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(51) International Patent Classification: HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, C12N 15/52 (2006.01) Cl 2N 15/70 (2006.01) KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, C12N 9/00 (2006.01) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (21) International Application Number: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, PCT/US20 19/023792 TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (22) International Filing Date: (84) Designated States (unless otherwise indicated, for every 24 March 2019 (24.03.2019) kind of regional protection available) . ARIPO (BW, GH, (25) Filing Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (26) Publication Language: English TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (30) Priority Data: EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 62/649,836 29 March 2018 (29.03.2018) US MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, (71) Applicant: WILLIAM MARSH RICE UNIVERSITY KM, ML, MR, NE, SN, TD, TG). [US/US]; 6100 Main Street, Houston, Texas 77005 (US). (72) Inventors: GONZALEZ, Ramon; 6100 Main Street, Published: Houston, Texas 77005 (US). TAN, Zaigao; 6100 Main — with international search report (Art. 21(3)) Street, Houston, Texas 77005 (US). CLOMBURG, James M.; 6100 Main Street, Houston, Texas 77005 (US). (74) Agent: VALOIR, Tamsen; 2603 Augusta Drive, Suite 1350, Houston, Texas 77057 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available) : AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,

(54) Title: BIOSYNTHESIS OF OLIVETOLIC ACID (57) Abstract: We present a biosynthetic approach to engineer novel microbes, such as as E . s¬ β- in ng ic s.

Figure 1. BIOSYNTHESIS OF OLIVETOLIC ACID

PRIOR RELATED APPLICATIONS

[0001] This application claims priority to U.S. Serial No. 62/649,836, filed March 29, 2018, and incorporated by reference in its entirety for all purposes.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] Not applicable.

FIELD OF THE DISCLOSURE

[0003] This invention allows the efficient microbial production of olivetolic acid, divarinolic acid, orsellinic acid and other aromatic polyketides, as well as various downstream products that can be made from them.

BACKGROUND OF THE DISCLOSURE

[0004] A broad diversity of natural products can be synthesized by type III polyketide synthases (PKS Ills). Many of these products have been found to benefit human health, with PKS III products and derivatives garnering significant research interest in recent years. For instance, anthocyanins, the water-soluble pigments from mulberry fruits, have been reported to be useful in treating obesity, inflammation, and cancer. Hyperforin, which is one of the primary active constituents from extracts of Hypericum perforatum, can be used for the treatment of depression. The monoaromatic compound olivetolic acid, a member of the PKS III product class, holds promise for pharmacological properties such as antimicrobial, cytotoxic, and photoprotective activities. In addition, olivetolic acid is a central intermediate in the synthesis of an important class of pharmacological compounds, as it serves as the alkylresorcinol moiety during the biosynthesis of cannabinoids, a class of products that are becoming increasingly important due to their numerous pharmacological properties.

[0005] Currently, the production of olivetolic acid and its derivatives is primarily through direct extraction from plants. However, given that plants grow relatively slowly and require at least several months for the accumulation of these compounds, direct extraction suffers from long cycles. While plant biotechnology offers the opportunity to improve natural product synthesis in native species, it is difficult to precisely control the expression level of transgenes in plants and adapt to industrial-scale production.

[0006] While the chemical synthesis of olivetolic acid is another alternative that has recently been reported, the structural complexity of most natural products dictates inherent inefficiencies with total chemical synthesis, which suffers from low yield and high energy waste. In contrast to these approaches, construction of microbial cell factories for production of these value-added plant natural products is a promising strategy.

[0007] Despite the structural complexity of the end product, the starting and extending units for polyketide biosynthesis are often tractable acyl-coenzyme A (CoA) intermediates. In the case of olivetolic acid biosynthesis, 3 iterations of 2 carbon addition (via decarboxylative condensation with malonyl-CoA as the donor) to an initial hexanoyl-CoA primer results in the formation of 3,5,7-trioxododecanoyl-CoA, which can be subsequently cyclized to form olivetolic acid (FIG. 1).

[0008] While it was initially thought that the polyketide synthase (OLS) from Cannabis sativa was solely responsible for olivetolic acid biosynthesis, recombinant OLS was found to only synthesize olivetol, the decarboxylated form of olivetolic acid.(Taura et a , 2009) It has since been shown that olivetolic acid biosynthesis requires a polyketide cyclase, i.e. olivetolic acid cyclase (OAC), in addition to OLS, which catalyzes a C2-C7 intramolecular aldol condensation of the 3,5,7-trioxododecanoyl-CoA intermediate with carboxylate retention.(Gagne et a , 2012)

[0009] Expression of OLS and OAC in Saccharomyces cerevisiae, along with feeding of sodium hexanote, enabled the synthesis of 0.48 mg/L olivetolic acid after a 4 day fermentation. (Gagne et a , 2012) This represents a promising first step toward the development of microbial cell factories for the production of olivetolic acid that can be built upon to improve product synthesis from biorenewable feedstocks.

[0010] A potential bottleneck in improving product synthesis in S. cerevisiae is compartmentalization of acetyl-CoA metabolism, which results in the requirement for significant engineering efforts for the production of acetyl-CoA-derived products. Given the need for hexanoyl-CoA and malonyl-CoA in olivetolic acid synthesis, which are both commonly derived from acetyl-CoA, we engineered a recombinant Escherichia coli capable of producing olivetolic acid. In addition to its well-known physiology, metabolic network, and the ease of genetic manipulation, E. coli has been engineered to produce a wide range of products from the acetyl-CoA node, including those derivatives directly from malonyl-CoA.

[0011] We have now created a novel recombinant E. coli strain with a pathway for olivetolic acid production by incorporating validated PKS and cyclase genes in addition to auxiliary enzymes for generating the required precursors. The expression of these enzymatic components with the required modules of a β-oxidation pathway reversal to supply hexanoyl- CoA, resulted in a functional biological pathway for the synthesis of olivetolic acid from a principle carbon source without supplying exogenous hexanoate and further identified precursor supply as a major limiting factor for product synthesis.

[0012] The use of auxiliary enzymatic components aimed at increasing hexanoyl-CoA and malonyl-CoA and systematic metabolic engineering efforts enabled the synthesis by fermentation of olivetolic acid at high titer, further demonstrating the viability of developing microbial cell factories for the synthesis of plant-based natural products.

SUMMARY OF THE DISCLOSURE

[0013] Type III polyketide synthases contribute to the synthesis of many economically important natural products, many of which are currently produced by direct extraction from plants or chemical synthesis. Olivetolic acid is a type III polyketide with various pharmacological activities, also recognized as a key intermediate in cannabinoid biosynthesis. To demonstrate the potential for microbial cell factories to circumvent limitations of plant extraction or chemical synthesis for type III polyketide synthesis, here we utilize a biosynthetic approach to engineer Escherichia coli for the production of olivetolic acid.

[0014] In vitro characterization of polyketide synthase and cyclase enzymes, olivetolic acid synthase and olivetolic acid cyclase, respectively, validated their requirement as enzymatic components of the olivetolic acid pathway and confirmed the ability for these eukaryotic enzymes to be functionally expressed in E. coli. This served as a platform for the combinatorial expression of these enzymes with auxiliary enzymes aimed at increasing the supply of hexanoyl-CoA and malonyl-CoA starting units. Through combining olivetolic acid synthase and olivetolic acid cyclase expression with the required modules of a β-oxidation reversal for hexanoyl-CoA generation, we demonstrate the synthesis of olivetolic acid without the addition of exogenous hexanoate. [0015] The integration of additional auxiliary enzymes for increased both hexanoyl-CoA and malonyl-CoA, along with evaluation of varying fermentation conditions enabled the synthesis of about 75 mg/L olivetolic acid. This is the first example of olivetolic acid production in a prokaryotic cell, including for example E. coli, adding a new example to the repertoire of valuable compounds synthesized in this industrial workhorse.

[0016] The process involves performing traditional fermentations using industrial organisms (e.g., E. coli, B. subtilis, S. cerevisiae and the like) that convert different feedstocks into longer-chain polyketides. Media preparation, sterilization, inoculum preparation, fermentation and product recovery from the cells, or the medium, or both, are the main steps of the process.

[0017] The microorganisms can be used as living chemical manufacturing systems, or can be harvested and used as bioreactors for as long as the enzymes remain functional in the non-growing cells. Alternatively, the enzymes can be purified and used in an in vitro system reconstituted from the purified enzymes. Such an embodiment may be preferred as allowing the most control over the synthesis of complicated polyketides. However, living systems may be preferred as allowing continuous production and would be suitable for simple polyketides or polyketide precursors for downstream products.

[0018] The pathways in a living system are generally made by transforming the microbe with an expression vector encoding one or more of the proteins, but the genes can also be added to the chromosome by recombineering, homologous recombination, and similar techniques. Where the needed protein is endogenous, as is the case in some instances, it may suffice as is, but it is often overexpressed using an inducible promoter for better functionality and user- control over the level of active enzyme.

[0019] Reference to proteins herein can be understood to include reference to the gene encoding such protein. Thus, a claimed “permease” can include the related gene encoding that permease. However, it is preferred herein to refer to the protein by standard name per ecoliwiki.net or Human Genome Organisation (HUGO) since both enzymatic and gene names have varied widely, especially in the prokaryotic arts.

[0020] Once an exemplary protein is obtained, many additional examples of proteins with similar activity can be identified by BLAST search. Further, every protein record is linked to a gene record, making it easy to design overexpression vectors. Many of the needed enzymes are already available in vectors, and can often be obtained from cell depositories or from the researchers who cloned them. But, if necessary, new clones can be prepared based on available sequence information using e.g., RT-PCR techniques or de novo gene synthesis. Thus, it should be easily possible to obtain all of the needed enzymes for overexpression.

[0021] Another way of finding suitable enzymes/proteins for use in the invention is to consider other enzymes with the same EC number, since these numbers are assigned based on the reactions performed by a given enzyme. An enzyme can thus be obtained, e.g., from AddGene.org or from the author of the work describing that enzyme, and tested for functionality as described herein. In addition, many sites provide lists of proteins that all catalyze the same reaction. See e.g., BRENDA, UNIPROT, ECOPRODB, ECOLIWIKI, to name just a few.

[0022] ETnderstanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotide sequences that encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species. Rising such databases, a gene or cDNA may be “optimized” for expression in E. coli, yeast, algal or other species using the codon bias for the species in which the gene will be expressed.

[0023] Initial cloning experiments have proceeded in E. coli for convenience since most of the required genes are already available in plasmids suitable for bacterial expression, but the addition of genes to bacteria is of nearly universal applicability. Indeed, since recombinant methods were invented in the 1970’s and are now so commonplace, even school children perform genetic engineering experiments using bacteria. Such species include e.g., Bacillus, Streptomyces, Azotobacter, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, Streptococcus, Paracoccus, Methanosarcina, and Methylococcus, or any of the completely sequenced bacterial species. Indeed, hundreds of bacterial genomes have been completely sequenced, and this information greatly simplifies both the generation of vectors encoding the needed genes, as well as the planning of a recombinant engineering protocol. Such species are listed along with links at en.wikipedia.org/wiki/List_of_sequenced_bacterial_genomes.

[0024] Additionally, yeast, such as Saccharomyces, are common species used for microbial manufacturing, and many species can be successfully transformed. Indeed, yeast are already available that express recombinant thioesterases —one of the termination enzymes described herein—and the reverse beta oxidation pathway has also been achieved in yeast, as well as E. coli. Other species include but are not limited to Candida, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha (Pichia angusta), Kluyveromyces lactis, Pichia pastoris , and Yarrowia lipolytica, to name a few.

[0025] It is also possible to genetically modify many species of algae, including e.g., Spirulina, Chlamydomonas, Laminaria japonica, Undaria pinnatifida, Porphyra, Eucheuma, Kappaphycus, Gracilaria, Monostroma, Enteromorpha, Arthrospira, Chlorella, Dunaliella, Aphanizomenon, Isochrysis, Pavlova, Phaeodactylum, Ulkenia, Haematococcus, Chaetoceros, Nannochloropsis, Skeletonema, Thalassiosira, and Laminaria japonica. Indeed, the microalga Pavlova lutheri is already being used as a source of economically valuable docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), and Crypthecodinium cohnii is the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.

[0026] Furthermore, a number of databases include vector information and/or a repository of vectors and can be used to choose vectors suitable for the chosen host species. See, for example, AddGene.org which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues. See also Plasmid Information Database (plasmid.med.harvard.edu) and DNASU.org having over 191,000 plasmids. A collection of cloning vectors of E. coli is also kept at the National Institute of Genetics as a resource for the biological research community.

[0027] The enzymes can be added to the genome or via expression vectors, as desired. Preferably, multiple enzymes are expressed in one vector or multiple enzymes can be combined into one operon by adding the needed signals between coding regions. Further improvements can be had by overexpressing one or more, or even all of the enzymes, e.g., by adding extra copies to the cell via plasmid or other vector. Initial experiments may employ expression plasmids hosting multigene operons or 2 or more open reading frames (ORFs) encoding the needed genes for convenience, but it may be preferred to insert operons or individual genes into the genome for long term stability.

[0028] Still further improvements in yield can be had by reducing competing pathways, such as those pathways for making e.g., acetate, formate, ethanol, and lactate, and it is already well known in the art how to reduce or knockout these pathways. See e.g., the Rice patent portfolio by Ka-Yiu San and George Bennett (US7569380, US7262046, US8962272, US8795991) and patents by these inventors (US8129157 and US8691552) (each incorporated by reference herein in its entirety for all purposes).

