WO2017036740A1 - Préparation biologique de 5-aminovalérate - Google Patents

Préparation biologique de 5-aminovalérate Download PDF

Info

Publication number
WO2017036740A1
WO2017036740A1 PCT/EP2016/068907 EP2016068907W WO2017036740A1 WO 2017036740 A1 WO2017036740 A1 WO 2017036740A1 EP 2016068907 W EP2016068907 W EP 2016068907W WO 2017036740 A1 WO2017036740 A1 WO 2017036740A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
enzyme
aminovalerate
seq
recombinant cell
Prior art date
Application number
PCT/EP2016/068907
Other languages
English (en)
Inventor
Joao Jorge
Volker Wendisch
Original Assignee
Universität Bielefeld
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universität Bielefeld filed Critical Universität Bielefeld
Publication of WO2017036740A1 publication Critical patent/WO2017036740A1/fr

Links

Classifications

    • 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.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids

Definitions

  • the present invention relates generally to genetic and metabolic engineering and biological preparation of 5-aminovalerate, and more specifically to a recombinant cell genetically engineered to produce an increased amount of 5-aminovalerate compared to its wildtype, a method for preparing 5-aminovalerate using the recombinant cell, and the use of the recombinant cell for preparing 5-aminovalerate.
  • 5 -Amino valerate is a potential C5 platform chemical for synthesis of valerolactam, 5- hydroxyvalerate, glutarate, and 1,5-pentanediol. It is naturally produced from L-lysine through the 5-aminovalerate pathway, wherein L-lysine 2-monooxygenase (DavB) catalyzes L-lysine into 5-aminovaleramide and delta-aminovaleramidase (DavA) further converts 5- aminovaleramide into 5-aminovalerate.
  • DavB L-lysine 2-monooxygenase
  • DavA delta-aminovaleramidase
  • the present invention therefore relates to a recombinant cell, wherein said cell has been genetically engineered such that it produces an increased amount of 5- aminovalerate compared to its wildtype.
  • the recombinant cell is a microbial cell, preferably a microbial cell selected from the group consisting of Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, P
  • the recombinant cell has, compared to its wildtype, (1) an increased activity of an enzyme El that catalyzes the amino group transfer of cadaverine; and/or (2) an increased activity of an enzyme E2 that catalyzes the dehydrogenation of 5- aminopentanal.
  • enzyme El is a putrescine aminotransferase.
  • Enzyme El may be an EC 2.6.1.82 class enzyme, for example an PLP-dependent putrescine:2-oxoglutaric acid aminotransferase (PatA).
  • enzyme El a) has the amino acid sequence of SEQ ID NO: l ; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).
  • enzyme E2 is a ⁇ -aminobutyraldehyde dehydrogenase.
  • Enzyme E2 may be an EC 1.2.1.19 class enzyme, for example a ⁇ -aminobutyraldehyde dehydrogenase (PatD).
  • enzyme E2 a) has the amino acid sequence of SEQ ID NO:2; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).
  • the recombinant cell has, compared to its wildtype, an increased activity of an enzyme E3 that catalyzes the decarboxylation of lysine.
  • Enzyme E3 may be a lysine decarboxylase, e.g. an EC 4.1.1.18 class enzyme.
  • enzyme E3 is Escherichia coli lysine decarboxylase (LdcC).
  • enzyme E3 a) has the amino acid sequence of SEQ ID NO:3; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).
  • the recombinant cell of the invention is a Corymb acterium glutamicum cell, preferably a Corymb acterium glutamicum GRLysl cell, wherein (a) the sugR gene as set forth in SEQ ID NO: 8 or a fragment thereof is deleted in said cell; (b) the IdhA gene as set forth in SEQ ID NO: 9 or a fragment thereof is deleted in said cell; (c) the snaA gene as set forth in SEQ ID NO: 10 or a fragment thereof is deleted in said cell; (d) the lysE gene as set forth in SEQ ID NO: 11 or a fragment thereof is deleted in said cell; and/or (e) the lysG gene as set forth in SEQ ID NO: 12 or a fragment thereof is deleted in said cell.
  • the invention is directed to a method for preparing 5-aminovalerate, the method comprising culturing the recombinant cell in a growth medium under conditions allowing production of 5-aminovalerate, and recovering 5-aminovalerate from the medium.
  • the invention encompasses use of the recombinant cell for preparing 5-aminovalerate.
  • Figure 1 is a schematic diagram showing a pathway for 5-aminovalerate production, wherein lysine decarboxylase (LdcC) catalyzes the conversion of L-lysine into cadaverine, PatA catalyzes the conversion of cadaverine into 5-aminopentanal, and PatD catalyzes the conversion of 5-aminopentanal into 5-aminovalerate.
  • LdcC lysine decarboxylase
  • Figure 2 is a gene map of recombinant plasmid )EKEx3-patDA encoding PatA and PatD derived from Escherichia coli.
  • Figure 3 is a gene map of recombinant plasmid pVWExl -IdcC encoding LdcC derived from Escherichia coli.
  • FIG. 4 is a schematic representation of a new metabolic route from l-lysine to 5AVA. Enzymatic reactions and the involved proteins are depicted. Dashed arrows represent more than one enzymatic reaction. Enzymes in grey boxes were added by heterologous expression of the respective genes from E. coli and are unknown in C. glutamicum. Abbreviations: 5AVA, 5- aminovalerate; LdcC, l-lysine decarboxylase; PatA, putrescine transaminase; PatD, 5- aminopentanal dehydrogenase; LysE, 1-lysine efflux permease; CgmA, putative putrescine/cadaverine efflux permease.
  • 5AVA 5- aminovalerate
  • LdcC l-lysine decarboxylase
  • PatA putrescine transaminase
