DK2785849T3 - Gærstammer, der er modificeret til at producere ethanol fra eddikesyre og glycerol - Google Patents

Gærstammer, der er modificeret til at producere ethanol fra eddikesyre og glycerol Download PDF

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DK2785849T3
DK2785849T3 DK12798012.6T DK12798012T DK2785849T3 DK 2785849 T3 DK2785849 T3 DK 2785849T3 DK 12798012 T DK12798012 T DK 12798012T DK 2785849 T3 DK2785849 T3 DK 2785849T3
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Bont Johannes Adrianus Maria De
Aloysius Wilhelmus Rudolphus Hubertus Teunissen
Paul Klaassen
Wouter Willem Antonius Hartman
Beusekom Shimaira Van
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Dsm Ip Assets Bv
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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
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
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    • 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
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • 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
    • 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
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01006—Glycerol dehydrogenase (1.1.1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/0101—Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • 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

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Claims (15)

1. Fremgangsmåde til fremstilling af ethanol, hvilken fremgangsmåde omfatter trinet fermentering af et medium med en gærcelle, hvor mediet indeholder eller tilføres: a) en kilde af mindst én af en hexose og en pentose; b) en kilde af eddikesyre; og c) en kilde af glycerol, hvor gærcellen fermenterer edikesyre, glycerol og mindst én af hexosen og pentosen til ethanol, og eventuelt indvinding af ethanolen, hvor gærcellen omfatter et exogent gen, der koder for et enzym med acetaldehyddehydrogenase-aktivitet, hvilket gen bibringer cellen evnen til at omdanne eddikesyre til ethanol, og hvor gærcellen omfatter et bakteriegen, der koder for et enzym med NAD+-koblet glyceroldehydrogenase-aktivitet, og hvor gærcellen omfatter en genmodifikation, der øger den specifikke aktivitet af dihydroxyacetonekinase, hvor gærcellen ikke er en gærcelle, der omfatter en genmodifikation, der reducerer specifik NAD+-afhængig formatdehydrogenase-aktivitet i cellen, hvor en forøgelse eller reduktion af en specifik aktivitet af et bestemt enzym er en forøgelse eller reduktion sammenlignet med den specifikke aktivitet af enzymet i en ellers identisk vildtypegærcelle.
2. Fremgangsmåde ifølge krav 1, hvor det exogene gen, der koder for enzymet med acetaldehyddehydrogenase-aktivitet omfatter en nukleotidsekvens, der koder for en aminosyresekvens med mindst én af: i) mindst 64 % aminosyresekvensidentitet med SEQ ID NO: 1, ii) mindst 76 % aminosyresekvensidentitet med SEQ ID NO: 3 og iii) mindst 61 % aminosyresekvensidentitet med SEQ ID NO: 5; og hvor bakteriegenet, der koder for et enzym med NAD+-koblet glyceroldehydrogenase-aktivitet, omfatter en nukleotidsekvens, der koder for en aminosyresekvens med mindst 45 % aminosyresekvensidentitet med SEQ ID NO: 7.
3. Fremgangsmåde ifølge krav 1 eller 2, hvor gærcellen omfatter en genmodifikation, der reducerer den specifikke aktivitet af NAD+-afhængig glycerol-3-phosphatdehydrogenase i cellen.
4. Fremgangsmåde ifølge krav 3, hvor genmodifikationen, der reducerer den specifikke aktivitet af NAD+-afhængig glycerol-3-phosphatdehydrogenase i cellen, er en genmodifikation, der reducerer eller inaktiverer ekspressionen af et endogent gen, der koder for en glycerolphosphatdehydrogenase, der har en aminosyresekvens med mindst 70 % sekvensidentitet med SEQ ID NO: 16.
5. Fremgangsmåde ifølge krav 1 eller 2, hvor genmodifikationen, der øger den specifikke aktivitet af dihydroxyacetonekinase, er overekspression af en nukleotidsekvens, der koder for en dihydroxyacetonekinase, og hvor fortrinsvis nukleotidsekvensen, der koder for dihydroxyacetonekinasen, omfatter en nukleotidsekvens, der koder for en aminosyresekvens med mindst 50 % aminosyresekvensidentitet med mindst én af SEQ ID NO: 8, 9 og 25.