[0029] As used herein, “homolog” means an enzyme with at least 40% identity to one of the listed sequences and also having the same general catalytic activity, although kinetic parameters of the reactions can of course vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it is typical for bacterial sequences to diverge significantly (40-60% identity), yet still be identifiable as homologs, while mammalian species tend to diverge much less (80-90% identity). Unless specified otherwise, any reference to an enzyme herein also includes its homologs that catalyze the s ame reaction.

[0030] As used herein, references to cells or bacteria or strains and all such similar designations include progeny thereof. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations that have been added to the parent. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0031] The terms “operably associated” or “operably linked,” as used herein, refer to functionally coupled nucleic acid sequences.

[0032] As used herein “recombinant” or “engineered” is relating to, derived from, or containing genetically engineered material. In other words, the genome was intentionally manipulated by humans in some way.

[0033] “Reduced activity” or “inactivation” (indicated by “-”) is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species. Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%, aka a “knock-out” or “null” mutants, indicated by ∆) . Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, and the like. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein.

[0034] “Overexpression” or “overexpressed” (indicated by “+”) in a cell is defined herein to be at greater expression than in the same cell without the genetic modification. Preferably, it’s at least 150% of protein activity as compared with an appropriate control species, and preferably 200, 500, 1000%) or more, or any activity in a host that would otherwise lack that enzyme. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, by adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or upregulating the endogenous gene, and the like.

[0035] The term “endogenous” or “native” means that a gene originated from the species in question, without regard to subspecies or strain, although that gene may be naturally or intentionally mutated. Thus, genes from Clostridia would not be endogenous to Escherichia, but genes from E. coli would be considered to be endogenous to any species of Escherichia. By contrast, the term “wild type” means a functional native gene that is not modified from its form in the wild. “Heterologous” means the gene is from a different biological source (microbe, plant, or animal). “Heterologous” may also refer to an endogenous gene removed from its normal milieu, for example on a plasmid or inserted into the chromosome at a location other than its normal location. In these cases, the gene may be expressed either from its native promoter or from a heterologous promoter.

[0036] “Expression vectors” are used in accordance with the art-accepted definition of a plasmid, virus or other propagatable sequence designed for protein expression in cells. There are thousands of such vectors commercially available, and typically each has an origin of replication (ori); a multiple cloning site; a selectable marker; ribosome binding sites; a promoter and often enhancers; and the needed termination sequences. Most expression vectors are inducible, although constitutive expression vectors also exist and either can be used.

[0037] As used herein, “inducible” means that gene expression can be controlled by the hand-of-man, by adding e.g., a ligand to induce expression from an inducible promoter. Exemplary inducible promoters include the lac promoter, inducible by isopropylthio-P-D- galactopyranoside (IPTG), the yeast AOX1 promoter inducible with methanol, the strong LAC promoter inducible with lactate, and the like. Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters.

[0038] As used herein, an “integrated sequence” means the sequence has been integrated into the host genome, as opposed to being maintained on an expression vector. It will still be expressible, either inducibly or constitutively.

[0039] As used herein, “olivetolic acid synthase” (also known as “olivetol synthase”) is a type III polyketide synthase that catalyzes three iterations of 2-carbon addition (via decarboxylative condensation with malonyl-CoA as the donor) to an initial hexanoyl-CoA primer resulting in the formation of 3,5,7-trioxododecanoyl-CoA, which subsequently can cyclize spontaneously to form olivetol or be cyclized by “olivetolic acid cyclase” to form olivetolic acid. Olivetolic acid synthase may also utilize an initial butanoyl-CoA primer resulting in the formation 3,5,7-trioxodecanoyl-CoA, which subsequently can cyclize spontaneously to form divarin or be cyclized by olivetolic acid cyclase to form divarinolic acid. Olivetolic acid synthase may also utilize an initial acetyl-CoA primer resulting in the formation 3,5,7-trioxooctanoyl-CoA, which subsequently can cyclize spontaneously to form orcinol or be cyclized by olivetolic acid cyclase to form orsellinic acid.

[0040] As used herein, “acetyl-CoA carboxylase” (ACC) (EC 6.4. 1.2) catalyzes the carboxylation of acetyl-CoA in the presence of ATP and bicarbonate (HC0 3 ) to form malonyl- CoA.

[0041] As used herein, “malonyl-CoA synthetase” (EC 6.2. 1.14), catalyzes the formation of malonyl-CoA from malonate, CoA and ATP.

[0042] A “synthase” is a generic term that describes an enzyme that catalyzes the synthesis of a biological compound without the requirement of a nucleoside triphosphate, such as ATP, as cosubstrate. A “synthetase” catalyzes the synthesis of a biological compound and requires ATP or another nucleoside triphosphate cosubstrate. “Ligase” is a term that describes an enzyme that catalyzes condensation of biological molecules with simultaneous cleavage of ATP or other high energy substrate. However, in the scientific literature the terms “synthase”, “synthetase”, and “ligase” do not always follow these strict definitions. Accordingly, as used herein, these terms may be used interchangeably.

[0043] As used herein, a “fatty acyl-CoA synthetase” catalyzes the formation of a fatty acyl-CoA from a fatty acid, CoA, and ATP. A “fatty acyl-CoA transferase” catalyzes the transfer of CoA from a fatty acyl-CoA to a second fatty acid without the requirement for ATP.

[0044] As used herein, a “thiolase” is an enzyme capable of catalyzing the condensation of acetyl-CoA with a C -acyl-CoA to produce a C +2 β-ketoacyl-CoA and CoA.

[0045] As used herein, a “hydroxyacyl-CoA dehydrogenase” is an enzyme that catalyzes the reduction of a β-ketoacyl-CoA to a β-hydroxyacyl-CoA.

[0046] As used herein, an “enoyl-CoA hydratase” is an enzyme that catalyzes the dehydration of a β-hydroxyacyl-CoA to enoyl-CoA. [0047] As used herein, an “acyl-CoA dehydrogenase” or “enoyl-CoA reductase” is an enzyme that catalyzes the reduction of enoyl-CoA to acyl-CoA.

[0048] As used herein, a “prenol kinase” is an enzyme capable of catalyzing the conversion of prenol (3-methyl-2-buten-l-ol) and ATP to 3,3-dimethylallyl phosphate (DMAP).

[0049] As used herein, a “isprenol kinase” is an enzyme capable of catalyzing the conversion of isprenol (3-methyl-3-buten-l-ol) and ATP to 3-methyl-3-buten-l-yl phosphate (IP).

[0050] As used herein, a “DMAP kinase” is an enzyme capable of catalyzing the conversion of DMAP and ATP to 3,3-dimethylallyl diphosphate (DMAPP).

[0051] As used herein, an “IP kinase” is an enzyme capable of catalyzing the conversion of IP and ATP to 3-methyl-3-buten-l-yl diphosphate (IPP).

[0052] As used herein, an “isopentenyl diphosphate isomerase” is an enzyme catalyzing the interconversion of DMAPP and IPP.

[0053] As used herein, a “geranyl diphosphate synthase” is an enzyme capable of catalyzing the condensation of DMAPP and IPP to form geranyl diphosphate (GPP).

[0054] As used herein, a “polyketide prenyltransferase” is an enzyme that transfers a prenyl group to a polyketide to form a prenylated polyketide. By way of example, the polyketide prenyltranferase geranyl diphosphate :olivetolate geranyl transferase (also known as CBGA synthase) transfers the geranyl group of GPP to the 3 position of olivetolic acid to form cannabigerolic acid (CBGA).

[0055] As used herein, a “cannabinoid” is a prenylated aromatic compound naturally found in Cannabis sativa, or a derivative or analog thereof.

[0056] As used herein, a “cannabinoid synthase” is an enzyme capable of converting one cannabinoid to another.

[0057] The following is a non-exhaustive list of examples of some of the key enzymes used in this filing.

[0058] As used herein, “principle carbon source” means the most abundant source of carbon, on a molar basis, that is used as a biosynthetic precursor for fermentation products. In this work, glycerol has been the principle carbon source of choice, but those with knowledge of the art will recognize that any carbon source that will serve as a biosynthetic precursor to acetyl-CoA, for example glucose, can potentially serve as a principle carbon source as used herein and serve as a fermentation feedstock for the production of fermentation products.

[0059] Referring to biosynthesis of olivetolic acid from a principle carbon source indicates that the carbon atoms in the olivetolic acid molecule can all originate from acetyl- CoA produced biosynthetically from said carbon source, without requiring that portions of the olivetolic acid molecule are derived from exogenously added hexanoate, which can serve as a precursor to hexanoyl-CoA or exogenously added malonate, which can serve as a precursor for malonyl-CoA.

[0060] As used herein, the following chemical names have the corresponding molecular structure:

[0061] The invention includes any one or more of the following embodiments, in any combination(s) thereof:

[0062] 1. A recombinant microorganism, comprising the expression of heterologous genes encoding olivetolic acid synthase and olivetolic acid cyclase in an amount sufficient to synthesize one or more of the compounds selected from olivetol; divarin; orcinol; olivetolic acid; divarinolic acid; and orsellinic acid.

[0063] 2 . A recombinant microorganism comprising the expression of heterologous genes encoding olivetolic acid synthase and olivetolic acid cyclase in an amount sufficient to synthesize one or more of the compounds: olivetol or olivetolic acid.

[0064] 3 . A recombinant microorganism comprising the expression of heterologous genes encoding olivetolic acid synthase and olivetolic acid cyclase in an amount sufficient to synthesize olivetolic acid.

[0065] 4 . The recombinant microorganism of embodiments 1-3, wherein either or both of the genes for olivetolic acid synthase and olivetolic acid cyclase are from Cannabis sativa.

[0066] 5 The recombinant microorganism of embodiments 1-4, comprising the heterologous expression of at least one additional gene that results in the increased production of one or more of said compounds.

[0067] 6 . The recombinant microorganism of embodiments 1-4, comprising the heterologous expression of at least one additional gene that results in the increased production of one or more of olivetol or olivetolic acid. [0068] 7 . The recombinant microorganism of embodiments 1-4, comprising the heterologous expression of at least one additional gene that results in the increased production of olivetolic acid.

[0069] 8. The recombinant microorganism of embodiments 5-7, wherein the expressed heterologous gene(s) result(s) in increased synthesis of malonyl-CoA.

[0070] 9 . The recombinant microorganism of embodiment 8, comprising the expression of (a) heterologous gene(s) encoding one or more of acetyl-CoA carboxylase or malonyl-CoA synthetase.

[0071] 10. The recombinant microorganism of embodiments 1-9, comprising the heterologous expression of at least one additional gene that results in the increased synthesis of fatty acyl-CoA.

[0072] 11. The recombinant microorganism of embodiment 10, comprising the expression of (a) heterologous gene(s) encoding one or more fatty acyl-CoA synthetase(s) or fatty acyl-CoA transferase(s) capable of converting a fatty acid to a fatty acyl-CoA.

[0073] 12. The recombinant microorganism of embodiments 10-1 1, wherein the expressed heterologous gene(s) result(s) in increased synthesis of one or more of butanoyl- CoA or hexanoyl-CoA.

[0074] 13. The recombinant microorganism of embodiments 10-1 1, wherein the expressed heterologous gene(s) result(s) in increased synthesis of hexanoyl-CoA.

[0075] 14. The recombinant microorganism of embodiments 11-13, wherein at least one fatty acyl-CoA synthetase gene is isolated from or is a derivative of a gene isolated from Cannabis sativa.

[0076] 15. The recombinant microorganism of embodiments 10-14, further comprising the heterologous expression of the fadD gene from E. coli.

[0077] 16. The recombinant microorganism of embodiments 1-15, wherein the microorganism comprises (a) heterologous gene(s) encoding one or more the following enzymes: a) thiolase capable of catalyzing the conversion of a C -acyl-CoA to a C +2 β- ketoacyl-CoA; b) hydroxyacyl-CoA dehydrogenase capable of catalyzing the conversion of β- ketoacyl-CoA to β-hydroxyacyl-CoA; c) enoyl-CoA hydratase capable of catalyzing the conversion of β-hydroxyacyl-CoA to enoyl-CoA; or d) acyl-CoA dehydrogenase or enoyl-CoA reductase capable of catalyzing the conversion of enoyl-CoA to acyl-CoA. [0078] 17. The recombinant microorganism of embodiments 1-16, wherein the microorganism further comprises (a) heterologous gene(s) encoding one or more polyketide prenyltransferase(s) capable of producing a cannabinoid compound(s) by condensing GPP with one or more of the compounds selected from olivetol; divarin; orcinol; olivetolic acid; divarinolic acid; and orsellinic acid.

[0079] 18. The recombinant microorganism of embodiment 17, wherein said gene(s) for at least one polyketide prenyltransferase is from Cannabis sativa or Streptomyces.

[0080] 19. The recombinant microorganism of embodiments 17-18, further comprising (a) heterologous gene(s) encoding one or more of the following enzymes: a) prenol kinase capable of catalyzing the conversion of prenol to dimethylallyl phosphate (DMAP); b) isprenol kinase capable of catalyzing the conversion of isprenol to isopentenyl phosphate (IP); c) DMAP kinase capable of catalyzing the conversion of DMAP to dimethylally diphosphate (DMAPP); d) IP kinase capable of catalyzing the conversion of IP to isopentenyl pyrophosphate (IP); e) isopentenyl diphosphate isomerase capable of catalyzing the interconversion of DMAPP and isopentenyl diphosphate (IPP); or f) geranyl diphosphate (GPP) synthase capable of catalyzing the condensation of DMAPP and isopentenyl diphosphate (IPP) to form GPP.

[0081] 20. The recombinant microorganism of embodiments 17-19, wherein the microorganism further comprises (a) heterologous gene(s) encoding one or more cannabinoid synthase enzyme(s) capable of converting one cannabinoid compound into another cannabinoid compound.

