ENGINEERED MICROORGANISM FOR THE PRODUCTION OF CANNABINOID BIOSYNTHETIC PATHWAY PRODUCTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of and priority from United States

Provisional Patent Application No. 62/927,316, filed October 29, 2019.

FIELD

[0002] The present disclosure relates to genetically engineered microorganisms for production of cannabinoid biosynthetic pathway products and cell cultures comprising thereof. The genetically engineered microorganisms comprise nucleic acid molecules having nucleic acid sequences encoding cannabinoid biosynthetic pathway enzymes for producing cannabinoid biosynthetic pathway products.

BACKGROUND

[0003] The commercialization of valuable plant natural products (PNPs) is often limited by the availability of PNP producing-plants, by the low accumulation of PNPs in planta and/or the time-consuming and often inefficient extraction methods not always economically viable. Thus, commercialization of PNPs of commercial interest is often challenging. The recent progress in genetic engineering and synthetic biology makes it possible to produce heterologous PNPs in microbes such as bacteria, yeasts and microalgae. For example, engineered microorganisms have been reported to produce the antimalarial drug artemisinin and of the opiate (morphine, codeine) painkiller precursor reticuline (Keasling 2012; Fossati et al 2014; DeLoache ef a/ 2015). However, the latest metabolic reactions to yield the valuable end-products such as codeine and morphine in genetically modified yeast-producing reticuline have yet to be successfully achieved. In some cases, bacterial or yeast platforms do not support the assembly of complex PNP pathways. In comparison, microalgal cells have been suggested to possess advantages over other microorganisms, including the likelihood to perform similar post-translational modifications of proteins as plant and recombinant protein expression through the nuclear, mitochondrial or chloroplastic genomes (Singh et al 2009).

[0004] Cannabinoid biosynthetic pathway products such as D9- tetrahydrocanannabinol and other cannabinoids (CBs) are polyketides responsible for the psychoactive and medicinal properties of Cannabis sativa. More than 110 CBs have been identified so far and are all derived from fatty acid and terpenoid precursors (ElSohly and Slade 2005). The first metabolite intermediate in the CB biosynthetic pathway in Cannabis sativa is olivetolic acid that forms the polyketide skeleton of cannabinoids. A type III polyketide synthase (PKS; also known as tetraketide synthase (TKS) orolivetol synthase) enzyme condenses hexanoyl-CoA with three malonyl-CoA in a multi-step reaction to form trioxododecanoyl-CoA. From there, olivetolic acid cyclase (OAC) (OAC; also known as 3,5,7-trioxododecanoyl-CoA CoA-lyase) catalyzes an intramolecular aldol condensation to yield OA. In subsequent steps, CB diversification is generated by the sequential action of “decorating” enzymes on the OA backbone. The gene sequence for PKS and OAC have been identified and characterized in vitro (Lussier2012; Gagne ef a/2012; Marks et al 2009; Stout et a/ 2012; Taura et al 2009).

SUMMARY

[0005] The present disclosure describes an engineered microorganism for production of a plant natural product such as a cannabinoid biosynthetic pathway product.

[0006] A method has been developed for the genetic transformation of microalgae with genes encoding biosynthetic enzymes involved in the production of cannabinoid biosynthetic pathway products. The coding sequences, without and with introns, for genes encoding biosynthetic enzymes were codon-optimized for enhanced expression in the selected microalgae strains. In some embodiments, genes were optimized (e.g., SEQ ID NO: 1-14), synthesized, arranged in different construction cassettes, and inserted into transformation vectors. In some embodiments, the open reading frame of a construct comprising genes and other elements (e.g., reporters, tags, peptide linkers) was codon- optimized (e.g., SEQ ID NO:71-84), synthesized, and inserted into transformation vectors. Different constructs comprising elements including constitutive promoters, single or combined genes encoding biosynthetic enzymes, linker sequences, introns, reporter sequences, tag sequences, rubisco small subunit sequences, ribosome binding sites, etc., were created and used to transform microalgae cells.

[0007] Accordingly, the present disclosure provides a genetically engineered microorganism that is capable of producing a cannabinoid biosynthetic pathway product.

[0008] In an embodiment of the genetically engineered microorganism as described herein, the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase, a second polynucleotide sequence encoding olivetolic acid cyclase, and at least one linker sequence between the first and second polynucleotide sequences, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

[0009] In an embodiment of the genetically engineered microorganism as described herein, the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34- 37 and 107-108).

[0010] In an embodiment of the genetically engineered microorganism as described herein, the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

[0011] In an embodiment of the genetically engineered microorganism as described herein, the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116), a second polynucleotide sequence encoding tetraketide synthase, and a third polynucleotide sequence encoding olivetolic acid cyclase.

[0012] In an embodiment of the genetically engineered microorganism as described herein, the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116) and a second polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof.

[0013] In an embodiment of the genetically engineered microorganism as described herein, the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and a second nucleic acid molecule comprising a third polynucleotide sequence encoding an aromatic prenyltransferase, and wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65. [0014] In an embodiment of the genetically engineered microorganism as described herein, the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and a second nucleic acid molecule comprising a second polynucleotide sequence encoding an aromatic prenyltransferase, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65.

[0015] Further provided is a cell culture comprising the genetically engineered microorganism as described herein, and a medium that is substantially free of a sugar.

[0016] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific Examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The disclosure will now be described in relation to the drawings in which: [0018] Fig. 1 shows an exemplary cannabinoid biosynthetic pathway based on enzymes from Cannabis sativa.

[0019] Fig. 2 shows a part of the cannabinoid biosynthetic pathway from Cannabis sativa ending in the production of olivetolic acid.

[0020] Fig. 3 shows exemplary fusion genes of tetraketide synthase (TKS) and olivetolic acid cyclase (OAC). Construct 1 (top) is TKS fused to OAC by a FMDV linker. Construct 2 (bottom is TKS fused to OAC by a peptide linker comprising a BamHI restriction site.

[0021] Fig. 4 shows schematic representations of the different engineered fusion genes expressed in microalgae cells.

[0022] Fig. 5 shows the assembly and insertion of the synthetic constructions into pChlamy vectors. (A) The synthetic constructions were inserted into a default vector (pKan ^R high-copy) which is used to transform Escherichia coli. (B) The transformed E. coli was grown to bulk plasmids containing the transgenes (synthetic constructions) and positive colonies were confirmed using the colony PCR method. (C) Two vectors were used for the metabolic engineering of C. reinhardtir. pChlamy3 and pChlamy 4. (D and E) Example of gels of colony PCR results (the integrity of DNA sequences were confirmed with Sanger sequencing which confirmed successful in frame of all combination of synthetic constructions/vectors).

[0023] Fig. 6 shows the transformation of E. coli and extraction of the recombinant pChlamy vectors. (A) T ransformed colonies for pC3_1 , pC3_2, pC4_1 and pC4_2 vectors all grew on ampicillin plates. (B) Positive recombinant clones were grown, and vectors were their size were verified on agarose gel.

[0024] Fig. 7 shows Chlamydomonas transformation with recombinant linearized pChlamy vectors and screening by the colony PCR method. (A) Chlamydomonas transformed with recombinant pChlamy3 vectors (pC3_1 , pC3_2) were grown on media containing hygromycin. (B) Cells transformed with recombinant pChlamy 4 vectors (pC4_1 , pC4_2) were grown on media containing zeocin. (C-F) DNA gels of colony PCR confirms positive transformed Chlamydomonas colonies for (C) pC3_1 , (D) pC3_2, (E) pC4_1 and (F) pC4_2.

[0025] Fig. 8 shows qRT-PCR analysis of the relative expression of the OAC transgene in Chlamydomonas cells transformed with recombinant pChlamy vectors such as pC3_1 , pC3_2, pC4_1 and pC4_2.

[0026] Fig. 9 shows SDS-PAGE gel of proteins extracted from Chlamydomonas cells transformed with pChlamy4 vectors. (A) pC4_1 transformed cells do not show an increase of two bands at, 42 (TKS) and 12 kDa (OAC) compared to control cells (lane 2). (B) pC4_2 transformed cells do not show an increase of a band at 60 kDa (expected TKS- OAC fused protein) compared to control cells (lanel). (C) Western blot using anti-FMDV- 2A antibodies reveals the presence of fused and single protein construction in different C. reinhardtii positive transformants.

[0027] Fig. 10 shows Phaeodactylum tricornutum (Pt) episomal transformation with TKS and OAC fusion genes. (A) A map of the episome (Karas et al 2015) (Epi) empty (Epicontrol) and engineered with construction 2 of TKS and OAC genes (Epi ^TKS - ^FMDV -° ^AC ). (B) DNA gel of the PCR products for full fragment insert of E p i ^{T KS_ FM D V} -° ^AC construct amplified by primers annealing sites on the Epi backbone performed on Pt colonies shows the entire insert (FcpD promoter -> FcpD terminator) at the correct size of 2591 bp. (C) Transformed P. tricornutum colonies, with Epi ^contlOl and E p i ^{T KS_ FM D v} -° ^AC ,were grown on zeocin plates. (D) Multiplex PCR results for colonies of Epi transformed with Pt DNA show that DNA was extracted from 1 colony of P. tricornutum for each isolate of TKS-FMDV- OAC.

[0028] Fig. 11 shows diatoms after lysis.

[0029] Fig. 12 shows a chromatogram in selected time range in SIM mode (MS 425.3) of a diatom extract transfected with an empty control vector and spiked with an OA standard.

[0030] Fig. 13 shows a chromatogram in selected time range in SIM mode (MS

425.3) of a diatom extract transfected with an empty control vector.

[0031] Fig. 14 shows a chromatogram in selected time range in SIM mode (MS 425.3) of a diatom extract 1 transfected with TKS and OAC enzymes.

[0032] Fig. 15 shows a chromatogram in selected time range in SIM mode (MS

425.3) of a diatom extract 2 transfected with TKS and OAC enzymes.

[0033] Fig. 16 shows a chromatogram in selected time range in SIM mode (MS

425.3) of a diatom extract 3 transfected with TKS and OAC enzymes. [0034] Fig. 17 shows (A) UPLC-MS of a C.reinhardtii clone transformed with a construct comprising TKS and OAC enzymes, with a OA peak at 225m/z and (B) a fragmentation of the 225m/z peak.

[0035] Fig. 18 shows (A) HPLC of C.reinhardtii clones transformed with a construct comprising TKS and OAC enzymes (samples 5, 6, 7, 10, 11 , and 12) displaying a peak of an OA-derivative at ~17.7min that is not detected in samples of wild type or OA-spiked C.reinhardtii samples. (B) UPLC-MS of wild type C.reinhardtii spiked with OA. (C) UPLC- MS of wild type C.reinhardtii.

DETAILED DESCRIPTION

[0036] The present disclosure describes an engineered microorganism such as a microalga, a cyanobacterium, a bacterium, a protist, or a fungus for production of a plant natural product such as a cannabinoid biosynthetic pathway product.

[0037] Accordingly, the present disclosure provides a genetically engineered microorganism that is capable of producing olivetolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase, a second polynucleotide sequence encoding olivetolic acid cyclase, and at least one linker sequence between the first and second polynucleotide sequences, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

[0038] The present disclosure further provides a genetically engineered microorganism that is capable of producing olivetolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

[0039] The present disclosure further provides a genetically engineered microorganism that is capable of producing olivetol, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34- 37 and 107-108).

[0040] The present disclosure further provides a genetically engineered microorganism that is capable of producing olivetolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116), a second polynucleotide sequence encoding tetraketide synthase, and a third polynucleotide sequence encoding olivetolic acid cyclase.

[0041] The present disclosure further provides a genetically engineered microorganism that is capable of producing olivetol, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116) and a second polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof.

[0042] The present disclosure further provides a genetically engineered microorganism that is capable of producing cannabigerolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and a second nucleic acid molecule comprising a third polynucleotide sequence encoding an aromatic prenyltransferase, and wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65. [0043] The present disclosure further provides a genetically engineered microorganism that is capable of producing cannabigerol, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and a second nucleic acid molecule comprising a second polynucleotide sequence encoding an aromatic prenyltransferase, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65.

[0044] The present disclosure further provides a cell culture comprising the genetically engineered microorganism as described herein.

[0045] The present disclosure further provides a cell culture comprising a genetically engineered microorganism as described herein for production of cannabinoid biosynthetic pathway products, and a medium that is substantially free of a sugar, wherein the genetically engineered microorganism is a photosynthetic microalga or a cyanobacterium.

[0046] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0047] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.

[0048] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0049] In the absence of any indication to the contrary, reference made to a "%" content throughout this specification is to be taken as meaning % w/v (weight/volume).

[0050] As used here, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (/.e., % identity=number of identical overlapping positions/total number of positions multiplied by 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs ( e.g ., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. In a specific embodiment, the nucleic acids are optimized for codon usage in a specific microalgal or cyanobacterial species. In particular, the nucleic acid sequence encoding the cannabinoid biosynthetic pathway enzyme incorporates codon-optimized codons for GC-rich microalgae, such as Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chui, Nannochloropsis oculate, Scenedesmus obliquus, Acutodesmus dimorphus, Dunaliella tertiolecta, and Heamatococus plucialis ; diatoms, such as Phaeodactylum tricornutum and Thalassiosira pseudonana ; or cyanobacteria such as Arthrospira platensis , Arthrospira maxima, Synechococcus elongatus, and Aphanizomenon flos-aquae.

[0051] The sequences of the present disclosure may be at least 80% identical to the sequences described herein; in another example, the sequences may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical at the nucleic acid or amino acid level to sequences described herein. Importantly, the proteins encoded by the variant sequences retain the activity and specificity of the proteins encoded by the reference sequences. Accordingly, the present disclosure also provides a nucleic acid molecule comprising nucleic acid sequence encoding a cannabinoid biosynthetic pathway enzyme with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 1-14, 56-60, and 66-70 or a sequence encoding a cannabinoid biosynthetic pathway enzyme comprised in any of the constructs provided in examples 7 and 8. Also provided is an amino acid sequence of a cannabinoid biosynthetic pathway enzyme with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO:15-21 and 61-65. [0052] Nucleic acid and amino acid sequences described herein are set out in Table 1.

