WO2021049616A1 - Transformant et procédé de production de 1,3-butanediol l'utilisant - Google Patents

Transformant et procédé de production de 1,3-butanediol l'utilisant Download PDF

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WO2021049616A1
WO2021049616A1 PCT/JP2020/034469 JP2020034469W WO2021049616A1 WO 2021049616 A1 WO2021049616 A1 WO 2021049616A1 JP 2020034469 W JP2020034469 W JP 2020034469W WO 2021049616 A1 WO2021049616 A1 WO 2021049616A1
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transformant
butanediol
alcohol dehydrogenase
corynebacterium
family
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乾将行
平賀和三
須田雅子
加藤直人
渡邉彰
生出伸一
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公益財団法人地球環境産業技術研究機構
ＧｒｅｅｎＥａｒｔｈＩｎｓｔｉｔｕｔｅ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

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  • the present disclosure relates to 1,3-butanediol production technology.
  • the present disclosure relates to, in one aspect, a transformant subjected to a specific genetic manipulation, and a technique for producing 1,3-butanediol using the transformant.
  • 1,3-Butanediol is used as a base material for cosmetics, a solvent for food flavors, and a synthetic raw material for pharmaceuticals and agricultural chemicals. It is also used as a raw material for polyurethane and polystyrene resins.
  • 1,3-butanediol is produced by aldol condensation of acetaldehyde, but it is expected to develop a production technology by a biological process toward the realization of a sustainable society.
  • Patent Document 1 is a transforming bacterium into which acetyl CoA carboxylase, acetoacetyl CoA synthase, acetoacetyl CoA reductase, and 3-hydroxybutyryl CoA reductase are introduced using Escherichia coli, yeast, corinebacterium, and crostridium as hosts. Discloses a technique for producing 1,3-butanediol from glucose using. In the examples, Escherichia coli is used as the host.
  • Patent Document 2 is a transformation capable of producing 1,3-butanediol by introducing acetoacetyl CoA reductase, 3-hydroxybutyryl CoA reductase, and 3-hydroxybutylaldehyde reductase using a microorganism as a host.
  • Genes encoding acetoacetyl-CoA reductase include thrA, akthr2, hom6, hom1, hom2, dadB, dadJ, Hbd2, Hbd1, hbd, HSD17B10, phbB, phaB, Msed_1423, Msed_0399, Msed_0389, Msed_392, ad.
  • Adh-A, mdh, ldhA, ldh, bdh and the like are disclosed.
  • genes encoding 3-hydroxybutyryl CoA reductase, acr1, sucD, bphG, blend, adhE, Msed_0709, mcr, asd-2, Saci_2370, Ald, eutE and the like are disclosed.
  • genes encoding 3-hydroxybutyraldehyde reductase, allrA, ADH2, yqhD, bdhI, bdhII, adhA, 4hbd, adhI, P84067, mmsb, dhat, 3hidh and the like are disclosed.
  • Non-Patent Document 1 describes 1,3-butane from glucose using a transforming bacterium in which Ralstonia eutropha acetyltransferase, acetoacetyl CoA reductase, and Clostoridium saccharoperbutylacetonicum 3-hydroxybutyryl CoA reductase are introduced into Escherichia coli. Disclose the technology for producing diols.
  • Non-Patent Document 3 discloses a technique for producing 1,3-butanediol from glucose via 3-hydroxybutanal using a transformant obtained by introducing the ald-keto reductase of Non-Patent Document 2 into Escherichia coli. To do.
  • Non-Patent Document 4 discloses information on the close relationship between a medium-chain alcohol dehydrogenase belonging to the cinnamyl alcohol dehydrogenase family derived from plants and YjgB derived from Escherichia coli and AdhC of Mycobacterium bovis.
  • the present disclosure provides a transformant capable of biologically producing 1,3-butanediol using a saccharide as a raw material, and a method for producing 1,3-butanediol using this transformant.
  • a gene encoding an alcohol dehydrogenase belonging to the cinamyl alcohol dehydrogenase (CAD) family is introduced into a host bacterium, and the host bacterium is the following enzymes (A) to (D). With respect to a transformant in which a mutation or gene has been introduced so that at least one of the above can be expressed or induced.
  • C Acetoacetyl CoA reductase
  • D 3-Hydroxybutyryl CoA reductase
  • the present disclosure comprises, in another aspect, a reaction step of culturing a transformant in a reaction solution containing a saccharide or a compound in which the transformant can metabolize acetyl-CoA. Including a recovery step of recovering 1,3-butanediol in the reaction solution.
  • the transformant relates to a method for producing 1,3-butanediol, which is a transformant in which a gene encoding an alcohol dehydrogenase belonging to the cinamyl alcohol dehydrogenase (CAD) family has been introduced into a host bacterium.
  • CAD cinamyl alcohol dehydrogenase
  • 1,3-butanediol in one embodiment, it is possible to provide a transformant capable of producing 1,3-butanediol from a saccharide as a raw material. According to the present disclosure, in another aspect, 1,3-butanediol can be biologically produced (produced) using this transformant.