[0082] 21. The recombinant microorganism of embodiment 20 wherein the cannabinoid synthase enzyme(s) is(are) one or more enzymes selected from: a) cannabidiol synthase capable of catalyzing the conversion of cannabigerol to cannabidiol; b) cannabinodivarin synthase capable of catalyzing the conversion of cannabigerovarin to cannabinodivarin; c) cannabidiorcol synthase capable of catalyzing the conversion of cannabigerorcol to cannabidiorcol; d) cannabidiolic acid synthase capable of catalyzing the conversion of cannabigerolic acid to cannabidiolic acid; e) cannabinodivarinic acid synthase capable of catalyzing the conversion of cannabigerovarinic acid to cannabinodivarinic acid; f) cannabidiorcolic acid synthase capable of catalyzing the conversion of cannabigerorcolic acid to cannabidiorcolic acid; g) cannabichromene synthase capable of catalyzing the conversion of cannabigerol to cannabichromene; h) cannabichromevarin synthase capable of catalyzing the conversion of cannabigerovarin to cannabichromevarin; i) cannabichromenorcol synthase capable of catalyzing the conversion of cannabigerorcol to cannabichromenorcol; j) cannabichromenic acid synthase capable of catalyzing the conversion of cannabigerolic acid to cannabichromenic acid; k) cannabichromevarinic acid synthase capable of catalyzing the conversion of cannabigerovarinic acid to cannabichromevarinic acid; 1) cannabichromenorcolic acid synthase capable of catalyzing the conversion of cannabigerorcolic acid to cannabichromenorcolic acid; m) tetrahydrocannabinol synthase capable of catalyzing the conversion of cannabigerol to tetrahydrocannabinol; n) tetrahydrocannabivarin synthase capable of catalyzing the conversion of cannabigerovarin to tetrahydrocannabivarin; o) tetrahydrocannabiorcol synthase capable of catalyzing the conversion of cannabigerorcol to tetrahydrocannabiorcol; p) tetrahydrocannabinolic acid synthase capable of catalyzing the conversion of cannabigerolic acid to tetrahydrocannabinolic acid; q) tetrahydrocannabivarinic acid synthase capable of catalyzing the conversion of cannabigerovarinic acid to tetrahydrocannabivarinic acid; or r) tetrahydrocannabiorcolic acid synthase capable of catalyzing the conversion of cannabigerorcolic acid to tetrahydrocannabiorcolic acid.

[0083] 22. The recombinant microorganism of embodiments 20-21, wherein at least one cannabinoid synthase enzyme gene is from Cannabis sativa.

[0084] 23. The recombinant microorganism of embodiments 1-22 that is a prokaryote or eukaryote.

[0085] 24. The recombinant microorganism of embodiment 23 that is a prokaryote.

[0086] 25. The recombinant microorganism of embodiment 24 that is Escherichia coli.

[0087] 26. A method of producing one or more of the compounds: olivetol; divarin; orcinol; olivetolic acid; divarinolic acid; or orsellinic acid, said method comprising: a) culturing the recombinant microorganism of embodiments 1-25 in a culture medium under conditions in which some or all of the heterologous genes are expressed by inducing expression of said genes or constitutively expressing said genes; b) synthesizing in the microorganism one or more of the compounds: olivetol; divarin; orcinol; olivetolic acid; divarinolic acid; or orsellinic acid; and c) optionally isolating from the cell or supernatant, or both, one or more of said compounds.

[0088] 27. The method of embodiment 26, further comprising adding malonate; butanoate; or hexanoate to the culture medium. [0089] 28. A method of producing one or more cannabinoid compounds, comprising: a) culturing the recombinant microorganism of embodiments 17-22 in a culture medium, under conditions in which some or all of the heterologous genes are expressed by inducing expression of said genes or constitutively expressing said genes; b) synthesizing in the microorganism one or more cannabinoid compound(s); and c) optionally isolating one or more of the cannabinoid compounds from the cell or supernatant, or both.

[0090] 29. The method of embodiment 28, comprising adding prenol; isoprenol; malonate; butanoate; or hexanoate to the culture medium.

[0091] 30. The method of embodiments 28-29, wherein the cannabinoid compound(s) is(are) one or more of: cannabigerol; cannabigerolic acid; cannabigerovarin; cannabigerovarinic acid; cannabigerorcol; cannabigerorcolic acid; cannabidiol; cannabidiolic acid; cannabinodivarin; cannabinodivarinic acid; cannabidiorcol; cannabidiorcolic acid; cannabichromene; cannabichromenic acid; cannabichromevarin; cannabichromevarinic acid; cannabichromeorcol; cannabichromeorcolic acid tetrahydrocannabinol; tetrahydrocannabinolic acid; tetrahydrocannabivarin; tetrahydrocannabivarinic acid; tetrahydrocannabiorcol; or tetrahydrocannabiorcolic acid.

[0092] 31. The method of embodiments 26-30, where the microorganism is cultured in a fermentation medium comprising a principle carbon source selected from glycerol or glucose.

[0093] 32. The method of embodiments 26-31, wherein said compound is produced at a concentration greater than 40 mg/L, and preferably greater than about 50 mg/L, 60 mg/L, 70, or 75 mg/L.

[0094] 33. The method of embodiments 26-32, wherein said compound is produced at a rate greater than 0.5 mg/hr-g, and preferably greater than 1 mg/hr-g, 2 mg/hr-g, 3 mg/hr-g, 4 mg/hr-g, 5 mg/hr-g, 6 mg/hr-g, 7 mg/hr-g, 8 mg/hr-g, or 9 mg/hr-g.

[0095] 34. A method of producing olivetolic acid comprising: a) culturing the recombinant microorganism of embodiments 1-25 in a culture medium, under conditions in which some or all of the heterologous genes are expressed by inducing expression of said genes or constitutively expressing said genes; b) synthesizing in the microorganism olivetolic acid; and c) optionally isolating olivetolic acid from the cell or supernatant.

[0096] 35. The method of embodiment 34, comprising adding exogenously to the cell culture one or more of the compounds: prenol; isoprenol; malonate; or hexanoate. [0097] 36. The method of embodiments 34-35, where the microorganism is cultured in a fermentation medium comprising a principle carbon source selected from glycerol or glucose.

[0098] 37. The method of embodiments 34-36, wherein olivetolic acid is produced at a concentration greater than about 40 mg/L, and preferably greater than about 50 mg/L, 60 mg/L, 70 mg/L, or 75 mg/L.

[0099] 38. The method of embodiments 34-37, wherein olivetolic acid is produced at a rate greater than 0.5 mg/hr-g, and preferably greater than 1 mg/hr-g, 2 mg/hr-g, 3 mg/hr-g, 4 mg/hr-g, 5 mg/hr-g, 6 mg/hr-g, 7 mg/hr-g, 8 mg/hr-g, or 9 mg/hr-g.

[00100] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise.

[00101] The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.

[00102] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[00103] The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.

[00104] The phrase “consisting of’ is closed, and excludes all additional elements.

[00105] The phrase “consisting essentially of’ excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention.

[00106] The following abbreviations are used herein: DESCRIPTION OF FIGURES

[00107] FIG. 1. Production of olivetolic acid by recruiting OLS and OAC. (A) Biosynthetic pathway of olivetolic acid from hexanoyl-CoA. 3 malonyl-CoA extender units are added to the hexanoyl-CoA primer to form olivetolic acid through the Claisen condensation catalyzed by OLS and C2-C7 aldol cyclization catalyzed by OAC. Potential pathway byproducts, e.g. PDAL, HTAL and olivetol, can also be formed through hydrolysis of intermediate CoAs or spontaneous cyclization without carboxyl group retention. (B) In vitro production of olivetolic acid using recombinant and purified OLS and OAC core enzymes. Detailed mass spectrometric (MS) identification of these olivetolic acid and by-products can be seen in FIG. Sl. (C) In vivo production of olivetolic acid from biotransformations with E. coli BL21 (DE3). E. coli cells with the induced OLS and OAC from LB medium were collected and resuspended in fresh M9Y medium+2% (wt/v) glucose with 4 mM hexanoate, and cultured at 22 °C for 48 h . Blank, BL21 (DE3) with pETDuet-l empty vector. PDAL, pentyl diacetic acid lactone, HTAL, hexanoyl triacetic acid lactone.

[00108] FIG. 2. Impact of auxiliary enzymes for increasing hexanoyl-CoA and malonyl- CoA supply for olivetolic acid production in BL21 (DE3). Left, auxiliary enzymes employed for increasing hexanoyl-CoA (FadD/FadK) and malonyl-CoA (MCS/ACC). FadD and FadK were employed to form hexanoyl-CoA from hexanoate and CoA. MCS and ACC can form the malonyl-CoA extender unit through two different mechanisms. Middle, enzyme organization of OLS/OAC core enzymes and FadD/FadK, MCS/ACC auxiliary enzymes. The vector expressing OLS and OAC was constructed using multiple cloning site 1 (Mcsl) and multiple cloning site 2 (Mcs2) of the pETduet-l plasmid respectively. FadD/FadK were constructed at the Mcsl of pCDFduet-l and MCS/ACC were constructed at the Mcs2 of pCDFduet-l. Right, olivetolic acid production using different combinations of auxiliary enzymes with OLS and OAC expression. E. coli cells expressing the indicated enzymes grown in LB medium were collected and resuspended in fresh M9Y medium with 2% (wt/v) glucose and 4 mM hexanoate. For strains harboring MCS, 12 mM malonate sodium was also included. Values represent the average of at least three biological replicates with error bars indicating standard deviation. FadD, long chain fatty acyl-CoA synthetase; FadK, short chain fatty acyl-CoA synthetase; MCS, malonyl-CoA synthetase; ACC, acetyl-CoA carboxylase.

[00109] FIG. 3. Production of olivetolic acid in engineered . coli JST10 (DE3). (A) In JST10 (DE3), activated r-BOX was achieved by overexpression of thiolase (TH) from Ralstonia eutropha, FadB hydroxyacyl-CoA dehydrogenase (HR) from E. coli, FadB enoyl- CoA hydratase (EH) and egTER enoyl-CoA reductase (ER) from Euglena glacilis. Fermentative by-product (lactate, succinate, ethanol, acetate) pathways were blocked through deletion of ldhA,frdA, adhE, pta, and poxB. OLS-OAC core enzymes and FadD-ACC auxiliary enzymes were recruited for olivetolic acid production. (B) Olivetolic acid titers in different strains. Engineered E. coli strains were grown in LB-like MOPS medium+2% (wt/v) glycerol supplemented with 4 mM hexanoate where indicated. For strains harboring MCS, 12 mM malonate sodium was also included. (C) Flaviolin biosynthesis pathway for measuring malonyl-CoA availability. ETpper, flaviolin biosynthesis pathway: 5 malonyl-CoA are condensed by RppA to form flaviolin, which has a specific absorbance at wavelength of 340 nm. Bottom, RppA was expressed with different MCS/ACC auxiliary enzymes for

characterization of malonyl-CoA availability in JST10 (DE3) strain. Engineered . coli strains were cultured in LB-like MOPS medium+2% (wt/v) glycerol. For strains harboring MCS, 12 mM malonate sodium was also included.

[00110] FIG. 4 . Optimization of fermentation conditions for olivetolic acid production by JST10-OLS-OAC -FadD-ACC. The engineered E. coli strain was cultured in LB-like MOPS medium+2% (wt/v) glycerol in 25 mL shake flasks. (A) Effects of different temperatures on olivetolic acid production with gene expression induced by 50 µΜ IPTG and 100 pM cumate. (B) Effects of different working volume (WV) on olivetolic acid production with gene expression induced by 50 pM IPTG and 100 pM cumate. (C) Effects of different IPTG dosages on olivetolic acid production with 100 pM cumate. (D) Effects of different cumate dosage on olivetolic acid production with 100 pM IPTG. For all experiments, inducers and 4 mM

hexanoate were added after strains reached an OD550 -0.4-0. 8.

[00111] FIG. 5 Olivetolic acid production and stability. (A) Olivetolic acid fermentation in bioreactor with controlled conditions. Fermentation was performed in 400 mL MOPS medium with 30 g/L glycerol in 500 mL bioreactor (Infors). Cultures were grown at 37 °C with

an initial OD550 of 0.07, 100 pM IPTG, 10 pM cumate and 4 mM hexanoate were added when

OD550 reached 0.4-0. 8, the pH was maintained at 7.0 by using 1.5 M H2SO4 and 3 M NaOH, the dissolved oxygen level was also monitored. (B) Olivetolic acid stability assays in the absence/presence of E. coli MG1655 (DE3) cells. The initial olivetolic acid titer was 110 mg/L. (C) Olivetol stability assay in the absence/presence of E. coli MG1655 (DE3) cells. All the olivetolic acid/olivetol stability assays were conducted in 400 mL MOPS medium with 30 g/L glycerol in 500 mL Infors bioreactor. In the presence of E. coli cells, E. coli initial inoculum was set as OD550 -0.07. OLA, olivetolic acid; OLO, olivetol.

[00112] FIG. 6. Biosynthetic pathway of divarinolic acid through recruiting OLS and OAC from butyryl-CoA. Three malonyl-CoA extender units are added to the butyryl-CoA primer through decarboxyl ative Claisen condensation to form 3,5,7-trioxodecanoyl-CoA, which can be further catalyzed by OAC via C2-C7 non-decarboxylative aldol cyclization to form divarinolic acid. Divarin can be formed by either spontaneous cyclization of 3,5,7- trioxodecanoyl-CoA in the absence of OAC or decarboxylation of divarinolic acid.