  • PatD 5- aminopentanal dehydrogenase
  • LysE 1-lysine efflux permease
  • Figure 5 shows the dependence of the growth rate of C. glutamicum on the 5AVA concentration added to glucose minimal medium.
  • Figure 6 shows the growth (filled circles) and 5AVA concentration (empty circles) in C. glutamicum wild type.
  • Cells were grown in CGXII medium with 4% (w/v) glucose and supplemented with about 20 mM of 5AVA after 9h of growth. Values and error bars represent the mean and the standard deviation of duplicate cultivations.
  • Figure 7 shows genes that are differentially expressed in Corynebacterium glutamicum grown in glucose minimal medium in the presence of 5AVA as compared to sodium chloride.
  • Figure 8 shows accumulation of 5AVA and side-products by different C. glutamicum strains.
  • the object of the present invention is to provide a means for biological preparation of 5- aminovalerate.
  • 5-aminovalerate production adopting a new pathway, i.e. L-lysine to cadaverine to 5-aminopentanal to 5-aminovalerate, is enabled by recombinant overexpression of certain enzymes essential for said pathway in a cell.
  • the term "cadaverine” refers to pentane- 1,5 -diamine
  • the term “5- aminovalerate” refers to 5-aminopentanoate.
  • the present invention relates to a recombinant cell, wherein said cell has been genetically engineered such that it produces an increased amount of 5-aminovalerate compared to its wildtype.
  • wildtype is preferably understood as meaning a cell whose genome has been generated naturally by evolution. The term is used both for the cell as a whole and for individual genes. Consequently, the term “wildtype” specifically does not include those cells and genes whose gene sequences have been modified at least partially by humans by means of recombinant processes.
  • the term "genetically engineered”, as used herein, refers to the use of molecular biology methods to manipulate nucleic acid sequences and introduce nucleic acid molecules into host organisms.
  • the term "recombinant cell”, as used herein, means a cell that has been subjected to recombinant DNA manipulations, such as the introduction of exogenous nucleic acid molecule, resulting in a cell that is in a form not found originally in nature. Recombinant cells are generated, for example, by transformation, transfection, transduction, conjugation or a combination of these methods, using a vector which contains the desired gene, an allele of said gene or parts thereof and a promoter enabling the gene to be expressed.
  • Overexpression is achieved in particular by integrating the gene or the alleles in the chromosome of the cell or in an extrachromosomally replicating vector.
  • Methods of creating recombinant cells are well known in the art. For example, see MOLECULAR CLONING: A LABORATORY MANUAL (Sambrook et al, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989), or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., eds., John Wiley & Sons, New York, 1987).
  • the term "increased amount”, as used herein in relation to the production of 5- aminovalerate by the described cells compared to wildtype cells, means that the recombinant cell produces at least twice, particularly preferably at least 10 times, additionally preferably at least 100 times, additionally still more preferably at least 1000 times and most preferably at least 10 000 times more 5-aminovalerate than the wildtype cell within a defined time interval.
  • the increase in product formation may be determined, for example, by culturing the recombinant and wildtype cells separately under the same conditions (same cell density, same growth medium, same culturing conditions) in a suitable growth medium for a particular time interval and then determining the amount of 5-aminovalerate in the cell supernatant.
  • the cell of the present invention may be a prokaryote or eukaryote cell and may be a mammalian cell, a plant cell or a microbial cell such as yeast, fungus or bacterium, with particular preference being given to microbial cells.
  • the recombinant cell is a microbial cell selected from the group consisting of Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas a
  • the recombinant cell has, compared to its wildtype, (1) an increased activity of an enzyme El that catalyzes the amino group transfer of cadaverine; and/or (2) an increased activity of an enzyme E2 that catalyzes the dehydrogenation of 5- aminopentanal.
  • an increased activity of an enzyme in a recombinant cell refers to an increase in the activity determined relative to the wildtype cell. Increased activities can be the result of, for example, increased amount of protein expressed by a recombinant cell (e.g., as the result of increased number of copies of DNA sequences encoding the protein, increased number of mRNA transcripts encoding the protein, and/or increased amount of protein translation of the protein from mRNA); changes in the structure of the protein (e.g., changes to the primary structure, such as, changes to the protein's coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters); and changes in protein stability (e.g., decreased degradation of the protein).
  • increased amount of protein expressed by a recombinant cell e.g., as the result of increased number of copies of DNA sequences encoding the protein, increased number of mRNA transcripts encoding the protein, and/or increased amount of protein translation of the protein from mRNA
  • changes in the structure of the protein
  • the coding polynucleotide sequences for the proteins described herein are codon optimized for expression in a particular cell.
