered variant lacks a native signal polypeptide.

[0478] Embodiment 1-28. The engineered variant of any one of Embodiments I-

25 to 1-27, wherein the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the C-terminus.

[0479] Embodiment 1-29. The engineered variant of any one of Embodiments I-

25 to 1-27, wherein the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus.

[0480] Embodiment 1-30. A nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29.

[0481] Embodiment 1-31. A nucleic acid comprising a nucleotide sequence encoding an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID

NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID

NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID

NO:145, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID

NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID

NO:175, SEQ ID NO: 177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO: 183, and SEQ ID NO:185.

[0482] Embodiment 1-32. The nucleic acid of Embodiment 1-30 or 1-31, wherein the nucleotide sequence is codon-optimized. [0483] Embodiment 1-33. A method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more nucleic acids of any one of Embodiments 1-30 to 1-32 into a host cell.

[0484] Embodiment 1-34. A vector comprising one or more nucleic acids of any one of Embodiments 1-30 to 1-32.

[0485] Embodiment 1-35. A method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more vectors of Embodiment 1-34 into a host cell.

[0486] Embodiment 1-36. A modified host cell for producing a cannabinoid or a cannabinoid derivative, wherein the modified host cell comprises one or more nucleic acids of any one of Embodiments 1-30 to 1-32.

[0487] Embodiment 1-37. The modified host cell of Embodiment 1-36, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide.

[0488] Embodiment 1-38. The modified host cell of Embodiment 1-37, wherein the GOT polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:17.

[0489] Embodiment 1-39. The modified host cell of Embodiment 1-37 or 1-38, wherein the modified host cell comprises two or more heterologous nucleic acids comprising the nucleotide sequence encoding the GOT polypeptide.

[0490] Embodiment 1-40. The modified host cell of Embodiment 1-36, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide.

[0491] Embodiment 1-41. The modified host cell of Embodiment 1-40, wherein the NphB polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:188.

[0492] Embodiment 1-42. The modified host cell of any one of Embodiments I-

36 to 1-41, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tetraketide synthase (TKS) polypeptide and one or more heterologous nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase (OAC) polypeptide. [0493] Embodiment 1-43. The modified host cell of Embodiment 1-42, wherein the TKS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:19.

[0494] Embodiment 1-44. The modified host cell of Embodiment 1-42 or 1-43, wherein the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide.

[0495] Embodiment 1-45. The modified host cell of any one of Embodiments I-

42 to 1-44, wherein the OAC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:21 or SEQ ID NO:48.

[0496] Embodiment 1-46. The modified host cell of any one of Embodiments I-

42 to 1-45, wherein the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide.

[0497] Embodiment 1-47. The modified host cell of any one of Embodiments I-

36 to 1-46, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acyl -activating enzyme (AAE) polypeptide. [0498] Embodiment 1-48. The modified host cell of Embodiment 1-47, wherein the AAE polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:23.

[0499] Embodiment 1-49. The modified host cell of Embodiment 1-47 or 1-48, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide.

[0500] Embodiment 1-50. The modified host cell of any one of Embodiments I-

36 to 1-49, wherein the modified host cell comprises one or more of the following: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMG-CoA synthase (HMGS) polypeptide; b) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a truncated 3 -hydroxy-3 -methyl-glutaryl-CoA reductase (tHMGR) polypeptide; c) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate kinase (MK) polypeptide; d) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a phosphomevalonate kinase (PMK) polypeptide; e) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate pyrophosphate decarboxylase (MVD1) polypeptide; or f) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a isopentenyl diphosphate isomerase (IDI1) polypeptide. [0501] Embodiment 1-51. The modified host cell of Embodiment 1-50, wherein the IDIl polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:25.

[0502] Embodiment 1-52. The modified host cell of Embodiment 1-50 or 1-51, wherein the tHMGR polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:27.

[0503] Embodiment 1-53. The modified host cell of any one of Embodiments I-

50 to 1-52, wherein the HMGS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:29.

[0504] Embodiment 1-54. The modified host cell of any one of Embodiments I-

50 to 1-53, wherein the MK polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:39.

