WO2017223025A1 - Escherichia coli modifiée pour la production d'acide butyrique - Google Patents

Escherichia coli modifiée pour la production d'acide butyrique Download PDF

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WO2017223025A1
WO2017223025A1 PCT/US2017/038234 US2017038234W WO2017223025A1 WO 2017223025 A1 WO2017223025 A1 WO 2017223025A1 US 2017038234 W US2017038234 W US 2017038234W WO 2017223025 A1 WO2017223025 A1 WO 2017223025A1
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bacterium
brevibacterium
genetically modified
streptomyces
genes encoding
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Brelan E. MORITZ
Liang Wang
Diane Sylvie CHAULIAC
Guimin ZHANG
Kesavalu Naidu LAKSHMYYA
Lonnie O. Ingram
Keelnatham T. Shanmugam
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University Of Florida Research Foundation, Incorporated
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    • C12N15/09—Recombinant DNA-technology
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    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • C12N9/88—Lyases (4.)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01157—3-Hydroxybutyryl-CoA dehydrogenase (1.1.1.157)
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    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01019—Phosphate butyryltransferase (2.3.1.19)
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    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02007—Butyrate kinase (2.7.2.7)
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01017—Enoyl-CoA hydratase (4.2.1.17), i.e. crotonase

Definitions

  • Butyric acid is a feedstock for synthesis of plastics, plasticizers and surfactants.
  • Various derivatives of butyric acid, such as esters, are used in food and perfume industries as well as in chemical and pharmaceutical industries.
  • Ethyl butyrate can also serve as an alternate octane enhancer in transportation fuels.
  • Butyric acid is a precursor of butanol, a next generation transportation fuel, and can potentially be the largest market for butyric acid.
  • butyric acid has been implicated in improving intestinal health in humans as well as in other animals; it is used as a feed additive, and is expected to become a food supplement.
  • Butyric acid also appears to have anti-neoplastic activity and is currently being evaluated for this application in prodrug formulations.
  • Butyric acid as a commodity chemical is produced on an industrial scale by chemical synthesis and by fermentation of starch.
  • Industrial scale butyric acid is produced by oxidation of butyraldehyde derived from propylene obtained during refining of petroleum.
  • Butyric acid production by fermentation utilizes anaerobic bacteria and is a sustainable way of producing this chemical for its various uses.
  • the chemical manufacturing process is preferred due to its lower production cost.
  • Butanol as a next generation biofuel is attractive due to its energy content, which is close to that of gasoline, as well as its other chemical properties.
  • the low titer and yield of butanol by native butanol producers combined with the toxicity of butanol limits its large scale fermentative production as a biofuel.
  • Recombinant E. coli strains engineered to produce butanol by fermentation are also limited in their use by the low titer of butanol due to their sensitivity to butanol.
  • An alternate approach is to construct recombinant E. coli strains that produce butyric acid at high titer and yield and chemically/electrochemically reduce it to butanol as a biofuel.
  • Bacteria that ferment sugars to butyric acid are found in various environments (for example, soil, anaerobic digesters and rumen) and are also natural inhabitants of human intestine. Based on the current list of cultured butyrate-producing bacteria isolated from various environments, fermentation of sugars to butyric acid appears to be restricted to strict anaerobes that ferment sugars to a mixture of products, including butyrate. Genetic modification of these bacteria for homo-butyrate fermentation, although in progress, is hampered by their strict anaerobic nature and very limited genetic tool set. In batch fermentations, the reported highest titer of butyric acid with native butyrate producers was about 40-45 g/L (Table 3).
  • butyrate production is accompanied by varying amounts of acetate, lactate and ethanol as co-products with acetate as the dominant co-product.
  • Butyric acid is also an intermediate in butanol producing Clostridia during growth at neutral pH. Clostridium acetobutylicum, a native butanol producer, has been engineered to produce butyric acid as a major product at a titer of about 33 g/L.
  • the invention provides a bio-based butyric acid production which provides a high titer of butyric acid (butyrate) not provided by the conventional methods.
  • Novel microorganisms for example, genetically modified Escherichia coli strains, that are useful for the production of butyrate are provided. Accordingly, the materials and methods of the subject invention can be used to produce butyrate for use in a variety of applications.
