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WO2015028583A2 - Cellules de conversion du glycérol et de l'acide acétique présentant un meilleur transport du glycérol - Google Patents

Cellules de conversion du glycérol et de l'acide acétique présentant un meilleur transport du glycérol Download PDF

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Publication number
WO2015028583A2
WO2015028583A2 PCT/EP2014/068325 EP2014068325W WO2015028583A2 WO 2015028583 A2 WO2015028583 A2 WO 2015028583A2 EP 2014068325 W EP2014068325 W EP 2014068325W WO 2015028583 A2 WO2015028583 A2 WO 2015028583A2
Authority
WO
WIPO (PCT)
Prior art keywords
glycerol
cell
seq
yeast
dehydrogenase
Prior art date
Application number
PCT/EP2014/068325
Other languages
English (en)
Other versions
WO2015028583A3 (fr
Inventor
Paul Klaassen
Wouter Willem Antonius HARTMAN
Original Assignee
Dsm Ip Assets B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Publication of WO2015028583A2 publication Critical patent/WO2015028583A2/fr
Publication of WO2015028583A3 publication Critical patent/WO2015028583A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • 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.)
    • C12N9/12—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
    • 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

  • yeasts that are capable of producing ethanol from acetic acid or acetate while retaining their abilities of fermenting hexoses (glucose, fructose, galactose, etc) as well as pentoses like xylose, and wherein these strains are used for the production of ethanol and/or other fermentation products.
  • Another object is to provide cells in particular yeast cells that can anaerobically ferment pentose, hexose, glycerol and acetic acid.
  • Another object is to provide strains that have an improved production of fermentation product.
  • Fig. 5 Nett acetic acid consumption, expressed in grams per liter, in time.
  • A no glycerol added
  • B 1 .3 grams of glycerol per liter
  • C 2.7 grams of glycerol per liter
  • D 7.2 grams of glycerol per liter
  • E 14.1 grams of glycerol per liter.
  • Fig. 10 Growth of RN 1041 -transformants in Mineral Medium supplemented with 2,3 grams urea per liter and containing 2.5% glucose, 2.5% xylose, 2 g/l HAc, 1 % glycerol and 200 G418/ml
  • SEQ ID NO: 37 Forward primer for the amplification of the glycerol transporter expression cassettes
  • Table 4 Description of protein function of proteins encoded by FPS1, GUP1, GUP2 and STL1.
  • GUP2 Probable membrane protein; possible role in proton symport of (YPL189w) glycerol; member of the MBOAT family of putative membrane-bound
  • glycerol uptake facilitator GlpF [Caloramator 33 WP_008907471.1 australicus] >emb
  • the cell of the invention may comprise an exogenous gene coding for an enzyme with the ability to reduce acetylCoA into acetaldehyde, which gene confers to the cell the ability to convert acetylCoA (and/or acetic acid) into ethanol.
  • An enzyme with the ability to reduce acetylCoA into acetaldehyde is herein understood as a bifuntional enzyme which catalyzes the following reactions (adhE): acetaldehyde + NADH ⁇ ethanol + NAD * (4)
  • the cell comprises one or more nucleotide sequence encoding a acetyl-CoA synthetase (E.C. 6.2.1.1 );
  • the crystal structures of a eukaryotic (e.g. from yeast) and bacterial (e.g. from Salmonella) form of this enzyme have been determined.
  • the yeast enzyme is trimeric, while the bacterial enzyme is monomeric.
  • the trimeric state of the yeast protein may be unique to this organism however, as the residues involved in the trimer interface are poorly conserved in other sequences.
  • the structures of the monomers are almost identical.
  • a large N-terminal domain (-500 residues) containing two parallel beta sheets is followed by a small (-1 10 residues) C-terminal domain containing a three- stranded beta sheet with helices.
  • the active site occurs at the domain interface, with its contents determining the orientation of the C-terminal domain.
  • the cell is a prokaryotic cell.
  • the cell is selected from the list consisting of Clostridium, Zymomonas, Thermobacter, Escherichia, Lactobacillus, Geobacillus and Bacillus.
  • the yeast cell thus acquires the ability to grow aerobically and/or anaerobically on xylose as sole energy and/or carbon source though direct isomerisation of xylose into xylulose (and further metabolism of xylulose).
  • direct isomerisation of xylose into xylulose occurs in a single reaction catalysed by a xylose isomerase, as opposed to the two step conversion of xylose into xylulose via a xylitol intermediate as catalysed by xylose reductase and xylitol dehydrogenase, respectively.
  • a given cell may comprise multiple copies of genes encoding unspecific aldose reductases as a result of di-, poly- or aneuploidy, and/or a cell may contain several different (iso)enzymes with aldose reductase activity that differ in amino acid sequence and that are each encoded by a different gene. Also in such instances preferably the expression of each gene that encodes an unspecific aldose reductase is reduced or inactivated.
  • the media used in the experiments was either YEP-medium (10 g/l yeast extract, 20 g/l peptone) or solid YNB-medium (6.7 g/l yeast nitrogen base, 15 g/l agar), supplemented with sugars as indicated in the examples.
  • YEP-medium 10 g/l yeast extract, 20 g/l peptone
  • solid YNB-medium 6.7 g/l yeast nitrogen base, 15 g/l agar
  • sugars as indicated in the examples.
  • solid YEP medium 15 g/l agar was added to the liquid medium prior to sterilization.
  • delta means chromosomal integration of the construct after recombination on the long terminal repeats of the Ty1 retrotransposon.
  • Strain RN1069 is derived from RN1041 : the GPD1 and GPD2 genes were disrupted by gene replacement. To this end, dominant antibiotic resistance markers, flanked by sequences homologous to the sequences just beside the open reading frame (ORF) of GPD1 or GPD2, were constructed by PCR and used to transform strain RN1041. These gene disruption cassettes have been included in the sequence listing as SEQ ID NO: 28 and 29 respectively. The construction of strain RN1069 is also described in detail in WO2013/081456.
  • the analyte concentrations are calculated based on the following signals ( ⁇ relative to DSS):