  • the increased enzyme activity is, for example, increased by at least 20 %, preferably at least 50 %, more preferably at least 100 % compared to the wildtype cell and may be determined by suitable methods known to those skilled in the art.
  • enzyme El that catalyzes the amino group transfer of cadaverine includes any enzyme that catalyzes the exchange of an amine group of cadaverine with a keto group of another molecule, and thereby converts cadaverine into 5-aminopentanal.
  • enzyme E2 that catalyzes the dehydrogenation of 5-aminopentanal includes any enzyme that catalyzes the removal of hydrogen atoms from the aldehyde group of 5-aminopentanal, and thereby converts 5-aminopentanal into 5 -amino valerate.
  • the enzyme El is a putrescine aminotransferase and/or (2) the enzyme E2 is a ⁇ -aminobutyraldehyde dehydrogenase.
  • Putrescine aminotransferases are best known in the art to catalyze the aminotransferase reaction from putrescine to 2-oxoglutarate, leading to glutamate and 4-aminobutanal, but it has been found that these enzymes can also catalyse transamination of cadaverine.
  • Dehydrogenases are a class of enzymes that catalyze the removal of hydrogen atoms from a particular molecule. It is known in the art that ⁇ -aminobutyraldehyde dehydrogenases from some species exhibit broad substrate specificity and have a marked preference for straight- chain aldehydes (up to 7 carbon atoms) as substrates.
  • enzyme El is an EC 2.6.1.82 class enzyme.
  • the abbreviation "EC” and accompanying notations, as used herein, are references to the enzyme classification as established by the nomenclature committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMD).
  • enzyme El is PLP-dependent putrescine:2- oxoglutaric acid aminotransferase (PatA). This enzyme can exchange the amine group of cadaverine with the keto group of alpha-ketoglutarate to produce 5-aminopentanal and glutamate.
  • enzyme El a) has the amino acid sequence of SEQ ID NO: l ; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).
  • said enzyme El has an amino acid sequence that has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% sequence identity to SEQ ID NO: l .
  • Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA.
  • the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity or similarity or homology and performs a statistical analysis of the identity or similarity or homology between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
  • the term "functional fragment”, as used herein, refers to a fragment of the enzyme that retains the characteristic catalytic activity of said enzyme.
  • enzyme E2 is an EC 1.2.1.19 class enzyme.
  • enzyme E2 is ⁇ -aminobutyraldehyde dehydrogenase (PatD). This enzyme acts on the aldehyde group of 5-aminopentanal with oxidized beta- nicotinamide adenine dinucleotide (NAD + ) as acceptor to produce 5-aminovalerate and reduced beta-nicotinamide adenine dinucleotide (NADH).
  • PatD ⁇ -aminobutyraldehyde dehydrogenase
  • enzyme E2 a) has the amino acid sequence of SEQ ID NO:2; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).
  • said enzyme E2 has an amino acid sequence that has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% sequence identity to SEQ ID NO:2.
  • the recombinant cell has, compared to its wildtype, an increased activity of an enzyme E3 that catalyzes the decarboxylation of lysine.
  • enzyme E3 that catalyzes the decarboxylation of lysine includes any enzyme that catalyzes the conversion of L-lysine into cadaverine.
  • enzyme E3 is a lysine decarboxylase.
  • Decarboxylases are a class of enzymes that catalyze the removal of a carbon dioxide from a carboxylic group.
  • Lysine decarboxylases are enzymes that catalyze the decarboxylation of L-lysine generating cadaverine.
  • enzyme E3 is an EC 4.1.1.18 class enzyme.
  • enzyme E3 is Escherichia coli lysine decarboxylase (ldcC).
  • enzyme E3 a) has the amino acid sequence of SEQ ID NO:3; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).
  • said enzyme E3 has an amino acid sequence that has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% sequence identity to SEQ ID NO:3.
  • a hybrid enzyme having the enzymatic activities of enzymes El and E2 and/or enzyme E3 and having the desired substrate specificity for 5-aminovalerate synthesis may be used, with such an enzyme being generated by recombinant DNA technology.
  • an enzyme which does initially not have the specific properties as defined herein can be modified, for example by altering the amino acid sequence thereof, in order to generate an enzyme having the enzymatic activities of enzymes El and E2 and/or enzyme E3 and having the desired substrate specificity.
  • the recombinant cell is further genetically engineered to increase the production of 5-aminovalerate, for example, by promoting the uptake or synthesis of a precursor thereof such as L-lysine or cadaverine, enhancing a positive feedback or reducing a negative feedback regulating biosynthesis thereof, reducing the catabolism or degradation thereof, or increasing the tolerance of the cell towards 5-aminovalerate and precursors thereof.
  • a precursor thereof such as L-lysine or cadaverine
  • enhancing a positive feedback or reducing a negative feedback regulating biosynthesis thereof reducing the catabolism or degradation thereof, or increasing the tolerance of the cell towards 5-aminovalerate and precursors thereof.