[0505] Embodiment 1-55. The modified host cell of any one of Embodiments I-

50 to 1-54, wherein the PMK polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:37.

[0506] Embodiment 1-56. The modified host cell of any one of Embodiments I-

50 to 1-55, wherein the MVD1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:33.

[0507] Embodiment 1-57. The modified host cell of any one of Embodiments I-

36 to 1-56, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide.

[0508] Embodiment 1-58. The modified host cell of Embodiment 1-57, wherein the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:31.

[0509] Embodiment 1-59. The modified host cell of any one of Embodiments I-

36 to 1-58, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a pyruvate decarboxylase (PDC) polypeptide. [0510] Embodiment 1-60. The modified host cell of Embodiment 1-59, wherein the PDC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:35.

[0511] Embodiment 1-61. The modified host cell of any one of Embodiments I-

36 to 1-60, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate synthetase (GPPS) polypeptide.

[0512] Embodiment 1-62. The modified host cell of Embodiment 1-61, wherein the GPPS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:41.

[0513] Embodiment 1-63. The modified host cell of any one of Embodiments I-

36 to 1-62, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[0514] Embodiment 1-64. The modified host cell of Embodiment 1-63, wherein the KAR2 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5.

[0515] Embodiment 1-65. The modified host cell of Embodiment 1-63 or 1-64, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[0516] Embodiment 1-66. The modified host cell of any one of Embodiments I-

36 to 1-65, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDI1 polypeptide.

[0517] Embodiment 1-67. The modified host cell of Embodiment 1-66, wherein the PDI1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9.

[0518] Embodiment 1-68. The modified host cell of any one of Embodiments I-

36 to 1-67, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IRE1 polypeptide.

[0519] Embodiment 1-69. The modified host cell of Embodiment 1-68, wherein the IRE1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 190.

[0520] Embodiment 1-70. The modified host cell of any one of Embodiments I-

36 to 1-69, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide.

[0521] Embodiment 1-71. The modified host cell of Embodiment 1-70, wherein the EROl polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7. [0522] Embodiment 1-72. The modified host cell of any one of Embodiments I-

36 to 1-71, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a FAD1 polypeptide.

[0523] Embodiment 1-73. The modified host cell of Embodiment 1-72, wherein the FAD1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:192.

[0524] Embodiment 1-74. The modified host cell of any one of Embodiments I-

36 to 1-73, wherein the modified host cell comprises a deletion or downregulation of one or more genes encoding a PEP4 polypeptide.

[0525] Embodiment 1-75. The modified host cell of Embodiment 1-74, wherein the PEP4 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15.

[0526] Embodiment 1-76. The modified host cell of any one of Embodiments I-

36 to 1-75, wherein the modified host cell comprises a deletion or downregulation of one or more genes encoding a ROT2 polypeptide.

[0527] Embodiment 1-77. The modified host cell of Embodiment 1-76, wherein the ROT2 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13.

[0528] Embodiment 1-78. The modified host cell of any one of Embodiments I-

36 to 1-77, wherein the modified host cell is a eukaryotic cell.

[0529] Embodiment 1-79. The modified host cell of Embodiment 1-78, wherein the eukaryotic cell is a yeast cell.

[0530] Embodiment 1-80. The modified host cell of Embodiment 1-79, wherein the yeast cell is Saccharomyces cerevisiae.

[0531] Embodiment 1-81. The modified host cell of Embodiment 1-80, wherein the Saccharomyces cerevisiae is a protease-deficient strain of Saccharomyces cerevisiae. [0532] Embodiment 1-82. The modified host cell of any one of Embodiments I-

36 to 1-81, wherein at least one of the one or more nucleic acids are integrated into the chromosome of the modified host cell.

[0533] Embodiment 1-83. The modified host cell of any one of Embodiments I-

36 to 1-81, wherein at least one of the one or more nucleic acids are maintained extrachromosomally. [0534] Embodiment 1-84. The modified host cell of any one of Embodiments I-

36 to 1-83, wherein at least one of the one or more nucleic acids are operably-linked to an inducible promoter.

[0535] Embodiment 1-85. The modified host cell of any one of Embodiments I-

36 to 1-83, wherein at least one of the one or more nucleic acids are operably-linked to a constitutive promoter.