  • FIG. 1 shows the results of a phylogenetic analysis showing that YjgB and AdhC belong to the same family as plant-derived cinamyl alcohol dehydrogenase (Plamt CAD).
  • FIG. 2 is a list of proteins and origins of the enzymes in the phylogenetic tree of FIG.
  • FIG. 3 shows the results of measuring the enzyme activity showing the 1,3-butanediol oxidizing activity of YjgB and AdhC.
  • FIG. 4 is a graph showing an example of 1,3-butanediol production of the 13BD78 strain.
  • FIG. 5 shows the results when the protein encoded by the adhC gene of Mycobacterium smegmatis is query in the search engine NCBI conserveed domain (CD) search.
  • FIG. 6 is a schematic diagram of the production pathway of 1,3-butanediol in the transformant according to the present disclosure.
  • the present disclosure is based on the finding that medium-chain alcohol dehydrogenases belonging to the cinamyl alcohol dehydrogenase (CAD) family have 1,3-butanediol oxidative activity and can be used in the production of 1,3-butanediol. ..
  • CAD cinamyl alcohol dehydrogenase
  • CAD Crohn's disease
  • MDR medium-chain dehydrogenase / reductase
  • Cinamil alcohol dehydrogenase (Cinamil ADH) a representative member of the CAD family, is an enzyme that plays an important role in the biosynthesis of lignin in the cell wall of plants. Enzymes belonging to the CAD family are known to exist not only in plants but also in yeast and prokaryotes (bacteria).
  • the "alcohol dehydrogenase belonging to the CAD family” in the present disclosure includes medium-chain alcohol dehydrogenase belonging to the CAD family.
  • “medium-chain alcohol dehydrogenase belonging to the CAD family” refers to an enzyme having a CAD domain in one or more embodiments.
  • the presence or absence of a CAD domain is determined by the search engine NCBI Conserved domain (CD) search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) in one or more embodiments.
  • CD NCBI conserveed domain
  • FIG. 5 shows the results when the protein encoded by the adhC gene of Mycobacterium smegmatis is query.
  • the "medium chain alcohol dehydrogenase belonging to the CAD family” in the present disclosure has an activity of oxidizing 1,3-butanediol using NADP + as a coenzyme in one or more embodiments.
  • " 1,3-butanediol oxidizing activity with NADP + as a coenzyme” can be measured by the method described in Examples.
  • the amino acid length of the "medium chain alcohol dehydrogenase belonging to the CAD family" in the present disclosure is, in one or more embodiments, more than 250 and less than 450, 260 or more and 440 or less, 270 or more and 430 or less, 280 or more and 420 or less, 290 or more. It is 410 or less, or 300 or more and 400 or less.
  • the "medium-chain alcohol dehydrogenase belonging to the CAD family" in the present disclosure includes enzymes of plants and microorganisms or enzymes derived from them in one or more embodiments, and improves the productivity of 1,3-butanediol. From this point of view, prokaryotes or enzymes derived from prokaryotes are preferable, and bacteria (bacteria) or enzymes derived from bacteria are more preferable.
  • immediate-chain alcohol dehydrogenases belonging to the CAD family include Cronobacter, Escherichia, Corynebacterium, Mycobacterium, and Bacillus.
  • the relevant enzymes of the genus (Bacillus) or Rhodococcus, or their orthologs, or enzymes derived from them are preferred.
  • "medium-chain alcohol dehydrogenases belonging to the CAD family” include Chronobacter sakazakii, Escherichia coli, Corynebacterium efficiens, and Corynebacterium terpenotabi. Corynebacterium terpenotabidum, Mycobacterium smegmatis, Bacillus subtilis, Bacillus megaterium, Rhodococcus erythropolis or Rhodococcus erythropolis, Rhodococcus erythropolis These orthologs, or enzymes derived from them, are preferred.
  • the enzymes shown in Table 1 of Examples, their orthologs, or enzymes derived from them are preferable.
  • AdhC of Bacillus megaterium, AdhC of Rhodococcus erythropolis, orthologs thereof, or enzymes derived from them are preferable.
  • medium chain alcohol dehydrogenase belonging to the CAD family in one or more embodiments, (1) Medium-chain alcohol dehydrogenase YjgB, which belongs to the cinnamyl alcohol dehydrogenase family of Cronobacter sakazakii, (2) The ortholog of (1) above in the genus Cronobacter and the genus Escherichia. (3) Medium-chain alcohol dehydrogenase gene AdhC belonging to the cinnamyl alcohol dehydrogenase family of Mycobacterium smegmatis, and (4) Mycobacterium, Bacillus, and Rhodococcus (4) The ortholog of (3) above in the genus Rhodococcus, Can be mentioned.
  • ortholog means a related protein having a homologous function existing in a different organism.
  • the term "derived enzyme” refers to, in one or more embodiments, the amino acid sequence of the original enzyme, eg, any of the amino acid sequences of SEQ ID NOs: 1 to 12 shown in Table 1 of the Examples. Refers to an enzyme having an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 97% or more identity.
  • the term “derived enzyme” means, in one or more embodiments, the same degree as the original enzyme, for example, the enzyme shown in Table 1 of Examples, "NADP + as a coenzyme 1,3-".