TABLE 1. Sequences

[0053] As used herein, the term “genetically engineered” and its derivatives refer to a microorganism whose genetic material has been altered using molecular biology techniques such as but not limited to molecular cloning, recombinant DNA methods, transformation and gene transfer. The genetically engineered microorganism includes a living modified microorganism, genetically modified microorganism or a transgenic microorganism. Genetic alteration includes addition, deletion, modification and/or mutation of genetic material. Such genetic engineering as described herein in the present disclosure increases production of plant natural products such as cannabinoid biosynthetic pathway products relative to the corresponding wild-type microorganism.

[0054] The term “cannabinoid” is generally understood to include any chemical compound that acts upon a cannabinoid receptor. Examples of cannabinoids include cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), tetrahydrocannabivarin (THCV), cannabichromanon (CBCN), cannabielsoin (CBE), cannbifuran (CBF), tetrahydrocannabinol (THC), cannabinodiol (CBDL), cannabicyclol (CBL), cannabitriol (CBT), cannabivarin (CBV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), cannabinerolic acid, cannabidiolic acid (CBDA), cannabinodiol (CBND), cannabinol propyl variant (CBNV), cannabitriol (CBO), cannabigerolic acid (CBGA), tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA), tetrahydrocannabivarinic acid (THCVA), cannabigerovarinic acid (CBGVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA), and derivatives thereof. Further examples of cannabinoids are discussed in PCT Patent Application Pub. No. WO2017/190249 and US Patent Application Pub. No. US2014/0271940.

[0055] A cannabinoid may be in an acid form or a non-acid form, the latter also being referred to as the decarboxylated form since the non-acid form can be generated by decarboxylating the acid form. Within the context of the present disclosure, where reference is made to a particular cannabinoid, the cannabinoid can be in its acid or non acid form, or be a mixture of both acid and non-acid forms.

[0056] A cannabinoid biosynthetic pathway product is a cannabinoid or a product associated with the production of a cannabinoid. Examples of cannabinoid biosynthetic pathway products include, but are not limited to, hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, cannabigerolic acid, cannabigerol, A9-tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol and cannabidiol. In an embodiment, the cannabinoid biosynthetic pathway product is at least one, two, three, four, five, six, seven, eight, nine, or ten of hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, cannabigerolic acid, cannabigerol, A9-tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol, or cannabidiol.

[0057] In one embodiment, the genetically engineered microorganism has increased production of at least one, two, three, four, five, six, seven, eight, nine, or ten cannabinoid biosynthetic pathway products relative to the corresponding wild-type microorganism. In another embodiment, the cannabinoid biosynthetic pathway product is at least one, two, three, four, five, six, seven, eight, nine, or ten of hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, cannabigerolic acid, cannabigerol, D9- tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol, or cannabidiol. For example, the genetically engineered microorganism may have increased production of olivetolic acid, or olivetolic acid and cannabigerolic acid, relative to the corresponding wild-type microorganism. In another example, the genetically engineered microorganism may have increased production of olivetol, or olivetol and cannabigerol, relative to the corresponding wild-type microorganism

[0058] The term "nucleic acid molecule" or its derivatives, as used herein, is intended to include unmodified DNA or RNA or modified DNA or RNA. For example, it is useful for the nucleic acid molecules of the disclosure to be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, it is useful for the nucleic acid molecules to be composed of triple- stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning. In some embodiments, the genetically engineered microorganism comprises at least one nucleic acid molecule described herein.

[0059] As used herein, the term “exogenous” refers to an element that has been introduced into a cell. An exogenous element can include a protein or a nucleic acid. An exogenous nucleic acid is a nucleic acid that has been introduced into a cell, such as by a method of transformation. An exogenous nucleic acid may code for the expression of an RNA and/or a protein. An exogenous nucleic acid may have been derived from the same species (homologous) or from a different species (heterologous). An exogenous nucleic acid may comprise a homologous sequence that is altered such that it is introduced into the cell in a form that is not normally found in the cell in nature. For example, an exogenous nucleic acid that is homologous may contain mutations, being operably linked to a different control region, or being integrated into a different region of the genome, relative to the endogenous version of the nucleic acid. An exogenous nucleic acid may be incorporated into the chromosomes of the transformed cell in one or more copies, into the plastid or mitochondrial DNA of the transformed cell, or be maintained as a separate nucleic acid outside of the transformed cell genome.

[0060] The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences.

[0061] Increased cannabinoid biosynthetic pathway products produced by a genetically engineered microorganism can be the result of increasing activity of one or more enzymes associated with cannabinoid biosynthetic pathway. Increase of activity of an enzyme in a microorganism can include, for example, the introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding the enzyme. In an embodiment, introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding an enzyme can be accomplished by transformation. Examples of cannabinoid biosynthetic pathway enzymes include, but are not limited to hexanoyl-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, geranyl pyrophosphate synthase, aromatic prenyltransferase, geranyl pyrophosphate:olivetolic acid geranyltransferase, cannabichromene synthase, tetrahydrocannabinolic acid synthase, and cannabidiolic acid synthase.

[0062] Figure 1 shows an exemplary cannabinoid biosynthetic pathway based on enzymes from Cannabis sativa : Tetraketide synthase (TKS) condenses hexanoyl-CoA and malonyl-CoA to form the intermediate trioxododecanoyl-CoA; Olivetolic acid cyclase (OAC) catalyzes an intramolecular aldol condensation to yield olivetolic acid (OA); aromatic prenyltransferase transfers a geranyldiphosphate (GPP) onto OA to produce cannabigerolic acid (CBGA); tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase catalyze the oxidative cyclization of CBGA into tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), respectively. Decarboxylation of THCA or CBDA to remove the carboxyl group will produce decarboxylated cannabinoids tetrahydrocannabinol (THC) or cannabidiol (CBD), respectively.

[0063] In addition to the exemplary cannabinoid biosynthetic pathway from Cannabis sativa shown in Figure 1 , alternative biosynthetic intermediates can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. For example, olivetol is an intermediate that lacks the carboxyl group of olivetolic acid. Use of olivetol instead of olivetolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly lack a carboxyl group such as cannabigerol (CBG), tetrahydrocannabinol (THC), or cannabidiol (CBD). In another example, tetraketide synthase (TKS) condenses butyryl-CoA and malonyl-CoA to form the intermediate trioxodecanoyl-CoA, and olivetolic acid cyclase (OAC) catalyzes an intramolecular aldol condensation of trioxodecanoyl-CoA to yield divarinolic acid. Divarinolic acid is an intermediate containing an n-propyl group in place of the n-pentyl group found in olivetolic acid. Use of divarinolic acid instead of olivetolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly contain an n-propyl group such as cannabigerovarinic acid (CBGVA), tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), or cannabichromevarinic acid (CBCVA). In another example, divarinol is an intermediate that lacks the carboxyl group of divarinolic acid, and contains an n-propyl group in place of the n-pentyl group found in olivetol. Use of divarinol instead of divarinolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly contain an n-propyl group and lack a carboxyl group such as cannabigerovarin (CBGV), tetrahydrocannabivarin (THCV), cannabidivarinic acid (CBDV), or cannabichromevarinic acid (CBCV).

[0064] In addition to the exemplary cannabinoid biosynthetic pathway from Cannabis sativa shown in Figure 1 , alternative enzymes can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. For example, in addition to the enzymes found in Cannabis sativa , alternative enzymes of a cannabinoid biosynthetic pathway may be found in other plants (e.g., Humulus lupulus), in bacteria (e.g., Streptomyces), or in protists (e.g., Dictyostelium discoideum). Enzymes that differ in structure, but perform the same function, may be used interchangeably in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. For example, the aromatic prenyltransferases CsPT1 (SEQ ID NO:18) and CsPT4 (SEQ ID NO:64) from Cannabis sativa , HIPT1 from Humulus lupulus (SEQ ID NO:65), and Orf2 (SEQ ID NO:63) from Streptomyces Sp. Strain CI190 are all aromatic prenyltransferases that catalyze the synthesis of CBGA from GPP and OA. In a further example, the Steelyl (SEQ ID NO:61) or Steely2 (SEQ ID NO:62) polyketide synthase from Dictyostelium discoideum , or a variant thereof, can be used to condense malonyl-CoA into olivetol, and may be used in place of TKS to produce olivetol in the absence of OAC.

[0065] In addition to the wild-type enzymes found in organisms discussed herein, modified variants of these enzymes can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. Variants of enzymes for use in a cannabinoid biosynthetic pathway can be generated by altering the nucleic acid sequence encoding said enzyme to, for example, increase/decrease the activity of a domain, add/remove a domain, add/remove a signaling sequences, or to otherwise alter the activity or specificity of the enzyme. For example, the sequence of Steelyl can be modified to reduce the activity of a methyltransferase domain in order to produce non-methylated cannabinoids. By way of example, this can be done by mutating amino acids G1516D+G1518A or G1516R relative to SEQ ID NO:61 as disclosed in WO/2018/148849, herein incorporated by reference. In a further example, the sequences of tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase can be modified to remove an N-terminal secretion peptide. By way of example, this can be done by removing amino acids 1-28 of SEQ ID NO:20 or 21 to produce a truncated enzyme as disclosed in WO/2018/200888, herein incorporated by reference.

[0066] An acyl-CoA synthetase is an acyl-activating enzymethat ligates CoA and a straight-chain alkanoic acid or alkanoate containing 2 to 6 carbon atoms to produce alkanoyl-CoA, wherein the alkanoyl-CoA is a thioester of coenzyme A containing an alkanoyl group of 2 to 6 carbon atoms. In one embodiment, the acyl-CoA synthetase is hexanoyl-CoA synthetase, which ligates CoA and hexanoic acid or hexanoate to produce hexanoyl-CoA. A hexanoyl-CoA synthetase may have the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence with at least 90% identity to SEQ ID NO: 19. In another embodiment, an acyl-CoA synthetase ligates CoA and butyric acid or butyrate to produce butyryl-CoA.

[0067] A type III polyketide synthase is an enzyme that produces polyketides by catalyzing the condensation reaction of acetyl units to thioester-linked starter molecules. A type III polyketide synthase may have the amino acid sequence of SEQ ID NO: 15, 61 or 62 or an amino acid sequence with at least 90% identity to SEQ ID NO: 15, 61 or 62. In an embodiment, a type III polyketide synthase condenses an alkanoyl-CoA with three malonyl-CoA in a multi-step reaction to form a 3,5,7-trioxoalkanoyl-CoA, wherein the 3, 5, 7-trioxoalkanoyl-CoA contains 8 to 12 carbon atoms. In another embodiment, the type III polyketide synthase is tetraketide synthase from Cannabis sativa which is also known in the art as olivetol synthase and 3,5,7-trioxododecanoyl-CoA synthase. In one embodiment, tetraketide synthase condenses hexanoyl-CoA with three malonyl-CoA in a multi-step reaction to form 3,5,7-trioxododecanoyl-CoA. In another embodiment, tetraketide synthase condenses butyryl-CoA with three malonyl-CoA in a multi-step reaction to form 3,5,7-trioxodecanoyl-CoA. In another embodiment, the type III polyketide synthase is Steelyl or Steely2 from Dictyostelium discoideum, comprising a domain with type III polyketide synthase activity, ora variant thereof (e.g., Steelyl (G1516D+G1518A) or Steelyl (G1516R) disclosed in WO/2018/148849). Steelyl is also known in the art as DiPKS or DiPKSI , and Steely2 is also known in the art as DiPKS37.

[0068] An olivetolic acid cyclase, as used herein, refers to an enzyme that catalyzes an intramolecular aldol condensation of a 3,5,7-trioxoalkanoyl-CoA to form a 2,4-dihydroxy-6-alkylbenzoic acid, wherein the alkyl group of the benzoic acid contains 1 to 5 carbons. In an embodiment, an olivetolic acid cyclase catalyzes the formation of olivetolic acid from 3,5,7-trioxododecanoyl-CoA. In another embodiment, an olivetolic acid cyclase catalyzes the formation of divarinolic acid from 3,5,7-trioxodecanoyl-CoA. An olivetolic acid cyclase may have the amino acid sequence of SEQ ID NO: 16 or 17 or an amino acid sequence with at least 90% identity to SEQ ID NO: 16 or 17. Olivetolic acid cyclase from Cannabis sativa is also known in the art as olivetolic acid synthase and 3,5,7-trioxododecanoyl-CoA CoA-lyase.

[0069] An aromatic prenyltransferase, as used herein, refers to an enzyme capable of transferring a geranyl diphosphate onto a 5-alkylbenzene-1 ,3-diol to synthesize a 2- geranyl-5-alkylbenzene-1 ,3-diol, wherein the alkyl group of the product contains 1 to 5 carbons. In one embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto olivetol to synthesize cannabigerol (CBG). In another embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto olivetolic acid (OA) to synthesize cannabigerolic acid (CBGA). In another embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto divarinolic acid to synthesize cannabigerovarin (CBGV). In another embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto divarinolic acid to synthesize cannabigerovarinic acid (CBGVA). An example of an aromatic prenyltransferase is aromatic prenyltransferase from Cannabis sativa which is also known in the art as CsPT1 , prenyltransferase 1 , geranylpyrophosphate-olietolic acid geranyltransferase, and geranyl-diphosphate: olivetolate geranytransferase. Further examples of aromatic prenyltransferase include HIPT1 from Humulus lupulus , CsPT4 from Cannabis sativa , and Orf2 (NphB) from Streptomyces Sp. Strain CI190. An aromatic prenyltransferase may have the amino acid sequence of SEQ ID NO: 18, 63, 64 or 65, or an amino acid sequence with at least 90% identity to SEQ ID NO: 18, 63, 64 or 65.

[0070] A tetrahydrocannabinolic acid synthase is also known in the art as D9- tetrahydrocannabinolic acid synthase, and synthesizes A9-tetrahydrocannabinolic acid by catalyzing the cyclization of the monoterpene moiety in cannabigerolic acid. A tetrahydrocannabinolic acid synthase may have the amino acid sequence of SEQ ID NO:

20 or an amino acid sequence with at least 90% identity to SEQ ID NO: 20.