[0536] Embodiment 1-86. The modified host cell of any one of Embodiments I-

36 to 1-85, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, grown under similar culture conditions for the same length of time.

[0537] Embodiment 1-87. The modified host cell of any one of Embodiments I-

36 to 1-86, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, grown under similar culture conditions for the same length of time.

[0538] Embodiment 1-88. The modified host cell of any one of Embodiments I-

36 to 1-87, wherein the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, grown under similar culture conditions for the same length of time.

[0539] Embodiment 1-89. The modified host cell of any one of Embodiments I-

36 to 1-88, wherein the modified host cell has a growth rate and/or higher biomass yield at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, grown under similar culture conditions for the same length of time.

[0540] Embodiment 1-90. The modified host cell of any one of Embodiments I-

36 to 1-89, wherein the modified host cell produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, grown under similar culture conditions for the same length of time. [0541] Embodiment 1-91. The modified host cell of any one of Embodiments I-

36 to 1-90, wherein the modified host cell produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500: 1, or greater than about 500: 1.

[0542] Embodiment 1-92. A method of producing a cannabinoid or a cannabinoid derivative comprising: a) culturing a modified host cell of any one of Embodiments 1-36 to I- 91 in a culture medium.

[0543] Embodiment 1-93. The method of Embodiment 1-92, wherein the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative.

[0544] Embodiment 1-94. The method of Embodiment 1-92 or 1-93, wherein the culture medium comprises a carboxylic acid.

[0545] Embodiment 1-95. The method of Embodiment 1-94, wherein the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid.

[0546] Embodiment 1-96. The method of Embodiment 1-95, wherein the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid.

[0547] Embodiment 1-97. The method of Embodiment 1-92 or 1-93, wherein the culture medium comprises olivetolic acid or an olivetolic acid derivative.

[00101] Embodiment 1-98. The method of Embodiment 1-92 or 1-93, wherein the cannabinoid is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin.

[0548] Embodiment 1-99. The method of any one of Embodiments 1-92 to 1-98, wherein the culture medium comprises a fermentable sugar.

[0549] Embodiment 1-100. The method of any one of Embodiments 1-92 to 1-98, wherein the culture medium comprises a pretreated cellulosic feedstock.

[0550] Embodiment I- 101. The method of any one of Embodiments 1-92 to 1-98, wherein the culture medium comprises a non-fermentable carbon source.

[0551] Embodiment 1-102. The method of Embodiment I- 101 , wherein the non- fermentable carbon source comprises ethanol. [0552] Embodiment 1-103. The method of any one of Embodiments 1-92 to 1-102, wherein the cannabinoid or the cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium.

[0553] Embodiment 1-104. The method of any one of Embodiments 1-92 to 1-103, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of any one of Embodiments 1-34 to 1-89, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, and wherein the modified host cell of any one of Embodiments 1-36 to 1-91 and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, are cultured under similar culture conditions for the same length of time.

[0554] Embodiment 1-105. The method of any one of Embodiments 1-92 to 1-104, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of any one of Embodiments 1-36 to 1-91, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, and wherein the modified host cell of any one of Embodiments 1-36 to 1-91 and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, are cultured under similar culture conditions for the same length of time.

[0555] Embodiment 1-106. The method of any one of Embodiments 1-92 to 1-105, wherein the cannabinoid is tetrahydrocannabinolic acid (THCA), and wherein the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of any one of Embodiments I- 36 to 1-91, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29, grown under similar culture conditions for the same length of time.

[0556] Embodiment 1-107. The method of any one of Embodiments 1-92 to 1-106, wherein the method produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

[0557] Embodiment 1-108. A method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of any one of Embodiments 1-1 to 1-29.

[0558] Embodiment 1-109. The method of Embodiment 1-108, wherein the method comprises recovering the produced cannabinoid or cannabinoid derivative. [0559] Embodiment 1-110. The method of Embodiment 1-108 or 1-109, wherein the cannabinoid is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin.

[0560] Embodiment 1-111. The method of any one of Embodiments 1-108 to 1-110, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of any one of Embodiments 1-1 to 1-29, wherein the engineered variant of any one of Embodiments 1-1 to 1-29, and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time.