  • the genetically engineered bacterium of the invention is genetically modified by the inactivation of genes encoding lactate dehydrogenase (ldhA) and alcohol dehydrogenase (adhE) and the introduction of one or more heterologous or homologous genes encoding acetoacetyl-CoA synthase (atoB; AtoB), hydroxybutyryl-CoA dehydrogenase (hbd; HBD), crotonase (crt; CRT), phosphotransbutyrylase (ptb; PTB), butyrate kinase (buk; BUK), and trans-enoyl-CoA reductase (ter; TER).
  • the butyryl-CoA dehydrogenase and associated electron transfer proteins (BCD/ETF) of the native butyrate pathway are replaced in the genetically engineered bacterium (e.g., Clostridium spp.) with a heterologous gene encoding a TER enzyme that catalyzes the same reaction.
  • Other embodiments include the further inactivation of genes encoding acetate kinase (ackA) and fumarate reductase (frd).
  • the genetically engineered bacterium is E. coli.
  • the gene encoding AtoB is obtained from E. coli
  • the genes encoding HBD, CRT, PTB and BUK are obtained from Clostridium acetobutylicum
  • the gene encoding TER is obtained from Treponema denticola.
  • Methods of producing and purifying butyrate by culturing the genetically engineered bacterium of the invention are also provided.
  • the bacterium is grown in a mineral salt medium. Additional advantages of this invention will become readily apparent from the ensuing description.
  • Figure 1 Primary fermentation pathway of anaerobic butyrate producing bacteria.
  • PFOR pyruvate ferredoxin oxidoreductase
  • THL thiolase
  • HBD hydroxybutyryl-CoA dehydrogenase
  • CRT crotonase
  • BCD/ETF butyryl-CoA dehydrogenase and associated electron transfer proteins
  • PTB phosphotransbutyrylase
  • BUK butyrate kinase
  • TES thioesterase
  • HYD hydrogenase.
  • Crotonyl-CoA reduction to butyryl-CoA by the BCD/ETF complex of native butyrate producing bacteria Catalysis of this reduction by a trans-enoyl-CoA reductase (Ter) from other microorganisms such as Treponema denticola can be used to reduce the amount of NADH required in this reaction by 50%.
  • Te trans-enoyl-CoA reductase
  • FIG. 3 An operational butyrate pathway in E. coli with appropriate competing pathways deleted.
  • An "X" over an arrow designates deletion/inactivation of the gene encoding an enzyme in the respective competing pathway.
  • FRD fumarate reductase
  • LDH lactate dehydrogenase
  • PFL pyruvate formate-lyase
  • PTA phosphotransacetylase
  • ACK acetate kinase
  • ADH-E aldehyde alcohol dehydrogenase
  • FHL formate hydrogen-lyase
  • AtoB acetoacetyl-CoA synthase.
  • Figure 4 Plasmid pBEM3 carrying a synthetic operon comprising atoB, hbd, crt and ter.
  • Figure 8 Distribution of fermentation products of E. coli strain BEM3-LB9-1. The results from Figure 7 were used in this calculation.
  • FIG. 9 Fed-batch fermentation of glucose to butyric acid.
  • E. coli strain BEM3- LB9 was cultured in LB medium with CaC0 3 (5 g/L). pH of the culture was maintained at 7.0 with KOH addition. Fermentation was started with 133 mM (24 g/L, w/v) glucose and at 16 hours, additional glucose (317 mM; 57 g/L) was added and fermentation continued.
  • Figure 10 Inhibition of growth of E. coli strain DC302 by formate. Luria broth with glucose (1%, w/v) and various amounts of formate was inoculated and incubated under anaerobic condition at 37°C. Cell density at the end of 25 hours of incubation was determined and plotted against initial formate concentration.
  • FIG. 11 Formate level in E. coli strain DC302 fermentation under different pH regimes. Cultures were grown in LB+glucose (50 g/L) with CaC0 3 (5 g/L) at 37°C. Culture pH was maintained at 7.0 (dash lines) or changed from pH 7.0 to 6.0 at 12 hours (indicated by the vertical arrow) and maintained at 6.0 (solid line).