[0071] A cannabidiolic acid synthase synthesizes cannabidiolic acid by catalyzing the stereoselective oxidative cyclization of the monoterpene moiety in cannabigerolic acid. A cannabidiolic acid synthase may have the amino acid sequence of SEQ ID NO:

21 or an amino acid sequence with at least 90% identity to SEQ ID NO: 21 .

[0072] In an embodiment, the nucleic acid molecule encodes at least one, two, three, four, five, or six of hexanoyl-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase; or encodes at least one, two, three, four, or five of type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase, without encoding hexanoyl-CoA synthetase. In another embodiment, the at least one nucleic acid molecule comprises nucleic acid sequence encoding hexanoyl-CoA synthetase comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence shown in SEQ ID NO:5 or 12. In another embodiment, the at least one nucleic acid molecule comprises nucleic acid sequence encoding type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2) comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence shown in SEQ ID NO:1 , 8, 56, 57, 66, or 67, or a sequence encoding type III polyketide synthase comprised in any of the constructs provided in examples 7 and 8. In another embodiment, the at least one nucleic acid molecule comprises nucleic acid sequence encoding olivetolic acid cyclase comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence shown in SEQ ID NO:2, 3, 9 or 10, or a sequence encoding olivetolic acid cyclase comprised in any of the constructs provided in examples 7 and 8. In another embodiment, the at least one nucleic acid molecule comprises nucleic acid sequence encoding aromatic prenyltransferase comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence shown in SEQ ID NO:4, 11 , 58, 59, 60, 68, 69, or 70 or a sequence encoding aromatic prenyltransferase comprised in any of the constructs provided in examples 7 and 8. In another embodiment, the at least one nucleic acid molecule comprises nucleic acid sequence encoding tetrahydrocannabinolic acid synthase comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence shown in SEQ ID NO:6 or 13, or a sequence encoding tetrahydrocannabinolic acid synthase comprised in any of the constructs provided in examples 7 and 8. In another embodiment, the at least one nucleic acid molecule comprises nucleic acid sequence encoding cannabidiolic acid synthase comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence shown in SEQ ID NO:7 or 14, or a sequence encoding cannabidiolic acid synthase comprised in any of the constructs provided in examples 7 and 8. In another embodiment, the nucleic acid molecule is comprised in a genetically engineered microorganism.

[0073] In an embodiment, the nucleic acid molecule comprising nucleic acid sequence encoding at least one of hexanoyl-CoA synthetase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO: 19, type III polyketide synthase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO: 15, 61 or 62, olivetolic acid cyclase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:16 or 17, aromatic prenyltransferase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:18, 63, 64, or 65, tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:20, and cannabidiolic acid synthase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:21. In another embodiment, the nucleic acid molecule does not comprise nucleic acid sequence encoding hexanoyl-CoA synthetase. In another embodiment, the nucleic acid molecule is comprised in a genetically engineered microorganism. [0074] As used herein, the term “vector” or “nucleic acid vector” means a nucleic acid molecule, such as a plasmid, comprising regulatory elements and a site for introducing transgenic DNA, which is used to introduce said transgenic DNA into a microorganism. The transgenic DNA can encode a heterologous protein, which can be expressed in and isolated from a microorganism. The transgenic DNA can be integrated into nuclear, mitochondrial or chloroplastic genomes through homologous or non- homologous recombination. The transgenic DNA can also replicate without integrating into nuclear, mitochondrial or chloroplastic genomes in an extra-chromosomal vector. The vector can contain a single, operably-linked set of regulatory elements that includes a promoter, a 5’ untranslated region (5’ UTR), an insertion site for transgenic DNA, a 3’ untranslated region (3’ UTR) and a terminator sequence. Vectors useful in the present methods are well known in the art. In one embodiment, the nucleic acid molecule is an episomal vector.

[0075] As used herein, the term “episomal vector” refers to a DNA vector based on a bacterial episome that can be expressed in a transformed cell without integration into the transformed cell genome. Episomal vectors can be transferred from a bacteria (e,g, Escherichia coli ) to another target microorganism (e.g. a microalgae) via conjugation.

[0076] In another embodiment, the vector is a commercially-available vector. As used herein, the term “expression cassette” means a single, operably-linked set of regulatory elements that includes a promoter, a 5’ untranslated region (5’ UTR), an insertion site for transgenic DNA, a 3’ untranslated region (3’ UTR) and a terminator sequence. In an embodiment, the at least one nucleic acid molecule is an episomal vector.

[0077] The term “operably-linked”, as used herein, refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. For example, a transcriptional regulatory sequence or a promoter is operably-linked to a coding sequence if the transcriptional regulatory sequence or promoter facilitates aspects of the transcription of the coding sequence. The skilled person can readily recognize aspects of the transcription process, which include, but not limited to, initiation, elongation, attenuation and termination. In general, an operably-linked transcriptional regulatory sequence joined in cis with the coding sequence, but it is not necessarily directly adjacent to it. [0078] The nucleic acid vectors encoding the cannabinoid biosynthetic pathway enzyme therefore contain elements suitable for the proper expression of the enzyme in the microorganism. Specifically, each expression vector contains a promoter that promotes transcription in microorganisms. The term “promoter,” as used herein, refers to a nucleotide sequence that directs the transcription of a gene or coding sequence to which it is operably-linked. Suitable promoters include, but are not limited to, pEF-1a, p40SRPS8, pH4-1 B, py-Tubulin, pRBCMT, pFcpA, pFcpB, pFcpC, pFcpD, HSP70A- RbcS2 (as shown in Table 1 as SEQ ID NO:38-45,112 and 114; see Slattery etal , 2018), and RbcS2. The skilled person can readily appreciate inducible promoters including chemically-inducible promoters, alcohol inducible promoters, and estrogen inducible promoters can also be used. Predicted promoters, such as those that can be found from genome database mining may also be used. In addition, the nucleic acid molecule or vector may contain one or more introns in front of the cloning site or within a gene sequence to drive a strong expression of the gene of interest. The one or more introns includes introns of FBAC2-1 TUFA-1 , EIF6-1 , RPS4-1 , RbcS2-1 , RbcS2-2 (as shown in Table 1 as SEQ ID NO:34-37 and 107-108). The nucleic acid molecule may contain more than one intron or more than one copy of the same intron. The nucleic acid molecule or vector also contains a suitable terminator such as tEF-1a, t40SRPS8, tH4-1 B, ty-Tubulin, tRBCMT, tFcpB, tFcpC, tFcpD, PAL, tFcpA, tRbcS2 (as shown in Table 1 as SEQ ID NO:46-53, 113 and 115). Seletectable marker genes can also be linked on the vector, such as the kanamycin resistance gene (also known as neomycin phosphotransferase gene II, or nptll), zeocin resistance gene, hygromycin resistance gene, Basta resistance gene, hygromycin resistance gene, or others.

[0079] As used herein, the term “tag” refers to an amino acid sequence that is recognized by an antibody. The tag amino acid sequence links to, for example, sequence of an enzyme, thereby allowing detection or isolation of the enzyme by the binding between the tag and the tag-specific antibody. For example, common tags known in the art include 6His, MYC, FLAG, V5, HA and HSV. These tags are useful when positioned at the N- or C-terminus. In some embodiments, the at least one nucleic acid molecule comprises one or more tag sequences encoding a tag with an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NQ:95-100. [0080] As used herein, the term “reporter” refers to a molecule that allows for the detection of another molecule to which the reporter is attached or associated, or for the detection of an organism that comprises the reporter. Reporters can include fluorescent molecules including fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP). In some embodiments, the at least one nucleic acid molecule comprises one or more reporter sequences encoding a reporter with an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO: 110-111.

[0081] In an embodiment, the nucleic acid molecule comprises a sequence encoding Rubisco small subunit. Rubisco small subunit may enable the targeting of a polypeptide to which it is attached to be exported to chloroplasts via an internal plastid- targeting signal (Hirakawa and Ishida 2010). Without being bound by theory, it is expected that exporting cannabinoid biosynthetic enzymes to the chloroplast compartment may enhance the exogenous cannabinoid biosynthetic pathway in microalgae because of the availability in the chloroplast of substrates including acetyl-CoA and malonyl-CoA. In some embodiments, the at least one nucleic acid molecule comprises a sequence encoding Rubisco small subunit with an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO: 116.

[0082] In an embodiment, the nucleic acid molecule or vector encoding the at least one cannabinoid biosynthetic pathway enzyme comprises a promoter nucleic acid sequence selected from SEQ ID NO:38-45, 112 and 114, wherein said promoter is operably-linked to a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence encoding a cannabinoid biosynthetic pathway enzyme. In another embodiment, the nucleic acid molecule comprises at least one intron sequence selected from SEQ ID NO:34-37 and 107-108. In another embodiment, the nucleic acid molecule comprises a terminator nucleic acid sequence selected from SEQ ID NO:46-53, 113 and 115. In another embodiment, the genetically engineered microorganism comprises a nucleic acid molecule comprising at least one sequence encoding a tag with an amino acid sequence selected from SEQ ID N0:95-100.

[0083] The nucleic acid molecule can be constructed to express at least one, two, three, four, five, or six enzymes associated with the cannabinoid biosynthetic pathway. In an embodiment, the nucleic acid molecule comprises two or more polynucleotide sequences, each of which encodes one cannabinoid biosynthetic pathway enzyme and is operably linked to the same promoter. Where at least two, three, four, five, or six enzymes are encoded in a construct, the construct can contain nucleotide sequence encoding a self-cleaving peptide linker, for example FMDV2a, extFMDV2a, or T2A peptide linker, which results in the enzymes being produced as separated proteins. The construct can also contain peptide linker sequences linking the enzymes as a fusion protein, for example 3(GGGGS) and FPL1 peptide linker, allowing substrate channelling in which the passing of the intermediary metabolic product of one enzyme directly to another enzyme or active site without its release into solution. The construct can also contain a combination of self-cleaving and non-self-cleaving linker sequences. In an embodiment, the nucleic acid molecule comprises at least one linker sequence between at least two polynucleotide sequences. In another embodiment, the linker sequence is SEQ ID NO:54 or 55 or a linker sequence disclosed in examples 7 and 8. In another embodiment, the nucleic acid molecule comprises one or more linker sequences encoding a peptide linker with an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO: 101- 106.

[0084] In another embodiment, the vector comprises a nucleic acid sequence as described herein. In another embodiment, a host cell is transformed with a vector or nucleic acid molecule comprising a nucleic acid sequence as described herein. In another embodiment, the host cell is any microorganism as described herein.

[0085] Nucleic acid sequences as described herein can be provided in vectors in different arrangements or combinations. Each individual sequence that encodes an enzyme of a cannabinoid biosynthetic pathway can be provided in separate vectors. Alternatively, multiple sequences can be provided together in the same vector. For example, nucleic acid sequences encoding a type III polyketide synthase and an olivetolc acid cyclase can be provided together in a first vector, a nucleic acid sequence encoding an aromatic prenyltransferase can be provided in a second vector, and nucleic acid sequences encoding a tetrahydrocannabinolic acid synthase and/or a cannabidiolic acid synthase can be provided in a third vector. Alternatively, sequences that encode all of the enzymes of a cannabinoid biosynthetic pathway can be provided together in the same vector. Where more than one sequence that encodes an enzyme is provided in the same vector, the sequences can be provided in separate expression cassettes, or together in the same expression cassette. Where two or more sequences are in the same expression cassette, they can be provided in the same open reading frame so as to produce a fusion protein. Two or more sequences that encode a fusion protein can be separated by linker sequences that encode restriction nuclease recognition sites or self-cleaving peptide linkers. Accordingly, a genetically modified microorganism for the production of cannabinoids can be engineered by stepwise transfection with multiple vectors that each comprises nucleic acid sequences that encode one or more enzymes of a cannabinoid biosynthetic pathway, or with a single vector that comprises nucleic acid sequences that encode all of the enzymes of a cannabinoid biosynthetic pathway.

[0086] As used herein, the term “microalgae” and its derivatives, include photosynthetic and non-photosynthetic microorganisms that are eukaryotes. As used herein, the term “cyanobacteria” and its derivatives, include photosynthetic microorganisms that are prokaryotes. In an embodiment, the microalga is a GC-rich microalga. As used herein, “GC-rich microalga” refers to a microalga wherein the DNA of the nuclear genome and/or the plastid genome comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% GC content. In an embodiment, the microalga is an oleaginous microalga. As used herein “oleaginous” refers to a microalga comprising a lipid conent of at least 35%, at least 40%, at least 45%, or at least 50% by weight. In an embodiment, the microalga is a cold-adapted microalga. As used herein, “cold-adapted” refers to a microalga that grows in temperate, sub-polar, or polar regions in nature, or that has been adapted in artificial growth conditions to grow at temperatures found in temperate, sub-polar, or polar regions. In some embodiments, the cold-adapted microalga grows at a temperature lower than 24°C, lower than 20°C, lower than 16°C, or lower than 12°C. In an embodiment, the microalga is a cold-adapted microalga that exhibits increased lipid content when grown at a temperature lower than 24°C, lower than 20°C, lower than 16°C, or lower than 12°C.

[0087] In an embodiment, the microalga is a green alga. In an embodiment, the microalga is from the phylum Chlorophyta. In an embodiment, the microalga is from the genera Ankistrodesmus, Asteromonas, Auxenochlorella, Basichlamys, Botryococcus, Botryokoryne, Borodinella, Brachiomonas, Catena, Carteria, Chaetophora, Characiochloris, Characiosiphon, Chlainomonas, Chlamydomonas, Chlorella,

Chlorochytrium, Chlorococcum, Chlorogonium, Chloromonas, Closteriopsis,

Dictyochloropsis, Dunaliella, Ellipsoidon, Eremosphaera, Eudorina, Floydiella, Friedmania, Haematococcus, Hafniomonas, Heterochlorella, Gonium, Halosarcinochlamys, Koliella, Lobocharacium, Lobochlamys, Lobomonas, Lobosphaera, Lobosphaeropsis, Marvania, Monoraphidium, Myrmecia, Nannochloris, Oocystis, Oogamochlamys, Pabia, Pandorina, Parietochloris, Phacotus, Platydorina, Platymonas, Pleodorina, Polulichloris, Polytoma, Polytomella, Prasiola, Prasiolopsis, Prasiococcus, Prototheca, Pseudochlorella, Pseudocarteria, Pseudotrebouxia, Pteromonas, Pyrobotrys, Rosenvingiella, Scenedesmus, Spirogyra, Stephanosphaera, Tetrabaena, Tetraedron, Tetraselmis, Trebouxia, Trochisciopsis, Viridiella, Vitreochlamys, Volvox, Volvulina, Vulcanochloris, Watanabea, Yamagishiella, Euglena, Isochrysis, Nannochloropsis. In an embodiment, the microalga is Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chui, Nannochloropsis oculate, Scenedesmus obliquus, Acutodesmus dimorphus, Dunaliella tertiolecta , or Heamatococus plucialis. In another embodiment, the microalga is a diatom, optionally Phaeodactylum tricornutum or Thalassiosira pseudonana.