  • Butanediol means an enzyme having "oxidizing activity". The same degree means that the difference in activity is within ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, or ⁇ 5% in one or more embodiments.
  • the transformant according to the present disclosure is a transformant in which a gene encoding "medium-chain alcohol dehydrogenase belonging to the CAD family" in the present disclosure has been introduced into a host bacterium.
  • the transformant according to the present disclosure is a transformant into which the gene has been introduced so that "medium-chain alcohol dehydrogenase belonging to the CAD family" can be expressed or induced in one or more embodiments.
  • the gene may be introduced by a plasmid or on a chromosome.
  • the gene is preferably introduced in one or more embodiments with a sequence of gene expression regulation (such as an appropriate promoter) to the extent that production of 1,3-butanediol is improved.
  • Host bacteria include Corinebacterium, Escherichia (especially Escherichia coli), Bacillus (especially Bacillus subtilis), Pseudomonas (especially Pseudomonas petitda), Brevibacterium, Streptococcus, Lactobacillus bacterium, Rhodococcus bacterium (especially Rhodococcus erythropolis, Rhodococcus opacus), Streptomyces bacterium, Saccharomyces yeast (especially Saccharomyces cerevisiae), Cryberomyces yeast, Sizosaccalomyces yeast , Tricosporon genus yeast, Rhodococcus yeast, Pikia genus yeast, Candida genus yeast, Neurospora genus mold, Aspergillus genus mold, Trichoderma genus mold and the like.
  • Coryneform bacteria are a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), under normal aerobic conditions. It is not particularly limited as long as it proliferates.
  • Examples of the coryneform bacterium include, in one or more embodiments, Corynebacterium spp., Brevibacterium spp., Earthlobactor spp., Mycobacterium spp., Micrococcus spp. And the like.
  • Coryneform bacteria Corynebacterium spp.
  • Corynebacterium spp. are Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkano. Examples include Corynebacterium alkanolyticum. From the viewpoint of improving the productivity of 1,3-butanediol, Corynebacterium glutamicum is preferable.
  • Corynebacterium glutamicum R strain (FERM BP-18976), ATCC13032 strain, ATCC13869 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 Examples thereof include strains, ATCC13287 strains, ATCC13655 strains, ATCC13745 strains, ATCC13746 strains, ATCC13761 strains, ATCC14020 strains, ATCC31831 strains, MJ-233 (FERM BP-1497), MJ-233AB-41 (FERM BP-1498) and the like.
  • These Corynebacterium glutamicum strains have been internationally deposited under the Budapest Treaty and are publicly available. From the same viewpoint, as the host bacterium, R strain (FERM BP-18976), ATCC13032 strain, and ATCC13869 strain are preferable.
  • the coryneform type such as Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, Corynebacterium lilium, etc.
  • Bacteria are also unified to Corynebacterium glutamicum [Liebl, W. et al., Transfer of Brevibacterium divaricatum DSM 20297T, "Brevibacterium flavum” DSM 20411, “Brevibacterium lactofermentum” DS , And Corynebacterium glutamicum and their distinction by rRNA gene restriction patterns.
  • Kazuo Komagata et al. Classification of Coryneform bacteria, Fermentation and industry, 45: 944-963 (1987) ].
  • Examples of the genus Brevibacterium include Brevibacterium ammoniagenes (for example, ATCC6782 strain) and the like.
  • Examples of the genus Arthrobacter include Arthrobacter globiformis (for example, ATCC8010 strain, ATCC4336 strain, ATCC21056 strain, ATCC31250 strain, ATCC31738 strain, ATCC35698 strain) and the like.
  • Examples of Mycobacterium genus include Mycobacterium bovis (for example, ATCC 19210 strain, ATCC 27289 strain) and the like.
  • Examples of Micrococcus spp. Are Micrococcus freudenreichii (for example, No. 239 strain (FERM P-13221)), Micrococcus leuteus (for example, No.
  • Micrococcus ureae for example, IAM1010 strain
  • Micrococcus roseus for example, IFO3764 strain
  • the production pathway of 1,3-butanediol in the transformant according to the present disclosure can be outlined as shown in FIG. 6 in one or more embodiments.
  • Glucol is metabolized to phosphoenolpyruvate, pyruvate, and acetyl-CoA to 3-hydroxybutyryl CoA and 3-hydroxybutyraldehyde (3-hydroxybutyraldehyde).
  • 3-Hydroxybutanal is then catalyzed by an alcohol dehydrogenase belonging to the CAD family to produce 1.3-butanediol.
  • the transformant according to the present disclosure further expresses at least one of the following enzymes (A) to (D) from the viewpoint of improving the productivity of 1,3-butanediol. It is preferable that the mutation or gene is introduced so as to be possible or induce expression, and it is more preferable that the mutation or gene is introduced so that expression can be enhanced or expression can be induced. preferable.