[00113] FIG. 7. In vitro production of divarinolic acid and divarin. (A). SDS-PAGE result of purified OLS and OLS+OAC. (B). Gas chromatography (GC) profile of samples. Upper, in the presence of only OLS. Middle, in the presence of both OLS and OAC. Lower, control without any OLS or OAC. (C). Mass spectra of divarin and divarinolic acid peaks.

[00114] FIG. 8. In vivo production of divarinolic acid and divarin in E. coli. In this example, the butyryl-CoA primer is provided by reversal of beta-oxidation (r-BOX) starting from acetyl-CoA. Alternatively, it can be provided by the action of a suitable fatty acyl-CoA synthetase or transferase on butanoate supplied exogenously (not shown). The malonyl-CoA extender units can be obtained from acetyl-CoA carboxylation, catalyzed by E. coli native acetyl-CoA carboxylase (ACC). Alternatively, malonyl-CoA can be produce from malonate, CoA, and ATP using malonyl-CoA synthetase (not shown).

[00115] FIG. 9. In vivo production of orsellinic acid and orcinol in E. coli. Acetyl-CoA serves as the primer and malonyl-CoA as the extender units. The malonyl-CoA can be obtained from acetyl-CoA carboxylation, catalyzed by E. coli native acetyl-CoA carboxylase (ACC). Alternatively, malonyl-CoA can be produced from malonate, CoA, and ATP using malonyl- CoA synthetase (not shown).

[00116] FIG. 10. In vivo production of cannabigerolic acid (CBGA) in E. coli. In this example, the prenyl transferase NphB catalyzes the prenylation of olivetolic acid, leading to CBGA. The GPP precursor comes from the condensation of IPP and DMAPP, which can come from the MEP pathway, a heterologous MYA pathway, or a novel pathway from prenol or isoprenol. Olivetolic acid is produced from hexanoyl-CoA and malonyl-CoA using OLS and OAC.

[00117] FIG. 11. In vivo production of cannabidiolic acid (CBDA) in E. coli. CBDA synthase (CBDAS) catalyzes the oxidative cyclization of CBGA into CBDA. The CBGA is produced by prenylation of olivetolic acid produced as described in Figure 10.

[00118] TABLE SI. Primers used in this study.

[00119] FIGURE SI. Mass spectrometry data

DETAILED DESCRIPTION

[00120] Strains and culture conditions. Uness otherwise specified, strains used in this study are listed in Table 1. E. coli BL21 (DE3) and JST10 (DE3) were employed as the host strains. Luria-Bertani (LB) medium was used for culturing E. coli cells for plasmid construction. Modified M9Y medium (6.7 g/L Na2HP0 4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L of NH4CI , 20 g/L glucose, 10 g/L yeast extract, 2 mM MgSCL, and 0.1 mM CaCL) was used for the biotransformation experiments for all BL21 (DE3) derived strains. LB-like MOPS medium used for JST10 (DE3) strains contains 125 mM MOPS, supplemented with 20 g/L glycerol (or 30 g/L in batch fermentation and olivetolic acid/olivetol stability assays), 10 g/L tryptone, 5 g/L yeast extract, 5 mM calcium pantothenate, 2.78 mM Na2HP0 4, 5 mM µΜ µΜ (NH4)2S0 4, 30 mMNFLCl, 5 sodium selenite, 100 pM FeS0 4, 100 biotin, and 1 mg/L thiamine-HCl. When necessary, ampicillin, spectinomycin and kanamycin were added at final concentrations of 100, 50 and 50 mg/L, respectively.

[00121] Construction of plasmids. All oligonucleotide primers used in this study are listed in Table Sl. Codon optimized OLS and OAC from Cannabis sativa for expression in E. coli were synthesized by GeneArt (Invitrogen) and then inserted into the first and second multiple cloning site of pETDuet-l, respectively, resulting into pET-Pl-OLS-P2-OAC. FadD and FadK were PCR-amplified from E. coli K-12 MG1655 genomic DNA and inserted into the first cloning site of pCDFDuet-l to obtain pCDF-Pl-FadD/FadK. The malonyl-CoA synthetase (MCS) gene from Bradyrhizobium japonicum was codon optimized and inserted into the second multiple cloning site of pCDFDuet-l to obtain pCDF-P2-MCS. AccA, AccB, AccC and AccD were PCR-amplified from MG1655 genomic DNA with a ribosome binding site (RBS) (underlined, Table Sl) and assembled into the second multiple cloning site of pCDFDuet-l through Gibson Assembly Cloning Kit (NEB) to obtain pCDF-P2-ACC. Table 1. Strains and plasmids used in this study.

Plasmids/strains Genetic characteristics Source Plasmids pETDuet-1 pBR322 ori with P T ; Amp R Novagen pCDFDuet-1 CDF ori with P T ; SmR Novagen pET-Pl -OLS pETDuet-1 carrying o/s This study pET-PI -OAC pETDuet-1 carrying oac This study pET-P 1-OLS-P2-OAC pETDuet-1 carrying OLS and oac This study pCDF-P1 -FadD pCDFDuet-1 carrying fadD This study pCDF-P1 -FadK pCDFDuet-1 carrying fadK This study pCDF-P2-MCS pCDFDuet-1 carrying mcs This study pCDF-P2-ACC pCDFDuet-1 carrying accA, accB, accC, accD This study pCDF-P1 -FadD-P2-MCS pCDFDuet-1 carrying fadD and mcs This study pCDF-P1 -FadK-P2-MCS pCDFDuet-1 carrying fadK and mcs This study pCDF-P1 -FadD-P2-ACC pCDFDuet-1 carrying fadD and accA, accB, accC, accD This study pCDF-P1 -FadK-P2-ACC pCDFDuet-1 carrying fadK and accA, accB, accC, accD This study pET-P 1-RppA pETDuet-1 carrying rppA This study E . coli Strains E . coli BL21 (DE3) Host strain for enzymes expression Lab collection AfrdA AldhA Apta AadhE ∆ροχΒ AyciA AybgC Aydil AtesA AfadM AtesB AfadE DE3 FRT-cymR-PC 5-fadB AfadAwzeo FRT-cymR- (Kim et al. , E . coli JST1 0 (DE3) PC 5-bktB AatoB FRT-cymR-PC 5-egTER at fabl chromosomal 201 5) location BL-OLS-OAC BL2 1 (DE3) with pET-P 1-OLS-P2-OAC This study BL-OLS-OAC-FadD BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -FadD This study BL-OLS-OAC-FadK BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -FadK This study BL-OLS-OAC-MCS BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P2-MCS This study BL-OLS-OAC-ACC BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -ACC This study BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -FadD-P2- BL-OLS-OAC-Fad D-MCS This study MCS BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -FadK-P2- BL-OLS-OAC-FadK-MCS This study MCS BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -FadD-P2- BL-OLS-OAC-Fad D-ACC This study ACC BL2 1 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -FadK-P2- BL-OLS-OAC-Fad K-ACC This study ACC JST 10-OLS-OAC JST1 0 (DE3) with pET-P 1-OLS-P2-OAC This study JST 10-OLS-OAC-FadD- JST1 0 (DE3) with pET-P1 -OLS-P2-OAC and pCDF-P1 -FadD-P2- This study MCS MCS JST 10-OLS-OAC-FadD- JST1 0 (DE3) with pET-P1 -OLS-P2-OAC and pCDF-P1 -FadD-P2- This study ACC ACC JST 10-OLS-OAC-FadD JST1 0 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P1 -FadD This study JST 10-OLS-OAC-ACC JST1 0 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P2-ACC This study JST 10-OLS-OAC-MCS JST1 0 (DE3) with pET-P 1-OLS-P2-OAC and pCDF-P2-MCS This study JST1 0-RppA JST1 0 (DE3) with pET-P1 -RppA This study JST 10-MCS-RppA JST1 0 (DE3) with pET-P1 -RppA and pCDF-P2-MCS This study JST 10-ACC-RppA JST1 0 (DE3) with pET-P1 -RppA and pCDF-P2-ACC This study [00122] In vitro production of olivetolic acid. E. coli BL21 (DE3) was used for expression of His-tagged OLS and OAC proteins, from their respective pET-Pl-OLS and pET- Pl-OAC constructs. BL21 (DE3) strains containing His-tagged OAC or OLS genes were grown at 37°C in 0.5 L LB medium with ampicillin. Enzyme expression was induced by addition of isopropy -β-D-thi ogal actosi de (IPTG) to a final concentration of 0.4 mM, when

OD550 of the culture was between 0.4-0. 8. After 18 h of induction at 37°C, cells were harvested by centrifugation at 12,000 rpm, 4°C, 10 min. The cell pellet was resuspended in lysis buffer (20 mM Tris-HCl, 0.5 MNaCl, 5 mM imidazole, 0.1% Triton-X 100, pH 8.0) and subjected to sonication using a Sonifier SFX250 (Branson). Following centrifugation (10,000 rpm, 4°C, 30 min), the supernatant containing soluble protein fraction was recovered and filtered through a 0.45 pm filter. Recombinant His-tagged proteins were purified using TALON metal affinity resin (Clontech). Soluble protein extract was applied to 1 ml packed column of the resin, and after washing the unbound proteins with wash buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) supplemented with 20 mM imidazole, the His-tagged enzymes were eluted from the column with elution buffer containing 250 mM imidazole. Purified His-tagged enzymes were concentrated to a final concentration of 2 mg/mL and elution buffer was exchanged with storage buffer (12.5 mM Tris-HCl, 50 mM NaCl and 2 mM DTT) at 4°C using Amicon ultrafiltration centrifugal devices. The concentrated enzymes were stored at -80°C for enzyme activity assays. Enzyme assays were performed in a 500 pL total reaction volume containing 100 mM potassium phosphate buffer (pH 7.0), 200 pM hexanoyl-CoA, 400 pM malonyl-CoA,

10 pg OLS, and 30 pg OAC (when included). (Gagne et al., 2012) The reaction mixture was incubated at 20°C for 16 h and 20 pL sulfuric acid (H2SO4) was added to terminate the reaction.

[00123] Biotransformation for olivetolic acid production. One milliliter ( 1 mL) of overnight cultures of recombinant . coli strains was inoculated in 50 mL fresh LB medium in

250 mL shake flask with ampicillin, and cultivated at 37°C, 200 rpm. When OD550 reached approximately 0.4-0. 8, 0.5 mM IPTG was added. The cultures were then incubated at 22°C for

15 h . Cells were then harvested by centrifugation, washed with fresh M9Y medium and resuspended in 50 mL M9Y medium to OD550 ~ 3 and supplied with 4 mM hexanote for biotransformation experiments. An additional 12 mM sodium malonate was added when malonyl-CoA synthetase (MCS) was expressed for malonyl-CoA synthesis. Following incubation at 22 °C for 48 h, the fermentation broth supernatants were extracted with an equal volume of ethyl acetate, evaporated with a stream of nitrogen and resuspended in 1 mL methanol for HPLC-MS analysis by using Agilent 1200 HPLC system and Bruker MicroToF

ESI LC-MS System. The column used was Shim-pack XR-ODS II C18, 2.0 mm><75 mm (Shimadzu). HPLC conditions were as follows: solvent A = 0.1% formic acid in H2O; solvent B = methanol; flow rate = 0.25 ml min 1 ; 0-2.5 min, 95% A and 5% B; 2.5-20 min, 95% A and 5% B to 5% A and 95% B ; 20-23 min, 5% A and 95% B; 23-24 min, 5% A and 95% B to 95% A and 5% B; 24-30 min, 95% A and 5% B .

[00124] Fermentation conditions for olivetolic acid production in shake flasks. Modified LB-like MOPS medium using glycerol as carbon source was used for all fermentations. (Kim et al., 2015) Fermentations were conducted in 25-mL Pyrex Erlenmeyer flasks (narrow mouth/heavy duty rim, Corning) filled with 5-20 mL of the MOPS medium with 20 g/L glycerol and fitted with foam plugs filling the necks. A single colony of the desired strain was cultivated overnight (14-16 h) in LB medium with appropriate antibiotics and used as the initial inoculum at the OD550 -0.07. After inoculation, flasks were incubated at 37 °C and 200 rpm until OD550 reached 0.4-0. 8, at which point IPTG (0-500 µΜ), cumate (0-500 pM) and hexanoate (4 mM) were added. 12 mM sodium malonate was also added when malonyl- CoA synthetase (MCS) was expressed for malonyl-CoA synthesis. Flasks were then incubated under the same conditions for 48 h post-induction unless otherwise stated.

[00125] Olivetolic acid fermentation in bioreactor with precise parameter control. Fermentations were performed in 400 mL MOPS medium with 30 g/L glycerol in a 500 mL bioreactor (Infors) at 37 °C. An overnight seed culture was used to inoculate the bioreactor to an OD550 of -0.07 and when the OD550 reached 0.4-0. 8, 100 pM IPTG, 10 pM cumate and 4 mM hexanoate were added. pH was maintained at 7.0 by using 1.5 M sulfuric acid (H2SO4) as acid solution and 3 M potassium hydroxide (NaOH) as base solution. The air flowrate was set at 50 mL/min.

[00126] Olivetolic acid/olivetol stability analysis. For olivetolic acid stability assays, 500 mL bioreactors with the above described media and conditions were utilized. The initial olivetolic acid titer was - 110 mg/L for olivetolic acid stability analysis. In the presence of E. coli cells, MG1655 (DE3) was employed as the testing strain. An overnight seed culture was used to inoculate the bioreactor an OD550 -0.07. pH was maintained at 7.0 by using 1.5 M H2SO4 and 3 M NaOH. The air flowrate was set at 50 mL/min.

[00127] Similar operation was performed for olivetol stability assay, only changing the initial olivetolic acid to olivetol (-80 mg/L).

