WO2011071204A2 - Procédé de production d'éthanol à partir de xylose à l'aide de saccharomyces cerevisiae recombiné mettant en jeu l'utilisation couplée de nadh et de nad+ - Google Patents

Procédé de production d'éthanol à partir de xylose à l'aide de saccharomyces cerevisiae recombiné mettant en jeu l'utilisation couplée de nadh et de nad+ Download PDF

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WO2011071204A2
WO2011071204A2 PCT/KR2009/007458 KR2009007458W WO2011071204A2 WO 2011071204 A2 WO2011071204 A2 WO 2011071204A2 KR 2009007458 W KR2009007458 W KR 2009007458W WO 2011071204 A2 WO2011071204 A2 WO 2011071204A2
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saccharomyces cerevisiae
ethanol
xylose
phosphate
xylitol
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PCT/KR2009/007458
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English (en)
Korean (ko)
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WO2011071204A3 (fr
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서진호
박용철
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서울대학교 산학협력단
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Priority to PCT/KR2009/007458 priority Critical patent/WO2011071204A2/fr
Priority to US12/815,837 priority patent/US8628944B2/en
Publication of WO2011071204A2 publication Critical patent/WO2011071204A2/fr
Publication of WO2011071204A3 publication Critical patent/WO2011071204A3/fr