[0561] Embodiment 1-112. The method of any one of Embodiments 1-108 to 1-111, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of any one of Embodiments 1-1 to 1-29, wherein the engineered variant of any one of Embodiments 1-1 to 1-29 and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time. [0562] Embodiment 1-113. The method of any one of Embodiments 1-108 to 1-112, wherein the cannabinoid is tetrahydrocannabinolic acid (THCA), and wherein the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instread of the engineered variant of any one of Embodiments 1-1 to 1-29, wherein the engineered variant of any one of Embodiments 1-1 to 1-29 and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time. [0563] Embodiment 1-114. The method of any one of Embodiments 1-108 to 1-113, wherein the method produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.

[0564] Embodiment 1-115. A method of screening an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, the method comprising: a) dividing a population of host cells into a control population and a test population; b) co-expressing in the control population a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 and a comparison tetrahydrocannabinolic synthase polypeptide, wherein the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 can convert cannabigerolic acid (CBGA) to a first cannabinoid, tetrahydrocannabinolic acid (THCA), and the comparison cannabinoid synthase polypeptide can convert the same CBGA to a different second cannabinoid; c) co-expressing in the test population the engineered variant and the comparison cannabinoid synthase polypeptide, wherein the engineered variant may convert CBGA to the same first cannabinoid, tetrahydrocannabinolic acid (THCA), as the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44, and wherein the comparison tetrahydrocannabinolic synthase polypeptide can convert the same CBGA to the second cannabinoid and is expressed at similar levels in the test population and in the control population; d) measuring a ratio of the first cannabinoid, tetrahydrocannabinolic acid (THCA), over the second cannabinoid produced by both the test population and the control population; and e) measuring an amount, in mg/L or mM, of the first cannabinoid produced by both the test population andthe control population.

[0565] Embodiment 1-116. The method of Embodiment 1-115, wherein the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein improved in vivo performance is demonstrated by an increase in the ratio of the first cannabinoid over the second cannabinoid produced by the test population compared to that produced by the control population under similar culture conditions for the same length of time.

[0566] Embodiment 1-117. The method of Embodiment 1-115 or 1-116, wherein the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 by producing the first cannabinoid in a greater amount, as measured in mg/L or mM, by the test population compared to the amount produced by the control population under similar culture conditions for the same length of time.

[0567] Embodiment 1-118. The method of any one of Embodiments 1-115 to 1-117, wherein the cannabinoid synthase polypeptide is a cannabidiolic acid synthase polypeptide. [0568] Embodiment 1-119. The method of Embodiment 1-118, wherein the cannabidiolic acid synthase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:3.

[0569] Embodiment 1-120. The method of any one of Embodiments 1-115 to 1-119, wherein the second cannabinoid is cannabidiolic acid (CBDA).

[0570] Embodiment 1-121. The method of any one of Embodiments 1-115 to 1-119, wherein the engineered variant is an engineered variant of any one of Embodiments 1-1 to I- 29.

[0571] Provided in Table 1 are amino acid and nucleotide sequences disclosed herein. Where a genus and/or species is noted, the sequence should not be construed to be limited only to the specified genus and/or species, but also includes other genera and/or species expressing said sequence. Orthologs of the sequences disclosed in Table 1 may also be encompassed by this disclosure. Nucleotide sequences indicated as codon optimized in Table 1 are codon optimized for expression in S. cerevisiae. In Table 1, “*” used as the end of a sequence denotes a stop codon. In reference to OAC*, “*” denotes a mutation is present in the sequence.

Table 1: Amino acid and nucleotide sequences of the disclosure

Examples

[0572] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); bp, base pair(s); nt, nucleotide(s); and the like.

