WO2017210381A1 - Bioconversion de matières premières de 1-carbone en produits chimiques et en combustibles - Google Patents

Bioconversion de matières premières de 1-carbone en produits chimiques et en combustibles Download PDF

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WO2017210381A1
WO2017210381A1 PCT/US2017/035364 US2017035364W WO2017210381A1 WO 2017210381 A1 WO2017210381 A1 WO 2017210381A1 US 2017035364 W US2017035364 W US 2017035364W WO 2017210381 A1 WO2017210381 A1 WO 2017210381A1
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coa
acyl
microorganism
coli
carbon
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Ramon Gonzalez
Alexander CHOU
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William Marsh Rice University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • 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
    • 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
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01054—Formate C-acetyltransferase (2.3.1.54), i.e. pyruvate formate-lyase or PFL

Definitions

  • PCT/US 15/58121 (WO2016069929) is incorporated by reference herein in its entirety for all purposes.
  • the invention relates to biotechnological methods for the production of industrially relevant chemicals from 1-carbon (CI) feedstocks.
  • CI 1-carbon
  • methods for the biological production of carbon-based products of interest directly by the assimilation of single carbon units are described.
  • Microbes have been designed and engineered to synthesize products of interest using feedstocks as diverse as sugars, glycerol, carbon dioxide, carbon monoxide, formate, methanol, and methane.
  • feedstocks as diverse as sugars, glycerol, carbon dioxide, carbon monoxide, formate, methanol, and methane.
  • C I feedstocks such conversions are made possible by a general network of metabolic pathways that are organized as shown in FIG. 1 and include specialized pathways for carbon fixation, central metabolism, and product synthesis. This type of metabolic architecture has been exploited in all metabolic engineering efforts conducted to date to develop microbes for industrial applications.
  • This 'top-down' engineering strategy is highly complex and suffers from inefficiencies arising from need to first produce common metabolic intermediates before eventually forming products.
  • This architecture is also commonly limited to elongation of a carbon backbone by a minimum of two carbons per step, which is a result of the aforementioned use of common metabolic intermediates.
  • This disclosure describes an alternative platform for the bioconversion of 1 - carbon substrates to carbon-based products of interest, which consists of a single engineered metabolic pathway that allows for the direct assimilation of one-carbon compounds.
  • the pathway uses single carbon extension units, which bypasses the need for the production of common metabolic intermediates and allows for elongation of a carbon backbone iteratively in single carbon increments.
  • the new synthetic pathway centers on the ability for formate acyltransferase enzymes, such as pyruvate formate lyase, to catalyze the condensation of a molecule of formate with an acyl-CoA.
  • formate acyltransferase enzymes such as pyruvate formate lyase
  • the resulting 2-ketoacid can then be converted back to an acyl- CoA, now one carbon longer than the originating acyl-CoA, resulting in overall carbon chain elongation and the production of useful products.
  • the reactions of the pathway are enabled by providing enzymes ito catalyze the needed reactions.
  • the necessary gene sequences are provided in an engineered microbial host, such that the microorganism synthesizes the enzymes that comprise the pathway, allowing for this engineered microorganism to synthesize carbon-based products of interest from single carbon molecules.
  • the enzymes that comprise the pathway are purified and combined in a reaction mixture, providing the ability to synthesize carbon-based products of interest from single carbon molecules.
  • the bacteria themselves can be harvested and used as non-growing bioreactors for the reactions. However, the use of living, growing systems is preferred.
  • carbon-based products of interest are produced solely from single carbon molecules.
  • products can be produced from a combination of single carbon molecules and multi-carbon molecules.
  • strain and the like may be used interchangeably and all such designations include their progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. 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.
  • a “cell,” “microbe,” etc. is generally understood to include a culture of such cells, as the work described herein is done in cultures having 10 9" 15 cells.
  • homolog means an enzyme with at least 40% identity to one of the listed sequences and also having the same general catalytic activity, although of course Km, Kcat, and the like can vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it is typical for bacterial sequences to diverge significantly (40-60%), yet still be identifiable as homologs, while mammalian species tend to diverge less (80-90%).
  • references to proteins herein can be understood to include reference to the gene encoding such protein.
  • a claimed "permease" protein can include the related gene encoding that permease.
  • proteins of similar activity can be identified by BLAST search. Further, every protein record is linked to a gene record, making it easy to design expression vectors. Many of the needed enzymes are already available in vectors, and can often be obtained from cell depositories or from the researchers who cloned them. But, if necessary, new clones can be prepared based on available sequence information using RT-PCR techniques or gene synthesis. Thus, it should be easily possible to obtain all of the needed enzymes for expression or overexpression. [0022] 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 that thus be obtained, e.g., from AddGene or from the author of the work describing that enzyme, and tested for functionality as described herein.
  • NCBITM provides codon usage databases for optimizing DNA sequences for protein expression in various species. Using 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.
  • % identity number of aligned residues in the query sequence/length of reference sequence. Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-250, and available through the NCBI website. The default parameters were used, except the filters were turned OFF.
  • “Operably associated” or “operably linked”, as used herein, refer to functionally coupled nucleic acid or amino acid sequences.
  • Recombinant is relating to, derived from, or containing genetically engineered material. In other words, the genome or genetic material was intentionally manipulated 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 (e.g., the wild type gene in the same host species). Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, by knock-out, by adding stop codons, by frame shift mutation, and the like.
  • null or “knockout” what is meant is that the mutation produces undetectable active protein.
  • a gene can be completely (100%) reduced by knockout or removal of part of all of the gene sequence.
  • Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can also completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein. All null mutants herein are signified by ⁇ .