[0088] In another embodiment, the cyanobacterium is from Spirulinaceae,

Phormidiaceae, Synechococcaceae, or Nostocaceae. In an embodiment, the cyanobacterium is Arthrospira plantesis , Arthrospira maxima, Synechococcus elongatus, or Aphanizomenon flos-aquae.

[0089] In another embodiment, the microorganism is a bacterium, for example from the genera Escherichia, Bacillus, Caulobacter, Mycoplasma, Pseudomonas, Streptomyces, or Zymomonas.

[0090] In another embodiment, the microorganism is a protist, for example from the genera Dictyostelium, Tetrahymena, Emiliania, or Thalassiosira.

[0091] In another emobodiment, the microorganism is a fungus, for example from the genera Aspergillus, Saccharomyces, Schizosaccharomyces, or Fusarium.

[0092] The present disclosure also provides a cell culture comprising a genetically engineered microorganism described herein for production of cannabinoid biosynthetic pathway products and a medium for culturing the genetically engineered microorganism. In an embodiment, the medium is substantially free of a sugar, i.e., the concentration of the sugar being less than 2%, less than 1.5%, less than 1 %, less than 0.5%, or less than 0.1% by weight. In another embodiment, the medium contains no more than trace amounts of a sugar, a trace amount commonly understood in the art as referring to insignificant amounts or amounts near the limit of detection. Sugars known to be required for culturing microorganisms that are not capable of photosynthesis include, but are not limited to, monosaccharides (e.g., glucose, fructose, ribose, xylose, mannose, and galactose) and disaccharides (e.g., sucrose, lactose, maltose, lactulose, trehalose, and cellobiose).

[0093] In another embodiment, the medium is substantially free of a fixed carbon source, i.e., the concentration of the fixed carbon source being less than 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1% by weight. In another embodiment, the medium contains no more than trace amounts of a fixed carbon source. The term “fixed carbon source”, as used herein, refers to an organic carbon molecule that is liquid or solid at ambient temperature and pressure that provides a source of carbon for growth, biosynthesis, and/or metabolism. Examples of fixed carbon sources include, but are not limited to, sugars (e.g. glucose, galactose, mannose, fructose, sucrose, lactose), amino acids or amino acid derivatives (e.g. glycine, N-acetylglucosamine, glycerol, floridoside, glucuronic acid, corn starch, depolymerized cellulosic material, plant material (e.g. sugar cane, sugar beet), and carboxylic acid (e.g. hexanoic acid, butyric acid and their respective salts). Sources of fixed carbon are disclosed in WO/2015/168458, the contents of which are herein incorporated by reference.

[0094] Microorganisms may be cultured in conditions that are permissive to their growth. It is known that photosynthetic microorganisms are capable of carbon fixation wherein carbon dioxide (which is not a fixed carbon source) is fixed into organic molecules such as sugars using energy from a light source. The fixation of carbon dioxide using energy from a light source is photosynthesis. Suitable sources of light for the provision of energy in photosynthesis include sunlight and artificial lights. Photosynthetic microorganisms are capable of growth and/or metabolism without a fixed carbon source. Microalgae can fix carbon dioxide from a variety of sources, including atmospheric carbon dioxide, industrially-discharged carbon dioxide (e.g. flue gas and flaring gas), and from soluble carbonates (e.g. NaHC03 and Na2C03), (see Singh et al 2014, the contents of which are hereby incorporated by reference). A non-fixed carbon source such as carbon dioxide can be added to a culture of microalgae by injection or by bubbling of a carbon dioxide gas mixture into the culture medium. Photosynthetic growth is a form of autotrophic growth, wherein a microorganism is able to produce organic molecules on its own using an external energy source such as light. This is in contrast to heterotrophic growth, wherein a microorganism must consume organic molecules for growth and/or metabolism. Heterotrophic organisms therefore require a fixed carbon source for growth and/or metabolism. Some photosynthetic organisms are capable of mixotrophic growth, wherein the microorganism fixes carbon by photosynthesis while also consuming fixed carbon sources. In mixotrophic growth, the autotrophic metabolism is integrated with a heterotrophic metabolism that oxidizes reduced carbon sources available in the culture medium. Photosynthetic microalgae are commonly cultivated in mixotrophic conditions by adding fixed carbon sources as described herein to the culture medium. Common sources of fixed caron that are used include glucose, ethanol, or waste products from industry such as acetate or glycerol (see Cecchin et al 2018, the contents of which are hereby incorporated by reference). Microorganisms such as microalgae and cyanobacteria may be cultured using methods and conditions known in the art (see, e.g., Biofuels from Algae, eds. Pandey et al., 2014, Elsevier, ISBN 978-0-444-59558-4, the contents of which are hereby incorporated by reference). Some microorganisms are capable of chemoautotrophic growth, Similar to photosynthetic microorganisms, chemoautotrophic organisms are capable of carbon dioxide fixation but using energy derived from chemical sources (e.g. hydrogen sulfide, ferrous iron, molecular hydrogen, ammonia) rather than light.

[0095] Microalgae can be grown in organic conditions without the use of chemicals or additives that contravene the standards for organically-produced products. Microalgae can be grown organically, for example, by growing them in conditions that comply with jurisdictional standards such as the standards set by the United States (US Organic Food Production Act; USDA National Organic Program Certification; USDA Organic Regulations), the European Union (Regulation No 834/2007 prior to January 1 , 2021 ; Regulation 2018/848 from January 1 , 2021), and Canada (Canadian Food Inspection Agency Canadian Organic Standards). Growing microalgae in organic conditions permits the production of organic plant natural products in microalgae.

[0096] The present disclosure also provides a nucleic acid molecule comprising a nucleotide sequence encoding at least one, two, three, four, five, or six cannabinoid biosynthetic pathway enzymes, wherein the nucleic acid molecule comprises at least one polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 1 -14, 56-60, 66-94. In one embodiment, the nucleic acid molecule comprises nucleic acid sequences encoding at least one, two, three, four, five or six of hexanoyl-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase. In another embodiment, the nucleic acid molecule comprises nucleic acid sequences encoding at least one, two, three, four, or five of type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase without encoding hexanoyl-CoA synthetase.

[0097] In an embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1 or a sequence encoding type III polyketide synthase comprised in any of the constructs provided in example 8 and a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 or a sequence encoding olivetolic acid cyclase comprised in any of the constructs provided in example 8. In another embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1 or a sequence encoding type III polyketide synthase comprised in any of the constructs provided in example 8 and a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3. In an embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 66 or 67. In another embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4, 68, 69, or 70 ora sequence encoding aromatic prenyltransferase comprised in any of the constructs provided in example 8, and optionally a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5. In another embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6 or a sequence encoding tetrahydrocannabinolic acid synthase (THCAS) comprised in any of the constructs provided in example 8 and/or a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7 or a sequence encoding cannabidiolic acid synthase (CBDAS) comprised in any of the constructs provided in example 8. In another embodiment, the nucleic acid molecule is comprised in a genetically engineered microorganism, optionally a GC-rich microalga, optionally Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chui, Nannochloropsis oculate, Scenedesmus obliquus, Acutodesmus dimorphus, Dunaliella tertiolecta , or Heamatococus plucialis.

[0098] In an embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8 or a sequence encoding type III polyketide synthase comprised in any of the constructs provided in example 7 and a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9 or a sequence encoding olivetolic acid cyclase comprised in any of the constructs provided in example 7. In another embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8 or a sequence encoding type III polyketide synthase comprised in any of the constructs provided in example 7 and a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:10. In an embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 56 or 57. In another embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11 , 58, 59, or 60 or a sequence encoding aromatic prenyltransferase comprised in any of the constructs provided in example 7, and optionally a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:12. In another embodiment, the nucleic acid molecule comprises at least a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13 or a sequence encoding tetrahydrocannabinolic acid synthase comprised in any of the constructs provided in example 7 and/or a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 or a sequence encoding cannabidiolic acid synthase comprised in any of the constructs provided in example 7. In another embodiment, the nucleic acid molecule is comprised in a genetically engineered microorganism, optionally a diatom, optionally Thalassiosira pseudonana or Phaeodactylum tricorn utum. [0099] The phrase “introducing a nucleic acid molecule into a microorganism” includes both the stable integration of the nucleic acid molecule into the genome of a microorganism to prepare a genetically engineered microorganism as well as the transient integration of the nucleic acid into microorganism. The introduction of a nucleic acid into a cell is also known in the art as transformation. The nucleic acid vectors may be introduced into the microorganism using techniques known in the art including, without limitation, agitation with glass beads, electroporation, agrobacterium-mediated transformation, an accelerated particle delivery method, i.e. particle bombardment, a cell fusion method or by any other method to deliver the nucleic acid vectors to a microorganism.

[00100] Further provided is a method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase, a second polynucleotide sequence encoding olivetolic acid cyclase, and at least one linker sequence between the first and second polynucleotide sequences, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

[00101] Further provided is a method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

[00102] Further provided is a method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34- 37 and 107-108).

[00103] Further provided is a method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116), a second polynucleotide sequence encoding tetraketide synthase, and a third polynucleotide sequence encoding olivetolic acid cyclase.

[00104] Further provided is a method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116) and a second polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof.

[00105] Further provided is a method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and a second nucleic acid molecule comprising a third polynucleotide sequence encoding an aromatic prenyltransferase, and wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65.

[00106] Further provided is a method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and a second nucleic acid molecule comprising a second polynucleotide sequence encoding an aromatic prenyltransferase, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65.

[00107] In an embodiment, the method involves at least one nucleic acid molecule comprising nucleic acid sequence encoding at least one, two, three, four, five, or six of hexanoly-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase. In another embodiment, the method involves at least one nucleic acid molecule comprising nucleic acid sequence encoding at least one, two, three, four, or five of type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase without encoding hexanoyl-CoA synthetase. [00108] In another embodiment, the method involves at least one nucleic acid molecule comprising nucleic acid sequence encoding at least one, two, three, four, five, or six of hexanoyl-CoA synthetase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO: 19, type III polyketide synthase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO: 15, 61 , or 62, olivetolic acid cyclase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:16 or 17, aromatic prenyltransferase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:18, 63, 64, or 65, tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:20, and cannabidiolic acid synthase comprises amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequence as shown in SEQ ID NO:21.

[00109] In an embodiment, the method involves a promoter nucleic acid sequence selected from SEQ ID NO:38-45, 112 and 114, wherein said promoter is operably-linked to a polynucleotide sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 1-14, 56-60, 66-70, 71-80 and 85-94. In another embodiment, the method involves at least one sequence encoding a tag with an amino acid sequence selected from SEQ ID N0:95-100, at least one intron sequence selected from SEQ ID NO:34-37 and 107-108, and/or a terminator nucleic acid sequence selected from SEQ ID NO:46-53, 113 and 115.

[00110] In an embodiment, the method involves at least two polynucleotide sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO:1-14, 56-60, and 66-70 or a polynucleotide sequence encoding a cannabinoid biosynthetic pathway enzyme comprised in a construct provided in examples 7 and 8. In another embodiment, the method involves at least one linker sequence between the at least two polynucleotide sequences. In another embodiment, the method involves a linker sequence that encodes a self-cleaving peptide, such as FMDV2a, extFMDV2a, or T2a. In another embodiment, the method involves a linker sequence that encodes a non-self-cleaving or fusion sequence, such as 3(GGGGS) or FPL1. [00111] In another embodiment, the method involves producing a cannabinoid biosynthetic pathway product in a microalga, wherein the microalga is a GC-rich microalga, optionally Chlamydomonas reinhardtii, or a diatom, optionally Phaeodactylum tricornutum or Thalassiosira pseudonana. In another embodiment, the method involves producing a cannabinoid biosynthetic pathway product in cyanobacteria, wherein the cyanobacteria are from Spirulinaceae, Phormidiaceae, Synechococcaceae, or Nostocaceae, optionally Arthrospira plantesis , Arthrospira maxima, Synechococcus elongatus, or Aphanizomenon flos-aquae. In another embodiment, the method involves introducing at least one nucleic acid molecule that is an episomal vector into the microorganism. In another embodiment, the method involves introducing at least one nucleic acid molecule described herein into the microorganism.

[00112] In another embodiment, the method involves production of at least one, two, three, four, five, six, seven, eight, nine, or ten cannabinoid biosynthetic pathway products including hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, cannabigerolic acid, cannabigerol, A9-tetrahydrocanannabinolic acid, cannabidiolic acid, D9- tetrahydrocanannabinol, or cannabidiol.

[00113] Particular embodiments of the disclosure include, without limitation, the following:

1. A genetically engineered microorganism that is capable of producing olivetolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase, a second polynucleotide sequence encoding olivetolic acid cyclase, and at least one linker sequence between the first and second polynucleotide sequences, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

2. The genetically engineered microorganism of embodiment 1 , wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

3. The genetically engineered microorganism of embodiment 1 or 2, wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

4. The genetically engineered microorganism of any one of embodiments 1 to

3, wherein the tetraketide synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO: 15, and the olivetolic acid cyclase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:16.

5. The genetically engineered microorganism of any one of embodiments 1 to 4, wherein the first nucleic acid molecule further comprises a third polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116).

6. The genetically engineered microorganism of embodiment 5, wherein the third polynucleotide sequence is 5’ to the first polynucleotide sequence.

7. The genetically engineered microorganism of any one of embodiments 1 to

6, wherein the first nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

8. The genetically engineered microorganism of any one of embodiments 1 to

7, wherein the first nucleic acid molecule is an episomal vector.

