PLASTIC POLYMERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application 63/395,739, filed on August 5, 2022, which is hereby incorporated by reference..

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under contracts DE-AC02- 05CH1 1231 and DE-FOA-0001916 awarded by the United Stated Department of Energy. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Type I polyketide synthases (PKSs) are multifunctional enzymes that contain discrete sets of enzymatic domains, i.e., modules, that can be engineered to produce a diversity of products, including pharmaceutical products, biofuels, and commodity chemicals. In production of PKS products, each module executes a cycle that begins with a Claisen condensation reaction between the growing chain on the ketosynthase (KS) domain and an extension molecule on the acyl carrier protein (ACP) loaded by the acyltransferase domain (AT). AT domains of Type-I PKSs can select a wide variety of extender units. After chain extension, the molecule’s carbonyl reduction state is determined by the reductive domains within a module, namely the ketoreductase (KR), dehydratase (DH), and enoylreductase

(ER), which generate the 0-hydroxyl, α-β alkene, or saturated β-carbons respectively when progressively combined. PKSs have variability in β-carbon reduction, which is a major source of polyketide diversity and another attractive feature for molecular design. Finally, a thioesterase (TE) domain typically releases the final product from the megasynthase via hydrolysis or cyclization. Due to their modularity, PKS systems have been extensively explored for production of “unnatural” natural products (Weissman and Leadlay. 2005. Nature Reviews Microbiology 3 :925-936). Hundreds of these molecules have been produced, ranging from basic lactones to modified versions of drugs and drug-like compounds. [0004] Plastics in use today are predominantly single-use and rarely recycled. This is not only wasteful from a resource and energy perspective, but also results in environmental stresses with >6 billion metric tons of plastic waste. A class of dynamic covalent polymer networks, known as vitrimers, are highly recyclable plastic polymers that combine the processing and recycling ease of thermoplastics with the performance advantages of thermosets. Most vitrimers are differentiated from classical thermosets, however, in that they can be chemically de-polymerized, typically into small molecules or short oligomers, including polydiketoenamines synthesized from BKDLs and diacids, and thus provide an opportunity to reduce environmental harm from waste plastics.

BRIEF SUMMARY

[0005] The present disclosure provides a platform to biologically produce β-keto-δ- lactones (BKDLs), which are important molecules for synthesis of vitrimer materials, including polydiketoenamine materials. For decades, microbial production of commodity chemicals is limited in the diversity of the molecules produced by natural or modified enzymes. The present disclosure describes polyketide synthases (PKSs) that can produce diversified BKDLs, which can be aliphatic or aromatic, with different substitutions at y and δ positions and no substitution at the α position. The substitutions include not only short carbon chains, but also long chains and branched chains with or without aromatics. The engineered modular PKSs and methods described herein provide high-purity products with specific and well-controlled biosynthesis.

[0006] Thus, in some aspects, provide herein is a genetically modified host cell comprising an engineered PKS that produces a β-keto-δ-lactone (BKDL), said PKS comprising a loading module comprising a loading domain, a first extension module, a second extension module and a thioesterase domain, wherein: the loading module comprises a loading domain that loads an acyl-CoA of the formula CoA- S-C(=O)R where R is an alkyl or aryl substituent comprising 1-20 carbon atoms; the first extension module that catalyzes the condensation of malonyl-CoA, methylmalonyl co A, or ethylmalonyl-CoA with an acyl group, wherein the first extension module comprises a ketosynthase (KS) domain; an acyltransferase (AT) domain specific for malonyl-CoA, methyhnalonyl-CoA, or ethylmalonyl-CoA; a ketoreductase (KR) domain; and an acyl carrier protein (ACP) domain; and the second extension module that catalyzes the condensation of malonyl-coA with an acyl groups, wherein the second extension molecule comprises a KS domain, a malonyl-CoA specific acyltransferase domain, and an acyl carrier protein domain. In some embodiments, the PKS does not comprise debsLM+debsMl+rifM2+debsTE. In some embodiments, the KR domain of the first extension module is inactive. In some embodiments, the second extension module comprises an inactive KR domain. In some embodiments, the KR domains in the first and the second extension modules are inactive. In some embodiments, the first and/or the second extension module comprises an inactive dehydratase module and/or an inactive enoyl reductase domain. In some embodiments, the loading domain loads acetyl- CoA, propionyl-CoA, butyryl-CoA,pentanoyl-CoA, isobutryl-CoA, 3 -methy 1-butyryl-CoA, hexanoyl-CoA, 4-methylpentanoyl-CoA, (S)-3-hydroxypentanoyl-CoA, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, L-alanine, glutamic acid, or trans-Cyclopentane-1, 2, dicarboxylic acid. In some embodiments, the PKS comprises a structure shown in column 2 of Table 1. In some embodiments, the PKS comprises a structure that produces the same BKDL a PKS in column 2 of Table 1, but comprises a different first or second extender molecule that has the same function. In some embodiments, a PKS comprises a loading module that loads the same acyl-CoA as a loading module shown in Table 1 , but is from a different PKS source. In some embodiments, one or more of the modules of a PKS that produces a desired BKDL is substituted with a corresponding module shown in Table 3.