  • “Overexpression” or “overexpressed” is defined herein to be at least 150% of protein activity as compared with an appropriate control species, or any expression in a species that lacks the activity altogether. Preferably, the activity is increased 100-500%). 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, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. All overexpressed genes or proteins are signified herein by "+".
  • endogenous 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, or placed under the control of a promoter that results in overexpression or controlled expression of said gene.
  • genes from Clostridia would not be endogenous to Escherichia, but a plasmid expressing a gene from E. coli or would be considered to be endogenous to any genus of Escherichia, even though it may now be overexpressed.
  • Native means having a wild type sequence from the species in question.
  • 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.
  • 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 operon inducible by IPTG, the yeast AOX1 promoter inducible with methanol, the strong LAC4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesis may still 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 extra-chromosomal expression vector. It will still be expressible, and preferably is inducible as well.
  • carbon based products of interest refers to products that can be made in microbes, including e.g., alcohols, such as ethanol, butanol, saturated and unsaturated fatty alcohols; diols, such as ethylene glycol, 1,3-propanediol, 1,4-butanediol; polyols, such as glycerol, erythritol; carboxylic acids, such as acetate, propionate, butyrate, crotonate, saturated and unsaturated fatty acids; hydroxycarboxylic acids, such as glycolate, lactate, 3-hydroxybutyrate, polyhydroxybutyrate; dicarboxylic acids, such as adipic acid, succinic acid; alkanes; alkenes; amines; polyketides; fatty acid esters.
  • alcohols such as ethanol, butanol, saturated and unsaturated fatty alcohols
  • diols such as ethylene glycol, 1,3-propanedi
  • FIG. 1 Current 'top-down' metabolic engineering approach based on editing existing architecture of natural metabolism.
  • FIG. 2 Single carbon manipulation reactions for the generation of formate and formyl-CoA. Representative enzymes for each reaction are given in the legend.
  • FIG. 3 A pathway for the assimilation of single carbon molecules into carbon based products of interest. Representative enzymes for each reaction are given in the legend. Pathway intermediates that are potential products of interest without additional modification are highlighted.
  • FIG. 4. An embodiment of the invention that results in the production of saturated or unsaturated carboxylic acids by the action of a thioesterase enzyme.
  • FIG. 5. An embodiment of the invention that results in the production of alcohols by the action of an alcohol dehydrogenase enzyme.
  • FIG. 6. An embodiment of the invention that results in the production of alkanes by the action of an aldehyde decarbonylase enzyme.
  • FIG. 7. An embodiment of the invention that results in the production of amines by the action of a transaminase enzyme.
  • FIG. 8 Vector construct containing the gene encoding formate acyl- transferase pflB and its activating enzyme pflA from E. coli for overexpression in E. coli.
  • FIG. 9 Vector construct containing the gene encoding N-terminal HIS-tagged acylating aldehyde reductase Lmol 179 from Lysteria monocytogenes for expression in E. coli.
  • FIG. 10 SDS-PAGE showing expression and purification of L. monocytogenes acylating aldehyde reductase Lmol 179 from E. coli.
  • FIG. 11 Time course of absorbance at 340 nm corresponding to the production of NADH in the assay of L. monocytogenes acylating aldehyde reductase Lmol 179.
  • FIG. 12 ESI-TOF MS data of the -CoA content of L. monocytogenes acylating aldehyde reductase Lmol 179 reaction assay mixtures after solid phase extraction.
  • FIG. 13 Vector construct containing the gene encoding LcdABC from Clostridium propionicum for expression in E. coli.
  • LcdABC is an example of a 2- hydroxyacyl-CoA dehydratase.
  • FIG. 14 Eadie-Hofstee plot for the determination of Euglena gracilis TER
  • egTER enzyme kinetics.
  • egTER is an example of transenoyl-CoA reductase.
  • FIG. 15 Time course of absorbance at 340 nm corresponding to the consumption of NADH in the assay of MhpF.
  • FIG. 16 Time course of absorbance at 340 nm corresponding to the consumption of NADH in the assay of FucO.
  • FIG. 17 Time course of absorbance at 340 nm corresponding to the production of NADH in the assay of KoPddABC coupled to acyl-CoA reductase.
  • FIG. 18 HPLC chromatogram of the in vitro assembly of the trans-2-enoyl-
  • the first function of the pathway is illustrated in FIG. 2.
  • Single carbon molecules of various reduction levels are interconverted by the illustrated reactions to produce formate, the single carbon unit used to extend a carbon backbone. Details regarding the reactions and exemplary enzymes that accomplish the first function can be found in TABLE 1.
  • Methane can be oxidized to methanol (FIG. 2, reaction 1) by a suitable methane monooxygenase.
  • Methanol can be oxidized to formaldehyde (FIG. 2, reaction 2) by a suitable methanol dehydrogenase.
  • Formaldehyde can be oxidized to formate (FIG. 2, reaction 3) by an aldehyde dehydrogenase.
  • Carbon dioxide can be reduced to formate (FIG. 2, reaction 4) by a formate dehydrogenase or by electrochemical methods.
  • single carbon molecules are the solely supplied carbon source.
  • a one-carbon acyl-CoA, formyl-CoA is produced.
  • Formate can be converted to formyl-CoA either directly (FIG. 2, reaction 7) by a suitable acetyl-CoA synthetase or through the intermediate formyl-phosphate (FIG. 2, reaction 5-6) by a suitable formate kinase and phosphate acetyl-transferase.
  • Formaldehyde can also be converted to formyl-CoA by a suitable acyl-CoA reductase.
  • Combinations of the above reactions can be used to generate formyl-CoA from other single carbon molecules.
  • an implementation that makes use of methane would include the expression of a methane monooxygenase, a methanol dehydrogenase, and an acyl-CoA reductase.