  • the recombinant cell of the invention is a Corynebacterium glutamicum cell, preferably a Corynebacterium glutamicum GRLysl cell, wherein (a) the sugR gene as set forth in SEQ ID NO: 8 or a fragment thereof is deleted in said cell; (b) the ldhA gene as set forth in SEQ ID NO: 9 or a fragment thereof is deleted in said cell; (c) the snaA gene as set forth in SEQ ID NO: 10 or a fragment thereof is deleted in said cell; (d) the lysE gene as set forth in SEQ ID NO: l 1 or a fragment thereof is deleted in said cell; and/or (e) the lysG gene as set forth in SEQ ID NO: 12 or a fragment thereof is deleted in said cell.
  • the deleted fragments of the nucleotide sequences set forth in SEQ ID Nos: 8-12 comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 80, at least 100, at least 130, at least 150, at least 180, at least 200 or at least 250 nucleotides.
  • Corynebacterium glutamicum GRLysl cell is well-known in art by Unthan et al. (Unthan et al., Biotechnol. J. 2015, 10, 290-301) and also characterized in Table 1 (B) of this document.
  • the invention is directed to a method for preparing 5-aminovalerate, the method comprising culturing the recombinant cell in a growth medium under conditions allowing production of 5-aminovalerate, and recovering 5-aminovalerate from the medium.
  • exogenous L-lysine is introduced to the culture in a suitable manner, for example added at the beginning or at a certain stage during culturing or fed continuously, to promote the synthesis of 5-aminovalerate by the recombinant cell.
  • cadaverine is directly introduced into the culture as the substrate.
  • the recombinant cell according to the invention may be cultured in growth medium in a continuous process or in a batch process (batch culture) or in a fed-batch process or repeated fed-batch process for the purpose of producing 5-aminovalerate.
  • a semi-continuous process, as described in GB-A-1009370 for example, is also conceivable.
  • a review of other known culturing methods is described in the textbook by Chmiel ("Bioreatechnik 1. Einbowung in die Biovonstechnik" [Bioprocessing 1.
  • the growth medium to be used must be suited to the requirements of the particular cell.
  • Culture media for various microorganisms are described in the "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D. C, USA, 1981).
  • Carbon sources which may be used are carbohydrates [such as, for example, monosaccharides (e.g. glucose, fructose, galactose, arabinose, xylose), oligosaccharides (e.g. maltose, saccharose, lactose), and polysacharides (e.g. starch, hydrolysed starch, cellulose, hydrolysed cellulose, hemicellulose, hydrolysed hemicellulose)], amino sugars (e.g. glucosamine, N-acetylglucosamine), and reaction products thereof such as, for example, sugar alcohols and polyhydroxy acids; carbon dioxide; organic mono-, di- and tricarboxylic acids optionally carrying 1 or more, e.g.
  • carbohydrates such as, for example, monosaccharides (e.g. glucose, fructose, galactose, arabinose, xylose), oligosaccharides (e.g. maltose, saccharose, lactose
  • hydroxyl groups e.g. acetic acid, tartaric acid, itaconic acid, succinic acid, propionic acid, lactic acid, 3-hydroxypropionic acid, fumaric acid, maleic acid, 2,5-furandicarboxylic acid, glutaric acid, laevulinic acid, gluconic acid, aconitic acid, succinic acid and diaminopimelic acid, citric acid; lipids; oils or fats such as, for example, rapeseed oil, soya oil, palm oil, sunflower oil, groundnut oil and coconut oil; saturated and unsaturated fatty acids, preferably with from 10 to 22 carbons, for example ⁇ -linolenic acid, dihomo-y-linolenic acid, arachidonic acid, palmitic acid, stearic acid, linoleic acid, eicosapentaenoic acid and docosahexaenoic acid; hydrocarbons such as methane, 1, 2, 3
  • butanol methanol, ethanol
  • diols preferably with from 3 to 8 carbons, e.g. propanediol and butanediol
  • polyhydric (also referred to as higher) alcohols with 3 or more, for example 3, 4, 5 or 6, OH groups, e.g. glycerol, sorbitol, mannitol, xylitol and arabinitol
  • ketones preferably with from 3 to 10 carbons and, where appropriate, 1 or more hydroxyl groups, e.g. acetone and acetoin
  • lactones e.g. ⁇ -butyrolactone, cyclodextrins, biopolymers, e.g.
  • polyesters e.g. polylactide, polysaccharides, polyisoprenoids, polyamides; aromatic compounds, e.g. aromatic amines, vanillin and indigo; proteins, for example enzymes such as amylases, pectinases, acidic, hybrid or neutral cellulases, esterases such aslipases, pancreases, proteases, xylanases and oxidoreductases such as laccase, catalase and peroxidase, glucanases, phytases; carotenoids, e.g.
  • lycopene (3-carotene, astaxanthin, zeaxanthin and canthaxanthin; proteinogenic and non-proteinogenic amino acids, e.g. lysine, glutamate, methionine, phenylalanine, aspartic acid, tryptophan and threonine; purine and pyrimidine bases; nucleosides and nucleotides, e.g. nicotinamide-adenine dinucleotide (NAD) and adenosine 5'-monophosphate (AMP); and also precursors and derivatives, for example salts of the acids mentioned, of the compounds mentioned above. [073] These substances may be used individually or as mixture. Particular preference is given to using carbohydrates, in particular glucose.
  • Nitrogen sources which may be used are organic compounds containing nitrogen, such as glucosamine, N-acetylglucosamine, peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • the nitrogen sources may be used individually or as mixture.
  • Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium salts.
  • the culture medium should furthermore contain metal salts such as, for example, magnesium sulphate or iron sulphate, which are required for growth.
  • Essential growth substances such as amino acids and vitamins may be used in addition to the substances mentioned above.
