WO2023093796A1 - 一种高产丁二酸的耐酸酵母菌株及其构建方法和应用 - Google Patents

一种高产丁二酸的耐酸酵母菌株及其构建方法和应用 Download PDF

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WO2023093796A1
WO2023093796A1 PCT/CN2022/133973 CN2022133973W WO2023093796A1 WO 2023093796 A1 WO2023093796 A1 WO 2023093796A1 CN 2022133973 W CN2022133973 W CN 2022133973W WO 2023093796 A1 WO2023093796 A1 WO 2023093796A1
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nucleic acid
gene
acid sequence
encoding
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张学礼
樊飞宇
郗永岩
徐洪涛
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中国科学院天津工业生物技术研究所
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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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/085—Bacillus cereus

Definitions

  • the invention relates to the field of biotechnology, in particular to a novel acid-resistant yeast strain for high-efficiency production of succinic acid and its construction method and application.
  • Succinic acid also known as succinic acid
  • succinic acid is an intermediate product of the tricarboxylic acid cycle in organisms and an important organic chemical raw material (Chae T U, Ahn J H, Ko Y S, et al. Metabolic engineering for the production of dicarboxylic acids and diamines[J]. Metabolic Engineering, 2019). Succinic acid is widely used in food, chemical, agricultural, pharmaceutical and other industries.
  • the traditional production method of succinic acid is chemical method, which is mainly synthesized from non-renewable petroleum resources, such as: paraffin oxidation method, maleic anhydride hydrogenation method, acrylic acid carbonyl synthesis method, etc.
  • the chemical synthesis method has disadvantages such as high energy consumption and high pollution, and it is difficult to meet the requirements of green manufacturing.
  • the production of succinic acid by biological fermentation has less pollution, lower energy consumption, and fixes carbon dioxide, which is more in line with the national sustainable development strategy. It is predicted that the global production of succinic acid produced by biological methods will reach 600,000 tons in 2020, and the market value will reach 539 million US dollars (Hyohak Song, Sang Yup Lee. Production of Succinic Acid by Bacterial Fermentation[J]. Enzyme and Microbial Technology, 2006, 39(3):352-361.).
  • the microbial strains used to synthesize succinic acid are mainly divided into two categories: the first category is fungal yeast, mainly including: Aspergillus niger, Aspergillus fumigatus, Byssochlamys nivea , Paecilomyces varioti, Saccharomyces cerevisiae, etc.; the second category is bacteria: including Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Natural producer strains such as Mannheimia succiniciproducens and Bacteroides fragilis and non-natural producer strains such as Corynebacterium glutamicum and Escherichia coli (Ahn J H, Jang Y S, Lee S Y.
  • the present invention uses an acid-resistant yeast Pichia kudriavzevii (Pichia kudriavzevii) CY902 strain isolated from the epidermis of wild fruits in Yunnan (preserved in the General Microorganism Center (CGMCC) of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCC) No.20885), through metabolic engineering to achieve efficient production of succinic acid.
  • CGMCC General Microorganism Center
  • the modified bacterial strain can achieve high-efficiency production of succinic acid under low pH conditions by means of fermentation with no or less addition of neutralizing agents.
  • the present invention provides a genetically engineered succinate-producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, optionally further having or having enhanced at least one of the following Activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase activity, (iii) fumarase activity, and (iv) succinate transporter activity.
  • the NDAPH-dependent malate dehydrogenase is from a plant, preferably a C4 plant, more preferably Gramineae, Cyperaceae, Compositae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae, or from Euglena or Thermobacter, more preferably from sorghum (Sorghum bicolor), corn (Zea mays), sugarcane (Saccharum officinarum), pea (Pisum sativum), chickpea (Cicer arietinum), spinach (Spinacia oleracea), Euglena gracilis or Methanothermobacter thermautotrophicus.
  • the genetically engineered succinate-producing yeast strain also has reduced or inactivated pyruvate decarboxylase and/or NAD-dependent glycerol-3-phosphate dehydrogenase.
  • the present invention provides a method of producing a genetically engineered succinate-producing yeast strain, comprising conferring or enhancing the NADPH-dependent malate dehydrogenase activity of said strain, optionally further comprising Confer or enhance at least one of the following activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase (EC 6.4.1.1) activity, (iii) fumarase (EC 4.2.1.2 ) activity, and (iv) succinate transporter activity.
  • the NDAPH-dependent malate dehydrogenase is from a plant, preferably a C4 plant, more preferably from the Gramineae, Cyperaceae, Compositae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae plants or from Euglena and Thermobacterium, more preferably sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena or thermoautotrophic Thermus methanosa.
  • the method further comprises attenuating or inactivating pyruvate decarboxylase and/or NAD-dependent glycerol-3-phosphate dehydrogenase in the strain.
  • the present invention provides a method for producing succinic acid, comprising (preferably in the range of pH ⁇ 3.5, such as 1.5-3.5 and/or under the condition of no or less addition of neutralizing agent) cultivating the present invention
  • the genetically modified succinic acid producing yeast strain and/or the genetically modified succinic acid producing yeast strain obtained by the method for producing a genetically modified succinic acid producing yeast strain according to the present invention.
  • the present invention provides a genetically engineered succinic acid producing yeast strain according to the present invention and/or a genetically engineered succinic acid producing yeast strain obtained by a method for producing a genetically modified succinic acid producing yeast strain described herein.
  • a succinic acid-producing yeast strain in the production of succinic acid (preferably in the range of pH ⁇ 3.5, such as 1.5-3.5 and/or without or less addition of neutralizing agent).
  • genetically modified refers to a strain artificially altered by biological means, which has one or more changes compared with the original strain before modification, such as gene deletion, amplification or mutation, thereby having an altered Biological properties such as improved productivity.
  • the initial strain may be a natural strain to which said genetic modification is to be performed or a strain with other genetic modifications.
  • a succinate-producing yeast strain refers to a yeast that, under appropriate conditions, can produce succinate (eg, via fermentation) and secrete succinate into the extracellular medium.
  • the succinic acid-producing yeast strain has a protein that can transport succinic acid to the extracellular space, so succinic acid can be secreted to the extracellular space after production.
  • Suitable succinate transporters for a given strain of yeast are known in the art and include, but are not limited to, the dicarboxylate transporter SpMAE1 of Schizosaccharomyces pombe and the dicarboxylate transporter of Aspergillus niger. Acid transporter AnDCT-02.
  • Yeasts known in the art for the production of succinic acid include, for example and without limitation, Zygosaccharomyces, Torulopsis, Candida, Pichia, Rhodotroula, Saccharomyces, Yarrowia, etc.
  • the succinic acid producing yeast strain is a Pichia, Saccharomyces, or Yarrowia strain.
  • the succinic acid-producing yeast strain is Pichia kudriazvii (for example strain CICC32244), Saccharomyces cerevisiae (for example strain BY4742) or Yarrowia lipolytica (for example strain Po1g), such as Pichia kudriazvii preserved in the General Microorganism Center (CGMCC) of the China Committee for Culture Collection of Microbial Cultures (CGMCC) in Beijing, China, with a preservation number of CGMCC No.20885.
  • CGMCC General Microorganism Center
  • CGMCC General Microorganism Center
  • having an activity means having a detectable activity compared to a reference that does not have the activity (eg, an initial strain or a wild-type strain).
  • has enhanced activity refers to an increase in activity of at least 5%, at least 10%, at least 20%, at least 30%, At least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or higher.
  • the activity of the protein can be produced or enhanced by any suitable means known in the art, such as including but not limited to expressing or overexpressing (for example, via a vector such as a plasmid) the corresponding gene encoding the protein in a bacterial strain, introducing the resulting Mutations that increase the activity of the protein, and the like.
  • one or more copies of the gene of interest or its homologous gene can be integrated into the genome (for example, by homologous recombination), any Select any site in the genome, (as long as the integration does not significantly negatively affect the growth and production of the strain), for example, one copy of any gene in the genome is replaced by one or more copies of the target gene or its homologous gene.
  • any Select any site in the genome for example, one copy of any gene in the genome is replaced by one or more copies of the target gene or its homologous gene.
  • reduced activity or inactivated means that the activity is reduced by at least 5%, at least 10%, at least 20%, at least 30% compared to a reference activity (such as the corresponding activity in the initial strain or wild-type strain). %, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even 100%.
  • the activity of a protein can be reduced or inactivated by any suitable means known in the art, such as including but not limited to using a weakened or inactivated corresponding gene encoding the protein, introducing a or inactivating mutations, using antagonists or inhibitors of the protein (eg, antibodies, ligands, etc.).
  • weakened or inactivated gene refers to the gene's activity, such as expression level (as a protein-coding gene) or regulatory performance (as a adjustment element) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable.
  • weakened or inactivated gene also encompasses that the activity level of the protein expressed by the gene is reduced compared to the activity level of the corresponding protein in the original strain or wild-type strain , for example by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even 100%.
  • the reference may be a wild-type microorganism or a microorganism prior to the desired genetic manipulation (for example, the initial microorganism for genetic manipulation to increase gene activity).
  • parental microorganism and initial microorganism are used interchangeably to refer to a microorganism to which a desired genetic manipulation (eg, enhancement or attenuation of gene or protein activity) is performed.