  • Even more combinations of the described reactions and accompanying enzymes can be used to allow for implementations that use a mixture of single carbon units, for example a combination of methane and carbon dioxide through all of the described reactions.
  • this function can be accomplished from either formaldehyde, by the expression of an acylating aldehyde dehydrogenase, or from formate, by a suitable acetyl-CoA synthetase or combined formate kinase and phosphate acetyl-transferase.
  • the second function of the pathway is the iterative elongation of a carbon backbone by the single carbon unit formate, known as an "extender unit” herein. This is illustrated in FIG. 3. Details regarding the reactions and exemplary enzymes that accomplish the second function can be found in TABLE 1.
  • formate is condensed with an acyl-CoA to give a 2-ketoacid that is one carbon longer than the initial acyl-CoA (FIG. 3, reaction 1) by a suitable formate acyl-transferase.
  • the 2-ketoacid is then reduced to a 2-hydroxyacid (FIG. 3, reaction 2) by a suitable 2-hydroxyacid dehydrogenase.
  • the 2-hydroxyacid is then converted to a 2-hydroxyacyl-CoA (FIG. 3, reaction 3) by a suitable acyl-CoA synthetase.
  • the 2-hydroxyacyl-CoA is reduced to a 2- hydroxyaldehyde (FIG. 3, reaction 4) by a suitable acyl-CoA reductase.
  • the 2- hydroxyaldehyde is further reduced to a 1,2-diol (FIG. 3, reaction 5) by a suitable 1,2-diol oxidoreductase.
  • the 1,2-diol is then converted to an aldehyde (FIG. 3, reaction 6) by a suitable diol dehydratase.
  • the resulting aldehyde is converted to an acyl-CoA (FIG. 3, reaction 7) that is one carbon longer than the initial acyl-CoA, by an acyl-CoA reductase.
  • This acyl-CoA can be used for further rounds of elongation.
  • the 2-hydroxyacyl-CoA produced earlier is converted to a trans-2-enoyl-CoA (FIG. 3, reaction 8) by a suitable 2-hydroxyacyl- CoA dehydratase.
  • the resulting trans-2-enoyl-CoA is converted to an acyl-CoA (FIG. 3, reaction 9) that is one carbon longer than the initial acyl-CoA, by a trans-2-enoyl-CoA reductase.
  • This acyl-CoA can be used for further rounds of elongation.
  • a combination of the above routes can be implemented at the same time such that for some carbon chain lengths, elongation takes place through FIG. 3 reactions 4-7, whereas for other carbon chain lengths, elongation takes place through FIG. 3 reactions 8 and 9.
  • both routes can be simultaneously present at the same time in the same system.
  • the carbon-based products of interest are the intermediates of the above reactions. Examples of these products are highlighted in FIG. 3 and include ketoacids, hydroxyacids, aldehydes, diols and polyols. In other embodiments of the invention, products are obtained by producing products of interest from the intermediates of the above reactions.
  • alcohols such as ethanol, butanol, saturated and unsaturated fatty alcohols
  • diols such as ethylene glycol, 1,3 -propanediol, 1,4-butanediol
  • polyols such as glycerol, erythritol
  • carboxylic acids such as acetate, propionate, butyrate, crotonate, saturated and unsaturated fatty acids
  • hydroxy carboxylic acids such as glycolate, lactate, 3-hydroxybutyrate, polyhydroxybutyrate
  • dicarboxylic acids such as adipic acid, succinic acid; alkanes; alkenes; amines; polyketides; and fatty acid esters.
  • products containing carboxylic acids can be produced by providing suitable thioesterase enzymes, which convert acyl-CoAs into carboxylic acids (FIG. 4).
  • Alcohols can be produced by providing a suitable alcohol dehydrogenase enzyme, which converts aldehydes into alcohols (FIG. 5).
  • Alkanes can be produced by providing suitable aldehyde decarbonylase enzymes, which convert aldehydes into alkanes (FIG. 6).
  • Amines can be produced by providing suitable transaminase enzymes, which convert aldehydes into amines (FIG. 7).
  • the described pathway is provided within the context of a microbial host.
  • the pathway in a living system is generally made by transforming the microbe with one or more expression vector(s) containing a gene encoding one or more of the enzymes, but the genes can also be added to the chromosome by recombineering, homologous recombination, gene editing, and similar techniques.
  • the needed protein is endogenous, as is the case in some instances, it may suffice as is, but is usually overexpressed for better functionality and control over the level of active enzyme.
  • one or more, or all, such genes are under the control of an inducible promoter.
  • Such species include e.g., Bacillus, Streptomyces, Azotobacter, Trichoderma, 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.
  • yeasts are common species used for microbial manufacturing, and many species can be successfully transformed. In fact, the alpha oxidation pathway is present in yeast and thioesterases, and the reverse beta oxidation pathways have been successfully expressed in yeast Saccharomyces .
  • Other species include but are not limited to Candida, Aspergillus, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha (Pichia angusta), Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica, to name a few.
  • Spirulina Spirulina, Apergillus, 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, and the like.
  • 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 expression vectors suitable for the chosen host species. See e.g., AddGene.org, which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues.
  • Plasmid Information Database (PlasmID) and DNASU 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. Furthermore, vectors (including particular ORFS therein) are usually available from colleagues.
  • 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 3 or more ORFs for convenience, but it may be preferred to insert operons or individual genes into the genome for stability reasons.
  • culturing of the developed strains can be performed to evaluate the effectiveness of the pathway at its intended goal— the production of products from single carbon compounds.
  • the organism can be cultured in a suitable growth medium, and can be evaluated for product formation on single carbon substrates, from methane to C0 2 , either alone or in combination with multi-carbon molecules.
