WO2019190945A1 - Biosynthèse d'acide olivetolique - Google Patents

Biosynthèse d'acide olivetolique Download PDF

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WO2019190945A1
WO2019190945A1 PCT/US2019/023792 US2019023792W WO2019190945A1 WO 2019190945 A1 WO2019190945 A1 WO 2019190945A1 US 2019023792 W US2019023792 W US 2019023792W WO 2019190945 A1 WO2019190945 A1 WO 2019190945A1
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coa
acid
synthase
microorganism
olivetolic acid
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PCT/US2019/023792
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English (en)
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Ramon Gonzalez
Zaigao TAN
James M. Clomburg
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William Marsh Rice University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • 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.
  • PKS Ills type III polyketide synthases
  • anthocyanins the water-soluble pigments from mulberry fruits
  • Hyperforin which is one of the primary active constituents from extracts of Hypericum perforatum
  • 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.
  • 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.
  • 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.
  • E. coli has been engineered to produce a wide range of products from the acetyl-CoA node, including those derivatives directly from malonyl-CoA.
  • 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.
  • 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.
  • 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.
  • industrial organisms e.g., E. coli, B. subtilis, S. cerevisiae and the like
  • Media preparation, sterilization, inoculum preparation, fermentation and product recovery from the cells, or the medium, or both, are the main steps of the process.
  • 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.
  • 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.
  • living systems may be preferred as allowing continuous production and would be suitable for simple polyketides or polyketide precursors for downstream products.
  • 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.
  • references to proteins herein can be understood to include reference to the gene encoding such protein.
  • a claimed“permease” can include the related gene encoding that permease.
  • 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.
  • many sites provide lists of proteins that all catalyze the same reaction. See e.g., BRENDA, UNIPROT, ECOPRODB, ECOLIWIKI, to name just a few.
  • NCBITM 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.
  • 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.
  • yeast such as Saccharomyces
  • 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.
  • 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.
  • Spirulina Chlamydomonas, Laminaria japonica, Undaria pinnatifida, Porphyra, Eucheuma, Kappaphycus, Gracilaria, Monostroma, Enteromorpha, Arthrospira, Chlorella, Dunaliella, Aphanizomenon, Isochrysis, Pavlova, Phaeo
  • 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.
  • DHA docosahexaenoic
  • EPA eicosapentaenoic acids
  • Crypthecodinium cohnii is the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.
  • 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.
  • the enzymes can be added to the genome or via expression vectors, as desired.
  • 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.
  • ORFs open reading frames
  • “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.
  • 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.
  • “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.
  • “Reduced activity” or“inactivation” 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 D). 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.
  • “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.
  • 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.
  • 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.
  • “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.
  • 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- -D- galactopyranoside (IPTG), the yeast AOX1 promoter inducible with methanol, the strong LAC 4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters.
  • 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.
  • “olivetolic acid 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.
  • 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.
  • malonyl-CoA synthetase (EC 6.2.1.14), catalyzes the formation of malonyl-CoA from malonate, CoA and ATP.
  • 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.
  • the terms“synthase”, “synthetase”, and“ligase” do not always follow these strict definitions. Accordingly, as used herein, these terms may be used interchangeably.
  • 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.
  • a“thiolase” is an enzyme capable of catalyzing the condensation of acetyl-CoA with a C n -acyl-CoA to produce a C n +2 b-ketoacyl-CoA and CoA.
  • a“hydroxyacyl-CoA dehydrogenase” is an enzyme that catalyzes the reduction of a b-ketoacyl-CoA to a b-hydroxyacyl-CoA.
  • an“enoyl-CoA hydratase” is an enzyme that catalyzes the dehydration of a b-hydroxyacyl-CoA to enoyl-CoA.
  • an“acyl-CoA dehydrogenase” or“enoyl-CoA reductase” is an enzyme that catalyzes the reduction of enoyl-CoA to acyl-CoA.
  • 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).
  • 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).
  • a“DMAP kinase” is an enzyme capable of catalyzing the conversion of DMAP and ATP to 3,3-dimethylallyl diphosphate (DMAPP).
  • an“IP kinase” is an enzyme capable of catalyzing the conversion of IP and ATP to 3 -methyl-3 -buten-l-yl diphosphate (IPP).