  • malate dehydrogenase (EC 1.1.1.82 (NADPH-dependent)), encoded by the MDH gene, is involved in the conversion between oxaloacetate and malate.
  • NADPH-dependent malate dehydrogenase is responsible for the conversion of oxaloacetate to malate, a reaction that consumes one molecule of NADPH.
  • NADPH-dependent malate dehydrogenase include C4 plants (e.g., Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Purslaneceae, and Amaranthaceae), Euglena, and Thermobacteria genera, etc., for example: sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena microglia, or thermoautotrophic Methanobacterium, etc.
  • having or having enhanced NDAPH-dependent malate dehydrogenase activity means that the strain has or has increased NDAPH-dependent malate dehydrogenase activity that catalyzes the conversion of oxaloacetate to malate.
  • soluble fumarate reductase (EC 1.3.1.6), encoded by the FRD gene, is an enzyme involved in the interconversion between fumarate and succinate, responsible for the conversion of fumarate to succinate.
  • Commonly used sources of soluble fumarate reductase are yeast and the order Kinetoplasma, especially Saccharomyces cerevisiae, Trypanosoma brucei, Leishmania mexicana, Trypanosoma cruzi Cruzi) and so on.
  • having or having enhanced fumarate reductase activity means that the strain has or has increased soluble fumarate reductase activity that converts fumarate to succinate.
  • fumarase (EC 4.2.1.2), encoded by the FUM gene, is an enzyme involved in the interconversion between fumarate and malate in the cytoplasm and mitochondria, responsible for the conversion of malate to fumarate.
  • Generally used sources include Actinobacillus succinogenes, Mannella succinogenes, Escherichia coli, Pichia Kudriazwi, Rhizopus oryzae, etc.
  • a FUM gene, FUM1 is known to exist in Pichia kuderia.
  • having or having enhanced fumarase activity means that the strain has or has increased fumarase activity to convert malate to fumarate.
  • a succinate transporter refers to a protein capable of transporting succinate in a cell to the outside of the cell, including for example but not limited to the SpMAE1 protein of Schizosaccharomyces pombe (Uniprot database accession number: P50537), Aspergillus niger (Aspergillus niger) dicarboxylic acid transporter AnDCT-02 (NCBI Reference Sequence: XP_001398131.1) and Escherichia coli dicarboxylic acid transporter EcDcuB (Gene ID: 948641) and EcDcuC (Gene ID: 945000), etc.
  • having or having enhanced succinate transporter activity means that the strain has or has increased activity of transporting succinate to the extracellular space.
  • the S. pombe dicarboxylic acid transporter SpMAE1 protein is encoded by the SpMAE1 gene, and the SpMAE1 protein is responsible for transporting intracellular dicarboxylic acid to the extracellular.
  • having or having enhanced SpMAE1 activity means that the strain has or has increased activity of transporting dicarboxylic acid extracellularly.
  • pyruvate carboxylase (EC 6.4.1.1), encoded by the PYC gene, is involved in the interconversion between oxaloacetate and pyruvate during gluconeogenesis and is responsible for converting pyruvate and carbon dioxide for oxaloacetate.
  • Commonly used sources of pyruvate carboxylase include fungi, especially yeast and filamentous fungi, preferably Saccharomyces cerevisiae, Pichia kudriazwi, Aspergillus oryzae, Kluyveromyces marxianus and the like. It is known that there is one PYC gene in Pichia kuderi Azwiya, that is, PYC1 gene.
  • having or having enhanced pyruvate carboxylase activity means that the strain has or has increased activity of converting pyruvate into oxaloacetate.
  • pyruvate decarboxylase (EC 4.1.1.43), encoded by the PDC gene, is involved in the decarboxylation of pyruvate in the synthetic ethanol pathway.
  • Pichia pastoris is known to have a PDC gene called PDC1 gene.
  • reduced activity or inactivated pyruvate decarboxylase means that the pyruvate decarboxylation activity of the enzyme is reduced or lost.
  • NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), encoded by the GPD gene, is involved in the interconversion between phosphoglyceroketone and glycerol-3-phosphate in the synthetic glycerol pathway.
  • Pichia pastoris is known to have a GPD gene, the GPD1 gene.
  • the reduced activity or inactivated NAD-dependent 3-phosphate glycerol dehydrogenase refers to the reduction or loss of the enzyme's activity of interconverting phosphoglyceroketone and 3-phosphate glycerol.
  • orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), encoded by the URA3 gene, is involved in the decarboxylation of orotidine-5-phosphate during pyrimidine synthesis. It is known that there is a URA3 gene in Pichia kuderia.
  • the reduced activity or inactivated 5'-orotidine phosphate decarboxylase refers to the reduction or loss of the activity of the enzyme in catalyzing the decarboxylation reaction of orotidine 5'-phosphate.
  • alcohol dehydrogenase 1 (EC 1.1.1.1), encoded by the ADH1 gene, is an enzyme involved in the interconversion between acetaldehyde and ethanol in the synthetic ethanol pathway.
  • the activity-reduced or inactivated alcohol dehydrogenase 1 means that the activity of the enzyme to catalyze the interconversion between acetaldehyde and ethanol is reduced or lost.
  • the ADH1 gene in the strain is knocked out, for example by homologous recombination.
  • monocarboxylate permease (NCBI Reference Sequence XP_029320775.1) is encoded by the MCH4 gene.
  • a reduced activity or inactivated monocarboxylate permease refers to a reduction or loss of the catalytic activity of the enzyme.
  • the MCH4 gene in the strain is knocked out, for example by homologous recombination.
  • the JEN2 gene encodes a dicarboxylic acid transporter involved in the process of transporting dicarboxylic acids in the medium into the cell. It is known that there are two JEN2 genes (JEN2-1 (encoding the polypeptide shown in SEQ ID NO: 14) and JEN2-2 (encoding the polypeptide shown in SEQ ID NO: 15)) in Pichia kudriazwhippia.
  • JEN2-1 encoding the polypeptide shown in SEQ ID NO: 14
  • JEN2-2 encoding the polypeptide shown in SEQ ID NO: 15
  • a reduced activity or inactivated dicarboxylic acid transporter is a reduced or lost activity of a cell to transport dicarboxylic acid in a culture medium into the cell.
  • a neutralizing agent refers to an agent that precipitates succinic acid from a fermentation system in the form of calcium succinate.
  • Substances known in the art that can be used as neutralizing agents include, for example but not limited to, calcium carbonate.
  • adding no or less neutralizing agent refers to at least 5%, at least 10%, at least 20%, at least 30% lower than the amount of neutralizing agent added when fermentatively producing succinic acid known in the art , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even 100%.
  • the amount of neutralizing agent added may be 0-30 g/L.
  • polypeptide As used herein, the terms "polypeptide,” “amino acid sequence,” “peptide,” and “protein” are used interchangeably herein to refer to a chain of amino acids of any length, which may contain modified amino acids and/or may be interrupted by non-amino acids .
  • the term also encompasses amino acid chains that have been modified naturally or by human intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component.
  • nucleic acid sequence As used herein, the expressions "gene”, “nucleic acid sequence”, “polynucleotide” and “nucleotide sequence” are used interchangeably and refer to a chain of nucleotides, including DNA and RNA. “Expression of a gene” refers to the transcription of a DNA region operably linked to an appropriate regulatory region, especially a promoter, into biologically active RNA and the translation of RNA into a biologically active protein or peptide.
  • a degenerate sequence refers to a nucleotide sequence that encodes the same amino acid sequence but differs in nucleotide sequence from a given sequence due to the degeneracy of the genetic code.
  • sequence identity can be detected by aligning the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide, as can be determined, for example, by standard alignment algorithm programs using default gap penalties established by each vendor . Whether two nucleic acid molecules have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" nucleotide sequences can be determined using known computer algorithms, such as BLASTN, FASTA, DNAStar and Gap (University of Wisconsin Genetics Computer Group (UWG), Madison WI, USA).
  • the percent identity of nucleic acid molecules can be determined, for example, by comparing sequence information using the GAP computer program (e.g., Needleman et al. J. Mol. Biol. 48:443 (1970), by Smith and Waterman (Adv. Appl. Math. 2:482 (revised 1981). Briefly, the GAP program defines similarity in terms of the number of similarly aligned symbols (ie, nucleotides) divided by the total number of symbols in the shorter of the two sequences.
  • GAP computer program e.g., Needleman et al. J. Mol. Biol. 48:443 (1970), by Smith and Waterman (Adv. Appl. Math. 2:482 (revised 1981).
  • the GAP program defines similarity in terms of the number of similarly aligned symbols (ie, nucleotides) divided by the total number of symbols in the shorter of the two sequences.
  • the Pk2365 gene encodes a bifunctional enzyme with oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation (EC 4.1.3.17 or 4.1.1.112).
  • a reduced or inactivated oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzyme means that the enzyme catalyzes oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolization The activity of diacid aldolization is reduced or lost.
  • the present invention provides a genetically engineered succinate-producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, optionally further having or having enhanced at least one of the following Activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase activity, (iii) fumarase activity, and (iv) succinate transporter activity.
  • the "at least one" includes activities selected from any 1, 2, 3 or all 4 of them.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity and soluble fumarate reductase activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity and pyruvate carboxylase activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity and fumarase activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity and succinate transporter activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity and acetone acid carboxylase activity.
