WO2008144791A2 - Procédé pour convertir du xylose ou des solutions de substrats contenant du xylose - Google Patents

Procédé pour convertir du xylose ou des solutions de substrats contenant du xylose Download PDF

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WO2008144791A2
WO2008144791A2 PCT/AT2008/000184 AT2008000184W WO2008144791A2 WO 2008144791 A2 WO2008144791 A2 WO 2008144791A2 AT 2008000184 W AT2008000184 W AT 2008000184W WO 2008144791 A2 WO2008144791 A2 WO 2008144791A2
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xylose
yeast strain
gene
yeast
ethanol
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PCT/AT2008/000184
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German (de)
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WO2008144791A3 (fr
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Barbara Petschacher
Bernd Nidetzky
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Vogelbusch Gesellschaft M.B.H.
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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
    • 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
    • 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
    • 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
    • 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
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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 invention relates to a method of converting xylose or xylose containing substrate solutions, e.g. Hydrolysates of lignocelluloses in ethanol by means of Saccharomyces cerevisiae.
  • bioethanol is considered to be an important, CO 2 -neutral alternative to petroleum-based fuels today.
  • Ethanol is produced today mainly by fermentation of glucose obtained from starch contained in plant materials such as wheat or corn.
  • sucrose from sugar beets or sugar cane is used as a substrate for ethanol production.
  • land u. forestry waste a much cheaper raw material for bioethanol production.
  • these vegetable waste materials may contain up to 30% of the pentose xylose.
  • the economic use of the xylose fraction of lignocellulosic starting material is critical to the economics of ethanol production.
  • yeast Saccharomyces cerevisiae lacks a functional metabolic pathway for the assimilation of xylose.
  • This metabolic pathway consists of the enzyme xylose reductase (XR) for the reduction of xylose to xylitol and the xylitol dehydrogenase (XDH) for the conversion of xylitol to xylulose.
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XK xylulose kinase
  • the hitherto mainly used scientific strategy to facilitate the ethanol fermentation of xylose by Saccharomyces cerevisiae was the heterologous expression of Pichia stipitis genes encoding xylose reductase and xylitol dehydrogenase, together with homologous overexpression of the gene for xylulose kinase from S. cerevisiae.
  • the first strains containing these enzymes (Ho et al., 1998, Eliasson et al. 2000, Toivari et al. 2001) showed xylose conversion, but the ethanol yield was low compared to the theoretically possible value and by-product formation, with xylitol and glycerol excretion mainly being high.
  • XDH is always NAD + specific, therefore, in the first two steps of xylose metabolism is genetically engineered Yeast NADH accumulates and NADPH is consumed. As a reaction of the cell, xylitol is excreted, the glycerol formation is increased, thereby decreasing the ethanol yield.
  • the ethanol yield in a co-fermentation of glucose and xylose was increased by 16-19%, which was mainly achieved by the reduction of xylitol formation by 44%.
  • Xylose isomerase (XI) is expressed, which converts xylose directly to xylulose, avoiding the co-factor imbalance. While expression of bacterial XI only resulted in a strain with low ethanol production from xylose (Wallfridsson et al., 1996), Kuyper et al. (2005) a Saccharomyces cerevisiae strain expressing XI from the fungus Piromyces.
  • a strain that combines XI expression with overexpression of xylulose kinase and enzymes of the pentose phosphate cycle with suppression of GRE3 activity in Saccharomyces cerevisiae shows high ethanol yields, low xylitol yields and anaerobic growth on xylose-containing media.
  • the good results in the ethanol yield expressing the redox-neutral XI illustrate the importance of coordinating the cofactor specificity of XR and XDH to allow for cofactor recycle.
  • a recombinant yeast strain with expression of XR and XDH shows higher xylose uptake rates than a strain with integration of the above-mentioned XI without any other genetic modification except XK overexpression (Karhumaa et al., 2007).
  • XR and XDH shows higher xylose uptake rates than a strain with integration of the above-mentioned XI without any other genetic modification except XK overexpression
  • the present invention is based on the object of developing a method and a yeast strain for converting xylose or xylose-containing substrates into ethanol by means of Saccharomyces cerevisiae, with which a correspondingly high yield of ethanol based on xylose is achieved.
  • this is achieved by using for conversion a yeast strain which comprises a per se known mutant of fungal xylose reductase in which a sequence motif Lys-Ser-Asn (homologous to Lys274-Ser-Asn276 in xylose reductase from Candida tenuis) was exchanged for Arg-Ser-Asp, and the wild-type xylitol dehydrogenase of eg Galactocandida mastotermitis, wherein the yeast strain optionally contains at least one additional gene copy of the xylulose kinase gene.