THCAS mutations, Construction, and Transformations

[0573] The THCAS mutations described herein were constructed based on the saturation mutagenesis of CBDAS enzyme performed earlier. See US Appln. No. 62/851,560 filed May 22, 2019, the contents of which are herein incorporated by reference in their entirety for all purposes. A selection of mutations that improved CBDAS were ported to the THCAS construct and tested in a strain that produces CBGA from OA. Table 2 shows data representing the performance of multiple, discrete integrations of the enzyme (n >10 for all genotypes). The control strain here, D488 contains wild-type THCAS and performs at expected levels. The performance results are depicted in Tables 2 and 3. Competition Assay Strains

[0574] In this initial set of 10 mutations tested, some, but not all mutations lead to increases in titer, indicating that there is not a 1 : 1 relationship between mutations in CBDAS and THCAS. Some beneficial mutations in CBDAS confer similar benefits to the THCAS enzyme. The magnitude of some improvements in THCA titer are surprisingly large, exceeding that observed for CBDAS. For example, the F371Y mutation in THCAS confers a titer increase of -81%, while the structurally equivalent F316Y mutation in CBDAS confers only a 43% increase in titer.

Table 2: THCAS mutants. All strains were assayed at n=3 or greater. NA = not applicable,

ND = not done.

Yeast transformation methods

[0575] Each DNA construct comprising one or more heterologous nucleic acids disclosed herein (e.g., constructs detailed in Table 4 was integrated into Saccharomyces cerevisiae (CEN.PK2, Strain S4) with standard molecular biology techniques in an optimized lithium acetate (LiAc) transformation. Briefly, cells were grown overnight in yeast extract peptone dextrose (YPD) media at 30 °C with shaking (200 rpm), diluted to an OD600 of 0.175 in YPD, and grown to an OD600 of 0.6 - 0.8. Transformations were conducted in 96 well plate format, using 1.67 mL of culture per well. The total culture volume was harvested by centrifugation, washed in equivalent volume of sterile water, spun down again, and washed in equivalent volume 100 mM LiAc. Cells were spun down again, the supernatant was removed, and the cells were resuspended in a transformation mix consisting of 80 pL 50% PEG, 12 pL 1M LiAc, 3.3 pL boiled salmon sperm DNA, 5 pL of PCR amplified library DNA, and 19.7 pL of water (scaled by number of transformations). Following a heat shock at 42 °C for 40 minutes, cells were recovered overnight in YPD media before plating on selective media. DNA integration was confirmed by colony PCR with primers specific to the integrations for a sample of colonies to confirm high rates of integration.

Yeast culturing conditions

[0576] Yeast colonies comprising library construct nucleic acids disclosed herein, modified host cells, were picked into 96-well microtiter plates containing 360 pL of YPD (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L dextrose (glucose)) and sealed with a breathable film seal. Cells were cultured at 30 °C in a high capacity microtiter plate incubator shaking at 1000 rpm and 80% humidity for 2 days (termed “pre-culture”) until the cultures reached carbon exhaustion. The growth- saturated cultures were subcultured into fresh plates containing YPGAL and either olivetolic acid or hexanoic acid, or an olivetolic acid derivative or a carboxylic acid other than hexanoic acid (10 g/L yeast extract, 20 g/L Bacto peptone, 20 or 40 g/L galactose, 1 g/L glucose and either 1 mM olivetolic acid or 2 mM hexanoic acid, or 1 mM of an olivetolic acid derivative or 2 mM of a carboxylic acid other than hexanoic acid), by taking 15 pL from the saturated cultures and diluting into 360 pL of fresh media and sealed with a breathable film seal. Modified host cells in the production media were cultured at 30 °C in a high capacity microtiter plate shaker at 1000 rpm and 80% humidity for an additional 5 days prior to extraction and analysis. Upon completion, 25 pL of whole cell broth was diluted into 975 pL of methanol, sealed with a foil seal, and shaken at 1200 rpm for 60 seconds to extract the cannabinoids or cannabinoid derivatives. After shaking, the plate was centrifuged at 1000 x g for 60 seconds to remove any solids. After centrifugation, the seal was removed and 10 pL of supernatant was transferred to a fresh assay plate containing 240 pL of methanol, sealed with a foil seal, shaken for 60 seconds at 900 rpm, and analyzed by LC-MS.

Analytical methods

[0577] Samples were analyzed by LC-MS mass spectrometer (Agilent 6470) using a Phenomenex Kinetex Phenyl-Hexyl 2.1 x 30 mm, 2.6 pm analytical column with the following gradient (Mobile Phase A: LC-MS grade water with 0.1% formic acid; Mobile Phase B: LC-MS grade acetonitrile with 0.1% formic acid):

[0578] The mass spectrometer was operated in negative ion multiple reaction monitoring mode. Each cannabinoid or cannabinoid derivative was identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition: Recovery and purifications

[0579] Whole-cell broth from cultures comprising modified host cells of the disclosure are extracted with a suitable organic solvent to afford cannabinoids or cannabinoid derivatives. Suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate.