  • the present invention provides a genetically engineered succinate-producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity and rich Maic acid enzyme activity.
  • the present invention provides a genetically engineered succinate-producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity and butane Diacid transporter activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, pyruvate carboxylase activity and fumarase activity active.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, pyruvate carboxylase activity and succinate transport protein activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, fumarase activity and succinate transporter active.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, acetone Acid carboxylase activity and fumarase activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, acetone Acid carboxylase activity and succinate transporter activity.
  • the present invention provides a genetically engineered succinate-producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, rich Purpurase activity and succinate transporter activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, pyruvate carboxylase activity, fumarase activity and succinate transporter activity.
  • the present invention provides a genetically engineered succinate producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, acetone Acid carboxylase activity, fumarase activity and succinate transporter activity.
  • having or having enhanced activity is achieved by expressing or overexpressing the corresponding coding gene in said strain.
  • the genetically engineered succinate-producing yeast strain expresses or overexpresses a gene encoding NADPH-dependent malate dehydrogenase.
  • the NADPH-dependent malate dehydrogenase is from a plant (preferably a C4 plant, more preferably Gramineae, Cyperaceae, Compositae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae plants) ), Euglena or Thermobacterium, preferably from sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena microglia or thermoautotrophic Methanobacterium, more preferably from sorghum.
  • NADPH-dependent malate dehydrogenase activity is produced or increased by expressing or overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase in said genetically engineered succinate-producing yeast strain , preferably, the MDH gene encoding the NADPH-dependent malate dehydrogenase is preferably from a plant, preferably a C4 plant, more preferably Poaceae, Cyperaceae, Compositae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae, or from Euglena or Thermobacterium, more preferably from sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena microglia or thermoautotrophic Methanobacterium, more preferably from sorghum.
  • the MDH gene encoding NADPH-dependent malate dehydrogenase comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or at least 75%, 80%, 85%, 90% of it , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and encodes a nucleoside of an amino acid sequence having NADPH-dependent malate dehydrogenase activity acid sequence.
  • the MDH gene encoding NADPH-dependent malate dehydrogenase is incorporated into the genome of a genetically engineered succinate-producing yeast (e.g. Pichia kudriazwii), e.g. in the Pk2365 gene seat position.
  • the MDH gene encoding NADPH-dependent malate dehydrogenase can be placed in a suitable promoter (such as the promoter of the FBA1 gene (such as shown in SEQ ID NO: 17)) and/or a terminator (such as the termination of the INO1 gene sub (such as shown in SEQ ID NO: 18)) under the control of.
  • the genetically engineered succinate producing yeast strain also has or has enhanced soluble fumarate reductase.
  • the soluble fumarate reductase is from yeast and the order Kinetoplastia, such as but not limited to Saccharomyces cerevisiae, Trypanosoma brucei, Leishmania mexicana, Trypanosoma cruzi.
  • soluble fumarate reductase activity is produced or increased by expressing or overexpressing a gene encoding soluble fumarate reductase in said genetically engineered succinic acid producing yeast strain
  • the gene encoding the soluble fumarate reductase is preferably from yeasts and kinetoplasma, such as but not limited to Saccharomyces cerevisiae, Trypanosoma brucei, Leishmania mexicana, Trypanosoma cruzi.
  • the 3' end glyoxylate cycle body localization peptide of the soluble fumarate reductase is partially or completely truncated, so that it exists freely in the cytoplasm.
  • the gene encoding soluble fumarate reductase comprises any sequence or degenerate sequence shown in SEQ ID NO: 3-5, or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and encoding an amino acid sequence having soluble fumarate reductase activity Nucleotide sequence.
  • the gene encoding a soluble fumarate reductase is incorporated into the genome of a genetically engineered succinate producing yeast (e.g. Pichia kudriazwigi), e.g. at the ADH1 locus s position.
  • the gene encoding soluble fumarate reductase can be placed in a suitable promoter (such as the promoter of ADH1 gene (such as shown in SEQ ID NO: 21)) and/or terminator (such as the terminator of ADH1 gene (Example shown in SEQ ID NO: 22)) under the control.
  • the genetically engineered succinate producing yeast strain also has or has enhanced succinate transporter activity.
  • the succinate transporter is selected from SpMAE1 protein, AnDCT-02 protein, EcDcuB and EcDcuC protein.
  • succinate transporter activity is produced or increased by expressing or overexpressing a gene encoding a succinate transporter, such as the SpMAE1 protein, in said genetically engineered succinate producing yeast strain.
  • the succinate transporter activity is produced or enhanced by expressing or overexpressing the SpMAE1 gene.
  • the SpMAE1 gene comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, A nucleotide sequence that is 94%, 95%, 96%, 97%, 98%, 99% or more identical and encodes an amino acid sequence (eg, from Schizosaccharomyces pombe) having succinate transporter activity.
  • the SpMAE1 gene is incorporated into the genome of a genetically engineered succinic acid producing yeast strain (eg Pichia kudriazwigi), for example at the location of the MCH4 locus.
  • the SpMAE1 gene can be placed in a suitable promoter (such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)) and/or a terminator (such as the terminator of the GAL2 gene (such as shown in SEQ ID NO: 20) )) under the control.
  • a suitable promoter such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)
  • a terminator such as the terminator of the GAL2 gene (such as shown in SEQ ID NO: 20)
  • the genetically engineered succinate producing yeast strain also has or has enhanced pyruvate carboxylase activity.
  • the pyruvate carboxylase activity can be produced or enhanced by expressing or overexpressing a gene encoding pyruvate carboxylase.
  • the pyruvate carboxylase can be derived from fungi, especially yeast and filamentous fungi, preferably Saccharomyces cerevisiae, Pichia kudriazwi, Aspergillus oryzae, Kluyveromyces marx and the like.
  • the gene encoding pyruvate carboxylase may be selected from the PYC gene of Aspergillus oryzae and the PYC1 gene of Pichia kudriazwi.
  • the pyruvate carboxylase comprises the amino acid sequence encoded by the sequence shown in SEQ ID NO: 6 or 7 or has at least 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and have pyruvate carboxylase activity amino acid sequence.
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence, or at least 75%, 80%, 85%, 90%, A nucleotide sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical and encodes an amino acid sequence having pyruvate carboxylase activity.
  • a gene encoding pyruvate carboxylase is incorporated into the genome of a genetically engineered succinate-producing yeast (eg, Pichia kuderiazwyi), eg at the location of the JEN2-1 locus.
  • the gene encoding pyruvate carboxylase can be placed in a suitable promoter (such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)) and/or a terminator (such as the terminator of the GAL2 gene (such as SEQ ID NO: 19) ID NO: 20 under the control of)).
  • the genetically engineered succinate producing yeast strain also has or has enhanced fumarase activity.
  • the fumarase activity can be produced or enhanced by expressing or overexpressing a gene encoding fumarase.
  • the genetically engineered succinate producing yeast strain expresses or overexpresses a gene encoding fumarase.
  • the fumarase is derived from, for example but not limited to, Actinobacillus succinogenes, Succinobacter mannheimii, Escherichia coli, Pichia kudriazwi, Rhizopus oryzae.
  • the fumarase comprises or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, as shown in SEQ ID NO: 95, Amino acid sequences that are 95%, 96%, 97%, 98%, 99% or more identical and have fumarase activity.
  • the 5' mitochondrial localization peptide of the fumarase is partially or completely truncated, so that it cannot be located in the mitochondria, but exists freely in the cytoplasm.
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or at least 75%, 80%, 85%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical and have fumarase activity.
  • the gene encoding fumarase is incorporated into the genome of a genetically engineered succinate-producing yeast (eg, Pichia kudriazwigi), eg at the position of the PDC1 locus.
  • the gene encoding fumarase can be placed in a suitable promoter (such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)) and/or a terminator (such as the terminator of the GAL2 gene (such as SEQ ID NO: 19) NO: 20 shown)) under the control.
  • a suitable promoter such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)
  • a terminator such as the terminator of the GAL2 gene (such as SEQ ID NO: 19) NO: 20 shown
  • the genes to be expressed or overexpressed can be integrated at a suitable position in the genome of the strain, as long as the integration does not negatively affect the growth, reproduction and/or production performance of the strain.
  • it may be integrated at any one or more genomic locations encoding the following proteins: (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8 ), (iii) 5'-phosphate orotidine decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) dicarboxylic acid transporter, (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzymes (EC 4.1.3.17 or 4.1.1.112).
  • the genetically engineered succinate producing yeast strain also has reduced or inactivated at least one of: (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent 3-phosphate glycerol dehydrogenase (EC 1.1.1.8), (iii) 5'-phosphate orotidine decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) di Carboxylate transporters, (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes (EC 4.1.3.17 or 4.1.1.112).
  • pyruvate decarboxylase EC 4.1.1.43
  • NAD-dependent 3-phosphate glycerol dehydrogenase EC 1.1.1.8
  • 5'-phosphate orotidine decarboxylase EC 4.1.1.23
  • the genetically engineered succinate producing yeast strain has reduced activity or inactivated pyruvate decarboxylase. Reduced activity or inactivated pyruvate decarboxylase can be achieved by attenuating or inactivating the gene encoding pyruvate decarboxylase in said strain.