[0007] In a further aspect, the disclosure provides a method of producing a BKDL comprising culturing the genetically modified host cell comprising an engineered PKS as described herein under conditions in which the engineered PKS is expressed and produces the BKDL. In some embodiments, the method further comprises purifying the BKDL. In an additional aspect, the disclosure provides a purified BKDL produced by said method.

[0008] In a further aspect, the disclosure provides a purifiedBKDL preparation comprising a BKDL having a structure of any one of the BKDLs shown in column 1 of Table 1 .

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 depicts four illustrative hybrid PKSs comprising a debs loading module, a debs extender module as a first extender module, and a second extender module from rif or mlsBl having a debs thioesterase (TE) domain and the BKDL compound each produces. The domain structure of each module is also shown. [0010] FIG. 2 depicts four illustrative hybrid PKSs comprising a lipomycin (lip) loading module, a debs extender module as a first extender module, and a second extender module fromrif ormlsBl having a debs TE domain and the BKDL compound each PKS produces. The domain structure of each module is also shown.

[0011] FIG. 3 depicts an example of the biosynthesis of 4-methyl 5-isobutyryl-BKDL (or y- methyl-δ-isobutyryl-BKDL) in Streptomyces albus using two PKSs integrated into the genome of S. albus JI 074. The first is lipomycin PKS1 (LipPKSl) and the second is myco lactone PKS Bl (MlsBl). The intermodular linker is the native linker between LipPKSl and LipPKS2.

[0012] FIG. 4 depicts the biosynthesis of 5-ethyl-BKDLand4-methyl 5-ethyl BKDL in E. coll.

[0013] FIG. 5 shows extension modules corresponding to “Extension Unit Subtype” entries in column X of Table 1.

DETAILED DESCRIPTION OF THE INVENTION

Terminology

[0014] It is to be understood that unless otherwise indicated, the disclosure is not limited to particular specific sequences, expression vectors, enzymes, host microorganisms, or methodology techniques, as they may vary.

[0015] As used herein, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a BKDL" includes a plurality of such BKDLs, reference to “a host cell” includes a plurality of host cells and so forth.

[0016] The terms “engineered”, “recombinant”, “non-natural”, or “non -naturally occurring” in the context of a PKS that produces a BKDL means that the PKS when considered as a whole does not occur in nature. Thus, a PKS may comprises naturally occurring modules and/or linker sequence that link modules that are naturally occurring, but the individual modules and linker do not occur in the same PKS in nature.

[0017] in the context of an engineered PKS, a module that “correspond to” to a given module of an engineered PKS as described herein, has the same function, i.e., for a loading module, comprises a loading domain that loads the same loading molecule; and for an extender module, employs the same extender mole

[0018] The term "acyl group" refers to any molecule comprising a carbonyl carbon bound to an oxygen, sulfur, or nitrogen, including, but not limited to, fatty acids, hydroxy fatty acids, acyl-coAs, and acyl-ACPs.

PKS subunits

[0019] Polyketide synthases (PKS) employ short chain fatty acyl Co As in Claisen condensation reactions to produce polyketides. PKSs are composed of discrete modules, each catalyzing the chain growth of a single step. A PKS of the present disclosure for production of a BLK typically comprises four components: a loading module, a first extension module, a second extension module, and a thioesterase (TE) domain. Modules for producing different BKDLs differ from each other in composition, so that, overall, a number of different starters and extenders, some of which contain stereospecific side chains, can be incorporated into a BKDL product. In some embodiments, the main chain is not greater than 12 carbons in length and the number of 5- or 6-member rings does not exceed 2. In typical embodiments, the following BKDL products are produced, where R ₁ =H, -CH ₃ , -CH ₂ CH ₃ , - (CH ₂ )nCH ₃ , -CHCH ₃ CH ₃ , -CH(CH ₂ )nCH ₃ CH ₃ , -CHCH ₃ NH ₂ , -CH(CH2)nNH ₂ COOH, - (CH) ₂ (CH2) _n COOH, where n=1-10.