  • the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures.
  • the depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
  • Clostridium spp. are native butanol producing bacteria.
  • butanol production by Clostridia is associated with the production of undesirable co-products due to the necessity to support optimal redox balance for growth and fermentation at the expense of butyrate yield.
  • Butyric acid production in native bacteria starts from acetyl-CoA generated by glycolysis of sugars and oxidative decarboxylation of pyruvate by pyruvate-ferredoxin oxidoreductase (PFOR) (Fig. 1).
  • the fermentation pathway listed in Fig. 1 utilizes three NADH molecules during the conversion of two acetyl-CoA molecules to one butyrate molecule.
  • the requirement of three NADH molecules in the butyrate production pathway from acetyl-CoA is due to the unique property of the BCD/ETF complex (Fig. 2).
  • the BCD/ETF complex catalyzes a two electron reduction step of crotonyl-CoA to butyryl-CoA but utilizes four electrons with associated release of one H 2 for every butyryl-CoA produced.
  • components of butyric acid fermentation pathways are transferred to other microbes such as E. coli to produce an engineered microbial biocatalyst for high titer and productivity of butyrate.
  • the subject invention provides materials and methods wherein unique and advantageous combinations of gene mutations are used to direct carbon flow to butyrate.
  • the techniques of the subject invention can be used to obtain products from native pathways as well as from recombinant pathways.
  • the subject invention provides a versatile platform for the production of these products with only mineral salts and sugar as nutrients in a defined medium.
  • bacteria that can be modified according to the present invention include, but are not limited to, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Bacillus subtilis, Brevibacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacter
  • the subject invention provides genetically modified bacterial strains (such as E. coli) that are suitable for the production of butyrate.
  • the microorganisms of the subject invention can employ a single step process using sugars as substrates, have high rates of product production, high yields, simple nutritional requirements ⁇ e.g., mineral salt medium), and a robust metabolism permitting the bioconversion of sugars to butyrate.
  • microorganisms produced according to the instant disclosure can have one or more target genes inactivated by various methods known in the art. Deletion/inactivation of a target gene indicates that the genetic modification of the target gene results in inactivation of the enzymatic activity of the polypeptide produced by the target gene.
  • a target gene can be inactivated by the introduction into the target gene of insertions, deletions, random mutations, frameshift mutations, point mutations, insertion of one or more stop codons or a combination thereof.
  • certain aspects of the invention provide for the insertion of at least one stop codon ⁇ e.g., one to ten or more stop codons) into the target gene.
  • Some aspects of the invention provide for the insertion or deletion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 or more bases in order to introduce a frameshift mutation into a target gene.
  • Yet other embodiments of the subject application provide for the introduction of one or more point mutations ⁇ e.g., 1 to 30 or more) within a target gene while other aspects of the invention provide for the total or complete deletion of a target gene from the microorganisms of the invention. Mutations and/or deletions in the promoter region of a gene resulting in the inactivation of target genes can also be performed. In each of these aspects of the invention, metabolic pathways are inactivated by the inactivation of the enzymatic activity of the polypeptide encoded by the target gene(s).
  • Target gene(s) refers to a gene which is to be inactivated/deleted from a bacterium.
  • target genes that are inactivated/deleted from a bacterium include IdhA, adhE,frd and ackA.
  • one or more genes selected from IdhA, adhE,frd and ackA are deleted/inactivated from a bacterium.
  • a "gene of interest” refers to a gene which is introduced into a bacterium from a heterologous or homologous source.
  • a gene from a heterologous source indicates that the gene is not native to the bacterium and is obtained from a source other than the bacterium being modified.
  • a gene from a homologous source indicates that the gene is naturally present in the bacterium being modified or from a different strain of bacterium of the same genus and species as the bacterium being modified.
  • one or more copies of a heterologous gene are introduced into the bacterium.
  • one or more copies of a homologous gene are introduced into a bacterium.
  • the bacterium contains an endogenous copy of the gene of interest which is naturally present in the bacterium in addition to the one or more copies of the gene of interest introduced into the bacterium.
  • a “native gene” or “native genes” is/are to be understood to be a gene (or genes) that is/are naturally found in a bacterium as opposed to a “heterologous gene” that is introduced into the bacterium which was obtained from any microorganism other than the bacterium.