  • the amount of products produced by the organism can be measured by UPLC or GC, and indicators of performance such as growth rate, productivity, titer, yield, or carbon efficiency can be determined.
  • Further evaluation of the interaction of the pathway enzymes with each other and with the host system can allow for the optimization of pathway performance and minimization of deleterious effects. Because the pathway is under synthetic control, rather than under the organism's natively evolved regulatory mechanisms, the expression of the pathway is usually manually tuned to avoid potential issues that slow cell growth or production and to optimize production of desired compounds.
  • pathway intermediates which can inhibit pathway enzymes or be cytotoxic.
  • Analysis of the cell cultures by HPLC or GC can reveal the metabolic intermediates produced by the constructed strains. This information can point to potential pathway issues.
  • a cell free in vitro version of the pathway can be constructed.
  • the overall pathway can be assembled by combining the necessary enzymes in a reaction mixture.
  • the pathway can be assessed for its performance independently of a host.
  • single carbon molecules such as carbon dioxide, formate, formaldehyde, methanol, methane, and carbon monoxide are solely used in the production of products containing at least one carboxyl group.
  • both formate and the priming acyl-CoA, in the form of formyl-CoA, are produced from single carbon molecules as described earlier.
  • the priming acyl-CoA can be derived from multi-carbon substrates.
  • the invention is implemented in an engineered microbial host that is designed to be able to convert the multi-carbon molecules into an acyl-CoA.
  • Some exemplary substrates include glucose and other sugars or glycerol and other sugar alcohols, which may be converted to acyl-CoAs via a pathway such as glycolysis, resulting in the production of acetyl-CoA or malonyl-CoA.
  • Still additional examples include substrates that contain carboxyl groups, which can be directly converted to acyl-CoAs by providing a suitable acyl-CoA synthetase.
  • the multi-carbon substrates may provide additional carbon and energy for microbial survival.
  • Enzymes of interest can be expressed from vectors such as pCDFDuet-1
  • the genes can be amplified by PCR using primers designed with 15-25 base pairs of homology for the appropriate vector cut site.
  • pCDFDuet-1 can be linearized with Ncol and EcoRI. Enzymes that will be purified by Ni-NTA column will make use of the 6X-HIS tag in pCDFDuet-1.
  • the vector can be linearized using only EcoRI in this case.
  • the PCR product can be inserted into the vector using e.g., the In-Fusion HD
  • Plasmid DNA can be isolated using any suitable method, including QIAprep Spin Miniprep Kit (QIAGEN, Limburg), and the construct confirmed by PCR and sequencing. Confirmed constructs can be transformed by e.g., electroporation into a host strain such as E. coli for expression, but other host species can be used with suitable expression vectors and possible codon optimization for that host species.
  • Expression of the desired enzymes from the constructed strain can be conducted in liquid culture, e.g., shaking flasks, bioreactors, chemostats, fermentation tanks and the like. Gene expression is typically induced by the addition of a suitable inducer, when the culture reaches an OD550 of approximately 0.5-0.8. Induced cells can be grown for about 4-8 hours, at which point the cells can be pelleted and saved to -20°C. Expression of the desired protein can be confirmed by running cell pellet samples on SDS-PAGE or by enzymatic assay.
  • the expressed enzyme can be directly assayed in crude cell lysates, simply by breaking the cells by chemical, enzymatic, heat or mechanical means. Depending on the expression level and activity of the enzyme, however, purification may be required to be able to measure enzyme activity over background levels. Purified enzymes can also allow for the in vitro assembly of the pathway, allowing for its controlled characterization. N-terminal or C-terminal HIS-tagged proteins can be purified using e.g., a Ni-NTA Spin Kit (Qiagen, Venlo, Limburg) following the manufacturer's protocol, or other methods could be used. The HIS-tag system was chosen for convenience only, and other tags are available for purification uses. Further, the proteins in the final assembled pathway need not be tagged if they are for in vivo use. Tagging was convenient, however, for the enzyme characterization work performed herein.
  • reaction conditions for enzyme assays can vary greatly with the type of enzyme to be tested. In general, however, enzyme assays follow a similar general protocol. Purified enzyme or crude lysate is added to suitable reaction buffer. Reaction buffers typically contain salts, necessary enzyme cofactors, and are at the proper pH. Buffer compositions often change depending on the enzyme or reaction type. The reaction is initiated by the addition of substrate, and some aspect of the reaction related either to the consumption of a substrate or the production of a product is monitored.
  • Spectrophotometric assays are convenient because they allow for the real time determination of enzyme activity by measuring the concentration dependent absorbance of a compound at a certain wavelength. There are not always compounds with a measureable absorbance at convenient wavelengths in the reaction, unfortunately. In these situations, other methods of chemical analysis may be necessary to determine the concentration of the involved compounds.
  • Gas chromatography (GC) is convenient for the quantification of volatile substances, of which fatty acids and aldehydes are of particular relevance. Internal standards, typically one or more molecules of similar type not involved in the reaction, is added to the reaction mixture, and the reaction mixture is extracted with an organic solvent, such as hexane.
  • Fatty acid samples for example, can be dried under a stream of nitrogen and converted to their trimethylsilyl derivatives using N,0-Bis(trimethylsilyl)trifluoroacetamide ("BSTFA") and pyridine in a 1 : 1 ratio. After 30 minutes incubation, the samples are once again dried and resuspended in hexane to be applied to the GC. Aldehyde samples do not need to be derivatized. Samples can be run e.g., on a Varian CP-3800 gas chromatograph (VARIAN ASSOC., CA) equipped with a flame ionization detector and HP-5 capillary column (AGILENT TECH., CA).