The suitable organic solvent, such as hexane, is added to the whole-cell broth from fermentations comprising modified host cells of the disclosure at a 10:1 ratio (10 parts whole-cell broth - 1 part organic solvent) and stirred for 30 minutes. The organic fraction is separated and extracted twice with an equal volume of acidic water (pH 2.5). The organic layer is then separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid or cannabinoid derivative crystals. The crude crystals may then be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate the crude cannabinoid or cannabinoid derivative. The crude crystalline product is re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered through a 1 pm filter to remove any insoluble material. The solvent is then removed e.g. by rotary evaporation, to produce pure crystalline product.

In vitro enzyme assay and cell-free production of cannabinoids or cannabinoid derivatives [0580] In some embodiments, modified host cells, e.g., modified yeast cells are cultured in 96-well microtiter plates containing 360 pL of YPD (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L dextrose (glucose)) and sealed with a breathable film seal. Cells are then cultured at 30 °C in a high capacity microtiter plate incubator shaking at 1000 rpm and 80% humidity for 3 days until the cultures reach carbon exhaustion. The growth-saturated cultures are then subcultured into 200 mL of YPGAL media to an OD600 of 0.2 and incubated with shaking for 20 hours at 30 °C. Cells are then harvested by centrifugation at 3000 x g for 5 minutes at 4 °C. Harvested cells are then resuspended in 50 mL buffer (50 mM Tris-HCl, 1 mM EDTA, 0.1 M KC1, pH 7.4, 125 units Benzonase) and then lysed (Emulsiflex C3, Avestin, INC., 60 bar, 10 min). Cells debris is removed by centrifugation (10,000 x g, 10 min, 4 °C). Subsequently, the supernatant is then subjected to ultracentrifugation (150,000 x g, 1 h, 4 °C, Beckman Coulter L-90K, TI-70). The resulting membrane fractions are then resuspended in 3.3 mL buffer (10 mM Tris-HCl, 10 mM MgCh, pH 8.0, 10% glycerol) and solubilized with a tissue grinder. Then, 0.02% (v/v) of the respective membrane preparations are then dissolved in reaction buffer (50 mM Tris-HCl, 10 mM MgCb, pH 8.5) and substrate (500 pM olivetolic acid, 500 pM GPP) to a total volume of 50 pL and incubated for 1 hour at 30 °C. Assays are then extracted by adding two reaction volumes of ethyl acetate followed by vortexing and centrifugation. The organic layer is evaporated for 30 minutes, resuspended in acetonitrile/H20/formic acid (80:20:0.05%) and filtered with Ultrafree® -MC columns (0.22 pm pore size, PVDF membrane material). Cannabinoids or cannabinoid derivatives are then detected via LC-MS and/or recovered and purified.

Yeast cultivation in a bioreactor

[0581] Single yeast colonies comprising modified host cells disclosed herein are grown in 15 mL of Verduyn medium (originally described by Verduyn et al, Yeast 8(7): 501-17) with 50 mM succinate (pH 5.0) and 2% glucose in a 125 mL flask at 30 °C, with shaking at 200 rpm to an OD600 between 4 to 9. Glycerol is then added to the culture to a concentration of 20% and 1 mL vials of the modified host cell suspension are stored at -80 °C. One to two vials of modified host cells are thawed and grown in Verduyn medium with 50 mM succinate (pH 5.0) and 4% sucrose for 24 hours, then sub-cultured to an OD600 reading of 0.1 in the same media. After 24 hours of growth at 30 °C with shaking, 65 mL of culture is used to inoculate a 1.3-liter fermenter (Eppendorf DASGIP Bioreactor) with 585 mL of Verduyn fermentation media containing 20 g/L galactose supplemented with hexanoic acid (2 mM), a carboxylic acid other than hexanoic acid (2 mM), olivetolic acid (1 mM), or an olivetolic acid derivative (1 mM). A poly-alpha-olefm may be added to the fermenter as an extractive agent. The fermenter is maintained at 30 °C and pH 5.0 with addition of NH4OH. In an initial batch phase, the fermenter is aerated at 0.5 volume per volume per minute air (VVM) and agitation ramped to maintain 30% dissolved oxygen. After the initial sugar is consumed, the rise in dissolved oxygen triggers feeding of galactose + hexanoic acid (800 g galactose per liter + 9.28 g hexanoic acid per liter) at 10 g galactose per liter per hour in pulses of 10 g galactose per liter doses (alternatively, rather than feeding the modified host cells disclosed herein hexanoic acid, olivetolic acid, an olivetolic acid derivative, or a carboxylic acid other than hexanoic acid is fed to the modified host cells).