  • a yeast strain which comprises a per se known mutant of fungal xylose reductase in which a sequence motif Lys-Ser-Asn (homologous to Lys274-Ser-Asn276 in xylose reductase from Candida tenuis) was exchanged for Arg-Ser-Asp, and the wild-type xylit
  • This invention provides a direct way to overcome the co-factoric imbalance between XR and XDH by expression of a xylose reductase
  • a co-factor use mutant which has a preference for NADH and thus allows for a higher degree of co-factor recycling between the XR and XDH catalyzed reactions.
  • the fungal xylose reductase used is that of Candida tenuis.
  • the invention covers the expression of a Candida tenuis xylose reductase K274R / N276D mutant (xylose reductase from Candida tenuis in which lysine 274 is replaced by arginine and asparagine 276 by aspartate) and a galactocandida mastotermitis xylitol dehydrogenase together with overexpression of the xylulose kinase gene from S. cerevisiae.
  • the K274R / N276D mutant has been found to be the best mutant of a whole set of rationally designed mutants in a thorough study of the possibilities of changing the co-factor specificity of CtXR (Candida tenuis xylose reductase) from NADPH to NADH was created (Petschacher et al., 2005).
  • the Candida tenuis xylose reductase was chosen as a good starting point due to the dual coenzyme specificity of the wild-type form.
  • the coenzyme preference of the K274R / N276D mutant calculated from the kinetic data shows a fivefold preference for NADH compared to a 33-fold preference for NADPH in the wild-type form.
  • the ethanol fermentation improved 1.2-fold from 0.29 to 0.36 g / g xylose in a batch fermentation on 50 g / L xylose at a carbon recovery rate of 99%.
  • Figure 1 shows schematically the cofactor recycle in the first two steps of xylose metabolism of recombinant yeast using NADH-specific XR.
  • Figure 2 shows in comparison the fermentation curves of the strain BP000 (wild type XR) and BPlOOOl (inventively provided with the K274R / N276D XR mutant Saccharomyces cerevisiae), where diagram A shows the fermentation of BP000 and B shows the diagram of fermentation of BPlOOOl.
  • filled squares mean xylose; Triangles ethanol; Circles xylitol; Asterisks glycerin; and open squares acetate.
  • the cell dry weight was about 1.5 g / L.
  • the Escherichia coli strain TOP10 (Invitrogen, Carlsbad, CA, USA) was used.
  • the template plasmids for the amplification of XR were pETII -CtXRWt carrying the xylose reductase gene of Candida tenuis (CBS4435, Ralpher et al., 1999) and pETI 1-CtXRDm carrying the K274R / N276D mutant gene of C. tenuis xylose reductase (Petschacher 2005).
  • the Galactocandida mastotermitis XDH gene was amplified from pBTacl (Habenicht 1999).
  • the plasmid pRS416GPD (Mumberg et al., 1995, a gift from Harald Pichler, Graz University of Technology) was used for the construction of gene cassettes with a yeast promoter and terminator.
  • Ylp5 purchased from DSMZ, Braunschweig, Germany
  • Saccharomyces cerevisiae strain CEN.PK 113-7-D (MATa MAL2-8c SUC2, a gift from Jochen Förster, Fluxome sciences, Denmark) was used.
  • the transformed bacteria were on Luria-Bertani medium agar plates with
  • yeast cells for transformation were grown in YPD medium.
  • the yeast transformants were selected on synthetic yeast complete medium agar plates made from nitrogen base ("yeast nitrogen base", Sigma, St. Louis, Mo., USA) with synthetic drop-out medium added without uracil (Sigma, St. Louis, MO, USA)
  • yeast nitrogen base Sigma, St. Louis, Mo., USA
  • synthetic drop-out medium added without uracil (Sigma, St. Louis, MO, USA)
  • a defined mineral medium containing vitamins and trace elements was selected The medium was supplemented with 0.01 g / L ergosterol and 0.42 g / L Tween 80, dissolved in boiling 96% by volume ethanol and supplemented with 100 mM citrate buffer pH 5.5. Construction of yeast integration vectors encoding the genes for WtXR or DmXR. XDH and XK included
  • Restriction enzymes were purchased from MBI Fermentas (ST.Lone-Roth, Germany) or New England Biolabs (Beverly, MA), the Pfu polymerase from Promega (Madison, WI, USA).
  • a Quiaprep Spin Miniprep Kit from Quiagen (Quiagen GmbH, Hilden, Germany) was used and a QUIAquick Gel Extraction Kit was used for DNA extraction from agarose. The handling and processing of nucleic acids was performed according to standard techniques.
  • the promoterless genes of the Candida tenuis wild-type and mutant xylose reductase, the Galctocandida mastotermitis XDH and the Saccharomyces cerevisiae XK from the template plasmids pETl l -CtXRWt, pETl l -CtXRDm, pBTacl and in the case of the XK of genomic Saccharomyces cerevisiae DNA was amplified using primers with a BamHI restriction site and reverse primers with a Sali restriction site, with both cleavage sites attached to the 5 'end of the primers (see Table 1).
  • PCR products were digested with BamHI and Sali, cloned into the multiple cloning site of pRS416GPD located between the GPD promoter and the CYCl terminator.
  • vectors were constructed for XRWt, XRDm, XDH and XK with a gene cassette containing the strong yeast constitutive GPD promoter, the target gene and the CYCl terminator. The correct insertion was verified by sequencing.
  • these gene cassettes were amplified with primers which have restriction sites at the 5 'ends which occur only once in Ylp5 (see Table 1).
  • the XK gene cassette was cloned into the AatII site, yielding the vector YK.
