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WO2021063958A1 - Moyens et procédés améliorés pour augmenter le rendement d'acétyl-coa à partir du glucose - Google Patents

Moyens et procédés améliorés pour augmenter le rendement d'acétyl-coa à partir du glucose Download PDF

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Publication number
WO2021063958A1
WO2021063958A1 PCT/EP2020/077253 EP2020077253W WO2021063958A1 WO 2021063958 A1 WO2021063958 A1 WO 2021063958A1 EP 2020077253 W EP2020077253 W EP 2020077253W WO 2021063958 A1 WO2021063958 A1 WO 2021063958A1
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Prior art keywords
microorganism
phosphate
activity
coa
encoding
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English (en)
Inventor
Romain Chayot
Alfredo ALARCÓN YÁÑEZ
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Global Bioenergies SA
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Global Bioenergies SA
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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
    • 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
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • 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
    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C12P7/28—Acetone-containing products
    • 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

  • US Patent 7,785,858 disclose a recombinant bacterium selected from the group consisting of the Enterobacteriaceae familiy, Coryneform bacterium, and Bacillus bacterium comprising increased phosphoketolase activity for the conversion of glucose to target molecules which are produced via the intermediate acetyl-CoA, including the group consisting of L-glutamic acid, L- glutamine, L-proline, L-arginine, L-leucine, L-cysteine, succinate and polyhydroxybutyrate.
  • These strains feature PEP-dependent glucose uptake with the EMPP operative.
  • the activity of phosphofructokinase in the bacterium of US Patent 7,785,858 is reduced compared to that of a wild-type or non-modified strain (see page 33).
  • FIG. 1 This is schematically illustrated in Figure 1 wherein the metabolic steps starting from glucose into fructose-6-phosphate are shown.
  • glucose is converted into glucose-6-phosphate by a glucose kinase (EC 2.7.1.2) and/or an N- acetylmannosamine kinase (EC 2.7.1.60).
  • Said glucose-6-phosphate is further converted into fructose-6-phosphate by a glucose-6-phosphate isomerase (EC 5.3.1.9).
  • No conversion from glucose-6-phosphate into 6-phosphate-gluconolacetate occurs when abolishing or reducing glucose-6-phosphate dehydrogenase (EC 1.1.1.49).
  • No conversion from fructose-6-phosphate into fructose-1 ,6-bisphosphate occurs when abolishing or reducing phosphofructokinase (EC 2.7.1.11 ).
  • a phosphoketolase does not necessarily have to be present.
  • the present invention relates to such a microorganism which is further characterized by having no phosphoketolase activity and, accordingly, has also no genetic modification to have an increased expression of a gene encoding a phosphoketolase and/or an increased phosphoketolase activity as compared to a non-modified microorganism.
  • fructose-6-phosphate is also a substrate for an enzyme of the Embden- Meyerhof-Parnas pathway, i.e. the phosphofructokinase
  • the recombinant microorganism has a reduced phosphofructokinase activity as compared to a non- mod if ied microorganism or the gene(s) encoding a phosphofructokinase has/have been inactivated.
  • activation in the context of the present invention preferably means complete inactivation, i.e. that the microorganism does not show phosphofructokinase activity. This means in particular that the microorganism does not show phosphofructokinase activity independent from the used growth conditions.
  • activation means that the gene(s) encoding phosphofructokinase which are present in the microorganism are genetically modified so as to prevent the expression of the enzyme.
  • step A a glucose kinase (glk) (EC 2.7.1.2) and/or N-acetylmannosamine kinase (nanK) (EC 2.7.1.60) capable of converting glucose into glucose-6-phosphate (step A as illustrated in Figure 1)

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un micro-organisme recombiné caractérisé en ce qu'il comprend : a) ayant une inactivation du/des gène(s) codant pour la phosphofructokinase ou présentant une activité de phosphofructokinase réduite par comparaison avec un micro-organisme non modifié; b) ayant une inactivation du/des gène(s) codant pour la glucose-6-phosphate déshydrogénase ou présentant une activité de glucose-6-phosphate déshydrogénase réduite par comparaison avec un micro-organisme non modifié; et c) ayant une modification génétique pour avoir une expression accrue du/des gène(s) codant pour la désoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) et/ou une activité accrue de désoxy-D-ribose-5-phosphate aldolase (EC 4.1.2.4) par rapport à un microorganisme non modifié. De plus, l'invention concerne l'utilisation de ce micro-organisme recombiné pour la conversion de l'acétyl-CoA en acétone, isobutène, propène et/ou isopropanol. En outre, l'invention concerne un procédé de production d'acétone, d'isobutène, de propène et/ou d'isopropanol à partir d'acétyl-CoA comprenant les étapes de culture du micro-organisme recombiné de la présente invention dans un milieu approprié.
PCT/EP2020/077253 2019-09-30 2020-09-29 Moyens et procédés améliorés pour augmenter le rendement d'acétyl-coa à partir du glucose Ceased WO2021063958A1 (fr)

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EP19200516 2019-09-30
EP19200516.3 2019-09-30

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4506462A4 (fr) * 2022-04-02 2025-11-19 Mint Biotechnologies Co Ltd Procédé de préparation de glycine, d'acétyl coenzyme a et d'acétyl coenzyme a en utilisant de la thréonine

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EP2295593A1 (fr) 2009-09-15 2011-03-16 Philippe Marliere Procédé de production enzymatique d'acide 3-hydroxy-3-méthylbutyrique à partir d'acétone et acétyl-CoA
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* Cited by examiner, † Cited by third party
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WO2014086781A1 (fr) 2012-12-07 2014-06-12 Global Bioenergies Production d'un hydrocarbure par fermentation
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WO2017071124A1 (fr) 2015-10-28 2017-05-04 同济大学 Système et procédé de prédiction d'informations d'emplacement et de comportement
WO2017085167A2 (fr) 2015-11-17 2017-05-26 Global Bioenergies Procédés de production d'isobutène à partir d'acide 3-méthylcrotonique
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4506462A4 (fr) * 2022-04-02 2025-11-19 Mint Biotechnologies Co Ltd Procédé de préparation de glycine, d'acétyl coenzyme a et d'acétyl coenzyme a en utilisant de la thréonine

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