  • the genetically engineered succinate producing yeast strain has a weakened or inactivated gene encoding pyruvate decarboxylase.
  • the gene encoding pyruvate decarboxylase such as the PDC1 gene, is knocked out in the genetically engineered succinate producing yeast strain.
  • the pyruvate decarboxylase comprises or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, as shown in SEQ ID NO: 10, Amino acid sequences that are 95%, 96%, 97%, 98%, 99% or more identical and have pyruvate decarboxylase activity.
  • the gene encoding pyruvate decarboxylase encodes a protein as shown in SEQ ID NO: 10 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or higher identity and an amino acid sequence having pyruvate decarboxylase activity.
  • the genetically engineered succinate-producing yeast strain also has reduced or inactivated NAD-dependent 3-phosphate glycerol dehydrogenase.
  • the genetically engineered succinate-producing yeast strain has a weakened or inactivated gene encoding NAD-dependent 3-phosphate glycerol dehydrogenase.
  • the gene encoding the NAD-dependent glycerol-3-phosphate dehydrogenase is knocked out in the genetically engineered succinate-producing yeast strain (eg, Pichia kuderiyazweig).
  • the NAD-dependent 3-phosphate glycerol dehydrogenase comprises or has at least 75%, 80%, 85%, 90%, 91%, 92%, Amino acid sequences that are 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical and have NAD-dependent glycerol-3-phosphate dehydrogenase activity.
  • the gene encoding said NAD-dependent 3-phosphate glycerol dehydrogenase encodes, for example, a protein shown in SEQ ID NO: 10 or has at least 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and an amino acid sequence having NAD-dependent 3-phosphate glycerol dehydrogenase activity.
  • the genetically engineered succinate producing yeast strain also has reduced or inactivated alcohol dehydrogenase 1 activity.
  • the genetically engineered succinate producing yeast strain has a weakened or inactivated gene encoding alcohol dehydrogenase 1 .
  • the gene encoding alcohol dehydrogenase 1 is knocked out in the genetically engineered succinate-producing yeast strain (eg, Pichia kuderiazwyi).
  • the alcohol dehydrogenase 1 comprises or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% as shown in SEQ ID NO: 12 , 95%, 96%, 97%, 98%, 99% or higher identity and amino acid sequence having alcohol dehydrogenase 1 activity.
  • the gene encoding said alcohol dehydrogenase 1 encodes a protein such as shown in SEQ ID NO: 12 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and amino acid sequence having alcohol dehydrogenase 1 activity.
  • the genetically engineered succinic acid producing yeast strain also has reduced or inactivated 5'-phosphate orotidine decarboxylase (EC 4.1.1.23), dicarboxylic acid transporter, Oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzyme and/or monocarboxylic acid permease.
  • 5'-phosphate orotidine decarboxylase EC 4.1.1.23
  • dicarboxylic acid transporter Oxaloacetate decarboxylation
  • 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzyme and/or monocarboxylic acid permease.
  • the gene encoding 5'-orotidine phosphate decarboxylase and/or the gene encoding a dicarboxylic acid transporter eg JEN2 gene such as JEN2 -1 gene, JEN2-2 gene
  • genes encoding monocarboxylic acid permeases and/or genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes are knockout.
  • the genetically engineered succinic acid producing yeast strain also has reduced or inactivated oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes.
  • the genetically engineered succinic acid producing yeast strain has weakened or inactivated genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzymes.
  • the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzymes in the genetically modified succinic acid producing yeast strain (such as Pichia kudriazweiwei) gene was knocked out.
  • the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzyme comprises the amino acid sequence shown in SEQ ID NO: 9 or at least 75%, 80% thereof , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with oxaloacetate decarboxylation and 3-hydroxy-3 - Amino acid sequence of methylglutaric acid aldol bifunctional enzymatic activity.
  • the gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzyme encodes a protein as shown in SEQ ID NO: 9 or at least 75%, 80% thereof , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with oxaloacetate decarboxylation and 3-hydroxy-3 - Amino acid sequence of methylglutaric acid aldol bifunctional enzymatic activity.
  • the genetically engineered succinic acid producing yeast strain also has reduced or inactivated 5'-phosphate orotidine decarboxylase.
  • the genetically engineered succinic acid producing yeast strain has a weakened or inactivated gene encoding 5'-orotidine phosphate decarboxylase.
  • the gene encoding 5'-phosphate orotidine decarboxylase is knocked out in said genetically engineered succinic acid producing yeast strain (eg Pichia kudriazweiwei).
  • the 5'-orotidine phosphate decarboxylase comprises an amino acid sequence as shown in SEQ ID NO: 13 or has at least 75%, 80%, 85%, 90%, 91%, 92% thereof , 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and amino acid sequence having 5'-orotidine phosphate decarboxylase activity.
  • the gene encoding said 5'-orotidine phosphate decarboxylase encodes a protein as shown in SEQ ID NO: 13 or has at least 75%, 80%, 85%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical and have 5'-orotidine phosphate decarboxylase activity.
  • the genetically engineered succinate producing yeast strain also has a reduced or inactivated monocarboxylate permease.
  • the genetically engineered succinic acid producing yeast strain has a weakened or inactivated gene encoding a monocarboxylic acid permease.
  • the gene encoding the monocarboxylic acid permease is knocked out in said genetically engineered succinic acid producing yeast strain (eg Pichia kudriazweiwei).
  • the monocarboxylate permease comprises the amino acid sequence shown in SEQ ID NO: 16 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93% thereof , 94%, 95%, 96%, 97%, 98%, 99% or higher identity and an amino acid sequence having monocarboxylic acid permease activity.
  • the gene encoding said monocarboxylate permease encodes, for example, a protein shown in SEQ ID NO: 16 or has at least 75%, 80%, 85%, 90%, 91%, 92%, Amino acid sequences that are 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical and have monocarboxylic acid permease activity.
  • the genetically engineered succinate producing yeast strain also has a reduced or inactivated dicarboxylate transporter.
  • the genetically engineered succinate producing yeast strain has a weakened or inactivated gene encoding a dicarboxylic acid transporter.
  • the gene encoding a dicarboxylate transporter such as the JEN2-1 or JEN2-2 gene, is knocked out in the genetically engineered succinic acid producing yeast strain (eg Pichia kudriazweiwei) .
  • the dicarboxylic acid transporter comprises an amino acid sequence as shown in SEQ ID NO: 14 or 15 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences and having dicarboxylic acid transporter activity.
  • the gene encoding said dicarboxylic acid transporter encodes a protein as shown in SEQ ID NO: 14 or 15 or has at least 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and an amino acid sequence having dicarboxylic acid transporter activity.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • MDH gene for hydrogenase optionally, endogenously encoding 5'-phosphate orotidine decarboxylase gene and/or endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldehyde bicondensation functions
  • the gene for the enzyme is knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • MDH gene for hydrogenase and overexpressed gene encoding soluble fumarate reductase optionally, endogenous gene encoding 5'-orotidine phosphate decarboxylase and/or endogenous encoding alcohol dehydrogenase 1
  • Genes and/or endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes were knocked out.
  • the MDH gene comprises a sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding soluble fumarate reductase comprises any sequence shown in SEQ ID NO: 3-5 or its degenerate sequence.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding the succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • MDH gene for hydrogenase and overexpressed gene encoding fumarase optionally, endogenous gene encoding 5'-orotidine phosphate decarboxylase and/or endogenous gene encoding pyruvate decarboxylase and/or The endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric aldol condensation bifunctional enzymes were knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • MDH gene for hydrogenase and overexpressed gene encoding pyruvate carboxylase optionally, endogenous gene encoding 5'-phosphate orotidine decarboxylase and/or endogenous gene encoding dicarboxylic acid transporter e.g.
  • the JEN2-1 or JEN2-2 genes and/or endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • a strain of genetically engineered succinate-producing yeast eg, Pichia kudriazvii, such as the CY902 strain
  • an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • the MDH gene for hydrogenase and the gene encoding pyruvate carboxylase, as well as the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase were knocked out, optionally endogenously Genes encoding 5'-orotidine phosphate decarboxylase and/or endogenously encoding dicarboxylic acid transporter genes (e.g. JEN2 gene such as JEN2-1 gene, JEN2-2 gene) and/or endogenously encoding oxaloacetate
  • the genes for the decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes were knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • a strain of genetically engineered succinate-producing yeast eg, Pichia kudriazvii, such as the CY902 strain
  • an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • MDH gene for hydrogenase gene encoding pyruvate carboxylase, gene encoding soluble fumarate reductase and gene encoding succinate transporter such as SpMAE1 gene, and endogenous gene encoding pyruvate decarboxylase and The endogenous gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase is knocked out, optionally, the endogenous gene encoding 5'-orotidine phosphate decarboxylase and/or the endogenous gene encoding dicarboxylate transporter Genes (eg JEN2 gene such as JEN2-1 gene, JEN2-2 gene) and/or endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzymes and/or endogenous
  • JEN2 gene such as JEN2-1 gene, JEN2-2 gene
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the gene encoding soluble fumarate reductase comprises any sequence shown in SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding succinate transporter comprises SEQ ID NO: 2 sequence or its degenerate sequence.