  • an“isopentenyl diphosphate isomerase” is an enzyme catalyzing the interconversion of DMAPP and IPP.
  • a“geranyl diphosphate synthase” is an enzyme capable of catalyzing the condensation of DMAPP and IPP to form geranyl diphosphate (GPP).
  • a“polyketide prenyltransferase” is an enzyme that transfers a prenyl group to a polyketide to form a prenylated polyketide.
  • 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).
  • a“cannabinoid” is a prenylated aromatic compound naturally found in Cannabis sativa, or a derivative or analog thereof.
  • a“cannabinoid synthase” is an enzyme capable of converting one cannabinoid to another.
  • “principle carbon source” means the most abundant source of carbon, on a molar basis, that is used as a biosynthetic precursor for fermentation products.
  • 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.
  • the invention includes any one or more of the following embodiments, in any combination(s) thereof:
  • 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.
  • 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.
  • 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.
  • microorganism comprises (a) heterologous gene(s) encoding one or more the following enzymes: a) thiolase capable of catalyzing the conversion of a C n -acyl-CoA to a C n +2 b- ketoacyl-CoA; b) hydroxyacyl-CoA dehydrogenase capable of catalyzing the conversion of b- ketoacyl-CoA to b-hydroxyacyl-CoA; c) enoyl-CoA hydratase capable of catalyzing the conversion of b-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.
  • 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.
  • 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.
  • DMAP
  • 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.
  • 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) cannabidiorcoli
  • a method of producing one or more of the compounds: olivetol; divarin; orcinol; olivetolic acid; divarinolic acid; or orsellinic 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 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.
  • 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.
  • 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 te
  • 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.
  • 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.
  • 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.
  • FIG. 2 Impact of auxiliary enzymes for increasing hexanoyl-CoA and malonyl- CoA supply for olivetolic acid production in BL21 (DE3).
  • auxiliary enzymes employed for increasing hexanoyl-CoA FadD/FadK
  • MCS/ACC malonyl-CoA
  • 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.
  • FadD long chain fatty acyl-CoA synthetase
  • FadK short chain fatty acyl-CoA synthetase
  • MCS malonyl-CoA synthetase
  • ACC acetyl-CoA carboxylase
  • FIG. 3 Production of olivetolic acid in engineered A. coli 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.
  • TH thiolase
  • HR FadB hydroxyacyl-CoA dehydrogenase
  • EH FadB enoyl- CoA hydratase
  • ER egTER enoyl-CoA reductase
  • 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.
  • RppA was expressed with different MCS/ACC auxiliary enzymes for characterization of malonyl-CoA availability in JST10 (DE3) strain.
  • Engineered A. coli strains were cultured in LB-like MOPS medium+2% (wt/v) glycerol. For strains harboring MCS, 12 mM malonate sodium was also included.
  • 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 mM 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.
  • WV working volume
  • 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.
  • inducers and 4 mM hexanoate were added after strains reached an OD550 -0.4-0.8.
  • 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.
  • 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.
  • 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.
  • FIG. 8 In vivo production of divarinolic acid and divarin in E. coli.
  • the butyryl-CoA primer is provided by reversal of beta-oxidation (r-BOX) starting from acetyl-CoA.
  • r-BOX beta-oxidation
  • 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).
  • ACC E. coli native acetyl-CoA carboxylase
  • malonyl-CoA can be produce from malonate, CoA, and ATP using malonyl-CoA synthetase (not shown).
  • 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).
  • ACC E. coli native acetyl-CoA carboxylase
  • malonyl-CoA can be produced from malonate, CoA, and ATP using malonyl- CoA synthetase (not shown).
  • FIG. 10 In vivo production of cannabigerolic acid (CBGA) in E. coli.
  • CBGA cannabigerolic acid
  • 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.
  • FIG. 11 In vivo production of cannabidiolic acid (CBDA) in E. coli. CBDA synthase (CBDAS) catalyzes the oxidative cyclization of CBGA into CBDA.
  • CBDA cannabidiolic acid
  • 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.