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une cellule génétiquement modifiée comprenant: a) une ou plusieurs séquences nucléotidiques codant pour une glycérole déshydrogénase (E.C. 1.1.1.6); b) une ou plusieurs séquences nucléotidiques codant pour une dihydroxyacétone kinase (E.C. 2.7.1.28 or E.C. 2.7.1.29) et c) une ou plusieurs séquences nucléotidiques codant pour un transporteur glycérol.
PCT/EP2014/068325 2013-08-29 2014-08-29 Cellules de conversion du glycérol et de l'acide acétique présentant un meilleur transport du glycérol WO2015028583A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP13182225 2013-08-29
EP13182225.6 2013-08-29

Publications (2)

Publication Number Publication Date
WO2015028583A2 true WO2015028583A2 (fr) 2015-03-05
WO2015028583A3 WO2015028583A3 (fr) 2015-04-23

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AR (1) AR097479A1 (fr)
WO (1) WO2015028583A2 (fr)

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016008819A1 (fr) * 2014-07-14 2016-01-21 Jacobs University Bremen Ggmbh Levure génétiquement modifiée présentant un catabolisme du glycérol amélioré
WO2016097202A1 (fr) * 2014-12-19 2016-06-23 Dsm Ip Assets B.V. Procédé de fermentation présentant une conversion de l'acide acétique et du glycérol améliorée
WO2016188813A1 (fr) 2015-05-22 2016-12-01 Dsm Ip Assets B.V. Cellule de levure consommant de l'acétate
WO2017060195A1 (fr) 2015-10-06 2017-04-13 Dsm Ip Assets B.V. Cellule eucaryote présentant une production accrue de produit de fermentation
WO2018114762A1 (fr) * 2016-12-23 2018-06-28 Dsm Ip Assets B.V. Production améliorée d'éthanol sans glycérol
WO2018172328A1 (fr) * 2017-03-21 2018-09-27 Dsm Ip Assets B.V. Production améliorée d'éthanol sans glycérol
WO2018176021A1 (fr) * 2017-03-24 2018-09-27 Danisco Us Inc Réduction d'acétate et de glycérol dans une levure modifiée ayant une voie de production d'éthanol exogène
WO2019063544A1 (fr) 2017-09-26 2019-04-04 Dsm Ip Assets B.V. Souche consommatrice d'acide acétique
CN111727196A (zh) * 2017-10-24 2020-09-29 丹尼斯科美国公司 具有改善的醇生产的酵母
US10913928B2 (en) 2016-12-20 2021-02-09 Dsm Ip Assets B.V. Yeast strains for ethanol production
WO2021089877A1 (fr) 2019-11-08 2021-05-14 Dsm Ip Assets B.V. Procédé de production d'éthanol
WO2022261003A1 (fr) 2021-06-07 2022-12-15 Novozymes A/S Micro-organisme génétiquement modifié pour une fermentation d'éthanol améliorée
WO2023285279A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285282A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285294A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285281A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285297A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285280A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinante
WO2023079050A1 (fr) 2021-11-04 2023-05-11 Dsm Ip Assets B.V. Cellule de levure recombinée

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008006037A2 (fr) * 2006-07-06 2008-01-10 Integrated Genomics, Inc. Compositions et procédés pour augmenter l'utilisation du glycérol
WO2010015122A1 (fr) * 2008-08-06 2010-02-11 Tianjin University Souches de levure et procédés pour fabriquer et utiliser de telles souches de levure
WO2010019882A1 (fr) * 2008-08-15 2010-02-18 Edeniq, Inc. Levure génétiquement modifiée et procédés de fabrication et d’utilisation associés
US20110250664A1 (en) * 2010-04-08 2011-10-13 Tianjin University Yeast having improved ethanol yield
US8772000B2 (en) * 2010-04-19 2014-07-08 Korea University Research And Business Foundation Transformant for enhancing bioethanol production, and method for producing ethanol by using said strain
WO2011149353A1 (fr) * 2010-05-27 2011-12-01 C5 Yeast Company B.V. Souches de levure manipulées pour produire de l'éthanol à partir d'acide acétique et de glycérol
EP2663645B1 (fr) * 2010-11-18 2014-12-17 DSM IP Assets B.V. Souches de levures modifiées pour produire de l'éthanol à partir de glycérol
AU2012346662B2 (en) * 2011-11-30 2016-07-28 Danisco Us Inc. Yeast strains engineered to produce ethanol from acetic acid and glycerol