  • the present invention provides a strain of genetically engineered succinate-producing yeast (eg, Pichia kudriazvii, such as the CY902 strain) with an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • a strain of genetically engineered succinate-producing yeast eg, Pichia kudriazvii, such as the CY902 strain
  • an overexpressed protein encoding an NADPH-dependent malate decapitate.
  • MDH gene for hydrogenase gene encoding pyruvate carboxylase, gene encoding soluble fumarate reductase, gene encoding fumarase and gene encoding succinate transporter such as SpMAE1 gene, and endogenous
  • the gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase are knocked out, optionally, the endogenous gene encoding orotidine 5'-phosphate decarboxylase and/or Genes endogenously encoding dicarboxylate transporters (e.g.
  • JEN2 genes such as JEN2-1 gene, JEN2-2 gene) and/or endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldehyde biscondensation
  • the gene for the functional enzyme and/or the endogenous gene encoding alcohol dehydrogenase 1 and/or the endogenous gene encoding monocarboxylate permease is knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the The gene encoding soluble fumarate reductase comprises the sequence shown in any one of SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or Its degenerate sequence
  • the gene encoding succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • the JEN2-1 gene encodes a protein as shown in SEQ ID NO: 14 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or higher identity and an amino acid sequence having dicarboxylic acid transporter activity.
  • the JEN2-2 gene encodes a protein as shown in SEQ ID NO: 15 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or higher identity and an amino acid sequence having dicarboxylic acid transporter activity.
  • the present invention provides a genetically engineered strain of Pichia kuderi Azwitzweig such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, optionally, The endogenous URA3 gene and/or the endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence.
  • the present invention provides a genetically engineered strain of Pichia kuderi Azwiya, such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed
  • the soluble fumarate reductase gene optionally, the endogenous URA3 gene and/or the endogenous ADH1 gene and/or the endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises a sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding soluble fumarate reductase comprises any sequence shown in SEQ ID NO: 3-5 or its degenerate sequence.
  • the present invention provides a genetically engineered strain of Pichia kuderi Azwiya, such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed
  • the genes of the succinate transporter such as the SpMAE1 gene, optionally the endogenous URA3 gene and/or the endogenous MCH4 gene and/or the endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding the succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • the present invention provides a genetically engineered strain of Pichia kuderi Azwiya, such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed
  • the genes for fumarase, optionally, the endogenous URA3 gene and/or the endogenous PDC1 gene and/or the endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence.
  • the present invention provides a genetically engineered strain of Pichia kuderi Azwiya, such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed
  • the genes for pyruvate carboxylase optionally, the endogenous URA3 gene and/or the endogenous JEN2-1 or JEN2-2 gene and/or the endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.
  • the present invention provides a genetically engineered strain of Pichia kudri Azwitzweig such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding pyruvate carboxylate
  • the gene of enzyme, as well as endogenous PDC1 gene and endogenous GPD1 gene are knocked out, optionally endogenous URA3 gene and/or endogenous JEN2 gene such as JEN2-1 gene, JEN2-2 gene and/or endogenous Pk2365 Genes are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.
  • the present invention provides a genetically modified strain of Pichia kuderi Azwiya, such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, encoding pyruvate carboxyl
  • the gene of lyase, the gene encoding soluble fumarate reductase and the gene encoding succinate transporter such as the SpMAE1 gene, as well as the endogenous PDC1 gene and the endogenous GPD1 gene are knocked out, optionally, endogenous
  • the source URA3 gene and/or the endogenous JEN2 gene such as JEN2-1 gene, JEN2-2 gene and/or the endogenous Pk2365 gene and/or the endogenous ADH1 gene and/or the endogenous MCH4 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the gene encoding soluble fumarate reductase comprises any sequence shown in SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding succinate transporter comprises SEQ ID NO: 2 sequence or its degenerate sequence.
  • the present invention provides a genetically engineered strain of Pichia kudriazwhiwei, such as CY902, with enhanced activities of sorghum NADPH-dependent malate dehydrogenase, Aspergillus oryzae pyruvate carboxylase , soluble fumarate reductase from Trypanosoma brucei, Pichia kudriazvii fumarase (5' mitochondrial localization peptide truncated) and succinate transporter SpMAE1, and endogenous pyruvate Decarboxylase and endogenous NAD-dependent glycerol-3-phosphate dehydrogenase activities are abolished.
  • CY902 a genetically engineered strain of Pichia kudriazwhiwei, such as CY902
  • sorghum NADPH-dependent malate dehydrogenase Aspergillus oryzae pyruvate carboxylase
  • the present invention provides a genetically modified strain of Pichia kuderi Azwiya, such as CY902, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, encoding pyruvate carboxyl
  • the genes encoding lyase, the gene encoding soluble fumarate reductase, the gene encoding fumarase, and the gene encoding succinate transporter such as the SpMAE1 gene, as well as the endogenous PDC1 gene and endogenous GPD1 gene were knocked out.
  • the endogenous URA3 gene and/or the endogenous JEN2 gene such as the JEN2-1 gene, the JEN2-2 gene and/or the endogenous Pk2365 gene and/or the endogenous ADH1 gene and/or the endogenous MCH4 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the The gene encoding soluble fumarate reductase comprises the sequence shown in any one of SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or Its degenerate sequence
  • the gene encoding succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • the present invention provides a method of producing a genetically engineered succinate-producing yeast strain comprising conferring or enhancing NADPH-dependent malate dehydrogenase (EC 1.1.1.82) activity in said strain .
  • imparting an activity refers to producing in a genetically engineered succinic acid producing yeast strain an activity that was not present in the original strain prior to genetic modification.
  • enhancing the activity means increasing the activity, for example by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% %, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or higher.
  • overexpression means that the expression level of a gene is increased, for example by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, relative to the level before genetic manipulation. At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or higher.
  • Methods for overexpressing genes are well known in the art, including but not limited to using strong promoters, increasing gene copy number, enhancers, and the like. Increasing gene copy number can be achieved, for example, but not limited to, by introducing one or more copies of an exogenous or endogenous gene, such as through an expression vector or integration into the genome.
  • exogenous gene refers to a gene from another cell or organism, eg, from the same species or a different species.
  • endogenous gene refers to a cell or organism's own genes.
  • the promoter can be selected from any suitable promoter known in the art, for example including but not limited to the promoter of the FBA1 gene encoding fructose 1,6-bisphosphate aldolase, the promoter encoding glyceraldehyde 3-phosphate dehydrogenase TDH3 gene promoter, PDC1 gene promoter encoding pyruvate decarboxylase, ADH1 gene promoter encoding alcohol dehydrogenase 1, PGK1 gene promoter encoding 3-phosphoglycerate kinase, TEF1 gene encoding transcription elongation factor Promoter, the promoter of the gene encoding phosphoglycerate mutase GPM1, the promoter of the gene encoding triosephosphate isomerase TPI1 and the promoter of the ENO1 gene encoding enolase (such as shown in SEQ ID NO: 100).
  • the NADPH-dependent malate dehydrogenase is from a plant, preferably a C4 plant, more preferably Gramineae, Cyperaceae, Compositae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae plants , or from Euglena and Thermobacterium, more preferably sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena microglia or thermoautotrophic Methanobacterium, more preferably sorghum.
  • NADPH-dependent malate dehydrogenase activity is produced or increased by expressing or overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase in said genetically engineered succinate-producing yeast strain .
  • the MDH gene encoding the NADPH-dependent malate dehydrogenase is preferably from a plant, preferably a C4 plant, more preferably Poaceae, Cyperaceae, Compositae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthus Plants of the family Euglenaceae, or from Euglena and Thermobacterium, more preferably sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena microglia or thermoautotrophic Methanobacterium, more preferably sorghum.
  • the MDH gene encoding NADPH-dependent malate dehydrogenase comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or at least 75%, 80%, 85%, 90% of it , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and encodes a nucleoside of an amino acid sequence having NADPH-dependent malate dehydrogenase activity sequence of acid sequences.
  • the MDH gene encoding NADPH-dependent malate dehydrogenase is incorporated into the genome of a genetically engineered succinate-producing yeast (e.g. Pichia kudriazwii), e.g. in the Pk2365 gene seat position.
  • the MDH gene encoding NADPH-dependent malate dehydrogenase is incorporated into the genome of yeast (eg, Pichia kudriazwigi) through homologous recombination, eg at the position of the Pk2365 locus.
  • the MDH gene encoding NADPH-dependent malate dehydrogenase can be placed in a suitable promoter (such as the promoter of the FBA1 gene (such as shown in SEQ ID NO: 17)) and/or a terminator (such as the termination of the INO1 gene sub (such as shown in SEQ ID NO: 18)) under the control of.
  • a suitable promoter such as the promoter of the FBA1 gene (such as shown in SEQ ID NO: 17)
  • a terminator such as the termination of the INO1 gene sub (such as shown in SEQ ID NO: 18)
  • the method further comprises conferring or enhancing soluble fumarate reductase activity in the strain.
  • the soluble fumarate reductase is from yeast and the order Kinetoplastia, such as but not limited to Saccharomyces cerevisiae, Trypanosoma brucei, Leishmania mexicana, Trypanosoma cruzi.