  • A Acetyl-CoA carboxylase that catalyzes the reaction that produces maronyl CoA using acetyl-CoA as a substrate
  • B Acetoacetyl-CoA synthase that catalyzes the reaction that irreversibly produces acetoacetyl-CoA from acetyl-CoA and malonyl CoA
  • C Acetoacetyl CoA reductase that catalyzes the reaction that produces 3-hydroxybutyryl CoA from acetoacetyl CoA
  • D 3-Hydroxybutyryl CoA that catalyzes the reaction that produces 3-hydroxybutanal from 3-hydroxybutyryl CoA.
  • the enzymes (A) to (D) above can express two, three, or all of them in one or more embodiments from the viewpoint of improving the productivity of 1,3-butanediol. It is preferable that the mutation or gene is introduced in such a manner or so that the expression can be induced.
  • One or more embodiments of introducing a mutation or gene such that an enzyme can be expressed (or upregulated) or induced (or upregulated) is a gene encoding the enzyme. It can be introduced by plasmid or on the chromosome, or it can be brought about by mutagenesis or base sequence substitution in the control sequence, gene coding region, or both of the enzyme gene present on the chromosome of the host bacterium. Can be mentioned.
  • the transformant according to the present disclosure suppresses the consumption of phosphoenolpyruvate, pyruvate, or acetyl-CoA by other pathways in one or more embodiments of 1,3-butanediol.
  • A' Phosphoenolpyruvate carboxykinase (PPC) that catalyzes the reaction that produces oxaloacetate using phosphoenolpyruvate as a substrate.
  • B' Phosphotransacetylase
  • C' Acetate Kinase
  • ACK Lactate dehydrogenase
  • LDH Lactate dehydrogenase
  • 1,3-Butanediol can be produced by reacting the transformant according to the present disclosure in a reaction solution containing a carbon source.
  • the transformants Prior to the growth reaction of the cells, it is preferable to culture the transformants under aerobic conditions at a temperature of about 25 to 38 ° C. for about 12 to 48 hours to grow them.
  • Culture medium As the medium used for aerobic culture of the transformant prior to the reaction, a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, other nutrients and the like can be used.
  • a carbon source sugars (monosaccharides such as glucose, flux, mannose, xylose, arabinose, and galax); disaccharides such as sucrose, malus, lax, cellobiose, xylose, and lehalose.
  • Polysaccharides such as starch; sugar alcohols such as manniose, sorbyl, xylose glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, gluconic acid; ethanol, Alcohols such as propanol; hydrocarbons such as normal paraffin can also be used.
  • concentration of the carbon source in the medium varies depending on the carbon source used, but is usually about 0.1 to 10 (w / v%). As the carbon source, one type may be used alone, or two or more types may be mixed and used.
  • inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate, urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like can be used.
  • nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ amine, protein hydrolyzate, and amino acid can also be used.
  • the nitrogen source one type may be used alone, or two or more types may be mixed and used.
  • the concentration of the nitrogen source in the medium varies depending on the nitrogen compound used, but is usually about 0.1 to 10 (w / v%).
  • inorganic salts include primary potassium phosphate, secondary potassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, calcium carbonate and the like.
  • inorganic salt one type can be used alone, or two or more types can be mixed and used.
  • concentration of the inorganic salt in the medium varies depending on the inorganic salt used, but is usually about 0.01 to 1 (w / v%).
  • the nutritional substance examples include meat extract peptone, polypeptone, yeast extract, dried yeast, corn steep liquor, skim milk powder, skim soybean hydrochloric acid hydrolyzate, animal and plant or microbial cell extracts and their decomposition products.
  • concentration of the nutrient substance in the medium varies depending on the nutrient substance used, but is usually about 0.1 to 10 (w / v)%.
  • vitamins can be added if necessary. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, wild boar, nicotinic acid and the like.
  • the pH of the medium is preferably about 6-8.
  • medium A As a specific preferable medium for corynebacterium glutamic acid, medium A [Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. : 182-196 (2004)], BT medium [Omumasaba, CA et al., Corynebacterium glutamicum glycolaldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8: 91-103 ( 2004)] and the like.
  • the sugar concentration may be set within the above range.
  • reaction solution water, a buffer solution, an inorganic salt medium or the like containing a saccharide or a compound capable of producing acetyl-CoA by metabolism of the transformant according to the present disclosure can be used.
  • saccharide glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, or mannitol are preferable.
  • Compounds capable of producing acetyl-CoA by metabolism of the transformant include organic acids such as pyruvic acid, acetic acid, lactic acid or gluconic acid, alcohols such as ethanol, inositol or glycerol, amino acids such as glutamine or glutamic acid, and naphthalene. Aromatic compounds such as, etc. can be used.
  • the concentration of the saccharide and the compound capable of producing acetyl-CoA by metabolism in the reaction solution varies depending on the saccharide and the compound used, but is usually about 0.1 to 10 (w / v%). Good.
  • the buffer solution include a phosphate buffer, a squirrel buffer, and a carbonic acid buffer.
  • the concentration of the buffer solution is preferably about 10 to 150 mM.