[00128] GC-FID/MS analysis. Quantification of olivetolic acid was conducted via GC- FID analysis using an Agilent 7890 B gas chromatograph equipped with an Agilent 5977 mass spectroscope detector (Agilent) and an HP-5ms capillary column (0.25 mm internal diameter, 0.25 µιη film thickness, 30 m length; Agilent). Sample preparation was conducted as follows: 2 mL culture samples were transferred to 5 mL glass vials (Fisher Scientific), 4-pentylbenzoic acid (final concentration 50 mg/L) was added as internal standard. Then 80 L of H2SO4 and 340 pL of 30 % (wt/v) NaCl solution were added for pH and ionic strength adjustment. Two milliliters of hexane was added for extraction. Vials were sealed with Teflon-lined septa (Fisher Scientific), secured with caps, and rotated at 60 rpm for 2 h . The samples were then centrifuged for 2 min at 6500 rpm to separate the aqueous and organic layers. After centrifugation, 1.5 mL of the top organic layer was transferred to new 5 mL glass vial and evaporated under a stream of nitrogen. Then, 100 pL pyridine and 100 pL of N,0-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) were added to the dried extract for derivatization at 70 °C for 1 h . After cooling to room temperature, 200 pL of derivatization product was transferred to vials (Fisher Scientific) for GC-MS analysis according to the following method: 1 pL were injected into the GC, which was run in splitless mode using helium gas as a carrier gas with a flow rate of 1 mL/min. The injector temperature was 280 °C and the oven temperature was initially held at 50 °C for 3 min and then raised to 250 °C at 10 °C/min and held for 3 min.

[00129] Recruiting OLS and OAC for olivetolic acid production. A synthetic pathway for olivetolic acid biosynthesis in E. coli requires at least two catalytic enzymes, olivetolic acid synthase (OLS) and olivetolic acid cyclase (OAC). OLS is a type III PKS (tetraketide synthase) from Cannabis trichomes that catalyzes the formation of 3,5,7-trioxododecanoyl-CoA from a hexanoyl-CoA primer and 3 malonyl-CoA extender units via decarboxylative Claisen condensation. This 3,5,7-trioxododecanoyl-CoA intermediate can then be cyclized by OAC via C2-C7 intramolecular aldol condensation to form olivetolic acid. In addition to the desired product, evidence suggests that pathway byproducts, e.g. pentyl diacetic acid lactone (PDAL), hexanoyl triacetic acid lactone (HTAL) and olivetol can also be formed through hydrolysis of intermediate polyketide CoAs or spontaneous cyclization (FIG. 1A).

[00130] To confirm the ability for OLS and OAC to synthesize olivetolic acid from hexanoyl-CoA and malonyl-CoA, in addition to evaluating potential by-products, we conducted in vitro analysis of these enzymatic components (FIG. IB). Codon optimized, His- tagged OLS and OAC were expressed and purified from E. coli and utilized to determine product formation in a reaction system including hexanonyl-CoA (primer) and malonyl-CoA (extender unit). As seen in Figure 1B, incubation of OLS and OAC in the presence of these substrates resulted in olivetolic acid synthesis. Pathway byproducts PDAL and olivetol were also detected in samples with OLS only or including both OLS and OAC (FIG. 1B). These by- products were the only products formed in the absence of OAC (i.e. assays with OLS only) confirming the indispensable nature of the OAC component for olivetolic acid formation.(Gagne et al., 2012)

[00131] We next evaluated the ability to produce olivetolic acid in vivo through the construction of plasmid pET-Pl-OLS-P2-OAC expressing codon-optimized versions of OLS

and OAC. This plasmid was transformed into . coli BL21 (DE3), and the resulting strain (BL- OLS-OAC) enabled the production of olivetolic acid following 48 h cultivation in biotransformation media with 4 mM hexanote (FIG. 1C). In addition to olivetolic acid, small amounts of olivetol were also detected, implying that while heterologous OAC cyclized the 3,5,7-trioxododecanoyl-CoA intermediate into olivetolic acid, the potential for by-product formation is also a concern in vivo. It should be noted that even with the small amounts of olivetolic acid produced (

[00132] Impact of precursor supply on olivetolic acid production in vivo. While the above results demonstrate the function of the required PKS and cyclase components in vivo, the low titers of olivetolic acid (< 0.1 mg/L) (FIG. 2) require additional assessment of the overall limitations for product synthesis. Given the functional expression and purification of OLS and OAC from E. coli, we reasoned a major limitation for olivetolic acid production may be the availability of required precursors. To determine the potential to improve product synthesis by increasing precursor supply, we expanded our synthetic approach through combinatorially expressing auxiliary enzymes for malonyl-CoA and/or hexanoyl-CoA generation with the OLS and OAC components.

[00133] For increasing malonyl-CoA supply, two classes of enzymes for biosynthesis of malonyl-CoA were evaluated. The first, malonyl-CoA synthetase (MCS) (EC 6.2.1.14), catalyzes the formation of malonyl-CoA from malonate, CoA and ATP. The MCS from Bradyrhizobium japonicum was codon optimized and expressed in conjunction with OLS and OAC in E. coli supplied with 12 mM sodium malonate. Consistent with our hypothesis, increasing malonyl-CoA supply using this approach resulted in increased olivetolic acid titer, from 0.1 mg/L to 0.65 mg/L (P < 0.05) (FIG. 2). While this shows the importance of increasing malonyl-CoA supply, MCS requires the addition of exogenous malonate. To generate increased malonyl-CoA without malonate supplementation, we evaluated the overexpression of acetyl-CoA carboxylase (ACC) (EC 6.4. 1.2) catalyzing the carboxylation of acetyl-CoA in

the presence of ATP and bicarbonate (HC0 3 )· In E. coli, ACC consists of four different subunits, e.g. AccA, AccB, AccC and AccD. Genes encoding the AccABCD complex were overexpressed (+ACC) in the BL-OLS-OAC strain (resulting in BL-OLS-OAC-ACC). However, the engineered +ACC strain did not improve olivetolic acid production in the absence of malonate (FIG. 2). ACC requires acetyl-CoA as catalytic substrate, which is one of the most important central metabolites in E. coli, participating in the TCA cycle, glyoxylate cycle, amino acid metabolism, and other important pathways. Although individual +ACC overexpression could channel more acetyl-CoA into malonyl-CoA available for olivetolic acid production, flux into other biosynthetic pathways and thus cellular growth may be impaired. Consistently, we observed lower growth with BL-OLS-OAC-ACC during the biotransformation (the highest OD550 was only 1.8) compared with BL-OLS-OAC (the highest

OD550 was 3.8). Impaired growth caused by ACC overexpression has been observed in prior studies, in both E. coli and S. cerevisiae.

[00134] While these initial experiments were conducted in the presence of hexanoate, the conversion of hexanoate to hexanoyl-CoA in these strains may be inefficient due to potential low expression levels of E. coli native fatty acyl-CoA synthetase(s). To evaluate the impact of the hexanoyl-CoA pool on olivetolic acid production, two different E. coli native fatty acyl- CoA synthetases were overexpressed individually. FadK has been reported as a fatty acyl-CoA synthetase which is primarily active on short chain fatty acids (C6-C8). However, we found that overexpression of FadK had no impact on olivetolic acid production under these conditions (FIG. 2). W e also explored another native E. coli fatty acyl-CoA synthetase, FadD, which has broad chain length specificity, with maximal activities associated with fatty acids ranging in length from C12 to C18. The overexpression of FadD in combination with OLS and OAC (strain BL-OLS-OAC -FadD) resulted in slight increases in olivetolic acid titer (FIG. 2). Combining this hexanoyl-CoA generating module with MCS, the highest olivetolic acid titer was achieved (0.71 mg/L) (FIG. 2).

[00135] Integration of the synthetic olivetolic acid pathway with a β-oxidation reversal for precursor supply. In contrast to the above approach which relied on native metabolite pools or exogenous acid addition for malonyl-CoA and hexanoyl-CoA supply, an alternative to further improve olivetolic acid production involves the integrated engineering of pathways leading to precursor synthesis. With malonyl-CoA generated directly from acetyl- CoA, the availability of this intermediate may play a critical role for olivetolic acid production. Furthermore, the role of acetyl-CoA becomes even more important when considering potential routes for generating hexanoyl-CoA. In prior studies, both fatty acid biosynthesis (FAB) and β-oxidation reversal (r-BOX) pathways have been employed for the production of hexanoic acid. However, the FAB pathway operates with acyl carrier protein (ACP) intermediates that are directly converted to carboxylic acid products through the expression of heterologous specific short-chain C6-ACP thioesterase. For conversion of hexanoic acid to hexanoyl-CoA, expression of a fatty acyl-CoA synthetase (or transferase), such as FadD, is required. Furthermore, the FAB pathway also requires malonyl-CoA as the extender unit during elongation resulting in increased competition for malonyl-CoA. In contrast, r-BOX operates with CoA intermediates, utilizes acetyl-CoA as extender unit and can directly generate hexanoyl-CoA. With this pathway initiating from acetyl-CoA and requiring an additional 2 acetyl-CoA molecules to generate hexanoyl-CoA, ensuring high intracellular levels of this acetyl-CoA intermediate are critical.

[00136] To this end, we sought to exploit an engineered strain (JST10 (DE3)) which has been previously utilized for hexanoic acid synthesis through r-BOX. In addition to containing chromosomal expression constructs for the required thiolase (BktB), β-ketoacyl-CoA reductase^-hydroxy acyl-CoA dehydratase (FadB), and enoyl-CoA reductase (egTER) r-BOX modules, this E. coli MG1655 derivative has fermentative product pathways (e.g. lactate, succinate, acetate and ethanol) and thioesterases (e.g. tesA and tesB among others) deleted to ensure adequate acetyl-CoA supply and minimize the loss of acyl-CoA intermediates. As such, this strain is a promising background strain for the expression of the synthetic olivetolic acid pathways (FIG. 3A). Furthermore, the increased acetyl-CoA supply in this strain may also provide a means of utilizing ACC, as opposed to MCS with exogenous malonate, for increasing malonyl-CoA availability.

[00137] Integration of r-BOX and the olivetolic acid biosynthesis pathway in JST10 (DE3) expressing OLS and OAC resulted in 2.8 mg/L olivetolic acid, nearly 30-fold higher than that in BL21 (DE3) (0.095 mg/L), even in the absence of hexanoate addition (FIG. 2). This result demonstrates the potential for olivetolic acid production from a principle carbon source (glycerol) through utilizing r-BOX for generating hexanoyl-CoA. To evaluate if hexanoyl-CoA supply was still a limiting factor in this strain, we also conducted experiments in which 4 mM hexanoate was supplied. Under these conditions, JST10-OLS-OAC produced ~2.3-fold higher olivetolic acid (6.5 mg/L) indicating that improving hexanoyl-CoA availability could potentially increase olivetolic acid production (FIG. 3B).

[00138] We then assessed the impact of malonyl-CoA supply on olivetolic acid production through the overexpression of auxiliary enzymes. Although MCS was identified as the most effective malonyl-CoA supply strategy for olivetolic acid production in BL21 (DE3), in JST10 (DE3) the overexpression of ACC resulted in the highest increase in olivetolic acid titer (FIG. 3B). Specifically, JSTlO-OLS-OAC-ACC produced 8.4 mg/L of olivetolic acid, 3-fold higher than JSTlO-OLS-OAC (2.8 mg/L) (P < 0.05). JSTlO-OLS-OAC-MCS (+12 mM sodium malonate) produced 3.6 mg/L of olivetolic acid, which is a 28% increase compared to JSTlO- OLS-OAC (P > 0.05) (FIG. 3B). This is likely caused by the distinct metabolic backgrounds of BL21 (DE3) and JST10 (DE3), as the deletion of acetyl-CoA competitive and consumption pathways in JST10 (DE3) is likely to result in increased availability of acetyl-CoA for malonyl- CoA generation. To confirm increased malonyl-CoA supply in this background, a heterologous malonyl-CoA availability indicator pathway was introduced. The polyketide synthase RppA from Streptomyces griseus, which iteratively condenses 5 molecules of malonyl-CoA to form flaviolin (which has a specific absorbance at the wavelength of 340 nm), was introduced into

JST10 (DE3) strains (FIG. 3C). While no significant increase in A34owas observed upon the combined overexpression of RppA and MCS (with 12 mM malonate supplementation) compared to RppA only, the overexpression of ACC with RppA lead to a significant increase in absorbance (FIG. 3D) (P < 0.05). These results provide further evidence that ACC overexpression is an effective strategy to increase malonyl-CoA supply in JST10 (DE3).

[00139] Given the impact of auxiliary enzymes and individually increasing hexanoyl-CoA and malonyl-CoA supply in JST10 (DE3), we next evaluated their combination in conjunction with the synthetic olivetolic acid pathway. As seen in FIG. 3B, additional hexanoate supplementation during fermentation with JSTlO-OLS-OAC-ACC resulted in a 2-fold increase in olivetolic acid titer to 16.6 mg/L (P < 0.05). Moreover, despite the overexpression of FadD having a negligible impact with hexanoate feeding and OLS/OAC overexpression, combined with ACC overexpression FadD significantly improved olivetolic acid titer (26.2 mg/L) (FIG. 3B). This indicates the importance of both hexanoyl-CoA and malonyl-CoA supply, as either can become the limiting factor as intracellular supply of each is increased. As such, coordinated increase in the supply of hexanoyl-CoA and malonyl-CoA is critical for producing olivetolic acid at high levels. While external addition to hexanoate was required here to increase titers, we also demonstrate a new application of r-BOX. Although prior studies of engineering of r- BOX primarily focused on production of short-chain fatty acids or alcohols, here we demonstrated that this pathway can also be employed for polyketide biosynthesis by supplying the starting CoA primer. [00140] Optimization of fermentation conditions for olivetolic acid production. Following the establishment of the best combination of enzymatic components for olivetolic acid production, we attempted to optimize the fermentation conditions for further titer improvement with strain JSTlO-OLS-OAC-FadD-ACC. This included evaluation of the impact of various temperatures, working volumes, and inducer concentrations on olivetolic acid production (FIG. 4). Results showed that 37°C was the optimal temperature for olivetolic acid production with a significant decrease in titer at both 30°C (17.6 mg/L) and 22°C (5.0 mg/L) (FIG. 4A). Based on the identified optimal temperature (37°C), we further studied the impact of working volume (WV, X mL in 25 mL flask, X/25 mL) as a means of altering aeration. We found that the engineered strain had the highest olivetolic acid titer of 26.8 mg/L at a WV of 15/25 mL (FIG. 4B). In addition to temperature and WV, we further investigated the impact of inducer concentrations (IPTG and cumate) on olivetolic acid production. In the engineered strain, genes encoding OLS, OAC, FadD, and ACC enzymes are expressed under the control of inducible T7 promoter, for which IPTG serves as inducer.