  • the 3' end glyoxylate cycle body localization peptide of the soluble fumarate reductase is partially or completely truncated, so that it exists freely in the cytoplasm.
  • soluble fumarate reductase activity is produced or increased by expressing or overexpressing a gene encoding soluble fumarate reductase in said genetically engineered succinic acid producing yeast strain
  • the gene encoding the soluble fumarate reductase is preferably from yeasts and kinetoplasma, such as but not limited to Saccharomyces cerevisiae, Trypanosoma brucei, Leishmania mexicana, Trypanosoma cruzi.
  • the gene encoding soluble fumarate reductase comprises any sequence shown in SEQ ID NO: 3-5 or its degenerate sequence, or at least 75%, 80%, 85% thereof , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and encoding amino acid sequence with soluble fumarate reductase activity the nucleotide sequence.
  • the gene encoding a soluble fumarate reductase is incorporated into the genome of a genetically engineered succinate producing yeast (e.g. Pichia kudriazwigi), e.g. at the ADH1 locus s position.
  • the gene encoding soluble fumarate reductase can be placed in a suitable promoter (such as the promoter of ADH1 gene (such as shown in SEQ ID NO: 21)) and/or terminator (such as the terminator of ADH1 gene (Example shown in SEQ ID NO: 22)) under the control.
  • the method further comprises conferring or enhancing succinate transporter activity in the yeast strain.
  • the succinate transporter is selected from SpMAE1 protein, AnDCT-02 protein, EcDcuB and EcDcuC protein.
  • succinate transporter activity is conferred or enhanced by expressing or overexpressing a gene encoding a succinate transporter, such as the SpMAE1 gene, in said genetically engineered succinate producing yeast strain.
  • the SpMAE1 gene comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or at least 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or higher identity and a nucleotide sequence encoding an amino acid sequence having succinate transporter activity, optionally, (for example, via homologous Recombinant) into the genome of a genetically engineered succinate producing yeast strain (eg Pichia kudriazweigi), for example at the location of the MCH4 locus.
  • a genetically engineered succinate producing yeast strain eg Pichia kudriazweigi
  • the SpMAE1 gene can be placed in a suitable promoter (such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)) and/or a terminator (such as the terminator of the GAL2 gene (such as shown in SEQ ID NO: 20) )) under the control.
  • a suitable promoter such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)
  • a terminator such as the terminator of the GAL2 gene (such as shown in SEQ ID NO: 20)
  • the method further comprises conferring or enhancing fumarase activity in the yeast strain.
  • the 5' mitochondrial localization peptide of the fumarase is partially or completely truncated, so that it cannot be located in the mitochondria, but exists freely in the cytoplasm.
  • fumarase activity is conferred or enhanced by expressing or overexpressing a gene encoding fumarase in said genetically engineered succinic acid producing yeast strain.
  • the fumarase is derived from, for example but not limited to, Actinobacillus succinogenes, Succinobacter mannheimii, Escherichia coli, Pichia kudriazwi, Rhizopus oryzae.
  • said fumarase comprises SEQ ID NO: 95 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98%, 99% or more identical and have fumarase activity.
  • the gene coding for said fumarase is as shown in SEQ ID NO: 95 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or higher identity and have fumarase activity amino acid sequence.
  • the gene encoding the fumarase comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or at least 75%, 80%, 85%, 90%, 91%, A nucleotide sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical and encodes an amino acid sequence having fumarase activity.
  • the gene encoding said fumarase is incorporated (e.g., via homologous recombination) into the genome of a genetically engineered succinate-producing yeast (e.g., Pichia kudriazwiii), e.g., in the PDC1 gene seat position.
  • the gene encoding the fumarase can be placed in a suitable promoter (such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)) and/or a terminator (such as the terminator of the GAL2 gene (such as SEQ ID NO: 19) NO: 20 shown)) under the control.
  • the method further comprises conferring or enhancing pyruvate carboxylase activity in the yeast strain.
  • conferring or enhancing pyruvate carboxylase activity is achieved by expressing or overexpressing a gene encoding pyruvate carboxylase in the strain.
  • the pyruvate carboxylase can be derived from fungi, especially yeast and filamentous fungi, preferably Saccharomyces cerevisiae, Pichia kudriazwi, Aspergillus oryzae, Kluyveromyces marx and the like.
  • the gene encoding the pyruvate carboxylase may be selected from the PYC gene of Aspergillus oryzae and the PYC1 gene of Pichia kudriazwi.
  • the pyruvate carboxylase comprises the amino acid sequence encoded by the sequence shown in SEQ ID NO: 6 or 7 or has at least 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and have pyruvate carboxylase activity amino acid sequence.
  • the gene encoding said pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence, or at least 75%, 80%, 85%, 90%, A nucleotide sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical and encodes an amino acid sequence having pyruvate carboxylase activity.
  • a gene encoding pyruvate carboxylase is incorporated into the genome of a genetically engineered succinate-producing yeast (eg, Pichia kuderiazwyi), eg at the location of the JEN2-1 locus.
  • the gene encoding said pyruvate carboxylase can be placed in a suitable promoter (such as the promoter of the TDH3 gene (such as shown in SEQ ID NO: 19)) and/or a terminator (such as the terminator of the GAL2 gene (such as SEQ ID NO: 19) ID NO: 20 under the control of)).
  • the method may integrate the gene to be expressed or overexpressed in the strain at an appropriate position in the genome of the strain, as long as such integration does not negatively affect the growth, reproduction and/or production performance of the strain.
  • the method includes integrating one or more of the above genes at any one or more genomic positions encoding the following proteins: (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent 3- Glycerol phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orotidine phosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) dicarboxylic acid transporter, (vi) Alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes (EC 4.1.3.17 or 4.1.1.112) .
  • the method further comprises attenuating or inactivating at least one of the following in the strain: (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent 3- Glycerol phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orotidine phosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) dicarboxylic acid transporter, (vi) Alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes (EC 4.1.3.17 or 4.1.1.112) .
  • the method further comprises reducing or inactivating pyruvate decarboxylase activity in the yeast strain.
  • reducing or inactivating the activity of a protein means reducing the activity of the protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% %, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable.
  • RNA for example, using small interfering RNA
  • a weak promoter when the gene is a polypeptide-encoding gene
  • gene knockout deletion Part or all of the gene or polypeptide sequence
  • mutation of certain sites in the gene or polypeptide such as the coding sequence or active domain, to reduce gene expression or regulatory activity or the activity of the expressed product
  • antagonists or inhibitors such as including but not limited to antibodies, interfering RNA, etc.
  • weakening or inactivating a gene refers to reducing the expression level (as a protein-coding gene) or regulatory performance (as a regulatory element) of the gene by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable.
  • weakening or inactivating genes including, for example, inhibiting gene expression such as knocking down (for example, using small interfering RNA), using weak promoters (when the gene is a polypeptide-encoding gene), etc.; gene knockout, deletion of part or The entire gene sequence; certain sites in the gene, such as the coding sequence, are mutated to reduce gene expression or regulatory activity or the activity of expressed products, etc.
  • reducing or inactivating pyruvate decarboxylase activity comprises attenuating or inactivating a gene encoding pyruvate decarboxylase.
  • weakening or inactivating the gene encoding pyruvate decarboxylase comprises knocking out the gene encoding pyruvate decarboxylase (such as encoding the amino acid sequence shown in SEQ ID NO: 10 or having at least 75%, 80%, 85% %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and have pyruvate decarboxylase activity amino acid sequence).
  • the method further comprises reducing or inactivating the activity of NAD-dependent glycerol-3-phosphate dehydrogenase in the yeast strain.
  • reducing or inactivating the activity of NAD-dependent glycerol-3-phosphate dehydrogenase comprises attenuating or inactivating a gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase.
  • weakening or inactivating the gene encoding NAD-dependent 3-phosphate glycerol dehydrogenase comprises knocking out the gene encoding NAD-dependent 3-phosphate glycerol dehydrogenase (for example, encoding the gene shown in SEQ ID NO: 11 Amino acid sequence or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith and Amino acid sequence with NAD-dependent glycerol-3-phosphate dehydrogenase activity).
  • the method further comprises reducing or inactivating 5'-phosphate orotidine decarboxylase and/or alcohol dehydrogenase 1 and/or monocarboxylic acid permease and/or in the yeast strain Or dicarboxylic acid transporter and/or oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzyme activity.
  • the method comprises attenuating or inactivating genes encoding 5'-orotidine phosphate decarboxylase, genes encoding dicarboxylic acid transporters (e.g. JEN2 gene such as JEN2-1 gene) in the yeast strain , JEN2-2 gene), gene encoding alcohol dehydrogenase 1, gene encoding monocarboxylate permease, gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes one or more of.
  • genes encoding dicarboxylic acid transporters e.g. JEN2 gene such as JEN2-1 gene
  • JEN2-2 gene e.g. JEN2 gene such as JEN2-1 gene
  • gene encoding alcohol dehydrogenase 1 e.g. JEN2 gene such as JEN2-1 gene
  • gene encoding monocarboxylate permease e.g., oxaloacetate decarboxylation
  • the gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation bifunctional enzyme (for example, encoding the amino acid sequence shown in SEQ ID NO: 9 or having at least 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and have oxaloacetate decarboxylation and 3-
  • the amino acid sequence of hydroxy-3-methylglutaric acid aldol condensation bifunctional enzymatic activity is knocked out, for example, by homologous recombination.