  • the pH of the culture is controlled by using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulphuric acid in a suitable manner.
  • Foaming can be controlled by using antifoams such as fatty acid polyglycol esters, for example.
  • Aerobic conditions may be maintained by introducing into the culture oxygen or oxygen-containing gas mixtures such as air, for example.
  • the culture temperature is usually from 20 °C to 45 °C and preferably from 25 °C to 40 °C.
  • the purification of 5 -amino valerate from the growth medium is preferably carried out continuously, it being furthermore preferred in this context also to produce 5-aminovalerate by fermentation in a continuous manner, so that the entire process from production of 5- aminovalerate up to its purification from the fermentation broth can be carried out continuously.
  • the preparation of 5-aminovalerate from the growth medium is continuously passed through a device for removing the cells employed during fermentation, preferably through a filter with a cut-off in the range from 20 to 200 kDa, where solid/liquid separation takes place.
  • centrifuge a suitable sedimentation device or a combination of these devices, it being especially preferred to first separate at least part of the cells by sedimentation and subsequently to feed the fermentation broth, which has been partly relieved of the cells, to ultrafiltration or to a centrifugation device.
  • the fermentation product which is enriched with regard to its 5-aminovalerate content is fed to a separation system, preferably a multistep separation system.
  • This separation system provides for a plurality of separation steps which are connected in series, from which steps in each case return lines lead away and back to the fermentation tank. Furthermore, exit pipes lead out of the respective separation steps.
  • the individual separation steps may operate by electrodialysis, reverse osmosis, ultrafiltration or nanofiltration. As a rule, the individual separation steps comprise membrane separation devices. The individual separation steps are selected due to the nature and the extent of the fermentation by-products and residual substrates.
  • 5- aminovalerate may also be removed by extractive methods from the fermentation solution which has been relieved of cells, in which case pure 5 -amino valerate may ultimately be obtained.
  • the 5-aminovalerate prepared by the method according to the invention may be neutralized before, during or after purification, for which purpose for example bases such as alkali metal or alkaline earth metal hydroxides, e.g. calcium hydroxide or sodium hydroxide or else N3 ⁇ 4 or NH4OH for example, can be employed.
  • bases such as alkali metal or alkaline earth metal hydroxides, e.g. calcium hydroxide or sodium hydroxide or else N3 ⁇ 4 or NH4OH for example, can be employed.
  • an in vitro cell-free system is established and utilized for the production of 5-aminovalerate.
  • cadaverine may be contacted with purified enzymes El and E2, or L- lysine may be contacted with purified enzymes El, E2 and E3 to produce 5-aminovalerate.
  • the invention encompasses use of the recombinant cell for preparing 5-aminovalerate.
  • lysE cgl424
  • lysG cgl425
  • snaA cgl722
  • Escherichia coli DH5a cells were used for gene cloning.
  • Corynebacterium glutamicum and Escherichia coli cells were routinely grown in lysogeny broth (LB) (10 g/L of tryptone, 5 g/L yeast extract and 10 g/L sodium chloride) in 500 mL baffled flasks on a rotary shaker (120 rpm) at 30 °C or 37 °C, respectively.
  • CGXII minimal medium was also used for general growth of Corynebacterium glutamicum. Growth was followed by measuring optical density at 600 nm. An optical density of 1 at this wavelength corresponds to 0.25 g/L cell dry weight.
  • the growth medium was supplemented with kanamycin (25 ⁇ g/mL), spectinomycin (100 ⁇ g/mL) and isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG) (1 mM).
  • C. glutamicum strains were cultured in 500 mL baffled flasks.
  • BHI seed culture was inoculated from an agar plate and grown overnight. The cells were harvested by centrifugation (4000 x g, 10 min) and washed once with CGXII medium lacking the carbon source. Subsequently, CGXII medium containing a given concentration of carbon along with the necessary supplements was inoculated to an optical density between 0.5-1.0.
  • C. as sole carbon or nitrogen source
  • glutamicum was inoculated to an optical density of 0.5 and grown in baffled flasks using CGXII medium with 30 mM of 5AVA as sole carbon source or in free-nitrogen CGXII medium supplemented with 90 mM of 5AVA as sole nitrogen source.
  • CGXII medium was supplemented with different 5AVA concentrations and grown in 48-well flower plate using the Biolector microfermentation system (m2p labs GmbH, Aachen, Germany).
  • samples were derivatised with ortho- phthaldialdehyde (OPA) and separated on a system consisting of a pre -column (LiChrospher 100 PvP8 EC- ⁇ , 40 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and a main column (LiChrospher 100 RP8 ⁇ 5 ⁇ , 125 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and detected with a fluorescence detector (FLD G1321A, 1200 series, Agilent Technologies). L-asparagine was used as internal standard.
  • OPA ortho- phthaldialdehyde
  • Elution buffer gradient consisted of 0.25% Na-acetate (pH 6), as the polar phase and methanol as the nonpolar phase were used at a flow rate of 1.0 mL min "1 and the injection volume was 5 ⁇ .
  • the product yield was calculated by dividing the 5AVA titer by the amount of glucose used.