[0582] Between pulses, the feed rate is lowered to 5 g galactose per liter per hour.

Upon a 10% rise in dissolved oxygen, the feed rate is resumed at 10 g L ^-1 hour ^-1 . As modified host cell density increases, dissolved oxygen is allowed to reach 0%, and the pulse dose is increased to 50 g galacose per liter. Oxygen transfer rate is maintained at rates representative of full-scale conditions of 100 mM per liter per hour by adjusting agitation as volume increased. Feed rate is adjusted dynamically to meet demand using an algorithm that alternates between a high feed rate and low feed rate. During the low feed rate, modified host cells should consume galactose and hexanoic acid, or, alternatively, olivetolic acid, an olivetolic acid derivative, or a carboxylic acid other than hexanoic acid, and any overflow metabolites accumulated during the high feed rate. A rise in dissolved oxygen triggers the high feed rate to resume. The length of time spent in the low feed rate reflects the extent to which modified host cells are over- or under-fed in the prior high feed rate pulse; this information is then monitored and used to tune the high feed rate up or down, keeping the low feed rate within a defined range.

[0583] Over time, the feed rate matches sugar and hexanoic acid, or, alternatively, olivetolic acid, an olivetolic acid derivative, or a carboxylic acid other than hexanoic acid, demand from modified host cells. This algorithm ensures minimal net accumulation of fermentation products other than cannabinoids or cannabinoid derivatives; biomass; and CO2. In some embodiments, the process continues for 5 to 14 days. In certain such embodiments, accumulated broth is removed daily and assayed for biomass and cannabinoid, or cannabinoid derivative concentration. A concentrated solution of NH4H2PO4, trace metals and vitamins are added periodically to maintain steady state concentrations.

Table 4: Constructs and strains used in the Examples

* If a strain has a parent strain, it is a child strain. All of the constructs present in the parent strain are also all present in the child strain.

** S4 is CEN.PK113-1A with genotype MATalpha; URA3; TRP1; LEU2; HIS3; MAL2-8C; SUC2

*** S487 is the base strain used to test THCA synthase constructs. In this strain, the nucleotide sequence encoding a pGALl tTDHl empty expression cassette is added and the i33 locus is returned to its native sequence by deleting the CBDAS construct there

Table 5: List of Regulatory and Other Elements

Table 5 Legend: Non-coding DNA regions (regulatory and other) referenced in the Figures are listed in Table 5. Flanking Homology regions direct recombination at specific genomic loci. Flanking homology upstream sequences are denoted with a “u”, and downstream with a "d"'. “I” indicates an intergenic integration site, e.g. ui7, di7 are the regions flanking intergenic region 7. Integrations that delete an open reading frame have flanking homology with the deleted gene indicated, e.g. uPEP4, dPEP4 are the regions flanking the PEP4 gene. Synthetic Recombination Sequences (SRS) direct internal recombination of two DNA constructs targeted for integration at the same locus. Linkers are short sequences used in assembly the DNA constructs, they are intervening between the indicated parts. Linkers Gl, G7, G10, RG1 and LTTDH1 contain the last 36 bp of the upstream DNA part; in cases where these linkers are used assume that the linker reconstitutes sequence omitted from the upstream part to create a seamless junction with the downstream part. Linkers DO and D9 are terminal linkers that direct entry of the DNA constructs into cloning vectors and are not integrated into the genome. Where no linker is shown between parts, the junction is also seamless.

[0584] Although the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.