  • VARIAN ASSOC., CA Varian CP-3800 gas chromatograph
  • AGILENT TECH., CA flame ionization detector
  • the pathway can be constructed in vivo with greater confidence.
  • the strain construction for the in vivo pathway operation should allow for the well-defined, controlled expression of the enzymes of the pathway.
  • E. coli, B. subtilus or yeast will be a host of choice for the in vivo pathway, but other hosts could be used.
  • the Duet system (MERCK KGaA, Germany), allows for the simultaneous expression of up to eight proteins by induction with IPTG in E. coli, and initial experiments will use this host.
  • Pathway enzymes can also be inserted into the host chromosome, allowing for the maintenance of the pathway without requiring antibiotics to ensure the continued upkeep of plasmids.
  • genes that can be placed on the chromosome can be placed on the chromosome, as chromosomal expression does not require separate origins of replication as is the case with plasmid expression.
  • DNA constructs for chromosomal integration usually include an antibiotic resistance marker with flanking FRT sites for removal, as described by Datsenko and Wanner, a well characterized promoter, a ribosome binding site, the gene of interest, and a transcriptional terminator.
  • the overall product is a linear DNA fragment with 50 base pairs of homology for the target site on the chromosome flanking each side of the construct.
  • the Flp-FRT recombination method is only one system for adding genes to a chromosome, and other systems are available, such as the RecBCD pathway, the RecF pathway, RecA recombinase, non-homologous end joining (NHEJ), Cre-Lox recombination, TYR recombinases and integrases, SER resolvases/invertases, SER integrases, PhiC31 Integrase, and the like. Chromosomal modifications in E. coli can also achieved by the method of recombineering, as originally described by Datsenko and Wanner.
  • the cells are prepared for electroporation following standard techniques, and the cells transformed with linear DNA that contains flanking 50 base pair targeting homology for the desired modification site.
  • a two-step approach can be taken using a cassette that contains both positive and negative selection markers, such as the combination of cat and sacB.
  • the cat-sacB cassette with targeting homology for the desired modification site is introduced to the cells.
  • the cat gene provides resistance to chloramphenicol, which allows for positive recombinants to be selected for on solid media containing chloramphenicol.
  • a positive isolate can be subjected to a second round of recombineering introducing the desired DNA construct with targeting homology for sites that correspond to the removal of the cat-sacB cassette.
  • the sacB gene encodes for an enzyme that provides sensitivity to sucrose.
  • growth on media containing sucrose allows for the selection of recombinants in which the cat-sacB construct was removed.
  • PI phage ly sates can be made from isolates confirmed by PCR and sequencing. The lysates can be used to transduce the modification into desired strains, as described previously.
  • Engineered strains expressing the designed pathway can be cultured under the following or similar conditions. Overnight cultures started from a single colony can be used to inoculate flasks containing appropriate media. Cultures are grown for a set period of time, and the culture media analyzed. The conditions will be highly dependent on the specifications of the actual pathway and what exactly is to be tested. For example, the ability for the pathway to be used for autotrophic growth can be tested by the use of formate or formaldehyde as a substrate in MOPS minimal media, as described by Neidhardt, supplemented with appropriate antibiotics, and inducers. Mixotrophic growth can be characterized by the addition of both single carbon compounds and glucose or glycerol.
  • Analysis of culture media after fermentation provides insight into the performance of the engineered pathway. Quantification of longer chain products can be analyzed by GC. Other metabolites, such as short chain organic acids and substrates such as glucose or glycerol can be analyzed by UPLC. Once the pathway is fully functional, the cultures can be grown in chemostat, providing continuous uninterrupted production of product in a living, growing system if desired.
  • Genome scale modeling allows for the identification of additional modifications to the host strain that might lead to improved performance. Deletion of competing pathways, for example, might increase carbon flux through the engineered pathway for product production.
  • Standard molecular biology techniques were used for gene cloning, plasmid isolation, and E. coli transformation.
  • Native E. coli genes were amplified from E. coli MG1655 genomic DNA using primers to append 15 bp of homology on each end of the gene insert for recombination into the vector backbone. Genes from other organisms were codon optimized and synthesized by either GeneArt (LIFE TECH., CA or GENSCRJPT, NJ). Plasmids were linearized by the appropriate restriction enzymes and recombined with the gene inserts using the In-Fusion HD Eco-Dry Cloning system (CLONTECH LAB. CA,). The mixture was subsequently transformed into Stellar competent cells (CLONTECH LAB.).
  • Plasmids also referred to as vectors in each case contain at least one promoter, a ribosome binding site for each gene, the gene(s) of interest, at least one terminator, an origin of replication, and an antibiotic resistance marker. Exemplary plasmids are shown in FIG. 8, 9, and 13.
  • a plasmid containing the codon optimized gene encoding 6X HIS-tagged Lmol l79 from E monocytogenes was constructed as described above.
  • the resulting construct, FIG. 9, was transformed into E. coli BL21(DE3) for expression.
  • the resulting strain was cultured in 50 mL of TB media containing 50 ⁇ g/mL spectinomycin in a 250 mL flask. When the culture reached an OD550 of approximately 0.6, expression was induced by the addition of 0.1 mM IPTG, and the cells were harvested by centrifugation after overnight incubation at room temperature. [0099] The resulting cell pellet was resuspended in Bacterial Protein Extraction
  • B-PER THERMO SCI, MA
  • SIGMA-ALDRICH CO., MO Benzonase nuclease
  • Lmol 179 was cloned (FIG. 9), expressed, and purified (FIG. 10) in E. coli as described above.