9. The genetically engineered microorganism of any one of embodiments 1 to

8, further comprising a second nucleic acid molecule comprising a fourth polynucleotide sequence encoding an aromatic prenyltransferase.

10. The genetically engineered microorganism of embodiment 9, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ I D NO: 18, or 63-65.

11. The genetically engineered microorganism of embodiment 9 or 10, wherein the second nucleic acid molecule further comprises a fifth polynucleotide sequence encoding tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase.

12. The genetically engineered microorganism of embodiment 11 , wherein the tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:20, and the cannabidiolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:21.

13. The genetically engineered microorganism of embodiment 11 or 12, wherein the fourth polynucleotide sequence is 5’ to the fifth polynucleotide sequence. 14. The genetically engineered microorganism of embodiment 13, wherein the second nucleic acid molecule further comprises at least one linker sequence between the fourth and fifth polynucleotide sequences.

15. The genetically engineered microorganism of embodiment 14, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

16. The genetically engineered microorganism of any one of embodiments 9 to

15, wherein the second nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

17. The genetically engineered microorganism of any one of embodiments 9 to

16, wherein the second nucleic acid molecule is an episomal vector.

18. A genetically engineered microorganism that is capable of producing olivetolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

19. The genetically engineered microorganism of embodiment 18, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

20. The genetically engineered microorganism of embodiment 18 or 19, wherein the first nucleic acid molecule further comprises at least one linker sequence between the first and second polynucleotide sequences.

21. The genetically engineered microorganism of embodiment 20, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

22. The genetically engineered microorganism of any one of embodiments 18 to 21 , wherein the tetraketide synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO: 15, and the olivetolic acid cyclase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:16.

23. The genetically engineered microorganism of any one of embodiments 18 to 22, wherein the first nucleic acid molecule further comprises a third polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116).

24. The genetically engineered microorganism of embodiment 23, wherein the third polynucleotide sequence is 5’ to the first polynucleotide sequence.

25. The genetically engineered microorganism of any one of embodiments 18 to 24, wherein the first nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

26. The genetically engineered microorganism of any one of embodiments 18 to 25, wherein the first nucleic acid molecule is an episomal vector.

27. The genetically engineered microorganism of any one of embodiments 18 to 26, further comprising a second nucleic acid molecule comprising a fourth polynucleotide sequence encoding an aromatic prenyltransferase.

28. The genetically engineered microorganism of embodiment 27, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ I D NO: 18, or 63-65.

29. The genetically engineered microorganism of embodiment 27 or 28, wherein the second nucleic acid molecule further comprises a fifth polynucleotide sequence encoding tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase.

30. The genetically engineered microorganism of embodiment 29, wherein the tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:20, and the cannabidiolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:21.

31. The genetically engineered microorganism of embodiment 29 or 30, wherein the fourth polynucleotide sequence is 5’ to the fifth polynucleotide sequence. 32. The genetically engineered microorganism of embodiment 31 , wherein the second nucleic acid molecule further comprises at least one linker sequence between the fourth and fifth polynucleotide sequences.

33. The genetically engineered microorganism of embodiment 32, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

34. The genetically engineered microorganism of any one of embodiments 27 to 33, wherein the second nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

35. The genetically engineered microorganism of any one of embodiments 27 to 34, wherein the second nucleic acid molecule is an episomal vector.

36. A genetically engineered microorganism that is capable of producing olivetol, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

37. The genetically engineered microorganism of embodiment 36, wherein the variant of Steelyl or Steely2 comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:61 or SEQ ID NO:62, respectively.

38. The genetically engineered microorganism of embodiment 36 or 37, wherein the first nucleic acid molecule further comprises a second polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116).

39. The genetically engineered microorganism of embodiment 38, wherein the second polynucleotide sequence is 5’ to the first polynucleotide sequence.

40. The genetically engineered microorganism of any one of embodiments 36 to 39, wherein the first nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID NQ:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

41. The genetically engineered microorganism of any one of embodiments 36 to 40, wherein the first nucleic acid molecule is an episomal vector.

42. The genetically engineered microorganism of any one of embodiments 36 to 41 , further comprising a second nucleic acid molecule comprising a third polynucleotide sequence encoding an aromatic prenyltransferase.

43. The genetically engineered microorganism of embodiment 42, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO: 18, or 63-65.

44. The genetically engineered microorganism of embodiment 42 or 43, wherein the second nucleic acid molecule further comprises a fourth polynucleotide sequence encoding tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase.

45. The genetically engineered microorganism of embodiment 44, wherein the tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:20, and the cannabidiolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:21.

46. The genetically engineered microorganism of embodiment 44 or 45, wherein the third polynucleotide sequence is 5’ to the fourth polynucleotide sequence.

47. The genetically engineered microorganism of embodiment 46, wherein the second nucleic acid molecule further comprises at least one linker sequence between the third and fourth polynucleotide sequences.

48. The genetically engineered microorganism of embodiment 47, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID NO:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

49. The genetically engineered microorganism of any one of embodiments 42 to 48, wherein the second nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

50. The genetically engineered microorganism of any one of embodiments 42 to 49, wherein the second nucleic acid molecule is an episomal vector.

51. A genetically engineered microorganism that is capable of producing olivetolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116), a second polynucleotide sequence encoding tetraketide synthase, and a third polynucleotide sequence encoding olivetolic acid cyclase.

52. The genetically engineered microorganism of embodiment 51 , wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

53. The genetically engineered microorganism of embodiment 51 or 52, wherein the second polynucleotide sequence is 5’ to the third polynucleotide sequence.

54. The genetically engineered microorganism of any one of embodiments 51 to 53, wherein the first nucleic acid molecule further comprises at least one linker sequence between the second and third polynucleotide sequences.

55. The genetically engineered microorganism of embodiment 54, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

56. The genetically engineered microorganism of any one of embodiments 51 to 55, wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

57. The genetically engineered microorganism of any one of embodiments 51 to 56, wherein the tetraketide synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO: 15, and the olivetolic acid cyclase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:16.

58. The genetically engineered microorganism of any one of embodiments 51 to 57, wherein the first nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

59. The genetically engineered microorganism of any one of embodiments 51 to 58, wherein the first nucleic acid molecule is an episomal vector.

60. The genetically engineered microorganism of any one of embodiments 51 to 59, further comprising a second nucleic acid molecule comprising a fourth polynucleotide sequence encoding an aromatic prenyltransferase.

61. The genetically engineered microorganism of embodiment 60, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ I D NO: 18, or 63-65.

62. The genetically engineered microorganism of embodiment 60 or 61 , wherein the second nucleic acid molecule further comprises a fifth polynucleotide sequence encoding tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase.

63. The genetically engineered microorganism of embodiment 62, wherein the tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:20, and the cannabidiolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:21.

64. The genetically engineered microorganism of embodiment 62 or 63, wherein the fourth polynucleotide sequence is 5’ to the fifth polynucleotide sequence.

65. The genetically engineered microorganism of embodiment 64, wherein the second nucleic acid molecule further comprises at least one linker sequence between the fourth and fifth polynucleotide sequences.

66. The genetically engineered microorganism of embodiment 65, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID NO:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

67. The genetically engineered microorganism of any one of embodiments 60 to 66, wherein the second nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID NQ:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

68. The genetically engineered microorganism of any one of embodiments 60 to 67, wherein the second nucleic acid molecule is an episomal vector.

69. A genetically engineered microorganism that is capable of producing olivetol, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116) and a second polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof.

70. The genetically engineered microorganism of embodiment 69, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

71. The genetically engineered microorganism of embodiment 69 or 70, wherein the second polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

72. The genetically engineered microorganism any one of embodiments 69 to 71 , wherein the variant of Steelyl or Steely2 comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:61 or SEQ ID NO:62, respectively.

73. The genetically engineered microorganism of any one of embodiments 69 to 72, wherein the first nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

74. The genetically engineered microorganism of any one of embodiments 69 to 73, wherein the first nucleic acid molecule is an episomal vector.

75. The genetically engineered microorganism of any one of embodiments 69 to 74, further comprising a second nucleic acid molecule comprising a third polynucleotide sequence encoding an aromatic prenyltransferase. 76. The genetically engineered microorganism of embodiment 75, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO: 18, or 63-65.

77. The genetically engineered microorganism of embodiment 75 or 76, wherein the second nucleic acid molecule further comprises a fourth polynucleotide sequence encoding tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase.

78. The genetically engineered microorganism of embodiment 77, wherein the tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:20, and the cannabidiolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:21.

79. The genetically engineered microorganism of embodiment 77 or 78, wherein the third polynucleotide sequence is 5’ to the fourth polynucleotide sequence.

80. The genetically engineered microorganism of embodiment 79, wherein the second nucleic acid molecule further comprises at least one linker sequence between the third and fourth polynucleotide sequences.

81. The genetically engineered microorganism of embodiment 80, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID NO:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

82. The genetically engineered microorganism of any one of embodiments 75 to 81 , wherein the second nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

83. The genetically engineered microorganism of any one of embodiments 75 to 82, wherein the second nucleic acid molecule is an episomal vector.

84. A genetically engineered microorganism that is capable of producing cannabigerolic acid, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and a second nucleic acid molecule comprising a third polynucleotide sequence encoding an aromatic prenyltransferase, and wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65.

85. The genetically engineered microorganism of embodiment 84, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

86. The genetically engineered microorganism of embodiment 84 or 85, wherein the first nucleic acid molecule further comprises at least one linker sequence between the first and second polynucleotide sequences.

87. The genetically engineered microorganism of embodiment 86, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

88. The genetically engineered microorganism of any one of embodiments 84 to 87, wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

89. The genetically engineered microorganism of any one of embodiments 84 to 88, wherein the tetraketide synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO: 15, and the olivetolicacid cyclase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:16.

90. The genetically engineered microorganism of any one of embodiments 84 to 89, wherein the first nucleic acid molecule further comprises a fourth polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116).

91. The genetically engineered microorganism of embodiment 90, wherein the fourth polynucleotide sequence is 5’ to the first polynucleotide sequence.

92. The genetically engineered microorganism of any one of embodiments 84 to 91 , wherein the first nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

93. The genetically engineered microorganism of any one of embodiments 84 to 92, wherein the first nucleic acid molecule is an episomal vector. 94. The genetically engineered microorganism of any one of embodiments 84 to 93, wherein the second nucleic acid molecule further comprises a fifth polynucleotide sequence encoding tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase.

95. The genetically engineered microorganism of embodiment 94, wherein the tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:20, and the cannabidiolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:21.

96. The genetically engineered microorganism of embodiment 94 or 95, wherein the third polynucleotide sequence is 5’ to the fifth polynucleotide sequence.

97. The genetically engineered microorganism of embodiment 96, wherein the second nucleic acid molecule further comprises at least one linker sequence between the third and fifth polynucleotide sequences.

98. The genetically engineered microorganism of embodiment 97, wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

99. The genetically engineered microorganism of any one of embodiments 84 to 98, wherein the second nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

100. The genetically engineered microorganism of any one of embodiments 84 to 99, wherein the second nucleic acid molecule is an episomal vector.

101. A genetically engineered microorganism that is capable of producing cannabigerol, wherein the genetically engineered microorganism comprises a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and a second nucleic acid molecule comprising a second polynucleotide sequence encoding an aromatic prenyltransferase, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65. 102. The genetically engineered microorganism of embodiment 101 , wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

103. The genetically engineered microorganism of embodiment 101 or 102, wherein the variant of Steelyl or Steely2 comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:61 or SEQ ID NO:62, respectively.

104. The genetically engineered microorganism of any one of embodiments 101 to 103, wherein the first nucleic acid molecule further comprises a third polynucleotide sequence encoding rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 116).

105. The genetically engineered microorganism of embodiment 104, wherein the third polynucleotide sequence is 5’ to the first polynucleotide sequence.

106. The genetically engineered microorganism of any one of embodiments 101 to 105, wherein the first nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

107. The genetically engineered microorganism of any one of embodiments 101 to 106, wherein the first nucleic acid molecule is an episomal vector.

108. The genetically engineered microorganism of any one of embodiments 101 to 107, wherein the second nucleic acid molecule further comprises a fourth polynucleotide sequence encoding tetrahydrocannabinolicacid synthase orcannabidiolic acid synthase.

109. The genetically engineered microorganism of embodiment 108, wherein the tetrahydrocannabinolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:20, and the cannabidiolic acid synthase comprises amino acid sequence with at least 90% sequence identity to sequence as shown in SEQ ID NO:21.

110. The genetically engineered microorganism of embodiment 108 or 109, wherein the second polynucleotide sequence is 5’ to the fourth polynucleotide sequence. 111. The genetically engineered microorganism of embodiment 110, wherein the second nucleic acid molecule further comprises at least one linker sequence between the second and fourth polynucleotide sequences.

112. The genetically engineered microorganism of embodiment 111 , wherein the linker sequence encodes a self-cleaving linker (e.g., amino acid sequence SEQ ID N0:101-103) or a fusion linker (e.g., amino acid sequence SEQ ID N0:104-106).

113. The genetically engineered microorganism of any one of embodiments 101 to 112, wherein the second nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NO:38-45, 112 and 114), a sequence encoding a tag (e.g., amino acid sequence SEQ ID N0:95-100), a sequence encoding a reporter (e.g., amino acid sequence SEQ ID NO: 110-111), and a terminator nucleic acid sequence (e.g., SEQ ID NO:46-53, 113, and 115).

114. The genetically engineered microorganism of any one of embodiments 101 to 113, wherein the second nucleic acid molecule is an episomal vector.

115. The genetically engineered microorganism of any one of embodiments 1 to 114, wherein the genetically engineered microorganism is a photosynthetic microalga or a cyanobacterium.

116. The genetically engineered microorganism of embodiment 115, wherein the genetically engineered microorganism does not comprise an exogenous nucleic acid molecule encoding hexanoyl-CoA synthetase.

117. The genetically engineered microorganism of embodiment 115 or 116, wherein the microalga is a GC-rich microalga, optionally Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chui, Nannochloropsis oculate, Scenedesmus obliquus, Acutodesmus dimorphus, Dunaliella tertiolecta , or Heamatococus plucialis.