  • the inorganic salt medium one or two kinds of inorganic salts such as primary potassium phosphate, secondary potassium phosphate, manesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. Examples include a medium containing the above. Of these, a medium containing magnesium sulfate is preferable. Specific examples of the inorganic salt medium include a BT medium and the like. The concentration of the inorganic salt in the medium varies depending on the inorganic salt used, but is usually about 0.01 to 1 (w / v%).
  • the pH of the reaction solution is preferably about 6-8.
  • the pH of the reaction solution is controlled to near neutral, particularly to about 7, with a pH controller (for example, manufactured by Able Inc., model: DT-1023) using an aqueous ammonia solution, an aqueous sodium hydroxide solution, or the like. It is preferable to react.
  • the reaction temperature that is, the survival temperature of the transformant during the reaction is preferably about 20 to 50 ° C, more preferably about 25 to 47 ° C. Within the above temperature range, 1,3-butanediol can be efficiently produced.
  • the reaction time is preferably about 1 to 7 days, more preferably about 1 to 3 days.
  • the culture may be a batch type, a fed-batch type, or a continuous type. Of these, the batch type is preferable.
  • the reaction may be carried out under reducing conditions or slightly aerobic conditions. Under either condition, Corynebacterium glutamic acid is carried out in a reaction that does not substantially proliferate, so that 1,3-butanediol can be produced more efficiently.
  • the reduction conditions are defined by the redox potential of the reaction solution.
  • the redox potential of the reaction solution is preferably about ⁇ 200 mV to ⁇ 500 mV, more preferably ⁇ 250 mV to ⁇ 500 mV.
  • the reduced state of the reaction solution can be easily estimated with a resazurin indicator (in the reduced state, decolorization from blue to colorless), but to be precise, a redox potentiometer (for example, ORP Electrodes manufactured by BROADLEY JAMES) is used. Can be measured.
  • a redox potentiometer for example, ORP Electrodes manufactured by BROADLEY JAMES
  • a known method can be used without limitation.
  • an aqueous solution for the reaction solution may be used instead of distilled water or the like.
  • the method for preparing the aqueous solution for the reaction solution is, for example, a culture solution for an absolutely anaerobic microorganism such as a sulfate-reducing microorganism.
  • distilled water or the like for about 1 to 60 minutes, preferably about 5 to 40 minutes under reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less.
  • the gas, especially the dissolved oxygen, can be removed to prepare an aqueous solution for the reaction solution under reducing conditions.
  • an appropriate reducing agent for example, thioglycolic acid, ascorbic acid, cystine hydrochloride, mel-powered acetic acid, thiol acetic acid, glutathione, sodium sulfide, etc.
  • An appropriate combination of these methods is also a method for preparing an aqueous solution for a reaction solution under effective reducing conditions.
  • the reaction system is made of an inert gas such as nitrogen gas or carbon dioxide gas.
  • a method of encapsulation can be mentioned.
  • the pH maintenance adjusting solution of the reaction system is added or various nutrient dissolving solutions are added. It may be necessary to add oxygen as appropriate, but in such a case, it is effective to remove oxygen from the added solution in advance.
  • the air volume should be set to a low value such as 0.5 vvm (volume per volume per minute) or less, and the stirring speed should be set to a low value such as 500 rpm or less. Can be made to.
  • the aeration can be cut off at an appropriate time, and the reaction can be carried out in combination with a state in which the anaerobic degree is improved under the condition of a stirring speed of 100 rpm or less.
  • 1,3-butanediol is produced in the reaction solution.
  • 1,3-butanediol can be recovered by recovering the reaction solution, 1,3-butanediol can also be separated from the reaction solution by a known method. Examples of such known methods include a distillation method, a membrane permeation method, an organic solvent extraction method, and the like.
  • a gene encoding an alcohol dehydrogenase belonging to the cinnamil alcohol dehydrogenase (CAD) family has been introduced into the host bacterium, and The host bacterium is a transformant into which a mutation or gene has been introduced so that at least one of the following enzymes (A) to (D) can be expressed or induced.
  • CAD cinnamil alcohol dehydrogenase
  • A Acetyl CoA carboxylase
  • B Acetoacetyl CoA synthase
  • C Acetoacetyl CoA reductase
  • D 3-Hydroxybutyryl CoA reductase
  • the host bacteria are described in the following (A') to (A') to ( The transformant according to [1], wherein at least one of the enzymes of D') is dysfunctional or deficient.
  • A' Phosphoenolpyruvate carboxykinase (PPC) that catalyzes the reaction that produces oxaloacetate using phosphoenolpyruvate as a substrate.
  • PPC Phosphoenolpyruvate carboxykinase
  • Alcohol dehydrogenases belonging to the CAD family are Cronobacter, Escherichia, Corynebacterium, Mycobacterium, Bacillus, or Rhodococcus. The transformant according to any one of [1] to [5], which is derived from the genus (Rhodococcus).
  • Alcohol dehydrogenases belonging to the CAD family include Cronobacter sakazakii, Escherichia coli, Corynebacterium efficiens, Corynebacterium terpenotabidum, and Mycobacterium.
  • Alcohol dehydrogenases belonging to the CAD family include YjgB from Cronobacter sakazakii, YjgB from Escherichia coli, AdhC from Corynebacterium terpenotabidum, and Mycobacterium mycobacterium.