  • C. glutamicum wild type was grown in CGXII minimal medium with 4% glucose and either 200 mM 5AVA or 200 mM sodium chloride. Cells were harvested in exponential phase of growth by centrifugation (4000 x g, 10 min, 4 °C) and kept at -80 °C.
  • RNA isolation was performed as described (Wendisch 2003) and the RNA was kept at - 80 °C until further use.
  • DNA microarray analysis, synthesis of fluorescently labelled cDNA from total RNA, DNA microarray hybridization, and gene expression analysis were performed as described previously (Netzer et al. 2004; Polen et al. 2007).
  • the data are available as Gene Expression Omnibus GSE83413 data set at http://www.ncbi.nlm.nih.gov/geo/.
  • Example 1 Construction of an expression vector encoding PatA and PatD [093] PCR primers listed in table 2 were obtained from Metabion GmbH (Martinsried, Germany). Escherichia coli DH5a cells were used for the preparation of plasmids. Genes were PCR amplified with KOD Hot Start DNA Polymerase (NOVAGEN, Darmstadt, Germany) using genomic DNA of Escherichia coli MG1655 cells as the template. The plasmids were isolated using the QIAprep spin miniprep kit (QIAGEN, Hilden, Germany), and the PCR products were purified using PCR purification kit or MinElute PCR purification kit (QIAGEN, Hilden, Germany). Escherichia coli cells were transformed by heat shock and Corynebacterium glutamicum cells were transformed by electroporation at 2.5 kV, 200 ⁇ and 25 uF. All cloned DNA fragments were verified by sequencing.
  • Start and stop codons are in bold, the ribosome binding sites are in italics, and the restriction sites are underlined.
  • patA and patD were PCR amplified using the respective primer pairs listed in Table 2.
  • the patD gene was first cloned into the PstVBamHl restriction sites in pEKEx3 to generate plasmid ⁇ 3-£> ⁇
  • the patA gene was cloned into the BamHVSacl restriction sites in pEKEx3-patD to generate the plasmid ⁇ )EKEx3-patDA as shown in Figure 2.
  • the start codon of the patA gene was changed from ttg to atg.
  • Example 2 Generation of recombinant Corynebacterium glutamicum cells overexpressing enzymes PatA, PatD and LdcC
  • Corynebacterium glutamicum DM1933 cells were transformed with the expression vector pVWExl -IdcC encoding lysine decarboxylase (LdcC) from Escherichia coli to generate Cadi cells, which are capable of converting L-lysine into cadaverine. These cells were further transformed with the expression plasmid pEKEx3 _patDA encoding PatA and PatD to generate 5AVA1 cells.
  • Example 3 Fermentative production of 5-aminovalerate using recombinant Corynebacterium glutamicum cells overexpressing enzymes PatA, PatD and LdcC
  • 5AVA1 cells were cultured in 50 mL of LB medium overnight. Then, the cells were harvested by centrifugation (4000 x g, 10 min) and washed twice with CGXII minimal medium without any carbon source. Finally, the cells were further inoculated into 50 mL of CGXII medium containing 4% (w/v) of glucose and the necessary supplements to grow to an optical density of 1.0.
  • Example 4 Quantification of 5-aminovalerate fermentatively produced using recombinant Corynebacterium glutamicum cells overexpressing enzymes PatA, PatD and LdcC
  • samples were derivatized with ortho- phthaldialdehyde (OPA) and separated on a system consisting of a pre -column (LiChrospher 100 RP18 EC- ⁇ , 40 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and a main column (LiChrospher 100 RP18 ⁇ 5 ⁇ , 125 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and detected with a fluorescence detector (FLD G1321A, 1200 series, Agilent Technologies). L-asparagine was used as internal standard.
  • OPA ortho- phthaldialdehyde
  • Elution buffer gradient consisted of 0.25% Na-acetate (pH 6), as the polar phase and methanol as the nonpolar phase were used at a flow rate of 0.7 mL min-1 and the injection volume was 5 ⁇ .
  • samples were derivatised with OPA and separated on an system consisting of a pre -column (LiChrospher 100 RP8 ⁇ 5 ⁇ , 40 x 4.6 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and the main column (LiChrospher 100 RP8 ⁇ 5 ⁇ , 125 x 4.6 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany).
  • 1,7- diaminoheptane was used as internal standard.
  • Elution buffer gradient consisted of 0.25% Na- acetate (pH 6), as the polar phase and methanol as the nonpolar phase were used at a flow rate of 1.0 mL min-1 and the injection volume was 5 ⁇ .
  • Example 5 Comparison of recombinant Corynebacterium glutamicum Cadi and 5A VA1 cells in the production of 5-aminovalerate
  • Cadi and 5AVA1 cells were used for the production of 5-aminovalerate, and 5- amino valerate produced by these cells were subjected to HPLC quantification. As shown in Table 3, Cadi cells expressing LdcC did not produce 5 -amino valerate to a detectable level. To the contrary, 5AVA1 cells expressing LdcC, PatA and PatD produced 5-aminovalerate at a concentration of 21.0 ⁇ 0.2 mM.
  • Example 6 5A VA tolerance assay, 5A VA uptake and 5A VA conversion
  • C. glutamicum wild type was grown in CGXII minimal medium with 30 mM of 5AVA as sole carbon source or in free-nitrogen CGXII minimal medium with 90 mM 5AVA as sole nitrogen source.
  • the cells did not grow indicating that C. glutamicum is not able to use 5AVA as sole carbon or nitrogen source (data not shown) and 5AVA re-uptake will not be a problem in 5AVA strain engineering.
  • Example 8 Heterologous expression of IdcC, patA and patD from E. coli enabled 5A VA production from glucose fermentation in Corynebacterium glutamicum wild type