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    • 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
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • 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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a method for producing ethanol from xylose using recombinant Saccharomyces cerevisiae , and more specifically, the coenzymes NADH and NAD + can be used to couple and Method for producing ethanol from xylose using recombinant Saccharomyces cerevisiae , which is transformed and transformed so that acetaldehyde dehydrogenase which produces acetic acid is not expressed. It is about.
  • Ethanol is the main ingredient of alcohol, and civilization has been drinking ethanol with the birth of alcohol. However, in modern times, ethanol has been spotlighted as an alternative clean energy to replace petroleum. In fact, ethanol has long been widely commercialized as a transport fuel in Brazil, and in the United States, ethanol with 10% ethanol added to gasoline Fuel is currently available.
  • Ethanol which is used as a fuel for transportation, is currently produced from sugar cane or corn.
  • Sugar cane is a raw material of raw sugar
  • corn is a food material. Therefore, when ethanol is widely used, sugar or It raises the side effects of rising prices for corn and the ethical problem of using grain as a source of fuel rather than food.
  • Xylose which is present in large quantities in waste wood or by-products of forest processing, is one of the potential candidate materials.
  • Xylose is a biomass (biomass) that is present in a lot of waste wood in the form of a polymer xylan. Xylose is finally separated through xylo-oligosaccharides and the like by hydrolysis of xylan, and is used for the production of xylitol which has caries prevention ability.
  • Xylose is a material that can be recovered semi-permanently on the earth because it can be recovered from the wood dispersion produced during the manufacture of pulp, etc., without inducing a rise in the price of alternative materials and free from ethical issues, Research is being done.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Saccharomyces cerevisiae is widely known as an ethanol-producing strain in the production of fermented liquor such as Takju, recently used as a host for the production of useful medicines, and also as a host for the production of ethanol It is studied a lot.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae X
  • Saccharomyces cerevisiae X is unable to metabolize xylose due to the absence of enzymes such as xylose reductase (XR) and xylitol dehydrogenase (XDH).
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • Saccharomyces cerevisiae produces ethanol as a by-product when it metabolizes xylose. Efforts have been made to use Saccharomyces cerevisiae .
  • the present invention in the production of ethanol (ethanol) from xylose using recombinant Saccharomyces cerevisiae , xylose to xylitol (xylitol) Transformed to express xylose reductase (XR) using NADH as a cofactor; Xylitol is converted to xylulose, and transformed to express xylitol dehydrogenase (XDH) using NAD + as a coenzyme; Transformed to overexpress xylulokinase (XK), which converts xylulose to xylulose 5-phosphate; Sedoheptulose 7-phosphate and glyceradehyde 3-phosphate were converted into erythrose 4-phosphate and fructose 6-phosphate.
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XK overexpress xylulo
  • TAL1 transaldolase 1
  • Saccharomyces cerevisiae is industrially used as an ethanol producing strain, but does not use xylose as a carbon source. This is because Saccharomyces cerevisiae does not have xylose reductase (XR) and xylitol dehydrogenase (XDH). xylulose) is because there is no metabolic activity. Therefore, in order to produce ethanol, XR and XDH enzymes need to be introduced into the host. XR generally uses NADPH as a coenzyme, and XDH generally uses NAD + as a coenzyme.
  • XR generally uses NADPH as a coenzyme
  • XDH generally uses NAD + as a coenzyme.
  • a feature of the present invention was to introduce NADH-dependent XR instead of NADPH to couple with NAD + -dependent XDH.
  • NADH-dependent XR instead of NADPH
  • NAD + -dependent XDH
  • xylose is converted to gyulose by NADH dependent XR and NAD + dependent XDH, Xylulose is further converted to xylulose 5-phosphate by XK introduced, and metabolism proceeds through the pentose phosphate cycle.
  • XK is an enzyme present in yeast, but if only XR and XDH are introduced into the strain without overexpressing it, it is possible to produce ethanol from xylose, but yield and productivity are remarkably low. Therefore, it is better to overexpress XK.
  • transaldolase 1 is an enzyme present on the pentose phosphate cycle, sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate (glyceradehyde 3-phosphate). ) Is converted to erythrose 4-phosphate and fructose 6-phosphate, which in the present invention can be improved by overexpressing ethanol productivity.
  • the recombinant Saccharomyces cerevisiae of the present invention is the integration of xylose reductase or xylitol dehydrogenase on the chromosome of Saccharomyces cerevisiae ( Saccharomyces cerevisiae ). If the gene is introduced on the chromosome has the advantage that the gene is not lost during the culture.
  • Saccharomyces cerevisiae of the present invention is that xylitol dehydrogenase (XDH) is inserted into the chromosome delta sequence of Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Good, because it can be inserted in multiple copies.
  • XDH xylitol dehydrogenase
  • the recombinant Saccharomyces cerevisiae of the present invention is additionally transformed to prevent acetaldehyde dehydrogenase from expressing acetaldehyde into acetic acid. It is better to prevent the production of by-product acetic acid to produce ethanol with high yield and high productivity.
  • the transformation so that the acetaldehyde dehydrogenase is not expressed may be achieved by crushing part or all of the gene encoding acetaldehyde dehydrogenase.
  • recombinant Saccharomyces cerevisiae which is further transformed to prevent the expression of acetaldehyde dehydrogenase, is preferably O 2 -limited fed-batch fermentation. It is better to perform this because the production of acetic acid as a by-product is minimized through fed-batch cultures that limit oxygen.
  • Ethanol production method of the present invention is Saccharomyces cerevisiae ( Saccharomyces cerevisiae NADH, a coenzyme of xylose reductase, and NAD, a coenzyme of xylitol dehydrogense + Is coupled to and utilized to remove acetaldehyde dehydrogenase, which mediates the production of by-product acetic acid, thereby producing ethanol in high yield and high productivity.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae NADH, a coenzyme of xylose reductase, and NAD, a coenzyme of xylitol dehydrogense + Is coupled to and utilized to remove acetaldehyde dehydrogenase, which mediates the production of by-product acetic acid, thereby producing ethanol in high yield and high productivity.
  • FIG. 1 is a flow chart showing a process of producing ethanol from xylose designed in the present invention.
  • 2 is a graph showing that the mutated XR has affinity for NADH higher than that for NADPH, unlike the wild-type XR.
  • 3 is a graph showing the effect of mutated XR and wild-type XR on the degree of metabolism of xylose (producing ability of xylitol).
  • Figure 4 is a graph showing the ethanol fermentation results of the SX3 strain and SX5 strain of the present invention compared.
  • Figure 5 is a graph showing the ethanol fermentation results of the SX5: ald6 ⁇ strain and SX5 strain inhibited the production of acetic acid of the present invention.
  • Saccharomyces cerevisiae D452-2 used as a host in this example was sold by Professor Makino of Kyoto University, Japan. (Seiya Watanabe, Ahmed Abu Saleh, Seung Pil Pack, Narayana Annaluru, Tsutomu Kodaki and Keisuke Makino. 2007. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis . Microbiol. 153: 3044- 3054).
  • Vectors YEpM4XR (WT), YEpM4XR (R276H) and pPGKXDH (WT) were also used by Professor Makino of Kyoto University, Japan, and point mutations for wild-type XR gave mutated XR (R276H) enzymes compared to wild-type NADPH. There is a higher affinity for NADH. (See FIG. 2, Seiya Watanabe, Ahmed Abu Saleh, Seung Pil Pack, Narayana Annaluru, Tsutomu Kodaki and Keisuke Makino. 2007.
  • YIpXR WT -XDH WT And parent vectors used in the production of YEpM4XR (R276H) YIp5 and ISXK were used by the former Seoul National University researcher Lee Tae-hee. Metabolic engineering studies on production of ethanol from xylose by recombinant Saccharomyces cerevisiae , Seoul National University Master's Thesis, 2000).
  • pAUR101 the parent vector used for the production of pAUR_d_ALD6, is a vector sold by Takara, Japan, and the ALD6 gene was cloned from S. cerevisiae CEN.PK2-1D.
  • the gene is Pichia stiphitis ( Pichia stipitis ) Cloning from CBS6054 was used.
  • Table 1 below shows the strains and genotypes thereof used in Examples 2 to 4 as prepared in this example.
  • strain D452-2 / pXR MUT / pXDH in which XR mut and XDH were epismally inserted in the form of plasmid was used.
  • Xylose was used as a substrate and aerobic fermentation was performed.
  • the control strain used wild-type NADPH dependent XR (hereinafter, also referred to as 'XR WT ') and D452-2 / pXR WT / pXDH in which the XDH was epitaxially inserted in the form of a plasmid.
  • Example 3 Ethanol fermentation using SX2, SX3 and SX5 strains prepared in Example 1
  • Ethanol fermentation was performed using SX2, SX3 and SX5 among the strains prepared in Example 1.
  • SX2 XR mut Wow XDH is a strain inserted into the chromosome by homologous recombination
  • SX3 is an additional insertion of XK into the delta sequence on the chromosome to increase ethanol productivity for SX2.
  • SX5 is a strain further overexpressing transaldolase 1 (TAL1 ) in order to improve the metabolic rate of the pentose phosphate cycle against SX3.
  • TAL1 transaldolase 1
  • Fermentation was carried out using a 1 L multi fermentation tank (manufactured by 'Brown'), the operating volume was 500 mL.
  • the temperature of the fermenter was maintained at 30 °C, pH of the fermentation broth was maintained at 5.5.
  • the fermentation was performed using a microaerobic fermentation method, which was stirred at 300 rpm and subjected to aeration at 0.1 vvm.
  • the SX3 strain was increased by 2.7 times in the xylose consumption rate compared to the SX2 strain, and the final ethanol concentration was about 50% higher.
  • the newly introduced XR and XDH enzymes were expressed in the strain, and the SX5 strain overexpressing the XK and TAL1 enzymes showed a twofold improvement in xylose consumption rate and a final ethanol concentration of 80% compared to the SX3 strain. .
  • the SX5 strain was about three times higher than the SX3 strain, and the yield was 47% higher.
  • Example 4 Strains SX5 and SX5 prepared in Example 1 above: ald6 ⁇ Fermentation with Ethanol
  • Ethanol fermentation was performed using SX5: ald6 ⁇ from which the ALD6 gene was removed from the SX5 and SX5 strains prepared in Example 1.
  • Fermentation was carried out using a 1 L multi fermenter (Brown Co.), the operating volume was 500 mL. The temperature of the fermenter was maintained at 30 °C, pH of the fermentation broth was maintained at 5.5. Fermentation was carried out using an O 2 limited fermentation method which was stirred at 200 rpm and aerated at 0.06 vvm.
  • the SX5: ald6 ⁇ strain in which the ALD6 gene encoding acetaldehyde dehydrogenase, was disrupted in recombinant yeast, was compared to the final ethanol as shown in Table 3 above. The concentration was improved by 35% and the yield of ethanol was improved by 70%.