[0020] PKS sequences and clusters have been extensively described and are readily available (see, e.g., the publicly available repository at www website clustercad.jbei.org.).

Loading modules

[0021] The term “loading module” as used herein refers to a module that comprises a domain that loads the starting molecule for synthesis of a BKDL, e.g., including, but not limited to any of the loading molecules, shown in Table 1. The starting molecule may be fed to a host cell or may be produced by the host cell. In some embodiments, a loading module comprises an acyltransferase (AT) domain and an acyl carrier protein (ACP) domain. In such loading modules, the AT domain determines the starting (“loading”) molecule for production of a desired BKDL. In some embodiments, the loading module can comprise an additional domain termed a KSQ domain, which is a ketosynthase-like decarboxylase domain. Thus, in some embodiments, a loading module comprises an AT domain comprising an arginine reside at the active site and a KSQ domain. In some embodiments, loading module may contain domains in addition to an AT and ACP domains, for example, a domain such as FkbM4, which comprises KS-AT-DH-KR-ACP. In some embodiments, the KS is inactivated, e.g., mutation of an active site Cys residue to Ala.

[0022] In still other embodiments, the loading module may not contain an AT or ACP domain from a PKS, but may employ another enzyme that loads a fatty acid onto an acyl carrier protein. Thus, for example, an acyl carrier protein from bacteria, such an E. coll ACP protein (acpP), may serve as the loading module carrier protein, and a bacterial acyl-ACP synthetase, such as VhAAS (Vibrio harveyi acyl-ACP synthetase) can be employed as alternative enzyme to load fatty acids onto ACP. Thus, loading molecule other than coA- containing loading modules may be employed to initiate production of a BKDL.

[0023] The choice of loading module is dictated by the desired functionality at the delta position in the BKDL compound. The acyltransferase domains within loading modules from different PKS sources are known to act on different acyl-CoAs of the basic formula CoA- S(=O)R, where R is an alkyl or aryl substituent of 1-20 carbons, i.e., a substituent of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons. Therefore, for example, if a -CH2-CH3 (ethyl group) is desired at the delta position, then selection of a loading module that loads CoA-S-C(=O)CH2-CH3, such as debsLM, would be appropriate.

[0024] Examples of the loading module of engineered PKSs for production of desired BKDLs are shown in Table 1. Examples of alternative loading modules for various loading modules in Table 1 are provided in Table 3. Accordingly, in these illustrative embodiment for the production of a BKDL shown in Table 1, the loading domain is from a PKS (or alternative PKS) that loads acetyl-CoA, propionyl-CoA, butyryl-CoA, pentanolyl-CoA, isobutryl, Co A, 3-methyl-butyryl-CoA, hexanoyl-CoA, 4-methylpentanoyl-CoA, (S)-3- hydroxypentanoyl-CoA, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, L-alanine, glutamic acid, or trans-Cyclopentane-1, 2, dicarboxylic acid. Extender modules

[0025] As used herein, the terms “extender module” or “extension module” are interchangeable. The engineered PKSs comprise two extender modules, Extender Module 1 , also referred to herein as the first extender module and Extender Module 2, also referred to herein as the second extender module.

[0026] In the context of the present disclosure, extender modules employed in a PKS for producing a BKDL minimally comprise a β-acyl ACP synthase, i.e., a ketosynthase (KS) domain, an AT domain, and an ACP domain that in concert carry out a single round of decarboxylative condensation. The KS domain conducts the decarboxylating condensation step to add the extender molecule to the chain. The acyltransferase (AT) domain of each extender module determines the extender molecule (for example, malonyl CoA or methylmalonyl CoA) for growth of the chain. The ACP domain of the extender module carries the growing acyl chain and in the case of the second extender module, can present the chain to a TE domain to terminate chain growth.