118. The genetically engineered microorganism of embodiment 117, wherein the microalga is Chlamydomonas reinhardtii.

119. The genetically engineered microorganism of embodiment 115 or 116, wherein the microalga is a diatom.

120. The genetically engineered microorganism of embodiment 119, wherein the microalga is Phaeodactylum tricornutum or Thalassiosira pseudonana. 121. The genetically engineered microorganism of embodiment 120, wherein the microalga is Phaeodactylum tricornutum.

122. The genetically engineered microorganism of embodiment 115 or 116, wherein the cyanobacterium is a Spirulinaceae, Phormidiaceae, Synechococcaceae, or Nostocaceae, optionally Arthrospira plantesis , Arthrospira maxima, Synechococcus elongatus or Aphanizomenon flos-aquae.

123. A cell culture comprising the genetically engineered microorganism of any one of embodiments 115 to 122, and a medium that is substantially free of a sugar.

124. The cell culture of embodiment 123, wherein the sugar is present in the medium at a concentration of less than 2% by weight.

125. The cell culture of embodiment 124, wherein the sugar is present in the medium at a concentration of less than 1 % by weight.

126. The cell culture of embodiment 125, wherein the sugar is present in the medium at a concentration of less than 0.5% by weight.

127. The cell culture of embodiment 126, wherein the sugar is present in the medium at a concentration of less than 0.1% by weight.

128. The cell culture of embodiment 127, wherein the sugar is present in the medium at no more than trace amounts.

129. The cell culture of any one of embodiments 123 to 128, wherein the sugar is a monosaccharide.

130. The cell culture of embodiment 129, wherein the monosaccharide is at least one of glucose, fructose, ribose, xylose, mannose, and galactose.

131. The cell culture of any one of embodiments 123 to 128, wherein the sugar is a disaccharide.

132. The cell culture of embodiment 131 , wherein the disaccharide is at least one of sucrose, lactose, maltose, lactulose, trehalose, and cellobiose.

133. The cell culture of any one of embodiments 123 to 132, wherein the medium is substantially free of a fixed carbon source.

134. The cell culture of embodiment 133, wherein the fixed carbon source is at least one of carboxylic acid and glycerol. 135. The cell culture of embodiment 134, wherein the carboxylic acid is hexanoic acid.

136. The cell culture of any one of embodiment 123 to 135, wherein the cell culture undergoes autotrophic growth.

137. The cell culture of embodiment 136, wherein the autotrophic growth is photosynthetic growth.

138. The cell culture of embodiment 137, wherein the photosynthetic growth occurs in the presence of a solar light source.

139. The cell culture of embodiment 137, wherein the photosynthetic growth occurs in the presence of an artificial light source.

140. The cell culture of any one of embodiments 123 to 139, wherein the cell culture undergoes growth in organic conditions

141. A method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase, a second polynucleotide sequence encoding olivetolic acid cyclase, and at least one linker sequence between the first and second polynucleotide sequences, wherein the first polynucleotide sequence is 5’ to the second polynucleotide sequence.

142. A method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

143. A method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NO:34-37 and 107-108).

144. A method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid SEQ ID NO: 116), a second polynucleotide sequence encoding tetraketide synthase, and a third polynucleotide sequence encoding olivetolic acid cyclase.

145. A method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding rubisco small subunit (e.g., amino acid SEQ ID NO: 116) and a second polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof.

146. A method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding tetraketide synthase and a second polynucleotide sequence encoding olivetolic acid cyclase, and a second nucleic acid molecule comprising a third polynucleotide sequence encoding an aromatic prenyltransferase, and wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65.

147. A method for producing a cannabinoid biosynthetic pathway product in a genetically engineered microorganism, comprising introducing into the microorganism a first nucleic acid molecule comprising a first polynucleotide sequence encoding Steelyl , Steely2, or a variant thereof, and a second nucleic acid molecule comprising a second polynucleotide sequence encoding an aromatic prenyltransferase, wherein the aromatic prenyltransferase comprises amino acid sequence with 90% sequence identity to sequence as shown in SEQ ID NO:63-65.

EXAMPLE 1

Genetic engineering of sequences and construction cassettes synthesis

[00114] The gene sequences encoding TKS and OAC were identified and the codons were optimized for maximal expression in Chlamydomonas reinhardtii. Genetic engineering of the DNA constructions was performed to increased expression of the transgenes.

Gene sequences [00115] It has been suggested that hexanoyl-CoA synthetase convert hexanoic acid to hexanoyl-CoA early in CB biosynthetic pathway (Fig. 1 ; modified from Gagne et al 2012). Another early metabolite intermediate in the CB biosynthetic pathway is olivetolic acid (OA) that forms the polyketide skeleton of cannabinoids. Without wishing to be bound by theory, OA is produced as follows (Fig. 2): First, a type III tetra/polyketide synthase (TKS) enzyme condenses hexanoyl-CoA with three malonyl-CoA in a multi-step reaction to form trioxododecanoyl-CoA. Then, the olivetolic acid cyclase (OAC) catalyzes an intramolecular aldol condensation to yield OA. In subsequent steps, CB diversification is generated by the sequential action of “decorating” enzymes on the OA backbone, which leads to cannabinoids A9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), each of which decarboxylates to yield A9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively (Fig. 1).

[00116] The gene sequence for TKS and OAC have been identified and characterized in vitro (Lussier 2012; Gagne ef a/2012; Marks et al 2009; Stout et al 2012; Taura et al 2009). The complete coding sequences for non-optimized TKS (GenBank: AB164375.1) and OAC (GenBank: JN679224.1) were obtained from public databases. The open reading frame of TKS (1158 bp) encodes for a protein of 385 amino acids with a calculated MW of 42 kDa (T aura et al 2009; Flores-Sanchez et al 2010). Whereas OAC is a relatively small sequence (485 bp) encoding for a small protein of 101 amino acids and a MW of 12 kDa (Marks et al 2009). Without wishing to be bound by theory, codon optimization is suggested to improve protein expression in a host organism by replacing the nucleic acids coding for a particular amino acid (i.e. a codon) with another codon which is purportedly better expressed in the host organism. This effect may arise due to different organisms showing preferences for different codons. In particular, microalgae and cyanobacteria may prefer different codons from plants and animals. The process of altering the sequence of a nucleic acid to achieve better expression based on codon preference is called codon optimization. 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 and Margalit 2003). Other modifications in codon usage to increase protein expression that are not dependent on codon bias have also been described (Welch et al 2009). Sequences optimized for the codon usage of Chlamydomonas reinhardtii are shown in SEQ ID NO: 1 -7, 22-27, and 54. These optimized sequences can also be used for other GC-rich microalgae. Genetic engineering of DNA constructions

[00117] Two engineered constructions for maximizing the expression of the transgenes are shown below.

[00118] Construction 1 :

[00119] First, two genes, TKS and OAC, were included on the same open reading frame. These genes were separated with the self-cleaving sequence FMDV2A from the foot-and-mouth disease virus. This construction was named Cons1_TKS-FMDV-OAC or Constructionl (Fig. 3). It is expected that in Chlamydomonas cells, Construction will express both genes on the same mRNA, at the same level, since they are under the regulation of the same strong promoter. During the translation of the mRNA into protein, the FMDV self-cleaves, thus resulting in TKS and OAC as separated proteins. It has been suggested that in Cannabis sativa, these two proteins do not need to interact to produce olivetolic acid (Gagne et al 2012). Therefore, Constructionl should mimic what happens in Cannabis.

[00120] Construction 2:

[00121] To increase the metabolic flow of reactions, Construction 2 was built which links TKS and OAC together using a peptide linker (Fig. 3). The strategy behind this construction is to increase the efficiency of reactions by having the two proteins in the same cellular space. Without wishing to be bound by theory, enzyme fusion is considered a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. This structural-functional complex between the sequential enzymes of CB biosynthetic metabolic pathway allows intermediate product from TKS to be passed (i.e. to promote substrate channeling) directly into the active site of the next consecutive enzyme, OAC. The restriction site BamHI was included in the sequence of Construction 2 as an additional tool and does not affect the expression of this transgene.

Gene synthesis

[00122] The sequences encoding Constructions 1 and 2 were sent for synthesis. The skilled person can readily recognize the methods for synthesizing nucleic acid molecules containing the sequences. Two genetic constructions containing the genes TKS and OAC were engineered for optimal expression and synthesized by the company DNA2.0 (USA). The more the genes are expressed, the more enzymes will be made to catalyze more substrates into the desired product, olivetolic acid. Fig. 4 shows a summary of the engineered constructions functioning in cells. In C. reinhardtii, the genes (DNA) for each construction is transcribed into mRNA and exported to the cytosol. There in the cytosol, the mRNA is translated into proteins (enzymes TKS and OAC) which will be able to catalyze the formation of the target metabolite, olivetolic acid.

EXAMPLE 2

Construction assembly, extraction and purification of the transformation vectors

[00123] The synthesized DNA constructions were assembled into integration and expression vectors to enhance expression of transgenes. Assembled vectors were transformed into E.coli , grown to bulk and large amount of pChlamy vectors were extracted and purified.

Assembly of the transformation vectors

The synthetic constructions were inserted into a default vector (KanR, high copy; Fig. 5A) which is used to transform Escherichia coli by electroporation. The transformed E. coli was grown to bulk plasmids containing the transgenes (synthetic constructions) and positive colonies confirmed by colony PCR (Fig. 5B). DNA gel of the colony-PCR from transformed E. coli shows the positive amplification of construction 1 (lane 1 and 2; 1213 bp), construction 2 (lane 4 and 5; 1192 bp), and lane 3 contains the DNA ladder from which the corresponding DNA size are labeled on the left of the gel (Fig. 5B). The plasmids were then extracted and ready for the synthetic constructions for assembly using the Gibson method (Gibson et at 2009). Two vectors were used for transformation of C. reinhardtii. pChlamy3 (pC3) and pChlamy 4 (pC4) (Fig. 5C). Each vector contains the strong hybrid promoter Hsp70A-RbcS2 and the intron 1 of RbcS2 in front of the cloning site to drive a strong expression of the synthetic construction (genes of interest) in C. reinhardtii (Schroda et at 2000; Diaz-Santos et al 2013). pChlamy 4 is a new generation of vector and, without wishing to be bound by theory, it contains additional features to allow a stronger expression. Such features include fusion (co-expression) of the selection marker zeocin resistance with the transgene, a 3' UTR for proper transcript termination and possible additional benefits like increased translation efficiency, mRNA stability, and polyadenylation signals (Fig. 5C). Thus, the synthetic constructions were PCR amplified with primers containing sequence for the Gibson assembly. The assembly was done using each synthetic construction into both pChlamy vectors. Table 2 summarizes the four possible combinations of construction/vector. Colony PCR coupled with Sanger sequencing confirmed correct reading frame of all combination of synthetic constructions/vectors (Fig. 5D and 5E). DNA gel of the colony-PCR from transformed E. coli shows the positive amplification of the Gibson assembled synthetic constructions into pChlamy3 (Fig. 5D) and pChlamy4 (Fig. 5E) vectors. In particular, positive assembly of pChlamy3 with construction 1 (lane 1 , 2 and 3; 1593 bp) and pChlamy3 with construction 2 (lane 4 and 5; 1557 bp) were confirmed (Fig. 5D). Also, positive assembly of pChlamy4 with construction 1 (lane 1 , 2 and 3; 1615 bp) and pChlamy4 with construction 2 (lane 4 and 5; 1579 bp) were also confirmed (Fig. 5E). Lane 6 on both gels (Fig. 5D and 5E) contains the DNA ladder from which the corresponding DNA MWs are labeled on the right of the gel.

Table 2. Summary of the combination between the synthetic constructions and the pChlamy vectors used.

Transformation of E. coli, bulking and purification of pChlamy vectors

[00124] Each of the successfully assembled pChlamy vectors (Fig. 5; T able 2) were used to transform E. coli using the heat shock method. Transformed E. coli was grown to bulk vectors in order to purified large amounts for the subsequent transformation of microalgae. Transformed colonies for pC3_1 , pC3_2, pC4_1 and pC4_2 vectors all grew on ampicillin plates (Fig. 6A) and positive colonies confirmed by colony PCR. Positive clones were grown and vectors were extracted and separated on agarose gel (Fig. 6B). Gel on the left shows pC3_1 at 6028 bp whereas the gel on the right shows PC4_2 at 5129 bp, and lane MM (Molecular Marker) contains the DNA ladder from which the corresponding DNA size are labeled on the left of the gel (Fig. 6B). Vectors were excised from gel and purified using columns from a vector purification kit (FroggaBio). Purified vectors were used for the transformation of C. reinhardtii cells as shown below. Large amount of purified Chlamydomonas vectors for four combinations were obtained. EXAMPLE 3

Chlamydomonas reinhardtii cells transformation and selection of positive transformants

[00125] Purified pChlamy vectors were used to transform C. reinhardtii through electroporation. Transformed cells were grown on antibiotic selection TAP solid media and the presence of the transgene was confirmed using the colony-polymerase chain reaction (PCR) method. Expression of transgenes was detected using real-time quantitative PCR (rt-qPCR) analysis and enzymes produced were detected using SDS- PAGE.

Transformation of C. reinhardtii with purified pChlamy vectors

[00126] C. reinhardtii cells were transformed with pChlamy vectors. Briefly, purified vectors were linearized using restriction enzyme Kpn1 and cells were electroporated in the presence of linear vector DNA. DNA was taken up by cells and integrated into the nuclear genome. Without wishing to be bound by theory, integration of exogenous DNA in C. reinhardtii is carried out by mechanisms involving non-homologous recombination (also known as non-homologous end joining), rather than homologous recombination (Plecenikova et al 2013). Homologous recombination is, however, the mechanism of choice when it comes to gene targeting since it allows insertion of the transgene in a very active part of the genome to bypass gene silencing mechanisms. Attempts to establish this method in Chlamydomonas have had limited success so far.