  • the purified enzyme was evaluated for its ability to convert formaldehyde into the extender unit formyl-CoA. Enzyme assays were performed in 23 mM potassium phosphate buffer pH 7.0, 1 mM CoASH, 0.5 mM NAD + , 20 mM 2-mercaptoethanol, and 50 mM formaldehyde. The reaction was monitored by measuring absorbance at 340 nm, corresponding to the production of NADH.
  • CoA compounds were extracted from the reaction mixture by solid phase extraction (SPE) using a CI 8 column, and the mass of the extracted CoAs were determined by ESI-TOF MS.
  • the inclusion of Lmol 179 in the reaction mixture resulted in the conversion of formaldehyde to formyl-CoA as indicated by the coproduction of NADH (FIG. 11).
  • Mass spectrometry analysis confirmed the production of formyl-CoA by Lmol 179.
  • a peak at the expected mass of formyl-CoA (796) was identified in the sample incubated with enzyme, as shown in FIG. 12. The peak was not present in the no-enzyme control (FIG. 12), indicating that Lmol 179 produced formyl-CoA.
  • the 2-hydroxyacyl-CoA undergoes dehydration to its corresponding trans-2-enoyl-CoA.
  • a 2-hydroxyacyl-CoA dehydratase was identified as LcdABC from C. propionicum.
  • LcdABC has been characterized by Hofmeister and Buckel for the dehydration of 2-hydroxybutyryl-CoA to crotonyl-CoA, with specific activity corresponding to 1.21 ⁇ 0.08 protein (Hofmeister & Buckel, 1992).
  • the genes encoding LcdABC were cloned as described above (FIG. 13).
  • trans-2-enoyl-CoA is then reduced to the saturated acyl-CoA by a trans-2- enoyl-CoA reductase.
  • EgTER E. gracilis
  • a variant from E. gracilis, EgTER was identified and cloned as described above.
  • in vitro assays were performed by monitoring the loss of NADH absorbance in the presence of 100 mM Tris HCL pH 7.5 and 0.2 mM NADH in a final volume of 200 [iL at 25°C. This revealed that the enzyme is capable of catalyzing the conversion of crotonyl-CoA to butyryl-CoA with specific activity corresponding to 1.21 ⁇ 0.08 ⁇ /min/mg protein (FIG. 14).
  • E. coli MhpF was expressed from an ASKA collection strain.
  • the ASKA collection is a set of ORFs cloned from E. coli K12 W3110 into the high copy number plasmid pCA24N. This plasmid has a modified pMBl replication origin (same as pQE30 from Qiagen) and CamR marker.
  • the cloned ORF is under control of the IPTG-inducible T5-lac promoter.
  • the cell debris and glass beads were pelleted by centrifugation and the supernatant comprising the cell extract was used for assays.
  • the assay mixture consisted of 100 mM MOPS pH 7.5, 6 mM DTT, 5 mM MgS0 4 , 0.3 mM Fe(NH 4 ) 2 (S0 4 ) 2 , 0.3 mM NADH, and 0.2 mM butyryl- CoA.
  • the reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of NADH, as shown in FIG. 15.
  • MhpF was capable of catalyzing the conversion with a specific activity of 0.009 ⁇ 0.003 protein.
  • E. coli FucO was assayed for its ability to perform an analogous reaction—the reduction of butyraldehyde to butanol.
  • FucO was expressed from an ASKA collection strain and purified as described above. Purified FucO was assayed in a buffer containing 100 mM Tris-HCl pH 7.5, 0.3 mM NADH, and 10 mM butyraldehyde. The reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of NADH, as shown in FIG. 16. FucO was capable of catalyzing the reduction of butyraldehyde to butanol with a specific activity of 5.08 ⁇ 0.08 ⁇ /min/mg protein.
  • FucO was tested for the ability to convert glycoaldehyde, a 2-hydroxyaldehyde, to ethane- 1,2-diol, as necessary for Scheme A. FucO was capable of catalyzing the reduction with a specific activity of 4.060 ⁇ 0.004 ⁇ /min/mg protein.
  • KoPddABC a diol dehydratase from Klebsiella oxytoca, was cloned, expressed, and purified as described.
  • Cell extracts of E. coli BL21(DE3) expressing KoPddABC were prepared by resuspending a pellet of said E. coli to an OD 550 of 40 in 60 mM potassium phosphate buffer pH 7.4 with 200 mM 1,2-ethanediol. 1 mL of the cell suspension was added to 0.75 g of glass beads and the cells were disrupted for 3 minutes using a cell disruptor (SCI. INDUS. NY). The cell debris and glass beads were pelleted by centrifugation and the supernatant comprising the cell extract was used for assays.
  • the cell extract was incubated at 30°C for 3 hours in the presence of 10 ⁇ coenzyme B 12. The reaction was terminated by the addition of 1% sulfuric acid, and the precipitant was pelleted by centrifugation. The supernatant was analyzed by HPLC. Acetaldehyde was detected in extracts of cells expressing KoPddABC, indicating that the dehydratase can convert 1,2-diols to their corresponding aldehydes.
  • Diol dehydratase was further assayed by coupling the dehydration of ethylene glycol to acetaldehyde to an acyl- CoA reductase (LmACR) to give acetyl-CoA with the reduction of NAD+ to NADH, which was monitored at 340 nm.
  • LmACR acyl- CoA reductase
  • the final assay mixture was 250 ⁇ . and contained 50 mM potassium phosphate pH 7.5, 5 mM CoASH, 0.5 mM NAD+, 0.2 M ethylene glycol, 7 iL purified LmACR (from frozen stock purified previously), 50 iL cell lysate, and 15 ⁇ coenzyme B 12.
  • the relevant controls included were no cell lysate (replaced with 50 ⁇ . of buffer) and no coenzyme B12.