[0027] In some embodiments, the ACP domain presents the chain to an active ketoreductase (KR) domain present in the first or second extension module for reduction of the β -carbonyl in an extension module. In some embodiments, neither the first nor the second extension module comprise an active KR domain. In some embodiments, the first and/or the second extension module can comprise an inactivating mutation in the KR domain, e.g., mutation of the active site Tyr to Phe. One of skill further understand that the stereochemistry of compounds is influenced by the different types of KR domain that may be present in the first extension molecule (see, columns 4 and 7 of Table and FIG. 5, as further discussed below).

[0028] In some embodiments, a native extension module may contain a KR, DH, or ER domain, but the natural product compound is not reduced at the corresponding position, accordingly, the domains are considered to be naturally inactive without any engineering. Thus, for example, an engineered PKS for production of a BKDL may also comprise a KR, DH, or ER domain that is inactive, but has not been mutated to render the domain inactive. For example the third extension module from the erythromycin PKS cluster has a KR like domain in its amino acid sequence, but incorporates a non-reduced ketone into the final product. As additional example, (see, e.g., Table 1 ) mlsB 1 contains a KR domain, but can be used as Extension Module 2. In the context of the present disclosure an “inactive” KR, DH, or ER domain refers to a domain that is mutated to render it inactive, a native domain that is not active because the natural product is not reduced at the corresponding position, or a domain that is inactive because the product it acts on is not produced by a preceding domain(s) in the PKS module.

[0029] The folio wing additional considerations thus apply to selection of first and second extender modules to produce a desired BKDL, if a candidate extender module contains KR, DH and ER. In the first extender module, if KR is active, DH must be inactivated by mutation, but it is not necessary to inactivate ER because no product is produced by DH. If KR is inactivated, DH and ER need not be inactivated. Mutations of PKS domains and inactivation of KR, DH, and ER is described, e.g., in the review by Drufva et al, Synthetic and Systems Biotechnol. 5:62-80, 2020.

[0030] In some embodiments, the first extension module comprises a malonyl-CoA, methylmalonyl coA, or ethylmalonyl-CoA loading AT. In some embodiments, the first extension module comprises an AT that loads (2S)-methylalonyl-CoA, (2S)-ethylalonyl-CoA, chloroethylmalonyl-CoA, (2R)-methoxymalonyl-CoA, (2R)-hydroxymalonyl-CoA, (2R)- aminomalonyl-CoA, or allyhnalonyl-CoA,

[0031] In some embodiments, the engineered PKS comprises a second extension module having a malonyl-CoA loading AT with KR inactivation.

[0032] With respect to the influence of KR domain on stereochemistry of desired production, FIG. 5 summarizes the stereochemistry of products produced by KR domains. Extension subtypes are designated Al , A2, Bl, B2, andC (see, e.g., Table 1, columns 4 and 7 of illustrative engineered PKSs), which refer to the stereochemistry of the product generated. In FIG. 5, R ₁ refers to H or CH ₃ , R ₂ refers to loading molecules, cleaved from thioester bond (for example loading molecules shown in Table 1). For type C, KR is inactivated whether KR is in extender module 1 or extender module 2. KR for type A1 generates product with a (2S, 3 S)-2-R2-3 -hydroxy group; A2 generates product with a (2R, 3S)-2-R2-3-hydroxy group; B1 generates product with a (2S, 3R)-2-R2-3-hydroxy group; and B2 generates product with a (2R, 3R)-2-R2-3-hydroxy group.

[0033] In some embodiments, a first extension module is selected from the first extension modules listed in Table 1 or an alternative listed in Table 3. [0034] In some embodiments, the second extension module is selected from the extension molecules listed as a second extension module in Table 1 or an alternative listed in Table 3.

TE domain

[0035] A fourth component of a PKS that produces a BKDL as described herein is a thioesterase that terminates synthesis. In some embodiments, the thioesterase is a thioesterase domain from erythromycin or pikromycin, or an equivalent PKS that comprises a TE domain that terminates chain elongation. In some embodiments, the cyclizing enzyme is a stand-alone thioesterase such as ‘Tes A from Escherichia coll. In some embodiments, the TE domain is a TE from a fatty acid synthase.

Illustrative PKSs

[0036] In some embodiments of a PKS of the present disclosure, no more than modules are from the same PKS source, i.e., in some embodiments, no more than two modulescome from the same native PKS. For example, in some embodiments, no more than modules are from debs.