[00141] Excessive IPTG addition has been reported to be toxic to E. coli cells expressing genes under the control of IPTG-inducible T7 promoters and will cause inclusion body formation for excessive proteins biosynthesis, resulting in inhibition of enzymatic activities and thus decreased product biosynthesis. To this end, optimization of IPTG dosage for olivetolic acid production is desirable. Results showed that the JSTlO-OLS-OAC-FadD-ACC strain still produced 3.1 mg/L of olivetolic acid without addition of IPTG, likely due to leaky expression under the T7 promoter (FIG. 4C). Upon induction by IPTG, olivetolic acid production increased significantly and a positive correlation was observed between IPTG dosage and olivetolic acid titer up to 100 µΜ . With 100 pM IPTG, the JSTlO-OLS-OAC- FadD-ACC strain produced 34.8 mg/L of olivetolic acid, which is 30% higher than the best titers achieved by using 50 pM IPTG. However, excessive dosage of IPTG (>100 pM) was found to decrease olivetolic acid production.

[00142] We also optimized the dosage effect of the inducer cumate, which activates the expression of enzymes in r-BOX by binding with CymR repressor. Results showed that, the JSTlO-OLS-OAC-FadD-ACC strain produced the highest level of olivetolic acid at 46.3 mg/L when cumate was added at 10 pM (FIG. 4D).

[00143] Olivetolic acid fermentation in bioreactor under controlled conditions. In order to obtain higher olivetolic acid titer, a batch fermentation with precise parameter control was conducted using the engineered strain JSTlO-OLS-OAC-FadD-ACC and the identified optimal fermentation conditions. Under these conditions, cell growth of JSTlO-OLS-OAC-

FadD-ACC reached the highest OD550 of 8 (corresponding cell mass is approximately 2.64 g/L) at 48 h . During the first 24 h, JSTlO-OLS-OAC-FadD-ACC consumed a total of -19 g/L of glycerol, 76 mg/L of hexanoate and produced 75.6 mg/L of olivetolic acid (FIG. 5A). During the first 24 hours, the average cell density was 0.34 grams dry cell weight per liter resulting in a productivity of 9.3 mg olivetolic acid per g dry cells per hour. This is the highest olivetolic acid titer and productivity achieved by fermentation of any wild type or engineered microbe.

[00144] However, we also observed that with the increase of fermentation time, olivetolic acid titer decreased. Specifically, from 24 h to 48 h olivetolic acid concentration decreased by about 50% to 37 mg/L. Two potential explanations were speculated for this observed decrease:

1) olivetolic acid is inherently unstable and will degrade spontaneously in aqueous environments; 2) olivetolic acid can be metabolized by E. coli cells. We further analyzed the olivetolic acid changes in the absence and presence of wild type E. coli cells (FIG. 5B) and determined that even in the absence of E. coli MG1655 (DE3), olivetolic acid levels decreased over time, with the majority of olivetolic acid decarboxyl ated to olivetol. In the presence of E. coli cells, although a portion was still observed to form olivetol, olivetolic acid was degraded more than in the absence of cells, indicating that at least a fraction of the olivetolic acid was metabolized by E. coli. Conversely, to further determine whether olivetol can spontaneously convert to olivetolic acid or be metabolized by E. coli cells, we conducted the similar experiments with olivetol. Results showed that olivetol can neither spontaneously convert to olivetolic acid nor be metabolized by E. coli cells under our experimental conditions (FIG. 5B). Olivetol seems more stable than olivetolic acid in aqueous environments, which might be related to the absence of carboxyl group.

[00145] Despite the structural complexity of type III polyketides, the tractable starting units required for their synthesis enables a synthetic approach for their production in which PKS and cyclase components can be integrated with pathways for the generation of priming and extending units. Here, functional expression and characterization of the Cannabis sativa olivetolic acid synthase and cyclase enzymes confirmed their requirement for the synthesis of olivetolic acid from hexanoyl-CoA and malonyl-CoA. Through the direct integration of OLS and OAC with modules of the β-oxidation reversal aimed at generating hexanoyl-CoA, we demonstrate the synthesis of the plant natural product olivetlic acid in engineered E. coli from a principle carbon source. By further combining these pathways with auxiliary enzymes for additional hexanoyl-CoA and malonyl-CoA generation, we also identified the supply of these precursors as a key limiting factor in olivetolic acid synthesis. Through combinational utilization these auxiliary enzymes and optimization of fermentation conditions, we achieved olivetolic acid titer of 75 mg/L and a productivity of 9.3 mg/g-hr. This represents the first report of olivetolic acid synthesis in E. coli, and further demonstrates the potential for microbial cell factories to overcome the limitations of direct plant extraction or chemical synthesis to produce plant-based natural products.

[00146] In vitro synthesis of divarin and divarinolic acid. The purpose of this experiment was to clone, express and purify OLS and OAC and test their activity for in vitro synthesis of divarinolic acid through OLS-mediated decarboxylative Claisen condensation and OAC-mediated cyclization. Butyryl-CoA is used as the primer, and malonyl-CoA serves as the extender unit.

[00147] Olivetol synthase (OLS) catalyzes three sequential decarboxylative Claisen condensation reactions with butyryl-CoA as the initial primer and malonyl-CoA as the extender unit. The first reaction condenses butyryl-CoA and malonyl-CoA to 3-oxohexanoyl-CoA, the second reaction condenses 3-oxohexanoyl-CoA and malonyl-CoA to a diketoacyl-CoA 3,5- dioxooctanoyl-CoA. The third reaction condenses 3,5-dioxooctanoyl-CoA and malonyl-CoA to a triketoacyl-CoA 3,5,7-trioxodecanoyl-CoA. Olivetolic acid cyclase OAC converts 3,5,7- trioxodecanoyl-CoA to divarinolic acid. Divarin can be formed by either spontaneous cyclization of 3,5,7-trioxodecanoyl-CoA in the absence of OAC or by decarboxylation of divarinolic acid. The divarinolic acid /divarin synthesis reaction is shown in FIG. 6 .

[00148] Genes encoding OLS and OAC were codon optimized and synthesized by GeneArt (Life Technologies, Carlsbad, CA, USA). These genes were then amplified by PCR using primers to append homology on each end for recombination into the pETduetl vector backbone (Novagen) with Phusion polymerase (Thermo Scientific, Waltham, MA). 6*His tag was fused at the N-terminal of each OLS or OAC. Plasmids were linearized by the appropriate restriction enzymes (New England Biolabs, Ipswich, MA, USA) and recombined with the gene inserts using the In-Fusion HD Eco-Dry Cloning system (Clontech laboratories, Mountain View, CA, USA). The mixture was subsequently transformed into Stellar competent cells (Clontech laboratories, Mountain View, CA, USA). Transformants that grew on solid media (LB+Agar) supplemented with the appropriate antibiotic were isolated and screened for the gene insert by PCR. Plasmids from verified transformants were isolated and the sequence of the gene insert was further confirmed by sequencing (Lone Star Labs, Houston, TX). The sequence-confirmed plasmids were introduced to BL2l(DE3) (Studier et al. 1986).

[00149] Primers used for genetic cloning in this example are shown in Table 8 below:

[00150] The codon-optimized OLS gene insert was PCR amplified with OLS-pET-For and OLS-pET-Rev primers and inserted into vector pETDuet-l (Novagen, Darmstadt, Germany) amplified by pET-For and pET-Rev primers through In-Fusion HD Eco-Dry Cloning system (Clontech laboratories, Mountain View, CA) to construct pET-ntH6-ols. The sequence of the OLS gene insert was further confirmed by sequencing (Lone Star Labs, Houston, TX) with usage of pET-seq-up and pET-seq-dn sequencing primers. The protein was expressed with an N-terminal 6 His-tag.

[00151] The codon-optimized OAC gene insert was PCR amplified with OAC-pET-For and OAC-pET-Rev primers and inserted into vector pETDuet-l (Novagen, Darmstadt, Germany) amplified by pET-For and pET-Rev primers through In-Fusion HD Eco-Dry Cloning system (Clontech laboratories, Mountain View, CA) to construct pET-ntH6-oac. The sequence of the OAC gene insert was further confirmed by sequencing (Lone Star Labs, Houston, TX) with usage of pET-seq-up and pET-seq-dn sequencing primers. The protein was expressed with an N-terminal 6 His-tag.

[00152] For expression of OLS/OAC, cultures were grown in 50 mL of LB media in 250 mL flasks (Wheaton Industries, Inc., Millville, NJ) at 37°C. A single colony of the desired strain was cultivated overnight (14-16 hrs) in 10 mL of LB medium in baffled flasks (Wheaton

Industries, Inc., Millville, NJ) with appropriate antibiotics and used as the inoculum ( 1 mL). The cells were induced with 0.1 mM IPTG at an OD550 ~ 0.6. After post-induction growth at 37°C for 4 h, the cells were collected and washed twice by 9 g/L sodium chloride solution. Cells were then re-suspended in lysis buffer (50 mM Na PC , 300 mM NaCl, 10 mM imidazole, pH 8.0) to an OD ~40. After re-suspension, the cells were disrupted using glass beads and then centrifuged at 4°C, 13000 g, 10 min in an Optima L-80XP Ultracentrifuge (Beckman-Coulter, Schaumburg, IL). The resultant supernatant is the crude enzyme extract.

[00153] The His-tagged enzymes were then purified from crude extract by using Ni-NTA spin kit (Qiagen, Valencia, CA). The crude extracts are centrifuged (270 g, 5 min) in spin columns which have been equilibrated with lysis buffer and then washed twice by wash buffer (50 mM NaH2P04, 300 mM NaCl, 20 mM imidazole, pH 8.0). After washing, the enzyme is eluted twice in elution buffer (50 mM NaH2P04, 300 mM NaCl, 500 mM imidazole, pH 8.0). Both washing and elution steps were by centrifugation at 890 g for 2 min. The purified enzyme extracts were then further concentrated and dialyzed through Amicon® Ultra 10K Device (Millipore, Billerica, MA). The enzymes were first filtered by centrifugation at 4°C, 14000 g, 10 min, and then washed with 100 mM potassium phosphate, pH 7 buffer under the same centrifugation conditions. Finally, the concentrated and dialyzed enzymes were recovered by 4°C, 1000 g, 2 min centrifugation.

[00154] The protein concentration was established using the Bradford Reagent (Thermo Scientific, Waltham, MA) using BSA as the protein standard. SDS-PAGE monitor of purified proteins was performed through XCell SureLockTM Mini-cell system (Invitrogen, Carlsbad, CA) with gels (12% acrylamide resolving gel and 4% acrylamide stacking gel) prepared through SureLockTM Mini-cell system (Invitrogen, Carlsbad, CA). The composition of the running buffer for SDS-PAGE was 3 g/L tris base, 14.4 g/L glycine and 1 g/L SDS in water.

[00155] Enzymatic assays for the formation of divarinolic acid through OLS condensation between butyryl-CoA and malonyl-CoA and subsequent OAC cyclization was performed. Enzyme assays were performed in a 500 L total reaction volume containing 100 mM potassium phosphate buffer (pH 7.0), 200 pM hexanoyl-CoA, 400 pM malonyl-CoA, 10 pg OLS, and 30 pg OAC (when included). The reaction mixture was incubated at 20 °C for 16 h and 20 pL sulfuric acid (H2S04) was added to terminate the reaction.

[00156] Quantification of divarinolic acid was conducted via GC-FID analysis using an Agilent 7890 B gas chromatograph equipped with an Agilent 5977 mass spectroscope detector (Agilent) and an HP-5 ms capillary column (0.25 mm internal diameter, 0.25 pm film thickness, 30 m length; Agilent). Sample preparation was conducted as follows: 2 mL culture samples were transferred to 5 mL glass vials (Fisher Scientific), 4-pentylbenzoic acid (final concentration 50 mg/L) was added as internal standard. Then 80 pL of H2SO4 and 340 pL of 30 % (wt/v) NaCl solution were added for pH and ionic strength adjustment. Two milliliters of hexane was added for extraction. Vials were sealed with Teflon-lined septa (Fisher Scientific), secured with caps, and rotated at 60 rpm for 2 h . The samples were then centrifuged for 2 min at 6500 rpm to separate the aqueous and organic layers. After centrifugation, 1.5 mL of the top organic layer was transferred to new 5 mL glass vial and evaporated under a stream of nitrogen. Then, 100 L pyridine and 100 pL of N,0-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) were added to the dried extract for derivatization at 70 °C for 1 h . After cooling to room temperature, 200 pL of derivatization product was transferred to vials (Fisher Scientific) for GC-MS analysis according to the following method: 1 pL were injected into the GC, which was run in splitless mode using helium gas as a carrier gas with a flow rate of 1 mL/min. The injector temperature was 280 °C and the oven temperature was initially held at 50 °C for 3 min and then raised to 250 °C at 10 °C/min and held for 3 min.