  • the gene encoding 5'-orotidine phosphate decarboxylase (which encodes the amino acid sequence shown in SEQ ID NO: 13 or has at least 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and an amino acid sequence having 5'-orotidine phosphate decarboxylase activity) is knocked out, for example Knockout by homologous recombination.
  • the gene JEN2-1 gene (coding such as SEQ ID NO: 14 shown in the gene JEN2-1 gene of coding dicarboxylic acid transporter or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and have dicarboxylic acid transporter activity amino acid sequence) knocked out, for example by means of homologous recombination knockout.
  • the gene JEN2-2 gene (coding such as SEQ ID NO: 15 shown in the gene JEN2-2 gene of coding dicarboxylic acid transporter or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and have dicarboxylic acid transporter activity amino acid sequence) knocked out, for example by means of homologous recombination knockout.
  • the gene encoding alcohol dehydrogenase 1 (which encodes the amino acid sequence shown in SEQ ID NO: 12 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and amino acid sequence having alcohol dehydrogenase 1 activity) are knocked out, for example by homologous recombination.
  • the gene encoding monocarboxylate permease (encoding such as the amino acid sequence shown in SEQ ID NO: 16 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and amino acid sequence having monocarboxylic acid permease activity) are knocked out, for example by homologous recombination .
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase in an acid-producing yeast strain, optionally including knockout of the endogenous gene encoding 5'-orotidine phosphate decarboxylase and/or the endogenous gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzyme genes.
  • a strain of genetically engineered succinate producing yeast eg, Pichia kudriazweigi, such as CY902
  • Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase in an acid-producing yeast strain optionally including knockout of the endogenous gene encoding 5'-orotidine phosphate decarboxylase and/or the endogenous gene
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding soluble fumarate reductase in an acid-producing yeast strain, optionally knocking out the endogenous gene encoding orotidine 5'-phosphate decarboxylase And/or endogenous gene encoding alcohol dehydrogenase 1 and/or endogenous gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolization bifunctional enzymes.
  • a strain of genetically engineered succinate producing yeast eg, Pichia kudriazweigi, such as CY902
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding a succinate transporter such as the SpMAE1 gene in an acid-producing yeast strain, optionally knocking out the endogenous gene encoding orotidine 5'-phosphate decarboxylase And/or an endogenous gene encoding a monocarboxylic acid permease and/or an endogenous gene encoding an oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzyme.
  • a strain of genetically engineered succinate producing yeast eg, Pichia kudriazweigi, such as CY902
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding fumarase in an acid-producing yeast strain, optionally knocking out the endogenous gene encoding 5'-orotidine phosphate decarboxylase and/or endogenous Source a gene encoding pyruvate decarboxylase and/or endogenously encode a gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes.
  • a strain of genetically engineered succinate producing yeast eg, Pichia kudriazweigi, such as CY902
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding pyruvate carboxylase in an acid-producing yeast strain, optionally knocking out the endogenous gene encoding 5'-orotidine phosphate decarboxylase and/or Endogenous genes encoding dicarboxylate transporters such as JEN2-1 or JEN2-2 genes and/or endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzymes.
  • a strain of genetically engineered succinate producing yeast eg, Pichia kudriazweigi, such as CY902
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding pyruvate carboxylase in an acid-producing yeast strain, as well as knockout of the endogenous gene encoding pyruvate decarboxylase and the endogenous encoding of NAD-dependent 3- Genes for glycerol phosphate dehydrogenase, optionally knockout endogenous genes encoding 5'-orotidine phosphate decarboxylase and/or endogenous genes encoding dicarboxylic acid transporters (e.g. JEN2 gene such as JEN2-1 gene, JEN2 -2 genes) and/or endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldol condensation
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902 strain), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase, the gene encoding pyruvate carboxylase, the gene encoding soluble fumarate reductase, and the gene encoding a succinate transporter in a diacid-producing yeast strain
  • the SpMAE1 gene as well as the knockout of the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent 3-phosphate glycerol dehydrogenase, optionally the endogenous gene encoding 5'-phosphate ortidine decarboxylase Genes and/or endogenous genes encoding dicarboxylate transporters (e.g.
  • JEN2 genes such as JEN2-1 gene, JEN2-2 gene) and/or endogenous encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutadiene Acid aldolase bifunctional enzyme gene and/or endogenous gene encoding alcohol dehydrogenase 1 and/or endogenous gene encoding monocarboxylic acid permease.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the The gene encoding soluble fumarate reductase comprises the sequence shown in any one of SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or Its degenerate sequence
  • the gene encoding succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • the present invention provides a method for producing a strain of genetically engineered succinate producing yeast (eg, Pichia kudriazweigi, such as CY902 strain), comprising Overexpression of the MDH gene encoding NADPH-dependent malate dehydrogenase, the gene encoding pyruvate carboxylase, the gene encoding soluble fumarate reductase, the gene encoding fumarase and A gene encoding a succinate transporter such as the SpMAE1 gene, and knockout of the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent 3-phosphate glycerol dehydrogenase, optionally, knockout of the endogenous gene encoding 5'-orotidine phosphate decarboxylase genes and/or endogenous genes encoding dicarboxylic acid transporters (e.g.
  • JEN2 genes such as JEN2-1 gene, JEN2-2 gene) and/or endogenous encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaric acid aldolase bifunctional enzyme gene and/or endogenous gene encoding alcohol dehydrogenase 1 and/or endogenous gene encoding monocarboxylate permease.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the The gene encoding soluble fumarate reductase comprises the sequence shown in any one of SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or Its degenerate sequence
  • the gene encoding succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • the present invention provides a method of producing a genetically engineered strain of Pichia kudriziwyi, such as CY902, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase, optionally , the endogenous URA3 gene is knocked out and/or the endogenous Pk2365 gene is knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence.
  • the present invention provides a method of producing a genetically engineered Pichia kudriazwhiwei, such as CY902 strain, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and encoding soluble
  • the gene of fumarate reductase, optionally, endogenous URA3 gene and/or endogenous ADH1 gene and/or endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises a sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding soluble fumarate reductase comprises any sequence shown in SEQ ID NO: 3-5 or its degenerate sequence.
  • the present invention provides a method of producing a genetically engineered Pichia kudriazwhiwei, such as the CY902 strain, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the An acid transporter gene such as the SpMAE1 gene, optionally knocking out the endogenous URA3 gene and/or the endogenous MCH4 gene and/or the endogenous Pk2365 gene.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding the succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • the present invention provides a method of producing a genetically engineered strain of Pichia kuderiyazwiya, such as CY902, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding fuma Acidase gene, optionally, endogenous URA3 gene and/or endogenous PDC1 gene and/or endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence.
  • the present invention provides a method of producing a genetically engineered strain of Pichia kuderiyazwiya, such as CY902, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding pyruvate
  • the gene for carboxylase optionally, the endogenous URA3 gene and/or the endogenous JEN2-1 or JEN2-2 gene and/or the endogenous Pk2365 gene are knocked out.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.
  • the present invention provides a method of producing a genetically engineered strain of Pichia kuderiyazwiya, such as CY902, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding pyruvate Carboxylase gene, and knockout of endogenous PDC1 gene and endogenous GPD1 gene, optionally knockout of endogenous URA3 gene and/or endogenous JEN2 gene such as JEN2-1 gene, JEN2-2 gene and/or endogenous Pk2365 Gene.
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.
  • the present invention provides a method for producing a genetically engineered Pichia kuderi Azwitzii strain, such as CY902, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase, encoding pyruvate A gene for carboxylase, a gene encoding soluble fumarate reductase, and a gene encoding a succinate transporter such as the SpMAE1 gene, and knockout of the endogenous PDC1 gene and endogenous GPD1 gene, optionally, knockout of the endogenous Source URA3 gene and/or endogenous JEN2 gene such as JEN2-1 gene, JEN2-2 gene and/or endogenous Pk2365 gene and/or endogenous ADH1 gene and/or endogenous MCH4 gene.
  • CY902 a genetically engineered Pichia kuderi Azwitzii strain
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the gene encoding soluble fumarate reductase comprises any sequence shown in SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding succinate transporter comprises SEQ ID NO: 2 sequence or its degenerate sequence.
  • the present invention provides a method for producing a genetically engineered Pichia kuderi Azwitzii strain, such as CY902, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase, encoding pyruvate Genes for carboxylase, genes encoding soluble fumarate reductase, genes encoding fumarase, and genes encoding succinate transporters such as the SpMAE1 gene, as well as knockout of the endogenous PDC1 gene and endogenous GPD1 gene , optionally, knocking out endogenous URA3 gene and/or endogenous JEN2 gene such as JEN2-1 gene, JEN2-2 gene and/or endogenous Pk2365 gene and/or endogenous ADH1 gene and/or endogenous MCH4 gene is knocked out.
  • CY902 a genetically engineered Pichia kuderi Azwitzii strain
  • the MDH gene comprises the sequence shown in SEQ ID NO: 1 or its degenerate sequence
  • the gene encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence
  • the The gene encoding soluble fumarate reductase comprises the sequence shown in any one of SEQ ID NO: 3-5 or its degenerate sequence
  • the gene encoding fumarase comprises the sequence shown in SEQ ID NO: 8 or Its degenerate sequence
  • the gene encoding succinate transporter comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence.