[0037] Table 1 provides illustrative engineered PKSs for production of the BKDL shown in the first column. In Table 1 column 2, LM = loading module, Ml = Module 1 of the indicated, Mn = module n of the indicated BKS, and TE = thioesterase). One of skill understands that alternative PKSs may be employed for production of a BKDL, so long as the combination of starter and extension molecules provides the substrates for generating the desired BKDL . As noted above, a loading module is selected based on the desired functionality at the delta position in the BKDL compound. The acyltransferase domains within loading modules from different PKS sources act on different acyl-CoAs. Thus, for example, if one desires a -CH2-CH3 (ethyl group) at the delta position, then selection of a loading module that acts on CoA-S-C(=O)CH2-CH3, such as a debs loading module (debsLM,) is appropriate.

[0038] Source PKS names corresponding the abbreviations employed in Table 1 are provided in Table 2. Table 2 also illustrates the organism that is the source of the PKS. Thus, in some embodiments, a PKS that produces a BKDL in accordance with the present disclosure comprises a module from a source organism listed in Table 2.

[0039] One of skill understands, however, that comparable modules for producing a BDKL shown in Table 1 can be obtained from alternative PKS (see, e.g., Table 3). Table 3 shows illustrative PKS modules for the loading and extension molecules defined in Table 1 and alternative modules for each. The source organism for other PKS modules not listed in Table

2 are available in the art. The full name of the PKS for the abbreviations employed in Table

3 that are not provided in Table 2 are listed in the paragraph following Table 2.

Table 2 PKS module sources of illustrative PKSs

[0040] The PKS source of additional PKS modules referred to in Table 3 are abbreviated as follows: end, chondrochloren; sgn, natamycin; lob, lobophorin; LMlm, aldgamycin; ncm, nocomycin; mer, meridamycin; mon, monensin; nid, niddamycin; tam, irandamycin; aal; actinoallo lide; rev, reveromycin, zin, griseochelin / zincophorin; his, halstoctacosanolide; sei, selvamicin; fos, fostriecin; eta, cystothiazole A; baf, bafilomycin; ttm, tautomycin; del, delftibactin; sul, sulfazecin; fen, fengyein; srf, surfactin, pps, p lipastatin; bor, borrelidin; sch, calcimycin; fos, fostriecin; idn, incednine; spi, spirangien O; cro, crocacin; acu, aculeximycin; idm, indanomycin / X- 14547; lad, laidlomycin; nan, nanchangmycin; fsc, candicidin A; pte, filipin, gfs, FD-891; hg, hygrocin, his, halstoctacosanolide; amb, ambruticin; cmi, cremimycin, ebe, ebelactone A; ang, angolamycin; aju, ajudazol A;mxa, amyalamid; acu, aculeximycin; gul, gulmirecin A; lad, laidlomycin; nbr, brasilinolide; nys, nystatin; sin, salinomycin; gon, PM 100117/18; auf, aurafuron A; lad, laidlomycin; mak, maklamicin; ger, dihydrochalcomycin, gdm, geldanamycin; lankamycin; fkb, FK520; tau, tautomycetin; nem,nemadectin, phn, phenylnannolone A, rub, rubradirin, asm, ansamitocin; and chm, chalcomycin.

[0041] In some embodiments, a PKS for the production of a BKDL comprises a loading module operably linked to a first extension molecule, operably linked to a second molecule, operably linked to a TE domain of any one of the PKS module as shown by the linear sequence provided in column 2 of Table 1. In some embodiments, the PKS module comprises a first extension molecule that uses the same extension molecule shown in column 5 of Table 1. I some embodiments, the PKS module comprises a fir st extension molecule drat can employ an extension module of the type listed in column 4 of Table 1 (see, FIG. 5). In some embodiments, the PKS module comprises a second extension molecule that uses the same extension molecule shown in column 8 of Table 1. In some embodiments, the PKS module comprises a second extension moleculethat can employ an extension module of the type listed in column 7 of Table 1 (see, FIG. 5).

[0042] The domain structure of illustrative first and second extension modules are shown in

Table 3.

Alternative BKDL synthesis modules

[0043] In some embodiments, a desired BKDL is synthesized using an alternative configuration in which a host cell is fed a beta hydroxy acid. The host cell is genetically modified to express a nucleic acid that expresses a loading module that can activate and load hydroxy acids directly and a single extension module that selects malonyl-CoA and lacks an active KR domain, e.g., an extension module as shown in Table 1 or Table 3. Alternatively, the host cell is genetically modified to express a co A ligase and a loading module that can accept hydroxy acyl CoAs and a single extension module that selects malonyl-CoA and lacks an active KR domain, e.g., an extension module as shown in Table 1 or Table 3.

