EP2235167A1 - Production accrue d'acétyl-coenzyme a - Google Patents

Production accrue d'acétyl-coenzyme a

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Publication number
EP2235167A1
EP2235167A1 EP09703058A EP09703058A EP2235167A1 EP 2235167 A1 EP2235167 A1 EP 2235167A1 EP 09703058 A EP09703058 A EP 09703058A EP 09703058 A EP09703058 A EP 09703058A EP 2235167 A1 EP2235167 A1 EP 2235167A1
Authority
EP
European Patent Office
Prior art keywords
host cell
coenzyme
acetyl
pyruvate
formate
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP09703058A
Other languages
German (de)
English (en)
Inventor
Gunter Festel
Jelena Simona Duvnjak
Eckhard Boles
Christian Weber
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Butalco GmbH
Original Assignee
Butalco GmbH
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 Butalco GmbH filed Critical Butalco GmbH
Publication of EP2235167A1 publication Critical patent/EP2235167A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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.)
    • C12N9/0008—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
    • 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.)

Definitions

  • the present invention relates to the development of enzymatic bypass reactions of pyruvate dehydrogenase in order to produce from pyruvate, coenzyme A and NAD + the molecules acetyl coenzyme A, NADH + H + and CO 2 , without energy equivalents, such as in the form of ATP to consume.
  • This bypass reaction consists either of the activities of a pyruvate decarboxylase together with a coenzyme A-dependent aldehyde dehydrogenase or of the activities of a pyruvate formate lyase together with a formate dehydrogenase.
  • the invention relates to the expression of one or both of these bypassing reactions in the cytoplasm of living cells, preferably eukaryotic cells.
  • the invention further relates in particular to the expression in eukaryotic cells of coenzyme A-dependent aldehyde dehydrogenases, preferably coenzyme A-dependent acetaldehyde dehydrogenases [also acetaldehyde: NAD + oxidoreductase (CoA-acetylating) or acetaldehyde called dehydrogenase (acetylating)] and / or of pyruvate formate lyases together with their activating enzyme.
  • the invention further relates to host cells, in particular modified yeast strains containing the genes for
  • Coenzyme A-dependent acetaldehyde dehydrogenases together with those for pyruvate decarboxylases and / or express the genes for pyruvate formate lyases together with their activating enzyme and those for formate dehydrogenases and form the polypeptides.
  • the polypeptides may either be formed individually or as fusion polypeptides between pyruvate decarboxylase and coenzyme A-dependent acetaldehyde dehydrogenase and / or between pyruvate formate lyase and formate dehydrogenase.
  • the acetyl-coenzyme A production is increased in the cytoplasm of the cells. This can lead to an increased and more efficient production of secondary products, in particular acetoacetyl-CoA,
  • Mevalonate isoprenoids, crotonic acid, 1-butanol, sterols, malonyl-CoA, fatty acids, lipids, alkanes, malonate, flavonoids, stilbenoids, malonylated secondary metabolites, and other compounds derived from or derived from the aforementioned ( Figure 1) ).
  • Figure 1 Mevalonate, isoprenoids, crotonic acid, 1-butanol, sterols, malonyl-CoA, fatty acids, lipids, alkanes, malonate, flavonoids, stilbenoids, malonylated secondary metabolites, and other compounds derived from or derived from the aforementioned ( Figure 1) ).
  • the invention is important in the context of the production of bio-based chemicals and pharmaceutical agents, e.g. 1-butanol, crotonic acid, butyl acetate, malonic acid, oils, alkanes, terpenes, isoprenoids, various antibacterial and fungicidal agents, and in particular the precursor molecule amorphadiene of the antimalarial drug artemisinin.
  • bio-based chemicals and pharmaceutical agents e.g. 1-butanol, crotonic acid, butyl acetate, malonic acid, oils, alkanes, terpenes, isoprenoids, various antibacterial and fungicidal agents, and in particular the precursor molecule amorphadiene of the antimalarial drug artemisinin.