[00157] The SDS-PAGE gel of purified OLS and OAC in the assay of in vitro divarinolic acid synthesis, is shown in FIG. 7A. As shown in FIG. 7B, in the presence of only OLS, only divarin can be formed, and the MS identification information of divarin can be seen in FIG. 7C. This result confirmed that the intermediate 3,5,7-trioxodecanoyl-CoA can cyclize spontaneously and be decarboxyl ated to yield divarin. In the presence of both OLS and OAC (FIG. 7A), both divarin and divarinolic acid can be formed (FIG. 7B), and the M S identification result can be seen in FIG. 7C, which demonstrates that OAC is indispensable for the C2 C7 non-decarboxylative aldol cyclization reaction of 3,5,7-trioxodecanoyl-CoA to yield divarinolic acid.

[00158] (Prophetic) in vivo synthesis of divarinolic acid and divarin. The purpose of this experiment is to clone and express olivetol synthase (OLS) (BAG14339.1) along with olivetolic acid cyclase OAC (AFN42527. 1) from Cannabis sativa in an Escherichia coli strain already overexpressing acetyl-CoA acetyltransferase AtoB from E. coli (NP_4 16728.1), 3- hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase multifunctional enzyme FadB from E. coli (NP 418288.1) and enoyl-CoA reductase Ter from Euglena gracilis (abbreviated egTER) (Q5EU90.1) for in vivo microbial synthesis of divarinolic acid.

[00159] Olivetol synthase (OLS) catalyzes three sequential decarboxyl ative Claisen condensation reactions with butyryl-CoA as the initial primer and malonyl-CoA as the extender unit. The first reaction condenses butyryl-CoA and malonyl-CoA to 3-oxohexanoyl-CoA, the second reaction condenses 3-oxohexanoyl-CoA and malonyl-CoA to a diketoacyl-CoA 3,5- dioxooctanoyl-CoA, the third reaction condenses 3,5-dioxooctanoyl-CoA and malonyl-CoA to a triketoacyl-CoA 3,5,7-trioxodecanoyl-CoA. Olivetolic acid cyclase OAC converts 3,5,7- trioxodecanoyl-CoA to divarinolic acid. Divarin can be formed by either spontaneous cyclization of 3,5,7-trioxodecanoyl-CoA in the absence of OAC or decarboxylation of divarinolic acid.

[00160] AtoB catalyzes the non-decarboxylative Claisen condensation reaction between acetyl-CoA and acetyl-CoA to supply 3-oxobutyryl-CoA (or acetoacetyl-CoA), which was subsequently catalyzed by FadB and egTER in the β-oxidation reversal pathway to form butyryl-CoA, serving as the primer for OLS catalysis. Acetyl-CoA is supplied through glycolysis from a carbon source such as glycerol or sugars. Malonyl-CoA is supplied through carboxylation of acetyl-CoA by E. coli native acetyl-CoA carboxylase complex ACC. Alternatively, malonyl-CoA can be produce from malonate, CoA, and ATP using malonyl- CoA synthetase (not shown). This pathway for divarinolic acid synthesis is shown in FIG. 8 .

[00161] JST06 (DE3) atoBCT5 fadBCT5 AfadA egterCTS @fabl serves as the host strain for the in vivo production of divarinolic acid. JST06 (DE3) (MG1655 (DE3) AldhA ApoxB Apia AadhE AfrdA AyciA AybgC Aydil AtesA AfadM AtesB) (Cheong et al. 2016) is an E. coli strain deficient in mixed-acid fermentation pathways due to deletions of genes IdhA, poxB, pta, adhE and f rdA which maximize the supply of acetyl-CoA, and deletion of genes encoding major thioesterases (yciA, ybgC, ydil, tesA,fadM and tesB), which minimize the hydrolysis of intermediate acyl-CoAs. As such, this strain is selected to maximize the flux of β-oxidation reversal for butyryl-CoA supply required for the synthesis of divarinolic acid via olivetol synthase OLS and olivetolic acid cyclase OAC.

[00162] AtoB, FadB and egTER are chromosomally expressed under pCT5 promoter with control by cumate. E. coli atoB and fadB genes were PCR amplified, digested with BglII and Notl, and ligated by T4 ligase (Invitrogen, Carlsbad, CA) into pUCBB-PCT5-ntH6-eGFP that was previously digested with BglII and Notl to produce pUCBB-PCT5-atoB and pUCBB- PCT5-fadB. The resulting ligation products were used to transform E. coli DH5a (Invitrogen, Carlsbad, CA), and positive clones identified by PCR were confirmed by DNA sequencing. To integrate the cumate-controlled atoB andfadB constructs into the chromosome, first the cumate repressor (cymR), promoter/operator regions (PCT5), and respective ORFs were PCR amplified, as were kanamycin and chloramphenicol drug constructs (via pKD4 and pKD3, respectively). These respective products were linked together via overlap extension PCR to create a final chromosomal targeting construct. Integration of the cumate-controlled constructs was achieved via standard recombineering protocols by using strain HME45 and selection on LB drug plates.

[00163] Construction of the strain serving as the PCR template for egTER was accomplished by first creating a kan-sacB fusion cassette via overlap extension PCR using pKD4 and genomic DNA, respectively. This kan-sacB cassette was integrated between fadB andfadA of the fadBACT5 strain formerly constructed (Vick et a , 2014) through subsequent recombineering. Seamless replacement of the kan-sacB cassette to create the cat-cymR-PCT5- egTER at the fadBA locus was done via recombineering and subsequent sucrose selection with codon optimized egter (Genscript, Piscataway, NJ) PCR product.

[00164] The fadA gene was separately deleted via recombineering in the HME45 derivative harboring the cumate-controlled fadBA construct by replacement of the fadA ORF with a zeocin resistance marker amplified from pKDzeo (Magner et al. 2007). For the creation of the cumate-controlled egTER, the cat gene, cymR repressor gene, hybrid cumate-controlled phage T5 promoter, and egTER gene are PCR amplified from genomic DNA of a strain with egTER seamlessly replacing fadBA at the cumate controlled fadBA locus (see below for details). This product is recombineered into strain HME45 at the end of thefabl locus, selecting on chloramphenicol (12.5 pg/ml) LB plates. Integration is done in a manner to duplicate the last 22 bp offabl (including stop codon) so as to retain an overlapping promoter for the next native downstream gene.

[00165] Codon-optimized genes encoding OLS were cloned together with the codon- optimized gene encoding OAC into appropriate vectors. These genes are amplified through PCR using appropriate primers to append homology on each end for recombination into the vector backbone with Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as the gene insert. Cloning and isolation of confirmed plasmids are conducted as described above.

[00166] MOPS minimal medium (Neidhardt et al., 1974) with 125 mM MOPS and Na2FfP04 in place of K2ITP04 (2.8 mM), supplemented with 10 g/L tryptone, 5 g/L yeast extract, 100 µΜ FeS04, 5 mM calcium pantothenate, 5 mM (NH4)2S04, and 30 mM NH4C1 is used for fermentations. Antibiotics (50 pg/mL carbenicillin and 50 pg/mL spectinomycin) were included when appropriate. All chemicals are obtained from Fisher Scientific Co. (Pittsburg, PA) and Sigma-Aldrich Co. (St. Louis, MO).

[00167] Fermentations are conducted in a SixFors multi-fermentation system (Infors HT, Bottmingen, Switzerland) with an air flowrate of 2 N L/hr, independent control of temperature (37°C), pH (controlled at 7.0 with NaOH and H2S04), and stirrer speed (660 rpm). The above fermentation media with 50 g/L glycerol, the inclusion of 5 µΜ sodium selenite, and 1 µΜ IPTG are used. Pre-cultures are grown as described above and incubated for 4 hours post- induction. An appropriate amount of this pre-culture is centrifuged, washed twice with fresh media, and used for inoculation with a target initial optical density of 0.05-0.1 (400 mL initial volume).

[00168] At various fermentation times samples are taken and 2 mL supernatant is collected through 5000 g, 5 min centrifuge in an Optima L-80XP Ultracentrifuge (Beckman-Coulter, Schaumburg, IL) of culture and is prepared for GC-FID analysis.

[00169] The supernatant aliquots of 2 mL are transferred to 5 mL glass vials (Fisher Scientific Co., Pittsburgh, PA) and extraction and derivatization with BSTFA conducted as described above. The quantification of divarinolic acid and divarin are performed in a Varian CP-3800 gas chromatograph (Varian Associates, Inc., Palo Alto, CA), equipped with a flame ionization detector (GC-FID) and an HP-INNOWax capillary column (0.32 mm internal diameter, 0.50 pm film thickness, 30 m length; Agilent Technologies, Inc., Santa Clara, CA), following the method: 100 °C initial column temperature, l5°C/min to 300°C, and 300°C held for 8 min. Helium ( 1 mL/min, Matheson Tri-Gas, Longmont, CO) is used as the carrier gas. The injector and detector are maintained at 280 and 300°C, respectively. A 1 L sample is injected in splitless injection mode.

[00170] (Prophetic) in vivo synthesis of orsellinic acid and orcinol. The purpose of this experiment is to clone and express olivetol synthase (OLS) (BAG14339.1) along with olivetolic acid cyclase OAC (AFN42527. 1) from Cannabis sativa in an Escherichia coli strain for in vivo microbial synthesis of orsellinic acid.

[00171] Olivetol synthase (OLS) catalyzes three sequential decarboxylative Claisen condensation reactions with acetyl-CoA as the initial primer and malonyl-CoA as the extender unit. The first reaction condenses acetyl-CoA and malonyl-CoA to 3-oxobutyryl-CoA (or acetoaceryl-CoA), the second reaction condenses 3-oxobutyryl-CoA and malonyl-CoA to a diketoacyl-CoA 3,5-dioxohexanoyl-CoA. The third reaction condenses 3,5-dioxohexanoyl- CoA and malonyl-CoA to a triketoacyl-CoA 3,5,7-trioxooctanoyl-CoA. Orcinol can be formed by either spontaneous cyclization of 3,5,7-trioxooctanoyl-CoA in the absence of OAC or decarboxylation of orsellinic acid. [00172] Olivetolic acid cyclase OAC converts 3,5,7-trioxooctanoyl-CoA to orsellinic acid. Acetyl-CoA is supplied through glycolysis from a carbon source such as glycerol or sugars. Malonyl-CoA is supplied through carboxylation of acetyl-CoA by E. coli native acetyl- CoA carboxylase (ACC). Alternatively, malonyl-CoA can be produce from malonate, CoA, and ATP using malonyl-CoA synthetase (not shown). This pathway for orsellinic acid synthesis is shown in FIG. 9 .

[00173] JC01 (DE3) serves as the host strain for the in vivo production of orsellinic acid. E. coli JC0l(DE3) (MGl655(DE3) AldhA ApoxB Apta AadhE AfrdA (Cheong et al. 2016) is an E. coli strain deficient in mixed-acid fermentation pathways due to deletions of genes IdhA, ροχΒ,ρ ία, adhE andfrdA , which maximize the supply of acetyl-CoA and malonyl-CoA for the synthesis of orsellinic acid via olivetol synthase OLS and olivetolic acid cyclase OAC.

[00174] The codon-optimized gene encoding OLS is cloned together with the codon- optimized gene encoding OAC into appropriate vectors. These genes are amplified through PCR using appropriate primers to append homology on each end for recombination into the vector backbone with Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as the gene insert. Cloning and isolation of confirmed plasmids are conducted as described above, MOPS minimal medium used for orsellinic acid production is prepared as described above, Fermentations for orsellinic acid production are conducted as described above, and qualitative and quantitative analysis (GC-FID) of orsellinic acid fermentation samples are conducted as described above.

[00175] (Prophetic) in vivo synthesis of cannabigerolic acid. The purpose of this experiment is to clone and express a prenyltransferase in an Escherichia coli strain that can produce geranyl pyrophosphate (GPP) and olivetolic acid for in vivo microbial synthesis of cannabigerolic acid. By way of example NphB from Streptomyces sp. (BAE00106.1) is the prenyltranferase used. However, any prenyltransferase capable of prenylating olivetolic acid may be used.

[00176] NphB catalyzes prenylation of olivetolic acid, leading to 3-geranyl olivetolic acid (or cannabigerolic acid, CBGA). This pathway for cannabigerolic acid synthesis is shown in FIG. 10

[00177] As described above, the olivetolic acid is generated via three sequential decarboxylative Claisen condensation reactions with hexanoyl-CoA as the initial primer and malonyl-CoA as the extender unit catalyzed by OLS. The triketoacyl-CoA 3,5,7- trioxododecanoyl-CoA will be catalyzed by olivetolic acid cyclase OAC to yield olivetolic acid.

[00178] The GPP is generated from condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) catalyzed by GPP synthase. The precursor of IPP and DMAPP can be supplied either from 2-methyl-(D)-erythritol-4-phosphate (MEP) pathway (or DXP pathway) or mevalonate pathway (or isoprenoid pathway or HMG-CoA reductase pathway) or via a novel pathway from prenol or isoprenol to GPP.

[00179] The MEP pathway starts from condensation of pyruvate and glyceraldehyde 3- phosphate catalyzed by DXP synthase (DXS) to form l-deoxy-D-xylulose5-phosphate (DXP). Then the DXP will be catalyzed sequentially by DXP reductoisom erase (DXR), 2-C-methyl- D-erythritol 4-phosphate cytidylyltransferase (IspD), 4-diphosphocytidyl-2-C-methyl-D- erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), HMB- PP synthase (IspG), HMB-PP reductase (IspH), and finally one IPP and one DMAPP are formed.