Accordingly, in such configurations, the genetically modified host cell expresses a loading module for loading the beta hydroxy acid fed to the cell and one extension module, rather than two extension modules.

Recombinant nucleic acids/host cells

[0044] The present invention provides recombinant nucleic acids that encode PKSs of the invention. The recombinant nucleic acids includenucleic acids that include a portion or all of a PKS module of the present disclosure. In

PKS of the present disclosure may additional comprise regulatory sequences, such as promoter and translation initiation and termination sequences, and sequences that facilitate stable maintenance in a host cell, e.g , sequences that provide the function of an origin of replication, or facilitate integration into host cell chromosomal or other DNA by homologous recombination. In some embodiments, the recombinant nucleic acid is stably integrated into a chromosome of a host cell. In some embodiments, the recombinant nucleic acid is a plasmid. Thus, the present invention also provides vectors, including expression vectors, comprising a recombinant nucleic acid of tire present invention. The present invention also provides host cells comprising any of the recombinant nucleic acid and/or PKS of the present invention. In some embodiments, the host cell, when cultured under suitable conditions, is capable of producing the a-olefin. These host cells include, for example and without limitation, prokaryotes such as Escherichia species, Bacillus species, Streptomyces species, Myxobacterial species, as well as eukaryotes including but not limited to yeast and fungal strains.

[0045] In some embodiments, the host cell is a bacterial host cell. Examples of bacterial host cells include, without limitation, species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Clostridium, Enterococcus, Lactobacillus, Lactococcu,

Oceanobaciilus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Staphococcus, Streptococcus, Streptomyces, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, andParacoccus taxonomical classes. In some embodiments, the prokaryotic host cells are E. coli, or a Bacillus sp. such as Bacillus sublilis. In some embodiments, a host cell is a cyanobacterial host cell or a microalgae host cell. In some embodiments, the host cell is a species of Escherichia, Corynebacterium, Pseudomonas. Streptomyces, or Bacillus. In some embodiments, the Escherichia species is E. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vidneris. In some embodiments, the Corynebacterium species is Corynebacterium ghrtanricum, Corynebacterium kroppenstedtii, Corynebacterium alimapuense, Coryn ebacterium amycolatum, Corynebacterium diphtheriae, Corynebacterium. efficiens, Corynebacterium jeikeiitm, Corynebacterium macginleyi, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale. In some embodiments, the Pseudomonas species is P. putida, P. aeruginosa, P. chlororaphis, P. fhtorescens, P. perlucinogena, P. stulzeri, P. syringae, P. cremoricolorala, P. entomophila, P. fidva, P. monteilii, P. rm plecoglossicida. In some embodiments, the Streptomyces species is S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. avermitilis, S. albus, or £ scabies. In some embodiments, the Bacillus species is B. subtilis, B. megaterium, B, Ucheniformis, B, anthracis, B. amyloliquefaciens , or B. pumihis.

[0046] In some embodiments, the host cell is a species of yeast. Examples of yeast host cells include species of Candida, Hansemda, Kluyveromyces, Pichla, Saccharomyces, Schizosaccharomyces , or Yarrowia species. In some embodiments, the yeast species is Saccharomyces carlsber gensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis. In some embodiments, the yeast species is a Candida species, including but not limited to C. tropicalis, C. maltosa, C. apicola. C. tropicalis, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. Hpolytica, C. panapsilosis or C. zeylenoides . In some embodiments, the yeast host cell is a non-oleaginous yeast, such as a Saccharomyces species. In some embodiments, the yeast species is an oleaginous yeast. In some embodiments, tire oleaginous yeast is a Rhodosporidium species such as Rhodosporidium toruloides. In some embodiments, the yeast species is Kluyveromyces lactis. In some embodiments, the yeast species is Yarrowia Hpolytica ..

Method of producing a BKDL

[0047] In another aspect, the present disclosure prorides methods for producing BKDLs in which the methods generally comprise providing a host cell engineered to contain a PKS as described here that is capable of producing a BKD1, and culturing said host cell in a suitable culture medium under suitable conditions such that the BKDL is produced. In some embodiments, the method further comprise purifying the BKDL. The present disclosure thus also provides compositions comprising a BKDL produced from a host cell as described herein.