  • AdhC from Mycobacterium smegmatis AdhC from Bacillus megaterium, AdhC from Rhodococcus erythropolis, orthologs thereof, or enzymes derived from them, preferably Chronobactor Sakazaki. YjgB from Cronobacter sakazakii, AdhC from Corynebacterium terpenotabidum, AdhC from Mycobacterium smegmatis, AdhC from Bacillus megaterium, AdhC from Bacillus megaterium, Rodcoccus Eris , Or any of these orthologs, or enzymes derived from them, according to any of [1] to [7].
  • Alcohol dehydrogenase belonging to the CAD family (1) Medium-chain alcohol dehydrogenase YjgB, which belongs to the cinnamyl alcohol dehydrogenase family of Cronobacter sakazakii, (2) The ortholog of (1) above in the genus Cronobacter and the genus Escherichia.
  • the 3-hydroxybutyryl CoA reductase is derived from Lactobacillus brevis, Escherichia coli, or Salmonella typhimurium, preferably Lactobacillus.
  • Corynebacterium glutamicum R-BD1 strain (Corynebacterium glutamicum R-BD1) (accession number: NITE BP-03015) Corynebacterium-type bacterial transformant.
  • CAD cinamyl alcohol dehydrogenase
  • the saccharide is selected from the group consisting of glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, and mannitol and combinations thereof of two or three or more.
  • the method for producing 1,3-butanediol according to any one of [14] to [16].
  • the compound capable of producing acetyl CoA by metabolism of the transformant is selected from the group consisting of organic acids, alcohols, amino acids and aromatic compounds, and combinations of 2 or 3 or more thereof, and the organic acid is selected from the group consisting of organic acids, alcohols, amino acids and aromatic compounds.
  • Example 1 Extraction from the database of medium-chain alcohol dehydrogenases belonging to the prokaryotic cinnamyl alcohol dehydrogenase (CAD) family (1) Search for YjgB derived from Escherichia coli and AdhC homologous protein derived from Mycobacterium bovis Using the BLASTP program, Using the amino acid sequences of SEQ ID NOs: 2 and 19 as query sequences, amino acid sequences having homology with YjgB derived from Escherichia coli or AdhC derived from Mycobacterium bovis were extracted from the GenBank database. Information on the extracted amino acid sequences is shown in Table 1.
  • CAD prokaryotic cinnamyl alcohol dehydrogenase
  • Table 2 shows the primer sequences used for isolation of the target enzyme gene.
  • GeneAmp PCR System 9700 manufactured by Applied Biosystems
  • Tks Gflex DNA Polymerase manufactured by Takara Bio Inc.
  • the PCR-amplified DNA fragment was introduced into the cloning vector Lldh4 having the PldhA promoter.
  • the names of the obtained plasmids are shown in Table 3.
  • a agar medium [(NH 2) 2 CO 2g , (NH 4) 2 SO 4 7g, KH 2 PO 4 0.5g, K 2 HPO 4 0.5g, MgSO 4 ⁇ 7H 2 O 0.5g, 0.06 % (w / v) Fe 2 SO 4 ⁇ 7H 2 O + 0.042% (w / v) MnSO 4 ⁇ 2H 2 O 1ml, 0.02% (w / v) biotin solution 1ml, 0.01% (w / v) 2 ml of an aqueous thiamine solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, 40 g of glucose, and 15 g of agar were suspended in 1 L of distilled water] and allowed to stand in a dark place at 30 ° C. for 24 hours.
  • the vector-introduced strain was similarly cultured as a control group.
  • Additional plates coli grown in Corynebacterium glutamicum R_CsYjgB, R_EcYjgB, R_CtAdhC, R_MsAdhC, R_BmAdhC, R_ReAdhC strain, and vectors introduced strain (EV),
  • a liquid medium containing kanamycin 50 ⁇ g / ml [(NH 2) 2 CO 2g , (NH 4) 2 SO 4 7g, KH 2 PO 4 0.5g, K 2 HPO 4 0.5g, MgSO 4 ⁇ 7H 2 O 0.5g, 0.06% (w / v) Fe 2 SO 4 ⁇ 7H 2 O + 0.042% (w / v) MnSO 4 ⁇ 2H 2 O 1ml, 0.02% (w / v) biotin solution 1ml, 0.01% (w / v ) thiamine solution 2 ml, yeast extract 2g, 7 g of vitamin assay casamino acid and 40 g of glucose were
  • the obtained cells were suspended in 1 ml of a buffer for extracting cells [20 mM Tris-hydrochloric acid buffer (pH 7.2), 5 mM dithiothreitol, 15% glycerol], and using a multi-bead shocker manufactured by Yasui Kikai Co., Ltd. The cells were crushed. The supernatant from which impurities were removed by centrifugation (4 ° C., 20,000 ⁇ g, 10 minutes) was used as a cell-free extract.
  • a buffer for extracting cells [20 mM Tris-hydrochloric acid buffer (pH 7.2), 5 mM dithiothreitol, 15% glycerol]
  • the cells were crushed.