  • the assays were performed at 28°C. The results are shown in FIG. 17.
  • a portion of the proposed invention was assembled in vitro to demonstrate the functionality of the combined pathway steps.
  • the trans-2-enoyl-CoA reductase and acyl-CoA reductase steps were combined to assess the overall conversion of crotonyl-CoA to butyraldehyde.
  • Experiments were carried out carried out in 50 mM Tris buffer, pH 7.5 containing 1 mM DTT at 37°C.
  • the reaction with MhpF contained 0.15 g/L TdTer, 0.1 g/L of MhpF, 7.5 mM NADH and 1.7 mM crotonyl-CoA was added to the media for use as a primer in the new reaction pathway.
  • an engineered E. coli strain MG1655(DE3) AfrmA AfdhFON serves as the host strain.
  • This strain contains deletions of genes that compete for one carbon substrates formate and formaldehyde.
  • Genes for overexpression are cloned into vectors or inserted into the chromosome using standard methods described above. Vectors and chromosomal constructs harbored by the host strain to give the engineered strains of interest.
  • an acyl-CoA synthetase is selected for overexpression to catalyze the conversion of formate into formyl-CoA.
  • a vector for example pETDuet-l-Pl-acs, expressing an acyl-CoA synthetase from E. coli, is constructed and transformed into the host strain.
  • a carbon dioxide reductase catalyzing the conversion of carbon dioxide to formate is added.
  • pETDuet-l-Pl-acs-P2-AwFdhF2-hycB2-hycB3-hydA2 expressing carbon dioxide reductase from Acetobacterium woodii is constructed and transformed into the host strain.
  • formaldehyde is the intended one carbon substrate
  • an acyl-CoA reductase is selected for overexpression to catalyze the conversion of formaldehyde to formyl-CoA.
  • Hydrolysis of formyl-CoA to formate is expected to occur spontaneously, but can be aided by expression of a thioesterase such as E. coli tesA.
  • a vector such as pETDuet-l-Pl-Lmol l79, expressing an acyl-CoA reductase from L. monocytogenes is constructed and transformed into the host strain.
  • a methanol dehydrogenase is added.
  • a vector such as pETDuet-l-Pl-Lmol 179-P2-BsMdh2, expressing a methanol dehydrogenase from Bacillus stearothermophilus is constructed and transformed into the host strain.
  • methane monooxygenase When methane is the intended one carbon substrate, a methane monooxygenase is added, giving for example pETDuet-l-Pl-Lmol l79-P2-BsMdh2-MtMmoXYZBC-orfY, which further expresses a methane monooxygenase from Methylosinus trichosporium OB3b.
  • a vector such as pRSFDuet-l-Pl-pflAB- P2-glcD expressing a 2-hydroxyacid dehydrogenase from E.
  • coli is constructed.
  • 1,2- diols are the desired product, overexpression of an acyl-CoA synthetase, acyl-CoA reductase, and 1,2-diol oxidoreductase are added.
  • a vector such as pRSFDuet-l-Pl-pflAB-P2-glcD-acs- Lmol l79-fucO is constructed containing an acyl-CoA synthetase from E. coli, an acyl-CoA reductase from J. monocytogenes, and a 1,2-diol oxidoreductase ⁇ ⁇ . coli.
  • an acyl-CoA must be regenerated for further condensation with formate by formate acyltransferase.
  • the acyl-CoA can be produced by overexpression of diol dehydratase and an acyl-CoA reductase.
  • a vector such as pRSFDuet-l-Pl-pflAB-P2-glcD-acs-Lmol l79-fucO-KoPddABC is constructed containing a diol dehydratase from K. oxytoca.
  • the acyl-CoA reductase Lmol l79 catalyzes two acyl-CoA reductase reactions.
  • Longer carbon chain products can also be obtained through a separate route, by overexpression of a 2-hydroxyacyl-CoA dehydratase and a trans-2-enoyl- CoA reductase to give the acyl-CoA.
  • a suitable example vector would be pRSFDuet-l-Pl- pflAB-P2-glcD-acs-CpLcdABC-EgTER, containing genes for the formate acyltransferase, 2- hydroxyacid dehydrogenase, acyl-CoA synthetase, along with a 2-hydroxyacyl-CoA dehydratase from C. propionicum, and a trans-2-enoyl-CoA reductase from E. gracilis.
  • any combination of a 'substrate conversion vector' and a 'product conversion vector' described above can be co-transformed into the host strain.
  • pETDuet-1 and pRSFDuet-1 are compatible for simultaneous maintenance an E. coli.
  • the host strain would be transformed with pETDuet-l-Pl-Lmol 179-P2-BsMdh2 and pRSFDuet-l-Pl-pflAB- P2-glcD to give the strain with genotype MG1655(DE3) AfrmA AfdhFON pETDuet-l-Pl- Lmol 179-P2-BsMdh2 pRSFDuet-l-Pl-pflAB-P2-glcD.
  • An exemplary substrate to product conversion is performed as follows. The engineered E. coli is grown overnight in LB medium at 37°C. 250 ⁇ .
  • MOPS-LB-Glycerol medium 125 mM MOPS, 4 mM Tricine, 39.52 mM H 4 C1, 5 mM ( H 4 ) 2 S0 4 , 0.276 mM K 2 S0 4 , 0.523 mM MgCl 2 , 50 mM NaCl, 0.01 mM FeS0 4 , 2.8 mM Na 2 HP0 4 , 0.5 ⁇ CaCl 2 , 0.004 ⁇ ( H 4 ) 6 Mo 7 0 24 , 0.4 ⁇ H 3 B0 3 , 0.03 uM CoCl 2 , 0.01 ⁇ CuS0 4 , 0.08 ⁇ MnCl 2 , 0.01 ⁇ ZnS0 4 , 15 uM thiamine, 10 g/L tryptone, 5 g/L yeast extract, 20 g/L glycerol) in a 125 mL baffled flask
  • MOPS minimal medium the composition of MOPS minimal medium is the same as MOPS-LB-Glycerol without the tryptone, yeast extract, and glycerol components.