  • C. glutamicum wild type is able to synthesize the amino acid 1-lysine, however, it is unable to use this amino acid for catabolic purposes (Bellmann et al. 2001), therefore, cannot produce naturally 5AVA.
  • C. glutamicum needs to be metabolic engineered by expressing heterologous genes to produce the 1-lysine-derived compounds cadaverine (Kind et al. 2010a; Mimitsuka et al. 2007) and 1-pipecolic acid (Perez-Garcia et al. 2016).
  • 5AVA production can be obtained by the decarboxylation of 1-lysine into the diamine cadaverine, which in turn can be transaminated and further dehydrogenated into the non-protein amino acid 5AVA (patent application, 10 2015 114 785.8). Therefore, the gene encoding the constitutive 1- lysine decarboxylase (LdcC) from E. coli was cloned into the IPTG-inducible expression plasmid pVWExl (Peters- Wendisch et al. 2001) and the genes encoding the putrescine transaminase (PatA) and the ⁇ -aminobutyraldehyde dehydrogenase (PatD) from E.
  • LdcC constitutive 1- lysine decarboxylase
  • the strain 5AVA2 produced 5AVA in C. glutamicum for the first time, however, the product titers were low meaning that the overexpression of three E. coli genes can only be the first step towards a competitive industrial strain.
  • 5AVA is derived from l-lysine
  • previous metabolic engineering strategies of C. glutamicum l-lysine production strains seem to be relevant and helpful.
  • the l-lysine overproducer strain GRLysl (Unthan et. al 2015) was transformed with the plasmids pVWExl-WcC and pEKEx3-patDA, yielding the strain 5AVA5 (Table 1 (B)).
  • the 5AVA5 strain produced almost 5 times more 5AVA than the strain 5AVA2 (Fig. 8).
  • SugR is DeoR-type transcriptional regulator that plays a key role in genetic control of a wide range of genes involved in sugar metabolism of C. glutamicum. Genes of the main glucose uptake system of C. glutamicum, the phosphoenolpyruvate phosphotransferase system (PTS), are repressed by SugR, as well as, other genes of glycolysis, namely the 1-lactate dehydrogenase encoded by IdhA that leads to 1-lactate formation (Engels et al. 2008; Engels and Wendisch 2007; Teramoto et al. 2011). More recently, deletion of sugR showed to increase l-lysine titers and volumetric productivities as well as the side -product 1-lactate (Perez-Garcia et al. 2016).
  • Strain 5AVA3 (5 A A5 AsugR, Table 1 (B)) was constructed under the assumption that an increase observed for the precursor l-lysine could result in the production of higher amounts of 5AVA. However, 5AVA titers were only slightly increased to 37.8 mM (Fig. 8), which might be due to the accumulation of 1-lactate as a side product.
  • CGXII minimal medium was used in 5AVA production experiments. This medium was originally optimized for l-lysine production (Kelle et al. 2005). l-lysine is an amino acid with two amino groups while 5AVA only contains one amino group. Reduction of the nitrogen amount in the CGXII medium has been successfully applied for the production of amino acids with only one amino group, like GAB A (Jorge et al. 2016) or proline (Jensen and Wendisch 2013), therefore, it was evaluated if the reduction of nitrogen amount of the medium affected 5AVA production. Experiments using CGXII medium with half concentration of nitrogen (CGXIIm, i.e.
  • Example 12 Avoid l-lysine export and the formation of N-acetylcadaverine
  • GRLysl strain contains two copies of the 1-lysine exporter gene lysE (Unthan et. al 2015). Deletion of one copy of lysE was done under the assumption that when only one copy of the lysE gene is present in the genome of C. glutamicum less 1-lysine would be exported and more 1-lysine would be available intracellularly to be converted into 5AVA. However, it had the contrary effect because when one copy of lysE was deleted, more 1-lysine and less 5AVA (36.8 mM) were observed extracellularly (see 5AVA6 strain, Fig. 8). The little amounts of 1-lysine detected were also detected for the WT strain that only contains a copy of the lysE gene, therefore, only by deleting both copies of this gene the loss of 1-lysine could be completely avoided.
  • N-acetylcadaverine is a well-known side product of cadaverine production (Kind et al. 2010a) and snaA (cgl722) was identified as the gene responsible for the acetylation of cadaverine and its deletion led to strains unable to produce N-acetylcadaverine (Kind et al. 2010b). N-acetylcadaverine was also observed as a side product of all the 5AVA production strains (Fig. 8), thus, the deletion of snaA was performed in the strain 5AVA6, yielding 5AVA7 (Table 1 (B)), which, as expected, did not produce anymore N-acetylcadaverine (Fig. 8). The 5AVA production increased to 39.2 mM as compared to the parental strain 5AVA6 (Fig. 8), however, the final 5AVA titer was lower as compared to the strain 5AVA4 (Fig. 8).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention décrit une cellule recombinante génétiquement modifiée pour produire une quantité accrue de 5-aminovalérate comparée à son type sauvage. L'invention concerne en outre un procédé de préparation de 5-aminovalérate, le procédé comprenant la culture de la cellule recombinante de l'invention dans un milieu de croissance sous des conditions permettant la production de 5-aminovalérate, et la récupération du 5-aminovalérate du milieu. Enfin, l'invention concerne l'utilisation de la cellule recombinante de l'invention pour la préparation de 5-aminovalérate.
PCT/EP2016/068907 2015-09-03 2016-08-08 Préparation biologique de 5-aminovalérate WO2017036740A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102015114785 2015-09-03
DE102015114785.8 2015-09-03