  • the supernatant from which impurities were removed by centrifugation (4 ° C., 20,000 ⁇ g, 10 minutes) was used as a cell-free extract.
  • the enzyme activity was measured by using 1 ml of a 50 mM Tris-hydrogen buffer (pH 7.2) containing 50 mM 1,3-butanediol, 0.2 mM NADP +, and a cell-free extract at 30 ° C.
  • the enzyme activity of reducing 1 ⁇ mol of NADP + to NADPH in 1 minute was defined as 1 unit.
  • 1,3-butanediol oxidizing activity was detected in all the YjgB and AdhC homologous protein expression strains analyzed. No 1,3-butanediol oxidizing activity was detected in the vector-introduced strain (EV) -derived sample used as the control group.
  • EV vector-introduced strain
  • Example 2 Construction of 1,3-butanediol-producing strain (1) Preparation and acquisition of chromosomal DNA [Example 1] 2. In addition to the chromosomal DNA prepared in (1), the chromosomal DNA of Corynebacterium glutamicum R (FERM P-18976) and Clostridium beijerinckii DSM 6422 was cultured according to the information of the strain acquisition institution, and then the DNA genome extraction kit (trade name: GenomicPrep Cells) was used. And Tissue DNA Isolation Kit (manufactured by Amasham).
  • Table 5 shows the primer sequences used for isolation of the target enzyme gene.
  • GeneAmp PCR System 9700 manufactured by Applied Biosystems
  • Tks Gflex DNA Polymerase manufactured by Takara Bio Inc.
  • the acetoacetyl CoA synthase gene aax from Streptomyces tendae and the 3-hydroxybutyryl CoA reductase gene eutes from Lactobacillus brevis are codon-optimal for expression in the corynebacterium glutamicum.
  • the converted genes were prepared by artificial synthesis (SEQ ID NOs: 55 and 56).
  • the 3-hydroxybutyryl CoA reductase encoded by eutE can reduce 3-hirodoxybutyryl-CoA using NAD (P) H as a coenzyme to form 3-hydroxybutanal.
  • NAD (P) H refers to NADH or NADPH.
  • the enzymes encoded by the genes related to 1,3-butanediol production are as follows. acc: Acetyl-CoA carboxylase aacs: Acetyl-acetyl-CoA synthase hbd: ⁇ -hydroxybutyryl CoA dehydrogenase eutE: 3-Hydroxybutyryl CoA reductase
  • the aacs gene, accBC gene, accD1 gene, and accE gene were introduced into the cloning vector Ltac5 containing the Ptac promoter.
  • the hbd gene and the eutE gene were introduced into the cloning vector Lgap10 having the PgapA promoter.
  • the obtained plasmid is shown in Table 6.
  • the yjgB gene and the adhC gene were introduced into the cloning vector Lldh4 having the PldhA promoter (Table 3 above).
  • the markerless chromosomal gene transfer vector LKSind1-4 cannot replicate in Corynebacterium glutamicum R.
  • the recombinant strain generated by the single crossover between the homologous region on the chromosome introduced into LKSind 1-4 and the plasmid derived from the vector is the kanamycin resistance gene on LKSind 1-4 and the sacR-sacB gene derived from Bacillus subtilis. By expression, it exhibits kanamycin resistance and plasmid sensitivity.
  • the double crossed strain derived from the recombinant strain exhibits kanamycin sensitivity and sucrose resistance due to the loss of the kanamycin resistance gene and the sacR-sacB gene derived from LKSind 1-4. Therefore, the target gene-introduced strain exhibits kanamycin sensitivity and sucrose resistance.
  • LKSind1-4Cbhbd an hbd chromosome-introduced strain was constructed by the above-mentioned method. Furthermore, based on the same principle, markerless disruption of ldhA, pta, ack, and ppc genes was performed using the markerless gene disruption vector pCRA725.
  • the enzymes encoded by these genes are: ldhA: Lactate dehydrogenase pta: Phosphotrans acetylase ac: Acetate kinase ppc: Phosphoenolpyrvate carboxylase The obtained strains are shown in Table 9.
  • Corynebacterium glutamicum 13BD78 strain is a Corynebacterium glutamicum R-BD1 strain, a patented microorganism of the National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818). Deposited at the Deposit Center (Deposit date: August 13, 2019, Deposit number: NITE BP-03015).
  • Each strain kanamycin 50 [mu] g / ml, and A agar medium [(NH 2) 2 CO 2g containing zeocin 25 ⁇ g / ml, (NH 4) 2 SO 4 7g, KH 2 PO 4 0.5g, K 2 HPO 4 0.
  • the strain grown on the above plate was inoculated into a test tube containing 2.5 ml of A liquid medium (same as above) containing 50 ⁇ g / ml of kanamycin and 25 ⁇ g / m of zeocin, and inoculated at 30 ° C. for 24 hours. , Aerobic shaking culture was performed.
  • the mixture was aerobically shaken and cultured for 17 hours.
  • Each strain was applied to A agar medium (same as above) containing 50 ⁇ g / ml of kanamycin and 25 ⁇ g / ml of zeocin, and allowed to stand in a dark place at 30 ° C. for 72 hours.