  • a heterologous or homologous gene introduced into a bacterium can be present in an extrachromosomal nucleic acid molecule and/or incorporated into the genome of the bacterium. In one embodiment, certain of the heterologous or homologous genes introduced into a bacterium are present on an extrachromosomal nucleic acid, whereas certain other heterologous or homologous genes are incorporated into the genome of the bacterium.
  • homologous gene When a homologous gene is introduced into a bacterium of the current invention, two or more copies of the homologous gene are present in the bacterium. Such multiple copies of the gene result in increased expression of the polypeptide encoded by the gene compared to a bacterium containing only the native copy of the gene.
  • An example of an extrachromosomal nucleic acid molecule is an expression vector.
  • An expression vector typically comprises an origin of replication and a selectable marker.
  • the gene to be expressed can be under the control of a promoter.
  • the promoter can be an inducible promoter.
  • One or more heterologous genes can be introduced into a bacterium in one or more expression vectors. Two or more genes can be included in an expression vector and can be under the control of the same/similar or different promoters.
  • An embodiment of the subject invention provides a genetically modified bacterial strain that comprises genetic modifications to one or more of the following target genes: a) IdhA, b) adhE, c) ackA and d) frd, said genetic modifications inactivating the enzymatic activity of the polypeptide produced by said target gene; the genetically modified bacterial strain further comprising one or more heterologous or homologous genes selected from atoB, hbd, crt, ptb, buk, and ter.
  • BCD/ETF of the genetically engineered bacterium is inactivated and/or replaced with a heterologous gene encoding a TER enzyme (for example, in Clostridium spp.).
  • homologs of atoB, hbd, crt, ptb, buk, and ter genes in various bacteria are well known and a person of ordinary skill in the art can determine appropriate homologs of these genes or proteins to be used in particular embodiments of the invention.
  • Non-limiting examples of certain homologs of these genes based on their GenBank accession numbers are provided below:
  • AtoB NP_149242, WP_033127205, WP_017751772, CBK80359, WP_022244 334, WP_003845123, WP_004899541, WP015871508 WP_029095550, WP_005191525, WP_027275745, WP_024484014, WP_015432282, WP_042804044, WP_005764280, WP_032098613, WP_012341626, WP_039198002, WP_043489071, WP_037404676, WP_005585598.
  • hdb WP_017751920, WP_002582766, CBK80243, WP_022342909,
  • WP_002582659 WP_022220769, WP_022517597, WP_039635970, WP_029165510, WP_006300083, WP_014163832, WP_037975905, WP_028856397, WP_036225283, WP_014807666, WP_006583969, WP_013644276, YP_006919220, WP_005661078, WP_012209273, WP_016092292, WP_006930460, WP 013970015.
  • WP_044977782 WP_021686193, WP_016526423, WP_024466379, WP_006187999, WP_014545506, WP_012025447, WP_020613765, WP_011584463, WP_010601335, GAL85934, WP_O14678420, WP_010277416, WP_034059171, WP_026975375, WP_024267921, WP_035588750, WP_037437986, WP_031445069, WP_034040141.
  • Formate hydrogen lyase is a protein complex composed of a formate dehydrogenase component (FdhF) and a hydrogenase component (for example, HycE in E. coli) in addition to electron carrier proteins and membrane anchor proteins.
  • FdhF formate dehydrogenase component
  • HycE hydrogenase component
  • Non-limiting examples of certain homologs of the FdhF and HycE proteins, the critical components of the formate hydrogen lyase complex based on their Uniprot entry numbers are provided below:
  • FdhF Q7UMT4, A0A0C7KNF3, W9BDM5, Q66FE2, B7LMN4, D2TP34, B5QZB0, F8VMM4, C9YRG1, 10LGV1, L7EMF5.
  • HycE A0A0B7JIU6, A0A0D1LBN9, A0A0C3IC03, E1W686, A0A023DU44, A0A0C5SEP3, A0A060VI10, A0A0B3DZW2, A0A0C9CB33, A0A0C8TEL9, A0A087FVB0, A0A0A0ZFE0, A0A0C7MTZ6, A0A0C8FYK1, A0A0D0MNR2, A0A0C9ENN3, A0A0D1NB17, I6S8T0, M4LTG6, C9Y2U7, B7LW13, F8VHT9, A0A097QVU5, Q8THY6.