  • the pellet containing the engineered E. coli is resuspended in MOPS minimal medium and the one carbon substrate of choice is added.
  • the substrate is provided by bubbling into the cell suspension or by adding to the headspace of a sealed container. After 24 hours, the culture medium containing the desired product is recovered and saved for analysis or product isolation.
  • the purpose of this example is to demonstrate the biosynthesis of chemicals by implementation of the described invention.
  • Vectors expressing the necessary enzymes to catalyze the desired substrate to product conversions are prepared as described above.
  • E. coli strain BL21(DE3) is transformed with the necessary vectors to catalyze the desired substrate to product conversion using standard methods.
  • the resulting engineered strain is grown overnight in LB medium at 37°C.
  • the overnight culture is used to inoculate a larger volume of LB medium to 1%.
  • the culture is grown at 30°C to an OD550 of 0.4 to 0.6 at which point gene expression is induced by adding IPTG to a concentration of 0.1 mM. 24 hours after inoculation, cells are harvested by centrifugation and the pellet is saved by freezing.
  • the pellet When ready, the pellet is thawed and the cells are resuspended in 50 mM potassium phosphate buffer pH 7.4. The cells are broken by glass beads and the cell extract containing the expressed proteins is collected after centrifugation. The one carbon substrate, as well as any necessary cofactors, are added to the cell extract and the reaction mixture is incubated at room temperature for 16 hours. The reaction mixture is saved for analysis or product isolation.
  • a portion of the proposed invention was assembled in vitro to demonstrate the functionality of the combined pathway steps.
  • the trans-2-enoyl-CoA reductase and acyl-CoA reductase steps were combined to assess the overall conversion of crotonyl-CoA to butyraldehyde.
  • Experiments were carried out carried out in 50 mM Tris buffer, pH 7.5 containing 1 mM DTT at 37°C.
  • the reaction with MhpF contained 0.15 g/L TdTer, 0.1 g/L of MhpF, 7.5 mM NADH and 1.7 mM crotonyl-CoA was added to the media for use as a primer in the new reaction pathway.
  • WO2015191972 Omega-carboxylated carboxylic acids and derivatives
  • WO2015191422 Omega-hydroxylated carboxylic acids

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Abstract

La présente invention porte également sur un procédé de biosynthèse de produits à base de carbone d'intérêt par l'assimilation de matières premières de carbone uniques. Une voie de réaction qui consolide la fixation du carbone, le métabolisme central et la synthèse du produit est conçue et mise en œuvre sous la forme d'un micro-organisme génétiquement modifié ou d'un mélange de réaction enzymatique. La présente invention utilise un mécanisme de formation de liaisons carbone-carbone qui n'a pas été précédemment exploité à cet effet et qui est basé sur l'utilisation de formiate acyltransférases. Le résultat est un procédé qui permet la production de produits chimiques par l'utilisation soit uniquement de matières premières de carbone uniques, soit d'un mélange de matières premières mono- et multi-carbonées.
PCT/US2017/035364 2016-06-02 2017-06-01 Bioconversion de matières premières de 1-carbone en produits chimiques et en combustibles WO2017210381A1 (fr)

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WO2023287814A3 (fr) * 2021-07-12 2023-03-02 University Of South Florida Plateforme d'acide carboxylique pour la production de combustible et de produits chimiques à un rendement élevé en carbone et en énergie

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US20100235934A1 (en) * 2007-05-22 2010-09-16 Ls9, Inc. Hydrocarbon-producing genes and methods of their use
US20110250654A1 (en) * 2008-10-28 2011-10-13 Glycos Biotechnologies, Inc. Microaerobic cultures for converting glycerol to chemicals
US20130052705A1 (en) * 2010-06-21 2013-02-28 William Marsh Rice University Engineered bacteria produce succinate from sucrose
US20150037853A1 (en) * 2011-10-31 2015-02-05 Ginkgo Bioworks, Inc. Methods and Systems for Chemoautotrophic Production of Organic Compounds
WO2015084633A1 (fr) * 2013-12-03 2015-06-11 Genomatica, Inc. Microorganismes et procédés pour améliorer les rendements de produits sur le méthanol faisant appel à la synthèse de l'acétyl-coa

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US20100235934A1 (en) * 2007-05-22 2010-09-16 Ls9, Inc. Hydrocarbon-producing genes and methods of their use
US20110250654A1 (en) * 2008-10-28 2011-10-13 Glycos Biotechnologies, Inc. Microaerobic cultures for converting glycerol to chemicals
US20130052705A1 (en) * 2010-06-21 2013-02-28 William Marsh Rice University Engineered bacteria produce succinate from sucrose
US20150037853A1 (en) * 2011-10-31 2015-02-05 Ginkgo Bioworks, Inc. Methods and Systems for Chemoautotrophic Production of Organic Compounds
WO2015084633A1 (fr) * 2013-12-03 2015-06-11 Genomatica, Inc. Microorganismes et procédés pour améliorer les rendements de produits sur le méthanol faisant appel à la synthèse de l'acétyl-coa

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WO2023287814A3 (fr) * 2021-07-12 2023-03-02 University Of South Florida Plateforme d'acide carboxylique pour la production de combustible et de produits chimiques à un rendement élevé en carbone et en énergie

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