  • FIGURE SI Mass spectrometry data
  • 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 Na 2 HP0 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 Na 2 HP0 4 , 5 mM (NH 4 ) 2 S0 4 , 30 mM NFLCl, 5 mM sodium selenite, 100 pM FeS0 4 , 100 mM 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.
  • MCS malonyl-CoA synthetase
  • E. coli BL21 (DE3) Host strain for enzymes expression Lab collection
  • JST 10-OLS-OAC-FadD- JST10 (DE3) with pET-P1 -OLS-P2-OAC and pCDF-P1-FadD-P2-
  • JST 10-OLS-OAC-FadD JST10 (DE3) with pET-P 1 -OLS-P2-OAC and pCDF-P1-FadD
  • JST 10-OLS-OAC-ACC JST10 (DE3) with pET-P 1 -OLS-P2-OAC and pCDF-P2-ACC
  • JST 10-OLS-OAC-MCS JST10 (DE3) with pET-P 1 -OLS-P2-OAC and pCDF-P2-MCS
  • JST10-RppA JST10 (DE3) with pET-P1-RppA
  • JST 10-MCS-RppA JST10 (DE3) with pET-P1-RppA and pCDF-P2-MCS
  • JST 10-ACC-RppA JST10 (DE3) with pET-P1-RppA and pCDF-P2-ACC
  • JST 10-ACC-RppA JST10 (DE3) with
  • 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 i sopropy 1 -b-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.
  • IPTG i sopropy 1 -b-D-thi ogal actosi de
  • 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.
  • H2SO4 sulfuric acid
  • MCS malonyl-CoA synthetase
  • 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.
  • OLS olivetolic acid synthase
  • OAC olivetolic acid cyclase
  • This 3,5,7-trioxododecanoyl-CoA intermediate can then be cyclized by OAC via C2-C7 intramolecular aldol condensation to form olivetolic acid.
  • 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).
  • 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)
  • MCS malonyl-CoA synthetase
  • ACC acetyl-CoA carboxylase
  • 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.
  • 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.
  • FadK had no impact on olivetolic acid production under these conditions (FIG. 2).
  • FadD 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 resulted in slight increases in olivetolic acid titer (FIG. 2).
  • SCS strain BL-OLS-OAC -FadD
  • FIG. 2 Combining this hexanoyl-CoA generating module with MCS, the highest olivetolic acid titer was achieved (0.71 mg/L) (FIG. 2).
  • FAB fatty acid biosynthesis
  • r-BOX b-oxidation reversal
  • 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.
  • ACP acyl carrier protein
  • FadD transferase
  • r-BOX operates with CoA intermediates, utilizes acetyl-CoA as extender unit and can directly generate hexanoyl-CoA.
  • 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.
  • 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.
  • 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.
  • a heterologous malonyl-CoA availability indicator pathway was introduced.
  • 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.
  • 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).
  • IPTG 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 mM.
  • 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.
  • excessive dosage of IPTG >100 pM was found to decrease olivetolic acid production.
  • Olivetol synthase 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.
  • 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).
  • 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.
  • 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.
  • lysis buffer 50 mM NaFbPCO, 300 mM NaCl, 10 mM imidazole, pH 8.0
  • lysis buffer 50 mM NaFbPCO, 300 mM NaCl, 10 mM imidazole, pH 8.0
  • 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.
  • 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.
  • 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.
  • 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 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). The reaction mixture was incubated at 20 °C for 16 h and 20 pL sulfuric acid (H2S04) was added to terminate the reaction.
  • H2S04 sulfuric acid
  • FIG. 7A The SDS-PAGE gel of purified OLS and OAC in the assay of in vitro divarinolic acid synthesis, is shown in FIG. 7A.
  • 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.
  • FIG. 7C In the presence of both OLS and OAC (FIG. 7A), both divarin and divarinolic acid can be formed (FIG. 7B), and the MS 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.
  • coli NP 418288.1
  • egTER Euglena gracilis
  • Q5EU90.1 enoyl-CoA reductase Ter from Euglena gracilis
  • Olivetol synthase 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.
  • 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 b-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.
  • 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.
  • 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.
  • this strain is selected to maximize the flux of b-oxidation reversal for butyryl-CoA supply required for the synthesis of divarinolic acid via olivetol synthase OLS and olivetolic acid cyclase OAC.