  • Non-limiting examples of the genetically modified bacterial strain according to the subject invention include Escherichia coli, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum,
  • the genetically modified bacterium of the subject invention is metabolically evolved to produce strains that produce higher amounts of butyrate compared to the bacterium before such metabolic evolution.
  • Metabolic evolution refers to the selection process for strains with improved growth and butyrate production (examples of which are provided within the disclosed examples).
  • the genetically modified bacterium of the subject invention is E. coli.
  • the genetically modified E. coli comprises deletion/inactivation of genes encoding ldhA and adhE.
  • the genetically modified E. coli further comprises heterologous or homologous genes encoding one or more of AtoB, HBD, CRT, PTB, BUK, and TER.
  • the genetically engineered E. coli in addition to the deletion/inactivation of genes encoding D-LDH and ADH-E, further comprises inactivation of genes encoding AckA and FRD.
  • An embodiment of the current invention provides methods of producing butyrate by culturing the genetically engineered microorganisms according to the subject invention.
  • Butyrate can be purified from the cultured microorganisms and further converted to butanol.
  • the conversion of butyrate to butanol is performed by an electrochemical and/or chemical process.
  • the microorganism of the current invention is grown in a complex medium or a mineral salt medium.
  • the mineral salt medium can comprise between 2% and 20% (w/v) carbohydrate, for example, about 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% (w/v) of a sugar.
  • the mineral salt medium is NBS or AMI, the composition of which is reproduced below in Table 1.
  • NBS + 1 mM betaine NBS media amended with betaine (1 mM).
  • Calculation includes KOH used to neutralize betaine-HCl stock.
  • Trace metal stock (1000X) was prepared in 120 mM HC1.
  • the mineral salt medium is KTS1, the composition of which is reproduced below in Table 2.
  • Glucose, CaC0 3 (5g/L) and betaine (1 mM) are added as needed.
  • a genetically modified bacterium comprising:
  • TER trans-enoyl-CoA reductase
  • bacterium is selected from Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium
  • a method of producing butyric acid comprising culturing the genetically modified bacterium of embodiments 1-12 in a culture medium and purifying butyric acid from the culture medium.
  • the synthetic pathway presented in Fig. 3 was constructed by combining genes from various microorganisms. Butyric acid producing E. coli strains constructed to date produced a highest reported butyric acid titer of about 10 g/L compared to as high as 45 g/L produced by butyrate-producing Clostridia (Table 3).
  • the current invention provides a genetically engineered E. coli containing the inactivation/deletion of genes encoding D-LDH and ADH- E, and carrying the gene encoding AtoB from E. coli, the genes encoding HBD, CRT, PTB and BUK from Clostridium acetobutylicum, and the gene encoding TER from Treponema denticola.
  • E. coli produces acetate, ethanol and lactate during fermentation with a small amount of succinate. Redox balance to support anaerobic growth of this bacterium is achieved by judicious combination of the pathways leading to these fermentation products. Deletion of both IdhA and adhE genes renders a double mutant (strain BEM3) anaerobic growth negative due to the inability to oxidize NADH and maintain redox balance to support glycolysis and ATP production. Introduction of the redox-balanced butyrate pathway (Fig. 3) into strain BEM3 is expected to restore anaerobic growth. E. coli strain BEM3 was used as the platform organism for introducing the butyrate pathway.
  • Plasmid pBEM3 (Fig. 4) carries the genes atoB from E. coli, hbd and crt from C. acetobutylicum ATCC824 and ter from T. denticola ATCC35404 as a synthetic operon and transcribed from a tetracycline inducible promoter.
  • Appropriate gene sequences were amplified by PCR from the genomic DNA of the corresponding microorganisms using primers listed in Table 4.
  • Plasmid pBEM3 was constructed using these DNA sequences verified, amplified DNA fragments with the aid of a commercial StarGate cloning kit (IB GMBH, Goettingen, Germany).