[00180] The MVA pathway starts from condensation of 2 acetyl-CoA catalyzed by acetoacetyl-CoA thiolase (AtoB) to form HMG-CoA. Then the HMG-CoA will be catalyzed sequentially by HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR) to yield mevalonate, these first 3 enzymatic steps are called the upper mevalonate pathway. The lower mevalonate pathway which converts mevalonate into IPP and DMAPP has 3 variants. In eukaryotes, mevalonate is phosphorylated twice in the 5-OH position by mevalonate-5-kinase (M5K) and phosphomevalonate kinase (PMK), then decarboxylated by mevalonate-5- pyrophosphate decarboxylase (MVD) to yield IPP. In some archaea such as Haloferax volcanii, mevalonate is phosphorylated once in the 5-OH position by mevalonate-5-kinase (M5K), decarboxylated by mevalonate-5-pyrophosphate decarboxylase to yield isopentenyl phosphate (IP), and finally phosphorylated again by isopentenyl phosphate kinase to yield IPP (Archaeal Mevalonate Pathway I). A third mevalonate pathway variant found in Thermoplasma acidophilum, phosphorylates mevalonate at the 3-OH position by mevalonate-3 -kinase (M3K) and mevalonate-3-phosphate-5-kinase, followed by phosphorylation at the 5-OH position. The resulting metabolite, mevalonate-3, 5-bisphosphate, is decarboxylated to IP, and finally phosphorylated to yield IPP (Archaeal Mevalonate Pathway II).

[00181] The prenol pathway starts from non-decarboxylative Claisen condensation between two acetyl-CoAs to acetoacetyl-CoA catalyzed by E. coli thiolase AtoB (NP_4 16728.1). Then, S. aureus 3-hydroxy-3 -methylglutaryl-CoA synthase HMGS (BAU36102.1) condenses acetoacetyl-CoA with another acetyl-CoA to generate 3-hydroxy-3- methylglutaryl-CoA (HMG-CoA). HMG-CoA is dehydrated to 3-methylglutaconyl-CoA by M. xanthus enoyl-CoA hydratase LiuC (WP_0l 1553770.1). M. xanthus glutaconyl-CoA decarboxylase AibAB (WP_0l 1554267.1, WP_0 11554268.1) decarboxylates 3- methylglutaconyl-CoA to 3-methylcrotonyl-CoA. 3-Methylcrotonyl-CoA is converted to prenol by alcohol-forming acyl-CoA reductase or aldehyde forming acyl-CoA reductase and alcohol dehydrogenase or carboxylate reductase and the hydrolysis enzyme selected from the group consisting thioesterase, acyl-CoA synthase, acyl-CoA transferase and carboxylate kinase plus phosphotransacylase. Alcohol-forming acyl-CoA reductase is selected from the group consisting C. acetobutylicum AdhE2 (YP_009076789.l) and aquaeolei VT8 Maqu_2507 (YP 959769.1). CbjALD from C. beijerinckii aldehyde forming acyl-CoA reductase (AAT66436.1) is selected for conversion of 3-methylcrotonyl-CoA to prenol. Alcohol dehydrogenase is selected from the group consisting E. coli YahK (NP 414859.1), E. coli YjgB (NP 418690.4) and Acinetobacter sp. SE19 ChnD (BAC80217.1). Prenol produced in this manner or alternatively supplied exogenously is then converted to DMAPP by one or two steps of phosphorylation. The first step is catalyzed by E. coli hydroxyethylthiazole kinase ThiM (WP 001 195564.1) to yield dimethylallyl phosphate, and the second step is catalyzed by M. thermautotrophicus phosphate kinase MtIPK (AAB84554.1). E. coli isopentenyl

pyrophosphate isomerase Idi (NP 417365. 1) converts DMAPP to IPP. Then, DMAPP and IPP are condensed to GPP catalyzed by E. coli GPP synthase IspA (NP 414955.1, S80F) or A. grandis GPP synthase GPPS2 (AANO 1134.1, N-terminal 84 aa is truncated). The prenol pathway is described in W02017161041 MICROBIAL SYNTHESIS OF ISOPRENOID PRECURSORS, ISOPRENOIDS AND DERIVATIVES INCLUDING PRENYLATED AROMATICS COMPOUNDS, and is incorporated by reference in its entirety for all purposes.

[00182] Alternatively, isoprenol can be produced endogenously or supplied exogenously and then converted to IPP by one or two steps of phosphorylation. The first step is catalyzed by E. coli hydroxyethylthiazole kinase ThiM (WP 001 195564.1) to yield isopentenyl phosphate, and the second step is catalyzed by M thermautotrophicus phosphate kinase MtIPK (AAB84554.1). E. coli isopentenyl pyrophosphate isomerase Idi (NP 417365.1) converts IP to DMAPP. Then, DMAPP and IPP are condensed to GPP catalyzed by E. coli GPP synthase IspA (NP_4l4955.l, S80F) or A. grandis GPP synthase GPPS2 (AAN01 134.1, N-terminal 84 aa is truncated). The isoprenol pathway is described in W02017161041 MICROBIAL SYNTHESIS OF ISOPRENOID PRECURSORS, ISOPRENOIDS AND DERIVATIVES INCLUDING PRENYLATED AROMATICS COMPOUNDS, and is included herein by reference.

[00183] Codon-optimized gene encoding these NphB is cloned into appropriate vectors. The gene is amplified through PCR using appropriate primers to append homology on each end for recombination into the vector backbone with Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as the gene insert. Cloning and isolation of confirmed plasmids are conducted as described above, MOPS minimal medium used for cannabigerolic acid production is prepared as described above, fermentations for cannabigerolic acid production are conducted as described above, and analysis (GC-FID) of cannabigerolic acid fermentation samples is also as described above.

[00184] (Prophetic) in vivo synthesis of cannabidiolic acid. The purpose of this experiment is to clone and express cannabidiolic acid synthase (CBDAS, AKC34419.1) from Cannabis sativa in an Escherichia coli strain that can produce cannabigerolic acid (CBGA) for in vivo microbial synthesis of cannabidiolic acid (CBDA).

[00185] CBDAS catalyzes the oxidative cyclization of CBGA into CBDA. This in vivo pathway for CBDA synthesis is shown in FIG. 11.

[00186] Codon-optimized gene encoding the CBDAS is cloned into appropriate vectors. The gene is amplified through PCR using appropriate primers to append homology on each end for recombination into the vector backbone with Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as the gene insert. Experiments are conducted largely as described above.

[00187] Each of the following references is incorporated by reference in its entirety for all purposes:

[00188] WO20 17020043 BIOSYNTHESIS OF POLYKETIDES

[00189] W02017161041 MICROBIAL SYNTHESIS OF ISOPRENOID PRECURSORS, ISOPRENOIDS AND DERIVATIVES INCLUDING PRENYLATED AROMATICS COMPOUNDS

[00190] W02012109176 REVERSE BETA OXIDATION PATHWAY [00191] US201402731 10 FUNCTIONALIZED CARBOXYLIC ACIDS AND ALCOHOLS BY REVERSE FATTY ACID OXIDATION

[00192] US20 160340699 TYPE II FATTY ACID SYNTHESIS ENZYMES IN REVERSE B-OXIDATION

[00193] US20130067619 GENES AND PROTEINS FOR AROMATIC POLYKETIDE SYNTHESIS

[00194] US20140141476 GENES AND PROTEINS FOR ALKANOYL-COA SYNTHESIS

[00195] US20160010126 PRODUCTION OF CANNABINOIDS IN YEAST

[00196] Gagne, S. J., Stout, J . M., Liu, E., Boubakir, Z., Clark, S. M., Page, J . E., 2012. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proc Natl Acad Sci U S A . 109, 1281 1-6.

[00197] Kim, S., Clomburg, J . M., Gonzalez, R., 2015. Synthesis of medium-chain length (C6-C10) fuels and chemicals via beta-oxidation reversal in Escherichia coli. J Ind Microbiol Biotechnol. 42, 465-75.

[00198] Taura, F., Tanaka, S., Taguchi, C., Fukamizu, T., Tanaka, H., Shoyama, Y., Morimoto, S., 2009. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway. FEBS Lett. 583, 2061-6. WE CLAIM

1) A recombinant prokaryotic microorganism, said microorganism expressing a heterologous gene for an olivetolic acid synthase and a heterologous gene for an olivetolic acid cyclase. 2) A recombinant microorganism, said microorganism expressing i) a heterologous gene for an olivetolic acid synthase and ii) a heterologous gene for an olivetolic acid cyclase, and further expressing (a) a heterologous gene(s) encoding acetyl-CoA carboxylase or (b) a heterologous gene encoding malonyl-CoA synthetase or both (a) and (b). 3) The microorganism of claims 1-2, further expressing a heterologous gene encoding a fatty acyl-CoA synthetase or a fatty acyl-CoA transferase capable of converting a fatty acid to a fatty acyl-CoA. 4) The microorganism of claim 3, wherein the further expressed heterologous gene encodes a fatty acyl-CoA synthetase. 5) The microorganism of claim 4, wherein the heterologous gene encoding a fatty acyl-CoA synthetase is the Escherichia colifadD. 6) The recombinant microorganism of claims 1-5, said microorganism further expressing (a) heterologous gene(s) for one of more of the following enzymes:

a) thiolase capable of catalyzing the conversion of a C -acyl-CoA to a C +2 β-ketoacyl- CoA; b) hydroxyacyl-CoA dehydrogenase capable of catalyzing the conversion of β-ketoacyl- CoA to β-hydroxyacyl-CoA; c) enoyl-CoA hydratase capable of catalyzing the conversion of β-hydroxyacyl-CoA to enoyl-CoA; or d) acyl-CoA dehydrogenase or enoyl-CoA reductase capable of catalyzing the conversion of enoyl-CoA to acyl-CoA. 7) The microorganism of claims 1-6, wherein one or more of said heterologous genes are from Cannabis sativa. 8) The microorganism of claims 1-7, further expressing a heterologous gene encoding a polyketide prenyltransferase. 9) The microorganism of claim 8, wherein said heterologous gene encoding said polyketide prenyltransferase enzyme is from Cannabis sativa or Streptomyces. 10) The microorganism of claims 8-9, further expressing (a) heterologous gene(s) for one of more of the following enzymes: a) prenol kinase; b) isprenol kinase; c) DMAP kinase; d) IP kinase; e) isopentenyl diphosphate isomerase; or f) geranyl diphosphate (GPP) synthase. 11) The microorganism of claims 8-10, further expressing one or more heterologous genes encoding (a) cannabinoid synthase enzyme(s). 12) The microorganism of claim 11, wherein said heterologous gene(s) encode(s) (a) cannabinoid synthase enzyme(s) that is(are) selected from cannabidiol synthase, cannabinodivarin synthase, cannabidiorcol synthase, cannabidiolic acid synthase, cannabinodivarinic acid synthase, cannabidiorcolic acid synthase, cannabichromene synthase, cannabichromevarin synthase, cannabichromenorcol synthase, cannabichromenic acid synthase, cannabichromevarinic acid synthase, cannabichromenorcolic acid synthase, tetrahydrocannabinol synthase, tetrahydrocannabivarin synthase, tetrahydrocannabiorcol synthase, tetrahydrocannabinolic acid synthase, tetrahydrocannabivarinic acid synthase, and tetrahydrocannabiorcolic acid synthase. 13) The microorganism of claims 11-12, wherein one or more of said heterologous gene(s) encoding (a) cannabinoid synthase enzyme(s) is(are) from Cannabis sativa. 14) A method of producing a compound, said method comprising: a) inoculating the recombinant microorganism of claims 1-7 to a culture medium; b) growing said microorganism in said culture medium under conditions where said heterologous genes are expressed for a time sufficient to produce one or more compounds selected from olivetol, divarin, orcinol, olivetolic acid, divarinolic acid, and orsellinic acid; and c) optionally isolating said one or more compounds from said microorganism or the culture medium or both. 15) The method of claim 14, wherein said culture medium comprises one or more of added malonate, butanoate, or hexanoate.

16) The method of claims 14-15, wherein olivetolic acid is produced at a concentration greater than about 40 mg/L or at a rate greater than 0.5 mg/hr-g.

17) A method of producing one or more cannabinoid compounds, comprising: a) inoculating the recombinant microorganism of claim 8-13 to a culture medium, b) growing said microorganism in said culture medium under conditions where said heterologous genes are expressed for a time sufficient to produce one or more cannabinoid compounds; c) optionally isolating said one or more cannabinoid compounds from the microorganism or said culture medium or both. 18) The method of claim 17, wherein said culture medium comprises one or more of added prenol, isoprenol, malonate, butanoate, or hexanoate. 19) The method of claims 17-18, wherein said one or more cannabinoid compounds is(are) selected from cannabigerol, cannabigerolic acid, cannabigerovarin, cannabigerovarinic acid, cannabigerorcol, cannabigerorcolic acid, cannabidiol, cannabidiolic acid, cannabinodivarin, cannabinodivarinic acid, cannabidiorcol, cannabidiorcolic acid, cannabichromene, cannabichromenic acid, cannabichromevarin, cannabichromevarinic acid, cannabichromeorcol, cannabichromeorcolic acid tetrahydrocannabinol, tetrahydrocannabinolic acid, tetrahydrocannabivarin, tetrahydrocannabivarinic acid, tetrahydrocannabiorcol, or tetrahydrocannabiorcolic acid.

20) The method of claims 17-18, wherein olivetolic acid is produced at a rate greater than 0.5 mg/hr-g.

Form PCT/ISA/210 (second sheet) (January 2015) Form PCT/ISA/210 (continuation of first sheet (2)) (January 201 5)