  • 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.
  • T4 ligase Invitrogen, Carlsbad, CA
  • coli DH5a (Invitrogen, Carlsbad, CA), and positive clones identified by PCR were confirmed by DNA sequencing.
  • the cumate-controlled atoB and fadB 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 and fadA of the fadBACT5 strain formerly constructed (Vick et ah, 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.
  • 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).
  • 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 the fabl locus, selecting on chloramphenicol (12.5 pg/ml) LB plates. Integration is done in a manner to duplicate the last 22 bp of fabl (including stop codon) so as to retain an overlapping promoter for the next native downstream gene.
  • 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.
  • 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 mM 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).
  • 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 mM sodium selenite, and 1 mM 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).
  • Olivetol synthase catalyzes three sequential decarboxyl ative 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.
  • 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).
  • ACC E. coli native acetyl- CoA carboxylase
  • 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.
  • 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 , rocB,r ⁇ a, adhE and frdA , 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.
  • 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.
  • GC-FID qualitative and quantitative analysis
  • [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.
  • GPP geranyl pyrophosphate
  • olivetolic acid for in vivo microbial synthesis of cannabigerolic acid.
  • NphB from Streptomyces sp. (BAE00106.1) is the prenyltranferase used.
  • any prenyltransferase capable of prenylating olivetolic acid may be used.
  • 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
  • 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.
  • the GPP is generated from condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) catalyzed by GPP synthase.
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • 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.
  • MEP 2-methyl-(D)-erythritol-4-phosphate
  • mevalonate pathway or isoprenoid pathway or HMG-CoA reductase pathway
  • 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).
  • DXS DXP synthase
  • 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
  • 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.
  • HMGS HMG-CoA synthase
  • HMGR HMG-CoA reductase
  • the lower mevalonate pathway which converts mevalonate into IPP and DMAPP has 3 variants.
  • 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.
  • M5K mevalonate-5-kinase
  • PMK phosphomevalonate kinase
  • 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).
  • M5K mevalonate-5-kinase
  • IP isopentenyl phosphate
  • IPP isopentenyl phosphate
  • 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.
  • M3K mevalonate-3 -kinase
  • mevalonate-3-phosphate-5-kinase mevalonate-3-phosphate-5-kinase
  • the resulting metabolite, mevalonate-3, 5-bisphosphate is decarboxylated to IP, and finally phosphorylated to yield IPP (Archaeal Mevalonate Pathway II).
  • the prenol pathway starts from non-decarboxylative Claisen condensation between two acetyl-CoAs to acetoacetyl-CoA catalyzed by E. coli thiolase AtoB (NP_416728.1). 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_011554268.1) 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.
  • 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 001195564.1) to yield dimethylallyl phosphate
  • the second step is catalyzed by M. thermautotrophicus phosphate kinase MtIPK (AAB84554.1).
  • E. coli isopentenyl pyrophosphate isomerase Idi converts DMAPP to IPP.
  • DMAPP and IPP are condensed to GPP catalyzed by E. coli GPP synthase IspA (NP 414955.1, S80F) or A.
  • 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 001195564.1) to yield isopentenyl phosphate
  • the second step is catalyzed by M thermautotrophicus phosphate kinase MtIPK (AAB84554.1).
  • E. coli isopentenyl pyrophosphate isomerase Idi converts IP to DMAPP.
  • DMAPP and IPP are condensed to GPP catalyzed by E.
  • 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.
  • CBDAS cannabidiolic acid synthase
  • CBGA cannabigerolic acid
  • CBDDA cannabidiolic acid
  • CBDAS catalyzes the oxidative cyclization of CBGA into CBDA. This in vivo pathway for CBDA synthesis is shown in FIG. 11.
  • 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.