[0048] In further embodiments, the disclosure provides a purified BKDL preparation comprising a BDKL structure of any one ofthe BKDL structures shown in Table I of column 1. In some embodiments, the disclosure provides a purified BKDL preparation comprising a BKDL structure of any one of the BKDL structures shown in Table 1 of column 1 with the proviso that the BKDL is not the BKDL product shown in column 1 for the PKS debsLM+almM2+mlsB1+debsTE. Techniques

Biosynthesis of 4-methyl-5-isobutvryl-BKDL in 5. albus

[0049] Genes encoding two PKSs were integrated into the genome of 5. albus J 1074. One gene contained a nucleic acid encoding a lipomycin (Lip) loading module (LIPLM), a Lip Module 1 (Lip1 ), and a Lipsl linker. The second gene contained a nucleic acid sequence encoding a Lip 2 N-terminal linker, a KR inactivated myolactone B1 (MlsB) module 1 (MlsBl) and Debs thioesterase domain (DebsTE). The genes were assembled through yeast assembly, digested by Ndel and Xhol restriction enzymes (Thermo Fisher Scientific, Waltham, MA) and cloned into Biobrick vectors (Lee et al. , J. Biological Engineering 5:12, 2011 ). The gene encoding the LipLM-Lip1 -Lip1 linker was cloned into a p21 vector, which contains a gapdh promoter, apramycin resistance marker, phiC31 integrase and R4 replicon, while Lip2_linker-M1sBl(KR inactivated)-DebsTE was cloned into a pl5a vector, which contains a gapdh promoter, spectinomycin resistance marker, vwb integrase and R4 replicon. All PKS module -components nucleic acids were synthesized by Genscript (Piscataway, NJ).

[0050] The resulting plasmids were transformed into E. coli ET12567/pUZ8002 cells for biparental conjugation with integration of the two PKS genes in phiC31 and vwb integration sites of Streptomyces albus JI 074. The resultant strahis were sporulated in MS agar plate and spores were stored in 30% glycerol under -80 °C for long-term storage.

[0051] When growing the strains for PKS expression and BKDL production, stored spore was inoculated into TSB media and grown for 2 days and then 1% of TSB seed culture was inoculated in 30 mL fermentation media in 250 mL baffled flask, with addition of 104.5 mm sterile glass beads and 10 g/L Talc microparticles. The fermentation media employed was R5 media supplemented with 2.4 g/L L-Valine. Fermentation was performed at 30 °C for 7 days, 200 rpm, 50 % humidity. After the fermentation, the cell cultures were harvested by centrifugation. Biological triplicates were grown for each strain. 50 mg-'L apramycin and 25 mg/L nalidixic acid were added in all media and all chemicals were obtained from Sigma- Aldrich Co. (St. Louis, MO) and Fisher Scientific Co. (Pittsburg, PA).

[0052 ] For quantification and qualification of BKDLs, 100 μL of supernatant of harvested cell cultures were mixed with 100 μL acetonitrile with 50 μL internal standard triacetic acid lactone, filtered by 3 kDa filter and then analyzed by LC-MS/ESL Chemical standard of BKDLs were purchased (Enamine, USA) and prepared as 10, 50, 100 μL to determine the standard curve. [0053] Agilent LC-MS/ESI Q-TOF 6456 i x 100 mm column (Phenomenex, USA) before injecting 3 riL sample for analysis. Mobile phase A is water with 0.1 % formic acid. Mobile phase B is methanol with 0.1 % formic acid. At 0.42 mL/min flow rate, 20 % B is increased to 72 % B in 6.5 min and further increased to 95 % B in another 1.3 min. 95 % B is maintained for 1 min and decreased to 20 % B in 0.2 min, followed by holding 20 % B for 2.2 min. The column temperature is 25 °C and MS is in negative mode.

Example 2. Biosynthesis of 5-ethyl-BKDL and 4-methyl-5-ethyl-BLDL in E. coli.