  • Plasmid pi 85 (Fig. 5) carries the genes (ptb and buk) encoding the two terminal enzymes in the butyrate pathway, PTB and BUK (Fig. 3).
  • the butyrate pathway with these two enzymes generates an additional ATP per glucose (net 3 ATPs per glucose) and is expected to increase both growth rate and cell yield of the butyrate-producing E. coli.
  • the ptb and buk genes were amplified by PCR using appropriate primers ([AvaI]ptbF and [AhdI]bukR; Table 4) from the genomic DNA of C. acetobutylicum ATCC824 and cloned into plasmid vector pACYC184 that is compatible with plasmid pBEM3 in E. coli host strains. These two genes are expressed from their own native promoters.
  • E. coli strain BEM3 with plasmids pBEM3 and pl85 (strain BEM3-LB0) grew anaerobically, although at a very low rate and produced butyric acid ( ⁇ 1 mM). Since growth and butyrate production are coupled in this strain, an anaerobic growth-based selection was used to optimize expression of the introduced synthetic butyrate pathway to support higher growth rate and cell yield.
  • strain BEM3-LB9 A derivative isolated after 9 transfers in pH-controlled fermentations (about 40 generations) (strain BEM3-LB9) produced butyric acid at a concentration of about 190 mM (17 g/L) in about 72 hours (Fig. 6). This is the highest titer of butyric acid produced by a recombinant E. coli strain in batch fermentation (Table 3). The volumetric productivity of butyrate by strain BEM3-LB9 at 0.61 g/l.h was also the highest not only among the butyrate producing E. coli strains reported in the literature but also among the native Clostridium strains during the last 20 years.
  • the butyrate yield was about 70% of the theoretical yield of glucose consumed and this was due to the production of succinate and acetate as co-products (93 and 72 mM, respectively).
  • the total product yield that includes butyrate and the co- products was about 100% of the expected yield of products from fermented glucose.
  • strain BEM3 was further modified with deletions in the ackA and frd genes (strain BEM9).
  • Strain BEM9 with the synthetic butyrate pathway (plasmids pBEM3 and pi 85) produced about 99 mM butyrate (8.7 g/L) when cultured in rich medium under 0 2 -limiting conditions. Growth-based metabolic selection of this strain in a mineral salt medium for higher butyrate titer, yield and productivity as well as integration of the butyrate pathway into the chromosome are also provided.
  • the synthetic butyrate pathway starts from acetyl-CoA and this starting material is produced from pyruvate by the enzyme pyruvate formate-lyase (FUL) in the recombinant E. coli (Fig. 3). Therefore, formate is an integral part of butyrate production and two moles of formate for every mole of butyrate are produced.
  • the results presented in Fig. 6 show that the culture accumulated about 0.3 M formate, a concentration that is expected to be inhibitory to metabolism. Therefore, further increase in butyrate titer may be limited by the total acid concentration in the medium.
  • E. coli has a native enzyme complex encoded by fdhF and yc that metabolizes formate to H 2 and C0 2 but this requires a culture pH of less than 6.0 and presence of molybdate in the medium. Both formate and molybdate are positive effectors for the FhlA protein, an activator of the genes encoding the FUL complex (formate dehydrogenase-H, hydrogenase 3 and associated electron transport proteins).
  • F A165 A modified FHLA protein that lacks the amino acids 5-374 was reported to function as an effective activator of the fdhF and hyc operons without the need for formate, molybdate and low pH (Self et al., Microbiology, 2001, 147(Pt 11):3093-3104, the disclosure of which is hereby incorporated by reference in its entirety).
  • This mutation can be introduced into strain BEM9 to enable rapid removal of formate to non-inhibitory gaseous products, H 2 and C0 2 .
  • the culture can accumulate butyrate as the main organic acid in the medium without formate as a co-product.
  • strain BEM3-LB9-1 A metabolically adapted derivative of strain BEM3-LB9 (strain BEM3-LB9-1) was found to reduce the lag period before fermentation of glucose to butyrate (Fig. 7). Although several co-products were still present in the fermentation broth of strain BEM3-LB9-1, butyrate accounted for about 50% of the fermented glucose carbon (Fig. 8) accounting for a butyrate molar yield of 65% (Table 2). When this culture was grown in a fed-batch mode, the butyric acid titer increased to 350 mM (30 g/L), almost doubling the titer of 195 mM (17 g/L) in batch fermentation (Fig. 9; Table 2).