  • the XDH gene cassette was inserted into the CIaI site of YXKDl, resulting in YGmXDH / XKS1, and finally the gene cassette for either XRWt or XRDm was cloned into the EcoRI site of YGmXDH / XKSl resulting in YCtXRWt / GmXDH / XKSl and YCtXRDm / GmXDH / XK led.
  • those plasmids were selected for further use that gave positive PCR results, when a forward primer was used which was homologous to a sequence upstream of the cloning site in the target vector, and a reverse primer homologous to a part of the gene.
  • Table 1 Cloning strategy for the construction of yeast integration plasmids YCtXRWt / GmXDH / XKS1 and YCtXRDm / GmXDH / XKS1
  • a gene cassettes contain S. cerevisiae GPD promoter - target gene -S. cerevisiae CYC1 terminator
  • Transformation of plasmids into E. coli TOP10 cells was performed by electroporation.
  • the plasmids YCtXRWt / GmXDH / XKSl and YCtXRDm / GmXDH / XKSl to be integrated into the yeast were cut with Sdal within the URA3 gene.
  • the linearized vectors were used for the transformation of Saccharomyces cerevisiae CEN.PK 113-5D by the lithium acetate method (Gietz 1995).
  • strain BP000 expressing the wild-type CtXR and strain BP1001 expressing the CtXR K274R / N276D mutant were recovered.
  • the batch fermentations were carried out in baffled 300 mL shake flasks sealed with a rubber stopper. Two glass tubes were inserted into the plugs, one fitted with a valve for purging with nitrogen. The other was completed with a finally fused tube which is provided with a slit in the wall to the outlet of the fermentation gases. In the piston, a magnet was used.
  • the cells for the inoculum were grown in minimal medium with 20 g / L glucose overnight at 30 ° C., 110 rpm, then 10 min. centrifuged at 5000 rcf (relative centrifuge force) and washed twice with 0.9% NaCl.
  • the cells were transferred to shake flasks filled with 280 mL minimal medium containing 15 g / L xylose to give a final OD600 of approximately 4.
  • the medium in the flasks was filled with nitrogen 5.0 for 15 min. before and 5 min. rinsed after inoculation.
  • the flasks were sealed and incubated at 30 ° C and at 100 rpm.
  • the samples were drawn after mixing the contents of the flask on a magnetic stirrer under nitrogen purge.
  • Samples for HPLC measurements were taken through a Satorius Minisart RC4 Filter (Satorius, Göttingen, Germany) filtered and stored at -20 ° C for further analysis. The batch fermentations were carried out in triplicate for each strain.
  • the cell growth was observed optically at 600 nm.
  • the cell dry weight was determined by filtering 50 ml of fermentation broth through a pre-dried and weighed 0.45 ⁇ m cellulose acetate membrane filter (Satorius) and drying it overnight in a drying oven at 105 ° C.
  • Glucose, xylose, xylitol, glycerol and acetate were determined using a Merck-Hitachi LaChrome HPLC system with an Aminex HPX-87H column (Biorad, Richmond, CA, USA) and a Merck L-7490 RI detector. 5mM sulfuric acid was used as the eluent at a constant flow rate of 0.6 mL / h and 65 ° C operating temperature.
  • the cells were grown aerobically at 120 rpm and 30 ° C. in defined minimal medium with 20 g / L glucose and 20 g / L xylose as carbon source up to an OD 60O of 6.
  • the cells from 3 mL of the fermentation broth were centrifuged at 5000 rcf for 10 min. and then lysed with 250 ⁇ L of the Y-PER reagent (Pierce, Rockford, IL, USA) according to the instructions for use.
  • the protein content of the crude extract was determined using a protein determination according to Bradford (Roti-Quant, Carl Roth GmbH, Düsseldorf, Germany), using BSA fraction 5 as standard.
  • Xylose reductase activity was assessed by initial velocity measurement in a photometric assay containing 700 mM D-xylose and 300 ⁇ M or 7 ⁇ M of either NADH or NADPH as previously described (Petschacher 2005).
  • Xylitol dehydrogenase activity was measured according to a published photometric experiment (Mayer 2002) containing 150 mM xylitol and 2 mM NAD + .
  • Xylulose kinase activity was determined as previously described (De Luccio et al., 2005).
  • the standard assay contained 5 mM ATP and 4.3 mM D-xylulose. Blank values from XDH activity with xylulose were subtracted.
  • the mutant CtXR shows a change of co-factor use of a 33-fold preference of NADPH in the wild-type form to a 5-fold preference of NADH in the mutant enzyme, calculated on the enzymatic efficiency.
  • the change in co-factor preference is due to a dramatic 43-fold increase in the K m value for NADPH in the mutant and a 5-fold increase in the xylose K m value compared to the wild type and mutant NADH - Application remained essentially unchanged.
  • the functional integration of these genes has been verified by the ability to aerobically grow on xylose for both strains.
  • xylose reductase was validated by measurements of photometric activity from crude extracts of cells aerobically grown on glucose and xylose (see Table 2).
  • the measurements at low NADPH concentration (7 mM) confirm the integration of the mutant which has a higher K m value for NADPH and therefore lower activities (16% of v max ) at low NADPH values than the wild-type (79% of
  • Table 3 Results of oxygen-limited batch fermentations of two recombinant S. cerevisiae strains differing in their co-factor specificity to 20 g / L xylose.
  • the xylitol production could be significantly reduced by 53% to 0.17 g / g and the glycerol production by 30% to 0.063 g / g.
  • the acetate formation increased from 0.02 to 0.03 g / g.