[0054] Genes encoding two combinatory PKSs with one consisting Lip loading module (LipLM), Debs Module 1 (DebsMl), MlsA8 C-terminal communication linker and the other consisting MlsA9 N-terminal communication linker, Rif module 2 (RifM2) and Debs thioesterase domain (DebsTE) were assembled through yeast assembly, then digested by Ndel and Xhol restriction enzymes (Themio Fisher Scientific, Waltham, MA) and cloned into Biobrick vectors (Lee etaL, 2011) digested by the same enzymes by T4 ligase (Thermo Fisher). LipLM-DebsMl-MlsA8_linker was cloned into pBbA5a vector which contains lacUV5 promoter, ampicillin resistance marker and pl 5a replicon, while MlsA9_linker- RifM2 -DebsTE was cloned into pBbS5k and pBbS6k vectors which contain lacUVS promoter (pBbS5k) or lacO-1 promoter (pBbS6k), kanamycin resistance marker and pSC101 replicon. All PKS module component genes w ere E. coli codon-optimized and synthesized by Genscript (Piscataw ay, NJ).

[0055] The resulting constructs containing genes of each of two combinatory' PKSs were transformed into Escherichia coli K207-3 strain (Murli et al., J. Industrial Microbiol, and Biotech 30:500-509, 2003) through chemical transformation. The resultant strains were stored in 30% glycerol under -80 °C for long-term storage.

[0056] When growi ng the strains for PKS expression andBKDL production, a colony from LB plate was inoculated into LB media and grown for overnight and then 1% of LB seed culture was inoculated in 2.5 mL fermentation media containedin each well of 24-well plate. The fermentation media was EZ-rich media (Teknova, Hollister, CA) supplemented with 10 g'L tryptone. 5 g'L yeast extract, 20 g/L yeast extract and 5 mM calcium pantothenate. After three to four hours of growth under 30 °C, the cell cultures were induced by 100 mM IPTG and transferred to 18 °C. After 72 hours of post-induction growth, the cell cultures were harvested. Biological triplicates were grown for each strain. 50 mg/L kanamycin and 100 mg/L carbenicillin were added in all media

Aldrich Co. (St. Louis, MO) and Fisher Scientific Co. (Pittsburg, PA).

[0057] For quantification and qualification of BKDLs, 100 pL of supernatant of harvested cell cultures were mixed with 100 μL acetonitrile with 50 μL internal standard triacetic acid lactone, filtered by 3 kDa filter and then analyzed by LC-MS/ESI. Chemical standard of BKDLs were purchased (Enamine, USA) and prepared as 10, 50, 100 μM to determine the standard curve. LC-MS method is described in Example 1.

[0058] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0059] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications can be made thereto without departing from tire spirit or scope of the appended claims.

Illustrative amino acid sequence of an acyl carrier protein encoded by a bacterial acp gene:

MSTIEERVKKIIGEQLGVKQEEVTNNASFVEDLGADSLDTVELVMALEEEFDTEIPD E

EAEK1TTVQAA1EQ1PGIGALVPAPVV1AAGRTEE (E. coli acpP)

Illustrative amino acid sequence of an acyl-ACP synthetase protein encoded by a bacterial aas gene:

MNQYVNDPSNYQLLIKNLLFSPVAFNPEQEIVYANHRRHSYKTFHDRVRQFANALT

KMGVKKGDTVAVMDYDSHRYLECYFAIPMIGAKLHMINVRLSPEQILYTIDHAEDDI

ILIHEEFLPILDQIKGRIDTVTRYVVLRDDEECEYERLLEQESTEYNFPDFDENTVA TTF

YTTGTTGFPKGVFFTHRQLVLHTMGILSTTGTNASQGRLHQGDIYMPTTPMFHVHAW

GLPYMATMLGVKQVYPGKYVPDVLLNLIEQEKVTFSHCVPTILHLLLSSPKSKAMDF

SGWKWIGGAALPKALCKSALERDIDVFAGYGMSETGPILSIVQLTPEQLELDVDQQ

AEYRSKTGKKVALVEAYIVDEDMNKLPHDGETAGEIVVRAPWLTPNYYKDNKifSK

ALWRGGYLHTGDVAHIDDEGF1KITDRVKDM1KISGEWVSSLELED1LHQHQSVSEV AVIGMPHNKWGEVPLALVTLKEDAQV

EIAKTSVGKVDKKELRKLHL (Vibrio harveyi acyl-ACP synthetase)

Tables

[0060] Tables 1 and 3 are provided below. An expanded view of columns 1-3 is attached as part of Table 1 (Expanded view — columns 1-3).

Table 1 - Expanded View of Columns 1-3 o. r o

6

LZi

6

Table 3. interchangeable PKS Modules-loading Modules and Extension 1 Modules

Table 3. Interchangeable PKS Modules-Extension 2 Modules