6. Fremgangsmåde ifølge et hvilket som helst af kravene 2 til 5, hvor cellen yderligere omfatter en genmodifikation, der øger mindst én af: i) den specifikke acetyl-CoA-syntetaseaktivitet, hvor genmodifikationen er overekspression af en nukleotidsekvens, der koder for en acetyl-CoA-syntetase; og ii) transporten af glycerol ind i cellen ved overekspression af en nukleotidsekvens, der koder for mindst én af et glyceroloptagelsesprotein og en glycerolkanal, hvor fortrinsvis nukleotidsekvensen, der koder for glyceroloptagelsesproteinet, omfatter en nukleotidsekvens, der koder for en aminosyresekvens med mindst 50 % aminosyresekvensidentitet med mindst én af SEQ ID NO: 10 og 11, og hvor fortrinsvis nukleotidsekvensen, der koder for glycerolkanalen, omfatter en nukleotidsekvens, der koder for en aminosyresekvens med mindst 30 % aminosyresekvensidentitet med aminosyresekvensen mellem aminosyrerne 250 og 530 af SEQ ID NO: 12.
7. Fremgangsmåde ifølge krav 6, hvor nukleotidsekvensen, der koder for en acetyl-CoA-syntetase, er acetyl-CoA-syntetasen, der kodes af S. cerevisiae-ACSl- eller ACS2-genet.
8. Fremgangsmåde ifølge krav 6 eller 7, hvor genmodifikationen øger den specifikke acetyl-CoA-syntetaseaktivitet under anaerobe betingelser ved overekspression af en nukleotidsekvens, der koder for en acetyl-CoA-syntetase, hvor nukleotidsekvensen er operabelt koblet til en promotor, der er aktiv under anaerobe betingelser.
9. Fremgangsmåde ifølge et hvilket som helst af kravene 2 til 8, hvor gærcellen omfatter mindst: i) et funktionelt exogent xyloseisomerasegen, hvilket gen bibringer cellen evnen til at isomerisere xylose til xylulose; og ii) funktionelle exogene gener, der koder for en L-arabinoseisomerase, en L-ribulokinase og en L-ribulose-5-phosphat-4-epimerase, hvilke gener sammen bibringer cellen evnen til at omdanne L-arabinose til D-xylulose-5-phosphat, og hvor fortrinsvis gærcellen omfatter mindst én yderligere genmodifikation, der resulterer i en egenskab valgt fra gruppen, der består af: a) forøget xylulosekinasespecifik aktivitet; b) forøget strømning via pentosephosphatvejen; c) reduceret uspecifik aldosereduktasespecifik aktivitet; og d) forøget transport af mindst én af xylose og arabinose ind i værtscellen.
10. Fremgangsmåde ifølge et hvilket som helst af ovennævnte krav, hvor gærcellen er fra en slægt valgt fra gruppen, der består af Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces og Yarrowia.
11. Fremgangsmåde ifølge krav 10, hvor gærcellen tilhører en art valgt fra gruppen, der består af S. cerevisiae, S. exiguus, S. bayanus, K. lactis, K. marxianus og Schizosaccharomyces pombe.
12. Fremgangsmåde ifølge et hvilket som helst af ovennævnte krav, hvor mediet indeholder eller tilføres et lignocellulosehydrolysat.
13. Fremgangsmåde ifølge et hvilket som helst af ovennævnte krav, hvor gærcellen fermenterer under anaerobe betingelser.
14. Gærcelle, der omfatter: a) et exogent gen, der koder for et enzym med acetaldehyddehydrogenase-aktivitet, hvilket gen bibringer cellen evnen til at omdanne eddikesyre til ethanol; og b) et bakteriegen, der koder for et enzym med NAD+-koblet giyceroldehydrogenase-aktivitet, hvor gærcellen ikke er en gærcelle, der omfatter en genmodifikation, der reducerer specifik NAD+-afhængig formatdehydrogenase-aktivitet i cellen, hvor gærcellen omfatter en genmodifikation, der øger den specifikke aktivitet af dihydroxyacetonekinase, hvor en forøgelse eller reduktion af en specifik aktivitet af et bestemt enzym er en forøgelse eller reduktion sammenlignet med den specifikke aktivitet af enzymet i en ellers identisk vildtypegærcelle.
15. Gærcelle ifølge krav 14, hvor gærcellen yderligere er som defineret i et hvilket som helst af kravene 2 til 11.
DK12798012.6T 2011-11-30 2012-11-26 Gærstammer, der er modificeret til at producere ethanol fra eddikesyre og glycerol DK2785849T3 (da)

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US201161564932P 2011-11-30 2011-11-30
EP11191333 2011-11-30
PCT/NL2012/050841 WO2013081456A2 (en) 2011-11-30 2012-11-26 Yeast strains engineered to produce ethanol from acetic acid and glycerol

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US (2) US9988649B2 (da)
EP (2) EP2785849B1 (da)
JP (1) JP2015504309A (da)
KR (1) KR20140099251A (da)
CN (2) CN104126011B (da)
AU (1) AU2012346662B2 (da)
BR (1) BR112014012963B8 (da)
CA (1) CA2857247C (da)
DK (1) DK2785849T3 (da)
EA (1) EA201491056A8 (da)
ES (1) ES2648865T3 (da)
MX (1) MX2014006446A (da)
MY (1) MY169074A (da)
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