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Abstract

L'invention concerne un procédé pour convertir du xylose ou des substrats contenant du xylose, par exemple des hydrolysats de lignocellulose dans de l'éthanol au moyen de Saccharomyces cerevisiae. L'objectif de l'invention est d'améliorer le rendement d'éthanol et de réduire la production de sous-produits. A cet effet, l'invention comprend l'utilisation d'une souche de levure qui exprime un mutant connu de la xylose-réductase fongique, dans laquelle un motif de séquence Lys-Ser-Asn (homologue de Lys274-Ser-Asn276 dans la xylose réductase obtenue à partir de Candida tenuis) est substitué par Arg-Ser-Asp, ainsi que le type sauvage de la xylite déshydrogénase de la Galactocandida mastrotermitis par exemple. Ladite souche de levure contient éventuellement au moins une copie génique supplémentaire du gène pour la xylulose réductase.
PCT/AT2008/000184 2007-05-30 2008-05-29 Procédé pour convertir du xylose ou des solutions de substrats contenant du xylose WO2008144791A2 (fr)

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AT0084907A AT505251B1 (de) 2007-05-30 2007-05-30 Verfahren zum umwandeln von xylose oder xylose enthaltenden substratlísungen
ATA849/2007 2007-05-30

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8097440B1 (en) 2008-10-31 2012-01-17 Gevo, Inc. Engineered microorganisms capable of producing target compounds under anaerobic conditions
US10808262B2 (en) 2013-12-03 2020-10-20 Genomatica, Inc. Microorganisms and methods for improving product yields on methanol using acetyl-CoA synthesis

Citations (1)

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WO2005093041A1 (fr) * 2004-03-26 2005-10-06 Forskarpatent I Syd Ab Xylose reductase mutee lors de la fermentation du xylose par s. cerevisiae

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NIDETZKY BERND ET AL: "Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli." CHEMICO-BIOLOGICAL INTERACTIONS, Bd. 143-144, 1. Februar 2003 (2003-02-01), Seiten 533-542, XP002504770 ISSN: 0009-2797 *
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PETSCHACHER BARBARA ET AL: "Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae" MICROBIAL CELL FACTORIES, BIOMED CENTRAL, LONDON, NL, Bd. 7, Nr. 1, 17. März 2008 (2008-03-17), Seite 9, XP021036443 ISSN: 1475-2859 *
PETSCHACHER BARBARA ET AL: "The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography" BIOCHEMICAL JOURNAL, Bd. 385, Nr. Part 1, Januar 2005 (2005-01), Seiten 75-83, XP002504768 ISSN: 0264-6021 in der Anmeldung erwähnt *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8097440B1 (en) 2008-10-31 2012-01-17 Gevo, Inc. Engineered microorganisms capable of producing target compounds under anaerobic conditions
US10808262B2 (en) 2013-12-03 2020-10-20 Genomatica, Inc. Microorganisms and methods for improving product yields on methanol using acetyl-CoA synthesis

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AT505251B1 (de) 2009-09-15
WO2008144791A3 (fr) 2009-02-05

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