  • Acetyl coenzyme A is a key molecule in the metabolism of almost all living things. It is used in their cells in many biochemical reactions. Chemically, it is a thioester between acetic acid (acetate) and coenzyme A.
  • Coenzyme A also coenzyme A, short CoA or CoASH
  • Coenzyme A is a coenzyme that is used for the "activation” of alkanoic acids and their derivatives and is involved in the energy metabolism.
  • Coenzyme A acetyl-CoA for short
  • Acetyl-CoA is a central intermediate metabolite that is switched between catabolic and anabolic processes.
  • acetyl CoA is formed during the oxidative degradation of fatty acids, certain amino acids and carbohydrates. It serves as a precursor for the biosynthesis of a wide variety of Metabolites and biochemicals. Since cellular membranes are impermeable to acetyl-CoA, it must be produced separately in eukaryotic cells either in each membrane-bound compartment or transported across the membranes: these include, for example, mitochondria,
  • acetyl-CoA is provided above all by the pyruvate dehydrogenase reaction from pyruvate and in the oxidation of the fatty acids and introduced into the citric acid cycle.
  • acetyl-CoA may be required for the synthesis of a variety of compounds: acetoacetyl-CoA, mevalonate, isoprenoids, sterols, sesquiterpenes, polyprenols, terpenoids, malonyl-CoA, flavonoids, stilbenoids, malonate, malonylated secondary metabolites, D-amino acids, N -Malonyl-aminocyclopropanecarboxylic acid, fatty acids, lipids, oils, waxes, cysteine, glucosinolate, xenobiotics, pesticides, bioplastics, polyhydroxybutyrate, aroma-active esters of an alcohol and a fatty acid, butanol, alkanes,
  • acetyl-CoA can be formed only by a few reactions.
  • One possibility is the synthesis of citrate by means of ATP citrate lyase (e.g.
  • Vertebrates, insects and plants another possibility is the synthesis of pyruvate by means of the reaction sequence of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase (in yeasts). These reactions or Reaction sequences require energy, which is usually provided in the form of ATP.
  • the acetyl-CoA formed can then be carboxylated, for example, by the acetyl-CoA carboxylase and form malonyl-CoA.
  • Another possibility is the condensation of two molecules acetyl-CoA to acetoacetyl-CoA. There are many more besides
  • the resulting intermediates serve as starting materials for the synthesis of a variety of metabolites, as described above.
  • acetyl-CoA is formed, for example, during the oxidative degradation of fermentable carbon sources, in particular carbohydrates, above all by the reaction sequence of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase or by ATP citrate lyase.
  • these two reactions have the disadvantage that they consume energy, which is mostly provided in the form of ATP hydrolysis. This can have a negative effect on the energy balance of the cells, which can, for example, cause lower biomass formation or lower product formation. This is especially true under anaerobic conditions or in certain fermentations.
  • the beer, wine and baking yeast Saccharomyces cerevisiae has been used for centuries for bread, wine and beer production due to its ability to ferment sugar to alcohol and carbon dioxide.
  • S. cerevisiae is used in addition to the production of heterologous proteins, especially in the production of bio-based chemicals such as ethanol and lactate or pharmaceutical-pharmaceutical agents such as Artemisinin use.
  • bio-based chemicals such as ethanol and lactate or pharmaceutical-pharmaceutical agents
  • a recombinant yeast strain has been described that can obviously produce 1-butanol.
  • S. cerevisiae also has no ATP citrate lyase.
  • S. cerevisiae synthesizes its cytosolic acetyl-CoA from pyruvate using pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase reactions. But in the last reaction two energy equivalents in the form of ATP are consumed. However, this means that in the oxidation of 1 molecule of glucose to 2 molecules of acetyl-CoA a total of 2 molecules of ATP must be applied. If, on the other hand, it were possible to create an energy-independent bypass reaction, then there would be a gain of 2 molecules of ATP.
  • Acetyl-CoA in eukaryotic host cells in particular yeasts and thereby preferably Saccharomyces species, overcome and lead to an increased and energy-independent provision of acetyl-CoA in the cytosol.