  • the acetate concentration in the fermentation broth was reduced from 70 mM for BEM3-LB9 to about 25 mM for strain DC302.
  • the remaining acetate is apparently coming from the catalytic activity of pyruvate dehydrogenase and pyruvate oxidase.
  • E. coli and other members of the family enterobacteriaceae dissimilates formate to H 2 and C0 2 using the enzyme formate hydrogen-lyase (FELL) to remove formate from the medium since formate is inhibitory to growth (Fig. 10).
  • FELL formate hydrogen-lyase
  • Inhibition of growth of strain DC302 by formate is concentration dependent and is exponential until it reached about 0.2 M.
  • Formate at 0.3 M completely inhibited growth under both aerobic and anaerobic condition even in rich medium with glucose. Since the formate concentration at the end of fermentation of strain BEM3-LB9-1 is about 0.3 M (Table 2) the accumulating formate maybe restricting metabolic flux of glucose to butyric acid. Therefore, to maintain high glucose flux to butyrate, formate is metabolized by the culture to H 2 and C0 2 by modification of the fermentation condition used in this Example.
  • the rate of formate removal from the medium by FUL is higher at pH 6.0 compared to a culture at pH 7.0.
  • the growth and fermentation of glucose to butyrate is optimal at pH 7.0.
  • strain DC302 was started at pH 7.0; however, after 12 hours, when the culture density reached close to the highest value, the culture pH was lowered to 6.0 and maintained for the rest of the fermentation period (Fig. 11; Table 1).
  • lowering the culture pH to 6.0 after growth did not affect butyrate production; however, it greatly reduced formate concentration in the medium.
  • a combination of strain construction (BEM3-LB9 vs DC302) and process modification (pH shift from 7.0 to 6.0) can lower the formate concentration.
  • CoA crotonyl coenzyme A

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Abstract

L'invention concerne des micro-organismes génétiquement modifiés utiles pour la production de butyrate. La bactérie de l'invention comprend l'inactivation/la délétion de gènes codant pour la lactate déshydrogénase (ldhA) et l'alcool déshydrogénase (adhE). La bactérie comprend en outre un ou plusieurs gènes hétérologues ou homologues codant pour l'acétoacétyl-CoA synthase (AtoB), l'hydroxybutyryl-CoA déshydrogénase (HBD), la crotonase (CRT), la phosphotransbutylase (PTB), la butyrate kinase (BUK) et la trans-énoyl-CoA réductase (TER) Dans un autre mode de réalisation, la bactérie génétiquement modifiée comprend également l'inactivation de gènes codant pour l'acétate kinase (ackA) et la fumarate réductase (FRD) et, éventuellement, l'expression constitutive de gènes codant pour la formiate hydrogène-lyase (FHL).
PCT/US2017/038234 2016-06-21 2017-06-20 Escherichia coli modifiée pour la production d'acide butyrique WO2017223025A1 (fr)

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CN112608951A (zh) * 2021-01-15 2021-04-06 驻马店华中正大有限公司 一种提高丁酸产率的发酵方法
CN114774338A (zh) * 2022-03-29 2022-07-22 北京航空航天大学 一种产丁酸的益生菌及其构建方法和应用
CN116496936A (zh) * 2023-03-02 2023-07-28 湖南酵本生物医药有限公司 一种醋酸杆菌和含有该菌的菌剂、以及它们的应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112608951A (zh) * 2021-01-15 2021-04-06 驻马店华中正大有限公司 一种提高丁酸产率的发酵方法
CN114774338A (zh) * 2022-03-29 2022-07-22 北京航空航天大学 一种产丁酸的益生菌及其构建方法和应用
CN114774338B (zh) * 2022-03-29 2023-08-08 北京航空航天大学 一种产丁酸的益生菌及其构建方法和应用
CN116496936A (zh) * 2023-03-02 2023-07-28 湖南酵本生物医药有限公司 一种醋酸杆菌和含有该菌的菌剂、以及它们的应用

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