[00127] Transformed cells were grown on selection media. Chlamydomonas transformed with pChlamy3 vectors were grown on media containing hygromycin (Fig. 7A) whereas cells transformed with pChlamy 4 vectors were grown on media containing zeocin (Fig. 7B). Positive cells were used for colony PCR to confirm the presence of the transgene (Fig. 7C-F). DNA gels of colony PCR confirm transformed Chlamydomonas colonies for pC3_1 (band at 1.337 kb from partial amplification of TKS-OAC sequence; Fig. 7C), pC3_2 (band at 1.304 kb from partial amplification of TKS-OAC sequence; Fig. 7D), pC4_1 (band at 1.311 kb from partial amplification of TKS-OAC sequence; Fig. 7E) and pC4_2 (band at 1.278 kb from partial amplification of TKS-OAC sequence; Fig. 7F). Lane 1 is the molecular marker that contains the DNA ladder from which the corresponding DNA sizes are labeled on the left of the gel, and lanes 2-10 correspond to different colonies where circles highlight the transformed Chlamydomonas containing the transgenes (Fig. 7C-F). Thus, C. reinhardtii cells containing the transgene randomly inserted in the nuclear genome were successfully created.

Confirmation of the expression of TKS and OAC in C. reinhardtii

[00128] Using quantitative PCR (qPCR) analyses, the expression of OAC of 30 different colonies for each constructions was screened (Fig. 8). Colonies that were expressing OAC above 1X were detected. For pChlamy3 transformed cells, 5/30 pC3_1 colonies and 6/30 pC3_2 colonies were found to express detectable OAC transcript above the 1X. The same ratio was observed for pChlamy4 transformed cells where 5/30 (pC4_1) and 5/30 (pC4_2) colonies showed expression above 1X. Without wishing to be bound by theory, transgene expression from the Chlamydomonas nuclear genome via the pChlamy4 vector offers several advantages over pChlamy3, including better expression due to reduced silencing from the fusion of the transgene to the zeocin resistance gene, sh-ble. In addition, pChlamy4 vectors offer protein tags such as 6His TEV and V5-His epitopes that can be fused to the transgene for detection and purification of the translated proteins. Thus, Chlamydomonas transformed with pC4 vectors are candidates for production of olivetolic acid.

[00129] Total proteins were extracted from pChalmy4-transformed cells and separated by SDS-PAGE (Fig. 9) followed by Western blot with anti-FMDV-2A antibodies to detect TKS-FMDV2A-OAC proteins and/or the self-cleaved TKS-FMDV2A proteins produced. On SDS-PAGE gel, pC4_1 transformed cells do not show an increase of a band at 60 kDa (expected TKS-OAC fused protein) compared to control cells (lanel ) (Fig. 9A; lane 3 contains the protein molecular marker). pC4_2 transformed cells do not show an increase of a band at 12 kDa (OAC protein alone) compared to control cells (lane 2) (Fig. 9B; lane 1 contains the protein molecular marker). However, Western blot analysis using anti-FMDV-2A antibodies detected TKS-FMDV2A-OAC proteins (Fig. 9C; lanesl- 4) and the self-cleaved TKS-FMDV2A proteins (Fig. 9C; lanes 7-8).

[00130] Hence, this Example shows the successful transformation of Chlamydomonas and the transgene was integrated into the nuclear genome. Stable transformants were screen for expression of the OAC transgene and positive strains detected.

EXAMPLE 4 Episomal vectors construction and diatom Phaeodactylum tricornutum cells transformation

Engineered diatoms

[00131] Microalgae provide a promising but challenging platform for the bioproduction of high value chemicals. Compared with model organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by inefficient molecular tools. To date, many microalgae are transformable but the introduced DNA is integrated randomly into the nuclear genome. Without wishing to be bound by theory, since integration of exogenous DNA in Chlamydomonas reinhardtii is principally carried out by mechanisms involving non-homologous recombination, the chance to encounter gene silencing is high, not the least because Chlamydomonas may be considered to possess high-silencing mechanisms. Hence, molecular tools to circumvent these challenges are necessary to facilitate efficient genetic engineering. Recently, an episomal vector system for diatoms was developed and shown to be highly stable (Karas et at 2015). Since episomes should not be affected by gene silencing mechanism, a diatom strain was engineered with the OAC-TKS transgene. Sequences optimized for the codon usage of Phaeodactylum tricornutum are shown in SEQ ID NO:8-14, 29-34 and 57. These optimized sequences can also be used for other diatoms such as Thalassiosira pseudonana.

[00132] A map of the episome (Karas et at 2015) (Epi) empty (Epicontrol) and engineered with construction 2 of TKS and OAC genes (EpiTKS-FMDV-OAC) is shown (Fig. 10A). DNA gel of the PCR products for full fragment insert of E p i ^{T KS_ FM D V} -° ^AC construct amplified by primers annealing sites on the Epi backbone performed on Pt colonies shows the entire insert (FcpD promoter -> FcpD terminator) at the correct size of 2591 bp (Fig. 10B; also includes negative control and 1 kb ladders). P. tricornutum colonies were grown on zeocin plates (except negative control; Fig. 10C). Each construct plate contains on average 50 colonies, while the positive control contains 92. Multiplex PCR results for colonies of Epi transformed with Pt DNA show that DNA was extracted from 1 colony of P. tricornutum for each isolate of TKS-FMDV-OAC (Fig. 10D). All P. tricornutum colonies were extracted and all 7 colonies between TKS 5-1 and 5-2 were screened (TKS colony 5 was chosen for sequencing, and it shows the correct sequence). DNA for each correct colony was extracted and digested with BamHI (Fig. 10D). Positive control of TKS colony 5 was also digested with BamHI, showing the expected sizes 8,020, 5,656, 2,346 and 725 bp. All positive P. tricornutum colonies were sent to the CNETE for further metabolite analysis.

[00133] Thus, three engineered diatom P. tricornutum were generated using the episomal vector system. The products of these engineered cells were sent for olivetolic acid analysis.

EXAMPLE 5

Identification of Products from the diatom Phaeodactylum tricornutum

[00134] Pellets from engineered diatom Phaeodactylum tricornutum (either controls transfected with empty vector or transfected with EpiTKS-FMDV-OAC) were lysed. The lysis was validated by microscopic observations (Fig. 11).

[00135] Chromatogram in selected time range in SIM mode (MS 425.3) of samples are shown in Fig.12-16. Fig.12 shows a lysate of control diatoms spiked with an olivetolic acid (OA) control to identify the OA peak. Fig.13 shows the chromatogram of lysate from empty vector control diatoms indicating an absence of OA. Figs.14-16 show chromatograms from different lysates of diatoms transfected with EpiTKS-FMDV-OAC showing a peak corresponding to OA with the expected retention time and MS.

[00136] This shows that an engineered microorganism such as microalgae transformed with constructs for the expression of cannabinoid biosynthetic pathway enzymes can produce cannabinoid biosynthetic pathway product.

EXAMPLE 6

Constructions Optimized for Expression in diatoms and GC-rich microalqae

[00137] This Example provides constructions of nucleic acid sequences that are optimized for expression in GC-rich microalgae such as Chlamydomonas reinhardtii, and diatoms such as Thalassiosira pseudonana and Phaeodactylum tricornutum. In particular, these constructs provide the co-expression of tetraketide synthase (a type III polyketide synthase) and olivetolic acid cyclase, aromatic prenyltransferase and hexanoyl-CoA synthetase, and tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase . A genetically engineered microorganism can contain a combination of these constructs, and consequently, the microorganism can co-express tetraketide synthase (a type III polyketide synthase), olivetolic acid cyclase, aromatic prenyltransferase, hexanoyl-CoA synthetase, tetrahydrocannabinolic acid synthase and cannabidiolicacid synthase . The detection and isolation of these enzymes can be carried out by antibodies specific to the tags attached to these enzymes, which include 6His, HA, FLAG, HSV, myc and V5.

EXAMPLE 7

[00138] Constructs comprising 1 or more sequences were designed by aligning the sequences in an open reading frame from 5’ to 3’ and codon-optimizing the open reading frame for enhanced expression in Phaeodactylum tricornutum.

Example 7.1 (AC 1 / Ptrefl )

[00139] T o generate a microorganism that produces olivetolic acid (OA), a construct comprising sequences that encode TKS and OAC enzymes was transformed into P. tricornutum.

[00140] A construct (Ptrefl, SEQ ID NO:71) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a T2A self-cleaving peptide linker sequence (position 1156 to 1218); and an OAC-encoding sequence (position 1219 to 1521) was inserted into a modified pPtGE30 plasmid (Slattery et al 2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast.

[00141] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00142] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00143] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

Example 7.2 (AC 2 / Ptref2)

[00144] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into P.tricornutum.

[00145] A construct (Ptref2, SEQ ID NO:72) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a 3(GGGGS) peptide linker sequence (position 1156 to 1200); and an OAC-encoding sequence (position 1201 to 1503) was inserted into a modified pPtGE30 plasmid (Slattery ef a/2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast. [00146] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00147] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00148] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

[00149] Example 7.3 (AC 3 / Ptref3)

[00150] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into P.tricornutum.

[00151] A construct (Ptref3, SEQ ID NO:73) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a 6His tag (position 1156 to 1173); a T2A self cleaving peptide linker sequence (position 1174 to 1236); an OAC-encoding sequence (position 1237 to 1539); and a Myc tag sequence (position 1540 to 1569) was inserted was inserted into a modified pPtGE30 plasmid (Slattery et al 2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast.

[00152] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00153] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00154] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

Example 7.4 (AC 4 / Ptref4)

[00155] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into P.tricornutum.

[00156] A construct (Ptref4, SEQ ID NO:74) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a 6His tag sequence (position 1156 to 1173); a 3(GGGGS) peptide linker sequence (position 1174 to 1218); an OAC-encoding sequence (position 1219 to 1521); and a Myc tag sequence (position 1522 to 1551) was inserted was inserted into a modified pPtGE30 plasmid (Slattery et al 2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast. [00157] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00158] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00159] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

Example 7.5 (AC 5 / Ptref5)

[00160] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into P.tricornutum.

[00161] A construct (Ptref5, SEQ ID NO:75) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a glycine codon (position 1156 to 1158); a YFP reporter sequence (position 1159 to 1911); a T2A self-cleaving peptide linker sequence (position 1912 to 1974); an OAC-encoding sequence (position 1975 to 2277); a glycine codon (position 2278 to 2280); and a RFP reporter sequence (position 2281 to 2988) was inserted was inserted into a modified pPtGE30 plasmid (Slattery et al 2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast.

[00162] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00163] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00164] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing and selected for analysis of OA production by GC-MS.

Example 7.6 (AC 6 / Ptref6)

[00165] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into P.tricornutum.

[00166] A construct (Ptref6, SEQ ID NO:76) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a glycine codon (position 1156 to 1158); a YFP reporter sequence (position 1159 to 1911); a 3(GGGGS) peptide linker sequence (position 1912 to 1956); an OAC-encoding sequence (position 1957 to 2259); and a Myc tag sequence (position 2260 to 2289) was inserted was inserted into a modified pPtGE30 plasmid (Slattery et al 2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast. [00167] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00168] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00169] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

Example 7.7 (AC 7 / Ptref7)

[00170] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into P.tricornutum.

[00171] A construct (Ptref7, SEQ ID NO:77) comprising from 5’ to 3’: a YFP reporter sequence (position 1 to 753); a glycine codon (position 754 to 756); a TKS-encoding sequence (position 757 to 1911); a 3(GGGGS) peptide linker sequence (position 1912 to 1956); an OAC-encoding sequence (position 1957 to 2259); and a Myc tag sequence (position 2260 to 2289) was inserted was inserted into a modified pPtGE30 plasmid (Slattery ef a/2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast.

[00172] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00173] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00174] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

Example 7.8 (AC 8 / Ptref8)

[00175] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into P.tricornutum.

[00176]

[00177] A construct (Ptref8, SEQ ID NO:78) comprising from 5’ to 3’: a rubisco small subunit (Rbs)-encoding sequence (position 1 to 417); 6 glycine codons (position 418 to 435); a YFP reporter sequence (position 436 to 1188); a glycine codon (position 1189 to 1191); a TKS-encoding sequence (position 1192 to 2346); a 3(GGGGS) peptide linker sequence (position 2347 to 2391); an OAC-encoding sequence (2392 to 2694); and a Myc tag sequence (position 2695 to 2724) was inserted was inserted into a modified pPtGE30 plasmid (Slattery et al 2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast.

[00178] The construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00179] The PtGE30 episomal vector was conjugated to P.tricornutum from E.coli.

[00180] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

Example 7.9 (AC 9 and AC 10 / Ptref9 and Ptrefl 0)

[00181] To generate a microorganism that produces OA, a construct comprising a sequence that encodes TKS enzyme and a separate construct that encodes OAC enzyme were transformed into P.tricornutum.

[00182] A first construct (Ptref9, SEQ ID NO:79) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); and a 6His tag sequence (position 1156 to 1173), and a second construct (Ptrefl 0, SEQ ID NO:80) comprising from 5’ to 3’: an OAC- encoding sequence (position 1 to 303); and a Myc tag sequence (position 304 to 333), were each inserted into separate modified pPtGE30 plasmid (Slattery et al 2018) containing a Zeocin resistance gene for algae and a Chloremphenicol resistance gene for E.coli and His selection in yeast.

[00183] The first construct was operably linked to a 40SRPS8 promoter (SEQ ID NO:39) and a FcpA terminator (SEQ ID NO: 113).

[00184] The second construct was operably linked to a FcpD promoter (SEQ ID NO:45) and a FcpD terminator (SEQ ID NO:53).

[00185] Both PtGE30 episomal vectors were conjugated to P.tricornutum from E.coli.

[00186] 4 Zeocin-resistant lines of positive transformants were verified by PCR and full episome sequencing, and selected for analysis of OA production by GC-MS.

Example 7.10 (Ptrefl 1)

[00187] To generate a microorganism that produces THCA, a construct comprising sequences that encode aromatic prenyltransferase (APT) and tetrahydrocannabinolic acid synthase (THCAS) enzymes was generated. [00188] A construct (Ptref11 , SEQ ID NO:81) comprising from 5’ to 3’: FcpC promoter (position 1 to 599); an APT-encoding sequence (position 600 to 1784); a T2A self-cleaving peptide linker sequence (position 1785 to 1847); a THCAS-encoding sequence (position 1848 to 3482); and a FcpC terminator (position 3483 to 3977) was generated.