  • the object is achieved by providing nucleic acid molecules which in each case encode a polypeptide of a coenzyme A-dependent acetaldehyde dehydrogenase or of a pyruvate formate lyase.
  • pyruvate-formate lyase must also be added
  • Nukleinsauremolekul which encodes a polypeptide of a pyruvate formate lyase activating enzyme, are provided.
  • a nucleic acid molecule according to the invention is a recombinant nucleic acid molecule.
  • nucleic acid molecules of the invention include dsDNA, ssDNA, PNA, CNA, RNA or mRNA or combinations thereof.
  • nucleic acid construct as used below is to be understood in the context of the present invention as a sequence consisting of dsDNA, ssDNA, PNA, CNA, RNA or mRNA or combinations thereof which comprises the nucleic acid molecule according to the invention or comprise a plurality of further sequences which facilitate the incorporation of the nucleic acid molecule according to the invention into a host cell, which increase the reading rate of the nucleic acid molecule according to the invention or which are known to the person skilled in the art for reasons other than those suitable for the purpose of the invention.
  • Acetyl-CoA is a central metabolic intermediate of yeast cells and can be expressed in yeast cells, and in particular in recombinant yeast cells, to be further reacted to a variety of products.
  • a pyruvate decarboxylase In the case of the heterologously expressed coenzyme A-dependent acetaldehyde dehydrogenase, a pyruvate decarboxylase must be simultaneously expressed in order to form from pyruvate, coenzyme A and NAD + the molecules acetyl coenzyme A, NADH + H + and CO 2 .
  • the coenzyme A-dependent acetaldehyde dehydrogenase sets the acetaldehyde formed by the pyruvate decarboxylase together with coenzyme A and NAD + to acetyl coenzyme A and NADH + H + .
  • yeast alcohol dehydrogenases can be reduced or eliminated.
  • NADH + H + implement This formate dehydrogenase can either be a yeast enzyme of its own (Fdhl (SEQ ID.2) or Fdh2 (SEQ ID.4) or else expressed heterologously since FDH1 (SEQ ID.3) and FDH2 (SEQ ID. 5) of S. cerevisiae, expression or activity must be increased, for example, by using a promoter which is strong under the fermentation conditions, whereby the natural promoter of one or both of the formate dehydrogenase genes becomes strong Promoter (eg HXT7konst, PGKl, ADHl, ).
  • yeast pyruvate decarboxylases it may be useful to reduce or eliminate the expression or activity of the yeast pyruvate decarboxylases, to reduce or avoid the co-generation of ethanol, and to increase the availability of pyruvate for the pyruvate formate lyase.
  • nucleic acid sequences of the nucleic acid molecules according to the invention which each encode a polypeptide of a coenzyme A-dependent acetaldehyde dehydrogenase, are preferably genes from prokaryotes (for example from Escherichia coli), e.g. mhpF (SEQ ID 6), a mutated allele of adhE (SEQ ID 7) with coenzyme A-dependent acetaldehyde dehydrogenase activity, or a truncated version of the mutant allele of adhE (SEQ ID 8) containing only the Coenzyme A-dependent acetaldehyde dehydrogenase activity of the bifunctional enzyme encodes.
  • the respective nucleic acid sequence can either code for a coenzyme A-dependent acetaldehyde dehydrogenase or it is used as a nucleic acid sequence fusion with a gene that is responsible for a
  • Polypeptide coded with pyruvate decarboxylase activity wherein a polypeptide is encoded, which has both activities.
  • Nucleic acid molecules which each code for a polypeptide of a pyruvate formate lyase and a pyruvate formate lyase activating enzyme are preferably genes from prokaryotes or anaerobic chytridiomycetes or chlorophytes.
  • the nucleic acid sequence encoding a polypeptide of pyruvate formate lyase may either encode that polypeptide alone or be expressed as nucleic acid sequence fusion with a gene encoding a polypeptide having formate dehydrogenase activity a polypeptide is encoded which has both activities.