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Abstract

L'invention porte sur un procédé de production d'éthanol à partir de xylose à l'aide de Saccharomyces cerevisiae recombiné. Selon le procédé, du NADH comme cofacteur de la xylose réductase requise pour la production d'éthanol et du NAD+ comme cofacteur de la xylitol déshydrogénase sont couplés l'un avec l'autre et utilisés et l'acétaldéhyde déshydrogénase servant d'intermédiaire pour la production d'acide acétique comme sous-produit est enlevée et de l'éthanol peut ainsi être produit en une quantité élevée et avec un rendement de production élevé.
PCT/KR2009/007458 2009-12-12 2009-12-12 Procédé de production d'éthanol à partir de xylose à l'aide de saccharomyces cerevisiae recombiné mettant en jeu l'utilisation couplée de nadh et de nad+ WO2011071204A2 (fr)

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PCT/KR2009/007458 WO2011071204A2 (fr) 2009-12-12 2009-12-12 Procédé de production d'éthanol à partir de xylose à l'aide de saccharomyces cerevisiae recombiné mettant en jeu l'utilisation couplée de nadh et de nad+
US12/815,837 US8628944B2 (en) 2009-12-12 2010-06-15 Method for producing ethanol from xylose

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US9809831B2 (en) * 2010-10-28 2017-11-07 Total Research & Technology Feluy Monascus strains modified for organic acid production
CN103180451B (zh) * 2010-11-11 2014-12-31 丰田自动车株式会社 使用重组酵母的乙醇的制造方法
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CN108277244B (zh) * 2018-01-08 2020-09-08 深圳瑞德林生物技术有限公司 固定化酶级联反应制备景天庚酮糖及醛糖

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Title
BENGTSON, O. ET AL.: 'Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisia.' BIOTECHNOLOGY FOR BIOFUELS vol. 2, no. 9, 05 May 2009, *
PRASAD, S. ET AL.: 'Ethanol as an alternative fuel from agricultural, industrial and urban residues.' RESOURCES, CONSERVATION AND RECYCLING, 2007 vol. 50, no. 1, 01 January 1939, *

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US20110143409A1 (en) 2011-06-16
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