Publications (1)

Publication Number Publication Date
WO2017036740A1 true WO2017036740A1 (fr) 2017-03-09

Family

ID=56738097

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/068907 WO2017036740A1 (fr) 2015-09-03 2016-08-08 Préparation biologique de 5-aminovalérate

Country Status (1)

Country Link
WO (1) WO2017036740A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624632A (zh) * 2017-03-15 2018-10-09 中国科学院微生物研究所 5-氨基戊酸的生物转化方法
CN109868297A (zh) * 2019-03-19 2019-06-11 南京工业大学 利用大肠杆菌表达DavA、DavB、GabD、GabT和LGOX产戊二酸的方法
CN111019875A (zh) * 2019-12-25 2020-04-17 江南大学 一种转录因子SugR编码基因及其在N-乙酰氨基葡萄糖生产中的应用
CN111849845A (zh) * 2019-04-26 2020-10-30 中国科学院微生物研究所 全细胞催化生产5-氨基戊酸的工程菌及5-氨基戊酸的制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1115550A (en) * 1965-11-15 1968-05-29 Kyowa Hakko Kogyo Company Ltd Process for the preparation of 5-aminovaleric acid
WO2010068953A2 (fr) * 2008-12-12 2010-06-17 Metabolix Inc. Procédé et compositions écologiques pour la production de produits chimiques à base de poly(5hv) et de 5 carbones
EP2647718A2 (fr) * 2012-04-06 2013-10-09 Metabolic Explorer Procédé de production de 5-aminopentanoate en utilisant un micro-organisme recombinant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1115550A (en) * 1965-11-15 1968-05-29 Kyowa Hakko Kogyo Company Ltd Process for the preparation of 5-aminovaleric acid
WO2010068953A2 (fr) * 2008-12-12 2010-06-17 Metabolix Inc. Procédé et compositions écologiques pour la production de produits chimiques à base de poly(5hv) et de 5 carbones
EP2647718A2 (fr) * 2012-04-06 2013-10-09 Metabolic Explorer Procédé de production de 5-aminopentanoate en utilisant un micro-organisme recombinant

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SI JAE PARK ET AL: "Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals", METABOLIC ENGINEERING, vol. 16, 1 March 2013 (2013-03-01), US, pages 42 - 47, XP055310156, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2012.11.011 *
UNTHAN SIMON ET AL: "Chassis organism from Corynebacterium glutamicum - a top-down approach to identify and delete irrelevant gene clusters", BIOTECHNOLOGY JOURNAL, vol. 10, no. 2, Sp. Iss. SI, February 2015 (2015-02-01), pages 290 - 301, XP002762894 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624632A (zh) * 2017-03-15 2018-10-09 中国科学院微生物研究所 5-氨基戊酸的生物转化方法
CN109868297A (zh) * 2019-03-19 2019-06-11 南京工业大学 利用大肠杆菌表达DavA、DavB、GabD、GabT和LGOX产戊二酸的方法
CN111849845A (zh) * 2019-04-26 2020-10-30 中国科学院微生物研究所 全细胞催化生产5-氨基戊酸的工程菌及5-氨基戊酸的制备方法
CN111019875A (zh) * 2019-12-25 2020-04-17 江南大学 一种转录因子SugR编码基因及其在N-乙酰氨基葡萄糖生产中的应用

Similar Documents

Publication Publication Date Title
JP5180060B2 (ja) 有機酸生産菌及び有機酸の製造法
JP6881448B2 (ja) アルデヒドの製造方法
JP7380768B2 (ja) アルデヒドの製造方法
CN109154008B (zh) 3-羟基己二酸的制造方法
JPWO2007046389A1 (ja) コハク酸の製造方法
JP2005095169A (ja) コハク酸の製造方法
WO2018022440A2 (fr) Matériaux et procédés pour le guidage d'un flux de carbone et production accrue de produits chimiques à base de carbone
JP2008530978A (ja) ４−ハイドロキシ−ｌ−イソロイシン又はその塩の製造法
WO2018022595A1 (fr) Matériaux et procédés utilisant des hôtes mutants produisant de la biotine pour la fabrication de produits chimiques à 7 atomes de carbone
JP7063332B2 (ja) 目的物質の製造方法
RU2699516C2 (ru) Новая лизиндекарбоксилаза и способ получения кадаверина с ее использованием
WO2017036740A1 (fr) Préparation biologique de 5-aminovalérate
CN109196107B (zh) α-氢化己二烯二酸的制造方法
CN113710813A (zh) 通过逆向乙醛酸支路生产乙醇酸和甘氨酸的微生物和方法
CN111560410A (zh) 咪唑二肽的制备方法
MX2015004180A (es) Microorganismos recombinantes para producir acidos organicos organicos.
JP2015228804A (ja) ２，３−ブタンジオール生産能を有する微生物およびそれを用いた２，３−ブタンジオールの製造方法、１，３−ブタジエンの製造方法
JP5602982B2 (ja) コハク酸の製造方法
US10597686B2 (en) Polypeptide having the activity of exporting O-acetyl-homoserine
JP2008067623A (ja) 非アミノ有機酸の製造方法
JP4720114B2 (ja) オキザロ酢酸またはオキザロ酢酸誘導体の製造方法
JP4428999B2 (ja) 非アミノ有機酸の製造方法
JP5034395B2 (ja) 有機酸生産菌及び有機酸の製造方法
US20140107377A1 (en) Microorganisms And Methods For Producing Acrylate And Other Products From Propionyl-CoA
US10676512B2 (en) Microorganism with enhanced L-lysine producibility and method for producing L-lysine using the same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16753623

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16753623

Country of ref document: EP

Kind code of ref document: A1