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016008819A1 (fr) * 2014-07-14 2016-01-21 Jacobs University Bremen Ggmbh Levure génétiquement modifiée présentant un catabolisme du glycérol amélioré
US11667935B2 (en) 2014-12-19 2023-06-06 Dsm Ip Assets B.V. Fermentation process for improved glycerol and acetic acid conversion
WO2016097202A1 (fr) * 2014-12-19 2016-06-23 Dsm Ip Assets B.V. Procédé de fermentation présentant une conversion de l'acide acétique et du glycérol améliorée
EP3831950A1 (fr) * 2014-12-19 2021-06-09 DSM IP Assets B.V. Procédé de fermentation à conversion d'acide acétique et de glycérol améliorée
US10883122B2 (en) 2014-12-19 2021-01-05 Dsm Ip Assets B.V. Fermentation process for improved glycerol and acetic acid conversion
WO2016188813A1 (fr) 2015-05-22 2016-12-01 Dsm Ip Assets B.V. Cellule de levure consommant de l'acétate
EP3604506A1 (fr) 2015-05-22 2020-02-05 DSM IP Assets B.V. Cellule de levure consommant des acétates
WO2017060195A1 (fr) 2015-10-06 2017-04-13 Dsm Ip Assets B.V. Cellule eucaryote présentant une production accrue de produit de fermentation
US11136600B2 (en) 2015-10-06 2021-10-05 Dsm Ip Assets B.V. Eukaryotic cell with increased production of fermentation product
CN108603179B (zh) * 2015-10-06 2022-02-08 帝斯曼知识产权资产管理有限公司 发酵产物生产增加的真核细胞
CN108603179A (zh) * 2015-10-06 2018-09-28 帝斯曼知识产权资产管理有限公司 发酵产物生产增加的真核细胞
EP3620516A1 (fr) 2015-10-06 2020-03-11 DSM IP Assets B.V. Cellule eucaryote avec une production accrue de produit de fermentation
US10913928B2 (en) 2016-12-20 2021-02-09 Dsm Ip Assets B.V. Yeast strains for ethanol production
US11624057B2 (en) 2016-12-23 2023-04-11 Dsm Ip Assets B.V. Glycerol free ethanol production
CN110088275A (zh) * 2016-12-23 2019-08-02 帝斯曼知识产权资产管理有限公司 改进的无甘油的乙醇生产
US11203741B2 (en) 2016-12-23 2021-12-21 Dsm Ip Assets B.V. Glycerol free ethanol production
US10982195B2 (en) 2016-12-23 2021-04-20 Dsm Ip Assets B.V. Glycerol free ethanol production
WO2018114762A1 (fr) * 2016-12-23 2018-06-28 Dsm Ip Assets B.V. Production améliorée d'éthanol sans glycérol
WO2018172328A1 (fr) * 2017-03-21 2018-09-27 Dsm Ip Assets B.V. Production améliorée d'éthanol sans glycérol
WO2018176021A1 (fr) * 2017-03-24 2018-09-27 Danisco Us Inc Réduction d'acétate et de glycérol dans une levure modifiée ayant une voie de production d'éthanol exogène
WO2019063544A1 (fr) 2017-09-26 2019-04-04 Dsm Ip Assets B.V. Souche consommatrice d'acide acétique
CN111133111A (zh) * 2017-09-26 2020-05-08 帝斯曼知识产权资产管理有限公司 消耗乙酸的菌株
CN111727196A (zh) * 2017-10-24 2020-09-29 丹尼斯科美国公司 具有改善的醇生产的酵母
WO2021089877A1 (fr) 2019-11-08 2021-05-14 Dsm Ip Assets B.V. Procédé de production d'éthanol
CN114667347A (zh) * 2019-11-08 2022-06-24 帝斯曼知识产权资产管理有限公司 用于生产乙醇的方法
WO2022261003A1 (fr) 2021-06-07 2022-12-15 Novozymes A/S Micro-organisme génétiquement modifié pour une fermentation d'éthanol améliorée
WO2023285279A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285282A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285294A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285281A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285297A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée
WO2023285280A1 (fr) 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinante
WO2023079050A1 (fr) 2021-11-04 2023-05-11 Dsm Ip Assets B.V. Cellule de levure recombinée

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WO2015028583A3 (fr) 2015-04-23
AR097479A1 (es) 2016-03-16

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