  • the strain grown on the above plate was inoculated into a test tube containing 2.5 ml of A liquid medium (same as above) containing 50 ⁇ g / ml of canamycin and 25 ⁇ g / ml of zeocin, and inoculated at 30 ° C. for 24 hours. , Aerobic shaking culture was performed.
  • the mixture was aerobically shaken and cultured for 17 hours.
  • the cells were collected by centrifugation (4 ° C., 4000 xg, 7 minutes) and washed with BT-U medium.
  • Shake culture was performed aerobically. If necessary, glucose was added to the culture solution during the culture.
  • High performance liquid chromatography system [ACQUITY UPLC H-Class (Waters), Unison UK- C18 (Imtaket), separated using 20 mM phosphoric acid in the mobile phase] was used for analysis.
  • 13BD78 was applied to A agar medium (same as above) containing 50 ⁇ g / ml of kanamycin and 25 ⁇ g / ml of zeocin, and allowed to stand in the dark at 30 ° C. for 72 hours.
  • the strain grown on the above plate was inoculated into a test tube containing 2.5 ml of A liquid medium (same as above) containing 50 ⁇ g / ml of canamycin and 25 ⁇ g / ml of zeocin, and inoculated at 30 ° C. for 24 hours. , Aerobic shaking culture was performed.
  • FIG. 4 shows the time course of the 1,3-butanediol concentration from the start of the production experiment to 24 hours.
  • 1,3-butanediol can be produced from a sugar raw material using a microorganism.

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Abstract

L'invention concerne : un transformant qui peut produire du 1,3-butanediol d'une manière biologique ; et un procédé de production de 1,3-butanediol à l'aide du transformant. Dans un aspect, un transformant produit par l'introduction d'un gène codant pour une alcool déshydrogénase appartenant à la cinnamyl-alcool déshydrogénase (CAD) dans une bactérie hôte, la bactérie hôte ayant un mutant ou un gène introduit dans celle-ci de telle manière qu'au moins l'une des enzymes (A) à (D) peut être exprimée ou l'expression de la ou des enzymes peut être induite.　(A) Acétyl-CoA carboxylase ; (B) acétyl-CoA synthétase ; (C) acétyl-CoA réductase ; et (D) 3-3-hydroxybutyryl-CoA réductase.
PCT/JP2020/034469 2019-09-11 2020-09-11 Transformant et procédé de production de 1,3-butanediol l'utilisant WO2021049616A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
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JP2012525158A (ja) * 2009-04-30 2012-10-22 ゲノマチカ， インク． １，３−ブタンジオールの産生のための生物
WO2014045781A1 (fr) * 2012-09-24 2014-03-27 昭和電工株式会社 Procédé de fabrication de butanediol
JP2016500261A (ja) * 2012-12-12 2016-01-12 アールイージー ライフ サイエンシズ リミテッド ライアビリティ カンパニー 脂肪酸誘導体のａｃｐ媒介性生産方法
WO2016168247A1 (fr) * 2015-04-15 2016-10-20 William Marsh Rice University Biosynthèse, avec des thiolases dépendantes d'acp, d'acide gras modifié
JP2018519829A (ja) * 2015-07-21 2018-07-26 ガバニング カウンシル オブ ザ ユニバーシティ オブ トロント １，３−ブタンジオールの産生のための方法および微生物
JP2018530342A (ja) * 2015-10-13 2018-10-18 ランザテク・ニュージーランド・リミテッド エネルギー発生発酵経路を含む遺伝子操作細菌

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EP2503003A1 (fr) * 2011-03-24 2012-09-26 Volker Sieber Voie de synthèse pour la production d'alcohols et d'amines
WO2014176514A2 (fr) * 2013-04-26 2014-10-30 Genomatica, Inc. Micro-organismes et procédés de production du 4-hydroxybutyrate, du 1,4-butanediol, et composés associés
JP6982452B2 (ja) * 2017-09-29 2021-12-17 旭化成株式会社 遺伝的に操作された微生物による１，５−ペンタンジオールの製造方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012525158A (ja) * 2009-04-30 2012-10-22 ゲノマチカ， インク． １，３−ブタンジオールの産生のための生物
WO2014045781A1 (fr) * 2012-09-24 2014-03-27 昭和電工株式会社 Procédé de fabrication de butanediol
JP2016500261A (ja) * 2012-12-12 2016-01-12 アールイージー ライフ サイエンシズ リミテッド ライアビリティ カンパニー 脂肪酸誘導体のａｃｐ媒介性生産方法
WO2016168247A1 (fr) * 2015-04-15 2016-10-20 William Marsh Rice University Biosynthèse, avec des thiolases dépendantes d'acp, d'acide gras modifié
JP2018519829A (ja) * 2015-07-21 2018-07-26 ガバニング カウンシル オブ ザ ユニバーシティ オブ トロント １，３−ブタンジオールの産生のための方法および微生物
JP2018530342A (ja) * 2015-10-13 2018-10-18 ランザテク・ニュージーランド・リミテッド エネルギー発生発酵経路を含む遺伝子操作細菌

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