  • nucleic acid molecules of the invention preferably comprise nucleic acid sequences which are identical to the naturally occurring nucleic acid sequence, individual
  • Each amino acid is encoded at the gene level by a codon. However, there are several different codons that code for a single amino acid. The genetic code is therefore degenerate. The preferred codon choice for a corresponding amino acid differs from organism to organism. This may be the case for heterologously expressed genes
  • the present invention furthermore relates to expression cassettes comprising a nucleic acid molecule according to the invention.
  • Expression cassettes further preferably include promoter and terminator sequences.
  • promoter sequences are selected from HXT7, truncated HXT7, PFK1, FBA1, PGK1, ADH1, TDH3.
  • terminator sequences are selected from CYCl, FBA1, PGK1, PFK1, ADH1, TDH3.
  • the present invention also relates to
  • Expression vectors comprising a nucleic acid molecule according to the invention or a novel nucleic acid molecule
  • the expression vectors according to the invention furthermore preferably comprise a selection marker.
  • the selection marker is selected from a leucine marker, a uracil marker, the dominant antibiotic marker geneticin, hygromycin and nourseothricin.
  • the present invention also relates to host cells which contain a nucleic acid construct according to the invention, an expression cassette according to the invention or an expression vector according to the invention.
  • a nucleic acid molecule according to the invention, an expression cassette according to the invention or an expression vector according to the invention is stably integrated into the genome of the host cell.
  • a host cell according to the invention is preferably a fungal cell and more preferably a yeast cell, such as Saccharomyces species, Kluyveromyces sp., Hansenula sp., Pichia sp. or Yarrowia sp ..
  • the present invention further relates to a recombinant host cell containing such an isolated nucleic acid molecule.
  • the present invention relates to a recombinant host cell containing a nucleic acid construct encoding a polypeptide, which catalyzes a substrate-product conversion selected from the following two reaction:
  • At least one DNA molecule is heterologous to said host cell and wherein said host cell has increased acetyl-coenzyme A production.
  • the present invention further preferably relates to a
  • a host cell as defined above characterized in that the increased acetyl-CoA formation takes place in the cytoplasm of the host cell.
  • the present invention further preferably relates to a
  • a host cell as defined above characterized in that the polypeptide that catalyzes the conversion of acetaldehyde + coenzyme A + NAD + to acetyl coenzyme A + NADH + H + is a coenzyme A-dependent acetaldehyde dehydrogenase.
  • the present invention further preferably relates to a host cell as defined above, characterized in that the polypeptide which catalyzes the conversion of pyruvate + coenzyme A to acetyl coenzyme A + formate, a pyruvate formate lyase and by the simultaneous expression of a pyruvate Formate lyase activating enzyme is activated.
  • the present invention more preferably relates to a host cell as defined above, characterized in that the coenzyme A-dependent acetaldehyde dehydrogenase is expressed in a host cell which simultaneously expresses a pyruvate decarboxylase activity.
  • the present invention more preferably relates to a host cell as defined above, characterized in that the coenzyme A-dependent acetaldehyde dehydrogenase and the pyruvate decarboxylase are expressed in a host cell having a reduced or no activity of an alcohol dehydrogenase.
  • the present invention more preferably relates to a host cell as defined above, characterized in that the pyruvate-formate-lyase and pyruvate-formate-lyase activating enzyme are expressed in a host cell which simultaneously expresses a polypeptide which inhibits the transformation of formate + NAD + catalyzed to CO 2 + NADH + H 4 .
  • the present invention more preferably relates to a host cell as defined above, characterized in that the protein which catalyzes the conversion of formate + NAD + to CO2 + NADH + H + is a formate dehydrogenase.
  • the present invention further preferably relates to a host cell as defined above, characterized in that the pyruvate formate lyase and pyruvate formate lyase activating enzyme and the formate dehydrogenase are expressed in a host cell having a reduced or no activity of a pyruvate Having decarboxylase.
  • the present invention more preferably relates to a host cell as defined above, characterized in that the host cell is a yeast.
  • the present invention further preferably relates to a
  • Host cell as defined above, characterized in that the host cell is selected from the following group: Pichia, Candida, Hansenula, Kluyveromyces, Yarrowia and Saccharomyces.