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Abstract

L'invention concerne une approche biosynthétique pour l'ingénierie de nouveaux microbes, tels qu'Escherichia coli, pour la production d'acide olivetolique et autres polycétides aromatiques, ainsi que divers produits en aval qui peuvent être obtenus à partir de ceux-ci. La souche d'E. coli selon l'invention synthétise l'acide olivetolique à partir d'une source de carbone principale in vivo par expression d'acide olivetolique synthase et d'acide olivetolique cyclase à l'aide des modules requis d'inversion de la β-oxydation pour la génération d'hexanoyl-CoA. Une souche d'E. coli qui synthétise l'acide olivetolique et intègre des enzymes auxiliaires supplémentaires pour augmenter à la fois l'hexanoyl-CoA et le malonyl-CoA, permettant la synthèse d'environ 75 mg/L d'acide olivetolique et une productivité de 9,3 mg/hr d'acide olivetolique produit par gramme en poids sec de cellules est en outre décrite.
PCT/US2019/023792 2018-03-29 2019-03-24 Biosynthèse d'acide olivetolique WO2019190945A1 (fr)

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CN110713962A (zh) * 2019-09-06 2020-01-21 南京农业大学 一株高产丙二酰辅酶a的基因工程菌及其构建方法和应用
WO2021071439A1 (fr) * 2019-10-11 2021-04-15 National University Of Singapore Production durable de cannabinoïdes à partir de charges de précurseurs simples à l'aide de saccharomyces cerevisiae
WO2021071438A1 (fr) * 2019-10-11 2021-04-15 National University Of Singapore Biosynthèse de cannabinoïdes à partir d'acide cannabigérolique à l'aide de nouvelles synthases de cannabinoïdes
WO2021163042A1 (fr) * 2020-02-12 2021-08-19 Lygos, Inc. Préparation et modification de polycétides de méroterpènes, de cétones et de lactones pour la semi-synthèse de cannabinoïdes
WO2021225952A1 (fr) * 2020-05-08 2021-11-11 Lygos, Inc. Production à grande échelle d'olivétol, d'acide olivétolique et d'autres alkylrésorcinols par fermentation
WO2022051433A1 (fr) * 2020-09-01 2022-03-10 Biomedican, Inc. Production de sesquicannabinoïdes
US11274320B2 (en) 2019-02-25 2022-03-15 Ginkgo Bioworks, Inc. Biosynthesis of cannabinoids and cannabinoid precursors
WO2022099255A1 (fr) * 2020-11-04 2022-05-12 Northwestern University Plateforme de fabrication biologique acellulaire pour la production d'acides gras et de cannabinoïdes
WO2022251714A3 (fr) * 2021-05-27 2023-01-05 Cb Therapeutics, Inc. Cyclases d'acide olivetolique pour la biosynthèse des cannabinoïdes

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Publication number Priority date Publication date Assignee Title
US11274320B2 (en) 2019-02-25 2022-03-15 Ginkgo Bioworks, Inc. Biosynthesis of cannabinoids and cannabinoid precursors
CN110713962A (zh) * 2019-09-06 2020-01-21 南京农业大学 一株高产丙二酰辅酶a的基因工程菌及其构建方法和应用
WO2021071439A1 (fr) * 2019-10-11 2021-04-15 National University Of Singapore Production durable de cannabinoïdes à partir de charges de précurseurs simples à l'aide de saccharomyces cerevisiae
WO2021071438A1 (fr) * 2019-10-11 2021-04-15 National University Of Singapore Biosynthèse de cannabinoïdes à partir d'acide cannabigérolique à l'aide de nouvelles synthases de cannabinoïdes
WO2021163042A1 (fr) * 2020-02-12 2021-08-19 Lygos, Inc. Préparation et modification de polycétides de méroterpènes, de cétones et de lactones pour la semi-synthèse de cannabinoïdes
WO2021225952A1 (fr) * 2020-05-08 2021-11-11 Lygos, Inc. Production à grande échelle d'olivétol, d'acide olivétolique et d'autres alkylrésorcinols par fermentation
WO2022051433A1 (fr) * 2020-09-01 2022-03-10 Biomedican, Inc. Production de sesquicannabinoïdes
WO2022099255A1 (fr) * 2020-11-04 2022-05-12 Northwestern University Plateforme de fabrication biologique acellulaire pour la production d'acides gras et de cannabinoïdes
WO2022251714A3 (fr) * 2021-05-27 2023-01-05 Cb Therapeutics, Inc. Cyclases d'acide olivetolique pour la biosynthèse des cannabinoïdes

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