Example 7.11 (Ptref12)

[00189] To generate a microorganism that produces THCA, a construct comprising sequences that encode APT and THCAS enzymes was generated.

[00190] A construct (Ptref12, SEQ ID NO:82) comprising from 5’ to 3’: FcpC promoter (position 1 to 599); an APT-encoding sequence (600 to 1784); a 3(GGGGS) peptide linker sequence (position 1785 to 1829); a THCAS-encoding sequence (position 1830 to 3464); and a FcpC terminator (position 3465 to 3959) was generated.

Example 7.12 (Ptref13)

[00191] To generate a microorganism that produces CBDA, a construct comprising sequences that encode APT and cannabidiolic acid synthase (CBDAS) enzymes was generated.

[00192] A construct (Ptref13, SEQ ID NO:83) comprising from 5’ to 3’: FcpC promoter (position 1 to 599); an APT-encoding sequence (position 600 to 1784); a T2A self-cleaving peptide linker sequence (position 1785 to 1829); a CBDAS-encoding sequence (position 1830 to 3461); and a FcpC terminator (position 3462 to 3956) was generated.

Example 7.13 (Ptref14)

[00193] To generate a microorganism that produces CBDA, a construct comprising sequences that encode APT and CBDAS enzymes was generated. [00194] A construct (Ptref14, SEQ ID NO:84) comprising from 5’ to 3’: FcpC promoter (position 1 to 599); an APT-encoding sequence (position 600 to 1784); a 3(GGGGS) peptide linker sequence (position 1785 to 1829); a CBDAS-encoding sequence (position 1830 to 3461); and a FcpC terminator (position 3462 to 3956) was generated. EXAMPLE 8

Plasmid Growth and Extraction from E.coli [00195] Synthetic constructs for transformation into C.reinhardtii were first inserted into a default vector (KanR, high copy) and transformed into E.coli by electroporation. Transformed E.coli was grown to bulk the plasmids containing the constructs. Positive E.coli were confirmed by colony PCR. The plasmids were then extracted and prepared for Gibson assembly into pChlamy3. pChlamy3 contains the strong hybrid promoter HSP70-RbcS2 and the intron 1 of RbcS2 in front of the cloning site.

Gibson assembly and transformation in E.coli

[00196] Assembled pChlamy3 vectors were used to transform E.coli by heat shock. Positive colonies were grown on ampicillin plates and confirmed by colony PCR. Transformed E.coli was then grown in liquid media LB-amp100 to bulk the vector before extraction and purification (Biobasic, miniprep kit). After linearization (digestion with Seal) for 3h, linearized vectors were verified on agarose gel 1% and purified (Biobasic, PCR clean up kit). Purified vectors were used for the transformation of C. reinhardtii cells.

Transformation of C.reinhardtii by glass bead method

[00197] C.reinhardtii was transformed with the purified vectors by the exemplary glass bead method described herein:

(1) A loop of stock culture from the plate was transferred aseptically in the hood to the flask containing TAP medium 50 ml_ in a 250 ml_ Erlenmeyer flask

(2) The culture was grown for 2 days in the growth chamber in a shaker (160 rpm) with the irradiation level approximately 60-70 uE

(3) 2 ml_ of culture was removed from the flask aseptically in the hood and transferred to the flask containing 400 mL of TAP medium in a 1 L flask

(4) The culture was grown in the growth chamber to a density of 1-2 x 10 ^* 6 cells/ml and/or an OD750 value between 0.6-0.8

(5) 200 mL culture was collected aseptically in the hood in a 250 mL autoclaved centrifuge bottle (two bottles) and harvested in a pre-warmed centrifuge and rotor at 3000g at RT

(6) the pellet was washed once with the 200 mL fresh growth medium, centrifuged again to collect the cells, and the supernatant was removed

(7) the pelleted cells were in 4mL of TAP medium (8) In a sterile 15ml tube 0.6 ml cells was vortexed 30 (2x15) seconds in the presence of 0.2 ml 20% polyethylene glycol 8000 (PEG) (Sigma 89510), 0.6 g acid-washed 0.5mm glass beads (Sigma G8772), and 3 ug of pChlamy3

(9) After vortexing cells were transferred to three different flasks containing 50 ml_ of TAP medium in a 250 ml_ Erlenmeyer flask and kept in the growth chamber shaker for 18 hrs for the recovery

(10) cells were collected in sterile falcon tubes in the hood and centrifuged at 3000g for 5 mins at RT

(11) the supernatant was removed and the cell pellet was suspended in fresh TAP medium equal to the volume of the cells used in the transformation mix (pChlamy3 -1 .2 ml_)

(12) 600 uL of cells were spread on the TAP agar plates containing hygromycin 10 ug/mL using the sterile 1ml_ tips

Total protein extraction from C.reinhardtii for HPLC

[00198] The culture medium containing the algae is homogenized then a known volume is introduced into Falcon tubes, centrifuged for 5 minutes at 10,000 RPM. The supernatant is removed, the pellet is suspended in 2 ml of Methanol MS. The suspension is maintained at -20 ° C.

[00199] Sea sand is cleaned with MS methanol (1 :1) and incubated 2 days before being used, soaking methanol is removed. During the 2 days, the methanol is changed (~8 times) until the methanol has no colour. In a mortar, previously cleaned with methanol MS, a little liquid nitrogen is added to cool the mortar, and a tip of sand and the pellet. The tube of the suspension is rinsed with 2ml of methanol MS, the 4 ml of suspension is ground by friction The ground material is recovered with 4 ml of methanol in a Falcon tube, and the pestle and the mortar are rinsed during recovery. The product is kept at - 20 °C.

[00200] A Strata-X-AW cartridge is conditioned with 1ml of Methanol, then 1ml of pure water. Introduce all the sample. The cartridge is cleaned with 1ml of 25mM ammonium acetate pH 6-7. The cartridge is cleaned 3 times with 1 ml of Methanol to remove a large part of the chromophores, and dried between the three injections. The 3 ml of rinsing methanol are analyzed for verification. The sample is eluted with 2 ^c 500 mI of 5% NH 4 OH in a mixture of methanol MS and acetonitrile MS (50:50), recovered in an Eppendorf tube and stored at -20 ° C. in the dark. The sample is stored at -20 ° C in the dark for testing within 2 weeks.

UPLC-MS

-Column: Poroshell 120 EC-C18 2.7pm 3.0x100mm Heated at 50 ° C

-Solvent: A: Pure Water (Grade LCMS) + 0.1% formic acid. B: Methanol (Grade LC-MS) + 0.1% formic acid.

-Gradient: From 0 to 20 min: from 40% B to 60% B From 10 to 20 min: from 60% B to 40% B

-Debit: 0.3 ml_ / min -Injection volume: 10.00 pL -Detector: UV: 230 nm -Duration: 20.0 min -Sample volume required: 1.00 ml

-Preparation of standard: Dilution from the available powder.

-Sample preparation: ½ dilution in methanol + 5% formic acid.

Example 8.1

[00201] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into C.reinhardtii.

[00202] A construct (G1C1 , SEQ ID NO:85) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a FMDV2A self-cleaving peptide linker sequence (position 1156 to 1227); and an OAC-encoding sequence (position 1228 to 1530) was inserted into pChlamy3 plasmid.

[00203] The construct was operably linked to a HSP70A-RbcS2 Hybrid promoter (SEQ I D NO: 114) and a RbcS2 terminator (SEQ I D NO: 115).

[00204] The vector was transfected into C.reinhardtii strain C-137 by glass bead method.

[00205] A positive transformant was selected by hygromycin resistance and PCR, and grown for 7 days before harvesting and extracting for analysis by UPLC-MS. UPLC- MS revealed the presence of OA in the positive transformant (Fig. 17A and 17B) which corresponds to the OA peak of a wild type control spiked with OA (Fig.18B). [00206] 6 positive transformants were selected by hygromycin resistance and PCR, and grown for 14 days before harvesting and extracting for analysis by HPLC. HPLC revealed the presence of a modified OA-derivative in the 6 positive transformants (Fig. 18A, Samples 5, 6, 7, 10, 11 , 12) compared to a wild type control and a wild type control spiked with 20ppm OA (Fig.18A). The modified OA-derivative is likely OA with an added thiol group on the carbon ring. Without being bound by theory, this may be caused by the addition of any thiol group (SH) provided by any molecule with acetylCoA within C.reinhardtii.

Example 8.2

[00207] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into C.reinhardtii.

[00208] A construct (G1C2, SEQ ID NO:86) comprising from 5’ to 3’: a TKS- encoding sequence (position 1 to 1155); a FPL1 linker sequence (position 1156 to 1191 ); and an OAC-encoding sequence (position 1192 to 1494) was inserted into pChlamy3 plasmid.

[00209] The construct was operably linked to a HSP70A-RbcS2 Hybrid promoter (SEQ I D NO: 114) and a RbcS2 terminator (SEQ I D NO: 115).

[00210] The vector was transfected into C.reinhardtii by glass bead method.

Example 8.3

[00211] To generate a microorganism that produces OA, a construct comprising a sequence that encodes TKS enzyme and a separate construct that encodes OAC enzyme were generated.

[00212] A first construct (G2TKS, SEQ ID NO:87) comprising a TKS-encoding sequence which comprises RbcS2-1 intron (position 330 to 474) and RbcS2-2 intron (position 1012 to 1340); and a 6His tag sequence (position 1630 to 1647), and a second construct (G20AC, SEQ ID NO:88) comprising an OAC-encoding sequence which comprises RbcS2-1 intron (position 126 to 270); and a Myc tag sequence (position 449 to 478), were generated.

Example 8.4 (CreifD

[00213] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into C.reinhardtii. [00214] A construct (Creif 1 , SEQ ID NO:89) comprising from 5’ to 3’: a 6His tag sequence (position 1 to 21); a TKS-encoding sequence (position 22 to 1173); a extFMDV2A self-cleaving peptide linker sequence (position 1174 to 1293); an OAC- encoding sequence (position 1294 to 1596); and a Myc tag sequence (position 1597 to 1626) was inserted into a pC3 plasmid with a Hygromycin resistance gene for algae and an Ampicillin resistance gene for E.coli.

[00215] The plasmid was transfected into C.reinhardtii by electroporation and 5 positive colonies over 21 tested by PCR (over 105 kept after 3 rounds of culture) were selected.

Example 8.5 (Creif2)

[00216] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into C.reinhardtii.

[00217] A construct (Creif2, SEQ ID NO:90) comprising from 5’ to 3’: a 6His tag sequence (position 1 to 21); a TKS-encoding sequence (position 22 to 1463) comprising RbcS2-1 intron at position 413 to 557 and 953 to 1097; a extFMDV2A self-cleaving peptide linker sequence (position 1464 to 1728) comprising RbcS2-1 intron at position 1493 to 1637; an OAC-encoding sequence (position 1729 to 2031); and a Myc tag sequence (position 2032 to 2061) was inserted into a pC3 plasmid with a Hygromycin resistance gene for algae and an Ampicillin resistance gene for E.coli.

[00218] The plasmid was transfected into C.reinhardtii by electroporation and 12 positive colonies detected by colony PCR were selected over 21 colonies tested.

Example 8.6 (Creif3)

[00219] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into C.reinhardtii.

[00220] A construct (Creif3, SEQ ID NO:91) comprising from 5’ to 3’: a 6His tag sequence (position 1 to 21); a TKS-encoding sequence (position 22 to 1173); a FPL1 linker sequence (position 1174 to 1218); an OAC-encoding sequence (position 1219 to 1521); and a Myc tag sequence (position 1522 to 1551) was inserted into a pC3 plasmid with a Hygromycin resistance gene for algae and an Ampicillin resistance gene for E.coli.

[00221] The plasmid was transfected into C.reinhardtii by electroporation and 13 positive colonies detected by colony PCR were selected over 21 tested. Example 8.7 (Creif4)

[00222] To generate a microorganism that produces OA, a construct comprising sequences that encode TKS and OAC enzymes was transformed into C.reinhardtii.

[00223] A construct (Creif4, SEQ ID NO:92) comprising from 5’ to 3’: a 6His tag sequence (position 1 to 21); a TKS-encoding sequence (position 22 to 1463) comprising RbcS2-1 intron at postion 413 to 557 and 926 to 1070; a FPL1 linker sequence (position 1464 to 1653) comprising RbcS2-1 intron at position 1465 to 1609; an OAC-encoding sequence (position 1654 to 1956); and a Myc tag sequence (position 1957 to 1986) was inserted into a pC3 plasmid with a Hygromycin resistance gene for algae and an Ampicillin resistance gene for E.coli.

[00224] The plasmid was transfected into C.reinhardtii by electroporation and 8 positive colonies detected by colony PCR were selected over 21 tested.

Example 8.8

[00225] To generate a microorganism that produces THCA, a construct comprising sequences that encode Cannabis sativa prenyltransferase 4 (CsPT4) and THCAS was generated.

[00226] A construct (G2C21 , SEQ ID NO:93) comprising from 5’ to 3’: a FLAG tag sequence (position 1 to 28); a CsPT4-encoding sequence (position 29 to 1218); a extFMDV2A self-cleaving peptide linker sequence (position 1219 to 1338); a THCAS- encoding sequence (position 1339 to 2892); and a HA tag sequence (position 2893 to 2919) was inserted into a pC3 plasmid with a Paramomycin resistance gene for algae and an Ampicillin resistance gene for E.coli.

Example 8.9 (Cre C2.2)

[00227] To generate a microorganism that produces CBDA, a construct comprising sequences that encode Orf2 and CBDAS was generated.

[00228] A construct (CreC2.2, SEQ ID NO:94) comprising from 5’ to 3’: a FLAG tag sequence (position 1 to 27); a Orf2-encoding sequence (position 28 to 945); a extFMDV2A self-cleaving peptide linker sequence (position 946 to 1065); a CBDAS- encoding sequence (position 1066 to 2616); and a HA tag sequence (position 2617 to 2643) was inserted into a pC3 plasmid with a Paramomycin resistance gene for algae and an Ampicillin resistance gene for E.coli. [00229] While the present disclosure has been described with reference to what are presently considered to be the preferred example, it is to be understood that the disclosure is not limited to the disclosed Examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00230] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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