  • the present invention more preferably relates to a host cell as defined above, characterized in that the host cell is Saccharomyces cerevisiae.
  • the present invention further preferably relates to a
  • a host cell as defined above characterized in that the host cell is an optionally anaerobic host cell.
  • the present invention further relates to a method for the increased delivery of acetyl-CoA in the cytosol in a host cell.
  • the inventive method comprises the expression of a erfmdungsgeloisen Nuklemsauremolekuls, an inventive expression cassette or erfmdungsge navalen expression vector in a host cell.
  • the method of the invention comprises providing a host cell as defined above and contacting the host cell with a fermentable carbon source under conditions in which the rate of formation of acetyl-CoA is increased.
  • the present invention further preferably relates to a method as defined above, characterized in that the fermentable carbon source is a C3 - C6 carbon source.
  • the present invention more preferably relates to a method as defined above, characterized in that the carbon source belongs to the group consisting of monosaccharides, oligosaccharides or polysaccharides.
  • the present invention more preferably relates to a method as defined above, characterized in that the carbon source belongs to the group consisting of glucose, fructose, sucrose, maltose, xylose or arabinose.
  • the present invention further preferably relates to
  • a method as defined above characterized in that the host cell is contacted with the carbon source in culture medium.
  • the present invention further preferably relates to
  • the present invention further preferably relates to
  • the present invention further preferably relates to
  • a method as defined above characterized in that the secondary product to the group consisting of mevalonate, isoprenoids, sterols, sesquiterpenes, polyprenols, terpenoids, flavonoids, stilbenoids, malonate, malonylated Secondary metabolites, D-amino acids, N-malonyl-aminocyclopropanecarboxylic acid, fatty acids, lipids, oils, waxes, cysteine, glucosinolate, xenobiotics, pesticides, bioplastics, polyhydroxybutyrate, flavor-active esters, 1-butanol, alkanes, butyl acetate.
  • the present invention further preferably relates to a process as defined above, characterized in that the production of the secondary product is increased.
  • the process is preferably carried out in a host cell according to the invention.
  • the present invention furthermore relates to a nucleic acid molecule, a novel nucleic acid molecule
  • Expression cassette an expression vector according to the invention, a host cell according to the invention with an increased acetyl-CoA production rate.
  • the present invention furthermore relates to the use of a nucleic acid molecule according to the invention, an expression cassette according to the invention, an expression vector according to the invention, a host cell according to the invention for the recombinant fermentation of biomaterial.
  • an expression cassette according to the invention, an expression vector according to the invention, a host cell according to the invention By using a nucleic acid molecule according to the invention, an expression cassette according to the invention, an expression vector according to the invention, a host cell according to the invention, the rate of formation of cytosolic acetyl-CoA is increased. The result is a faster, more efficient and increased production of acetyl-CoA in the cytoplasm of the host cell according to the invention and optionally an increased production of secondary products as described above.
  • the present invention further relates to a polypeptide having in vitro and / or in vivo coenzyme A-dependent acetaldehyde dehydrogenase activity and / or a polypeptide having in vitro and / or in vivo pyruvate formate lyase activity.
  • the present invention further relates to an isolated Nukleinsauremolekul that encodes a polypeptide according to the invention.
  • polypeptide of the invention isolated Nukleinsauremolekul and the host cell of the invention are preferably used to increase the production of cytosolic acetyl-CoA.
  • polypeptide according to the invention, isolated nucleic acid molecule and the host cell according to the invention are preferably used for increasing the production of cytosolic acetyl-CoA and derived derivatives thereof in host cells, in particular yeasts, wherein the host cells also contain one or more additional modifications, such as nucleic acid molecules.
  • such an additional modification according to the invention is, for example, a host cell which, on account of the provision of heterologous nucleic acid molecules, produces the following secondary products: acetoacetyl-CoA, mevalonate, isoprenoids, sterols, sesquiterpenes, polyprenols, terpenoids, malonyl-CoA, flavonoids, stilbenoids, Malonate, malonylated secondary metabolites, D-amino acids, N-malonyl
  • Aminocyclopropanecarboxylic acid fatty acids, lipids, oils, waxes, cysteine, glucosinolate, xenobiotics, pesticides, bioplastics, polyhydroxybutyrate, flavor-active esters of an alcohol and a fatty acid, butanol, alkanes, butyl acetate, and all other compounds derived from those cited to let.
  • polypeptide according to the invention isolated nucleic acid molecule and the host cell according to the invention are preferably used for increasing the production of cytosolic acetyl-CoA and secondary products in the fermentation of carbon sources.
  • nucleic acids according to the invention Possible uses of the nucleic acids according to the invention, expression cassettes, expression vectors and host cells are the production of high-quality precursor products for further biochemical and chemical syntheses.
  • Expression cassettes, expression vectors and host cells are available.
  • the invention enables an increased and energy-independent provision of acetyl-CoA in the cytosol of eukaryotic host cells, in particular yeasts and preferably Saccharomyces species. Since the provision of acetyl-CoA is a limiting reaction in the biotechnological production of a variety of
  • Secondary products also makes it possible to increase the production of these secondary products, e.g. 1- butanol, crotonic acid, mevalonate, isoprenoids, fatty acids, alkanes, or flavonoids, especially in host cells containing other corresponding heterologous nucleic acids. This applies in particular to fermentations of biomaterial under anaerobic or microaerobic conditions.
  • Antibiotic resistance was added to the medium after autoclaving 40 ⁇ g / ml ampicillin.
  • Solid nutrient media additionally contained 2% agar. The cultivation took place at 37 ° C. 1 . 2 yeasts
  • Synthetic Complete Selective Medium SC 0.67% yeast nitrogen base w / o anno acids, pH 6.3,
  • Synthetic mimic selective medium SM 0.16% yeast nitrogen base w / o amino acid and ammonium sulphate, 0.5% ammonium sulphate, 20 mM potassium dihydrogen phosphate, pH 6.3, carbon source in the respectively indicated concentration
  • Synthetic Fermentation Medium (Mammalian Medium) SFM: (Verduyn et al., 1992), pH 5.5
  • Salts (NH 4 ) 2 SO 4 , 5g / l; KH 2 PO 4 , 3g / l; MgSO 4 * 7H 2 O, 0.5 g / l trace elements: EDTA, 15 mg / l, ZnSO 4 M, 5 mg / l; MnCl 2 MH 2 O, 0, lmg / l; CoCl 2 * 6H 2 0, 0.3 mg / 1; CuSO 4 , 0.192 mg / 1; Na 2 Mo0 4 * 2H 2 O, 0.4 mg / L; CaCl 2 * 2H 2 O, 4.5 mg / L; FeSO 4 * 7H 2 O, 3 mg / 1; H 3 BO 3 , 1 mg / 1; KI, 0.1 mg / l
  • Vitamins biotin, 0.05 mg / 1; p-aminobenzoic acid, 0.2 mg / l; Nicotinic acid, 1 mg / 1; Calcium pantothenate, 1 mg / 1; Pyridoxine HCL, 1 mg / 1; Thiamine HCL, 1 mg / 1; Minositol, 25 mg / 1
  • Solid solid and selective media contained 1.8% agar in addition.
  • the cultivation of the yeast cells was carried out at 30 ° C.
  • the synthetic mineral medium used for the fermentations contained salts, trace metals and vitamins in the concentrations listed above and L-arabinose as carbon source. Of the trace metals and the vitamins, a master lottery was scheduled. Both solutions were sterile-filtered. Both were stored at 4 ° C.
  • the pH value was crucial. The various trace elements had to be completely dissolved in water sequentially in the above order. After each addition, the pH had to be adjusted to 6.0 with KOH before the next trace element could be added. At the end, the pH was adjusted to 4.0 with HCL.
  • Electroporation method according to Dower et al. (1988) and Wirth (1993) using an Easyject prima device (EQUIBO).
  • the isolation of plasmid DNA from E. coli was carried out by the alkaline lysis method of Birnboim and DoIy (1979), modified according to Maniatis et al. (1982) or alternatively with the "QIAprep Spin Miniprep Kit” from Qiagen.
  • the cells of a stationary yeast culture (5 ml) were harvested by centrifugation, washed and resuspended in 400 ⁇ l of Buffer Pl (Plasmid Mini Kit, Qiagen). After the addition of 400 ul buffer P2 and 2/3 volume of glass beads (0.45 mm 0) of the cells were disrupted by shaking for 5 minutes on a Vibrax (Vibrax-VXR from Janke & Kunkel or IKA). The supernatant was mixed with 2 volumes of buffer P3, mixed and incubated on ice for 10 min. After a 10-minute Centrifugation at 13000 rpm was precipitated by adding 0.75 ml of isopropanol to the supernatant, the plasmid DNA at room temperature. The DNA pelleted by centrifugation for 30 min at 13000 rpm was washed with 70% ethanol, dried and resuspended in 20 ⁇ l water. 1 ⁇ l of the DNA was used for transformation into E. coli.
  • Phusion TM High Fidelity PCR System The polymerase chain reaction was carried out in a total volume of 50 ⁇ l using the Finnzymes "Phusion TM High Fidelity PCR System" according to the manufacturer's instructions Each batch consisted of 1-lOng DNA or 1-2 yeast colonies as the synthesis template,
  • PCR reaction was carried out in a thermocycler from Techne and the PCR conditions were selected as needed as follows:
  • Oligonucleotides or adapted to the size of the expected product.
  • the PCR products were checked by agarose gel electrophoresis and then purified.
  • the purification of the PCR products was carried out with the "QIAquick PCR Purification Kit” from Qiagen according to the manufacturer.
  • the separation of DNA fragments with a size of 0.15-20kb was carried out in 0.5-l% agarose gels with 0.5 ug / ml ethidium bromide.
  • lxTAE buffer 40 mM Tris, 40 mM acetic acid, 2 mM EDTA was used (Mamatis et al., 1982).
  • the desired DNA fragment was excised from the TAE agarose gel under long-wave UV light (366 nm) and isolated using the "QIAquick Gel Extraction Kit” from Qiagen according to the manufacturer's instructions. 4. Enzymatic modification of DNA
  • Sequence-specific cleavage of the DNA with restriction endonucleases was performed under the manufacturer's recommended incubation conditions for 1 hour with 2-5U enzyme per ⁇ g of DNA.
  • Electroporation An alternative method of transforming bacteria with plasmid DNA.

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Abstract

L'invention concerne une cellule hôte recombinante qui contient au moins une molécule d'acide nucléique, qui code un polypeptide qui catalyse une transformation en substrat-produit qui est sélectionnée parmi les deux réactions suivantes : acétaldéhyde + coenzyme A + NAD+ en acétyl-coenzyme A + NADHH-H+, et ou pyruvate + coenzyme A en acétyl-coenzyme A + formiate; sachant qu'au moins une molécule d'ADN est hétérologue de ladite cellule hôte et sachant que ladite cellule hôte présente une production accrue d'acétyl-coenzyme A. L'invention concerne en outre un procédé pour un taux accru de formation d'acétyl-coA dans une cellule hôte.
EP09703058A 2008-01-14 2009-01-14 Production accrue d'acétyl-coenzyme a Withdrawn EP2235167A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200810004253 DE102008004253B4 (de) 2008-01-14 2008-01-14 Gesteigerte Produktion von Acetyl-Coenzym A
PCT/EP2009/000181 WO2009090050A1 (fr) 2008-01-14 2009-01-14 Production accrue d'acétyl-coenzyme a

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EP2277989A1 (fr) 2009-07-24 2011-01-26 Technische Universiteit Delft Production d'éthanol dépourvu de glycérol par fermentation
BR112012028290B1 (pt) 2010-05-05 2021-02-02 Lallemand Hungary Liquidity Management Llc. levedura recombinante, processo para converter biomassa em etanol e meio de fermentação compreendendo dita levedura
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

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