  • succinate-producing yeasts for genetic modification include, for example, but not limited to, Candida, Pichia, Rhodotorula, Saccharomyces, Yarrowia, Zygomyces ruckeri, Torulopsis spp.
  • the succinic acid producing yeast used for genetic modification is selected from a strain of Pichia, Saccharomyces, or Yarrowia.
  • the succinic acid-producing yeast used for genetic modification is Pichia kudriazweiwei, Saccharomyces cerevisiae or Yarrowia lipolytica, such as deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC), Pichia pastoris Kudriazwi with the preservation number of CGMCC No.20885.
  • the present invention provides a method for producing succinic acid, comprising cultivating the genetically modified succinic acid producing yeast strain according to the present invention or the succinic acid producing yeast strain according to the present invention under conditions suitable for fermentative production of succinic acid.
  • a genetically modified succinic acid-producing yeast strain prepared by the method for producing a genetically modified succinic acid-producing yeast strain, optionally including isolating and purifying the produced succinic acid.
  • the conditions for fermentatively cultivating succinic acid-producing yeast strains for fermentative production of succinic acid are known in the art, including but not limited to pH, temperature, medium composition, fermentation time and the like.
  • Culture media for the fermentative production of succinic acid by succinic acid-producing yeast strains are known in the art, including for example but not limited to inorganic salt medium (about 5-12% w/v glucose, optionally containing about 30 g/L CaCO ).
  • Temperatures for the fermentative production of succinic acid by succinic acid producing yeast strains are known in the art, for example about 25-37°C, for example about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C.
  • the succinic acid-producing yeast strain of the present invention is fermented at 30° C. to produce succinic acid.
  • the succinic acid producing yeast strain of the present invention can be fermented at a suitable pH value known in the art, such as less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, A pH of less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, less than about 1.0 (e.g.
  • the succinic acid producing yeast strain of the invention is fermented at a pH ⁇ about 3.0 to produce succinic acid.
  • the succinic acid producing yeast strain of the present invention can be fermented for a suitable time, such as about 12-96 hours, such as about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96 hours.
  • the succinic acid-producing yeast strain of the present invention is fermented for about 24-72 hours, such as about 30 hours.
  • the succinic acid-producing yeast strain of the present invention can be fermented under shaking conditions (for example, about 100-300 rpm, such as about 150, about 200, about 250 rpm) to produce succinic acid.
  • the succinic acid content in the fermentation broth can be determined by a suitable method known in the art, such as HPLC.
  • the present invention provides a method of producing succinic acid, comprising producing succinic acid at an acidic pH (less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, less than about 1.0, for example pH about 1.0-7.0, 1.0-6.0, 1.0-5.5, 1.0-5.0, 1.0-4.5, 1.0 -4.0, 1.0-3.5, 1.0-3.0, 2.0-7.0, 2.0-6.0, 2.0-5.5, 2.0-5.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 3.0-7.0, 3.0-6.0 , 3.0-5.5, 3.0-5.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 4.0-7.0, 4.0-6.0, 4.0-5.5, 4.0-5.0, 4.0-4.5) conditions, in inorganic salt medium ( A genetically engineered succinate
  • the method for producing succinic acid described herein does not require the addition of a neutralizing agent.
  • the present invention provides the genetically modified succinic acid producing yeast strain of the present invention or the genetically modified succinic acid producing yeast strain prepared according to the method of the present invention.
  • Use of diacid-producing yeast strains for the production of succinic acid particularly at acidic pH (less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, less than about 1.0, for example pH about 1.0-7.0, 1.0-6.0, 1.0-5.5, 1.0-5.0, 1.0-4.5, 1.0-4.0, 1.0 -3.5, 1.0-3.0, 2.0-7.0, 2.0-6.0, 2.0-5.5, 2.0-5.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 3.0-7.0, 3.0-6.0, 3.0-5.5 , 3.0-5.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 4.0
  • step means that the step is present or absent.
  • the term "about” refers to a range of values that includes the specified value that a person skilled in the art would reasonably consider to be similar to the specified value. In some embodiments, the term “about” means within standard error using measurements generally accepted in the art. In some embodiments, about refers to +/- 10% of a specified value.
  • a range disclosed herein should be considered to also specifically disclose all possible subranges and individual values within that range.
  • a description of the range 1 to 6 should be deemed to have explicitly disclosed the subranges 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. , and individual numbers in the range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the scope.
  • Example 1 Overexpression of SbMDH gene in Pichia pastoris CY902 ⁇ URA3 strain
  • the gene URA3 (CY902 locus number PK2075) encoding its orotidine 5'-phosphate decarboxylase (Orotidine 5'-phosphate decarboxylase) was knocked out by homologous recombination, and the ⁇ URA3 mutant (Xi,Y .; Zhan, T.; Xu, H.; Chen, J.; Bi, C.; Fan, F.; Zhang, X., Characterization of JEN family carboxylate transporters from the acid-tolerant yeast Pichia kudriavzevii and their applications in succinic acid production. Microb Biotechnol 2021.0(0),1–18. doi:10.1111/1751-7915.13781).
  • CRISPR/Cas9 plasmid pWSPK-Cas9 (GenBank accession number: MW296878.1) suitable for this strain.
  • This plasmid contains the URA3 screening marker, and positive transformants can be obtained through auxotrophic screening.
  • the sorghum-derived malate dehydrogenase SbMDH (Uniprot database retrieval number: P17606) (SEQ ID NO: 1) was overexpressed at the locus number Pk2365 of the CY902 genome, and the promoter and terminator used
  • the promoters are the promoter of CY902's own fructose 1,6-bisphosphate aldolase gene FBA1 (SEQ ID NO: 17) and the terminator of inositol-3-phosphate synthase gene INO1 (SEQ ID NO: 18).
  • the SbMDH gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and optimized according to the codon preference of CY902.
  • the specific construction method is as follows:
  • primers 1_UP_2365_F and 1_UP_2365_R1 were used to amplify CY902's own Pk2365 gene upstream homology arm fragment 1 (fragment 1); the amplification system and procedures refer to TAKARA Product instructions for DNA polymerase; use primers 2_P FBA1 _F and 2_P FBA1 _R (see Table 1) to amplify the CY902 self-FBA1 promoter sequence (fragment 2); use the plasmid containing the SbMDH synthetic sequence as a template, use primers 3_SbMDH_F and 3_SbMDH_R ( See Table 1) amplify the SbMDH coding sequence (fragment 3) of sorghum; take CY902 genomic DNA as a template, and use primers 4_T INO1 _F and 4_T INO1 _R (see Table 1) to amplify the CY902 self I
  • CY902 genomic DNA as a template, use primers 1_UP_2365_F and 1_UP_2365_R2 (see Table 1) to amplify CY902's own Pk2365 gene upstream homology arm fragment 2 (fragment 6); use primers 5_DW_2365_F2 and 5_DW_2365_R (see Table 1) to amplify CY902's own Pk2365 gene Downstream homology arm fragment 2 (fragment 7).
  • pWSPK-Cas9 plasmid GenBank retrieval number: MW296878.1
  • primers pWSPK-F and pWSPK-R see Table 1 to amplify a 9052bp plasmid backbone (referred to as pWSPK_backbone, SEQ ID NO : 23); use primers sgRNA-1F and 2365_sgRNA-1R (see Table 1) to amplify the 5' end sequence (abbreviated as 2365_sgRNA_1) of sgRNA (GenBank accession number: MW296878.1), the 3' end of the fragment contains the Pk2365 gene Special 20nt protospacer sequence.
  • sgRNA_2 Using the pWSPK-Cas9 plasmid as a template, use primers sgRNA-2F and sgRNA-2R (see Table 1) to amplify the 3' end sequence of the 500bp sgRNA (abbreviated as sgRNA_2, SEQ ID NO: 97).
  • the above-mentioned plasmid backbone pWSPK_backbone, sgRNA fragments 2365_sgRNA_1 and sgRNA_2 were ligated using a seamless cloning kit (Beyond Biotechnology Co., Ltd., Shanghai, product number: D7010S).
  • the seamless cloning product was transferred into Trans1-T1 competent cells (Quanshijin Biotechnology Co., Ltd., Beijing, product number: CD501-02), and the obtained positive plasmid was named pWSPK_2365.
  • the pWSPK_2365 plasmid and fragments 6 and 7 were transformed into the CY902 ⁇ URA3 strain according to the yeast electroporation method (doi:10.1111/1751-7915.13781), and a positive transformant was obtained after screening, which was named SA101-1 strain (genotype: CY902 ⁇ URA3, ⁇ Pk2365);
  • the pWSPK_2365 plasmid and fragments 1-5 were transformed into the CY902 ⁇ URA3 strain by yeast electroporation, and a positive transformant was obtained after screening, which was named SA101-2 strain (genotype: CY902 ⁇ URA3, Pk2365::P PkFBA1 -ORF SbMDH -TPkINO1 ) .
  • the soluble pyridine nucleotide transhydrogenase EcSthA (EC 1.6.1.1) (SEQ ID NO: 98) (SEQ ID NO: 98) (SEQ ID NO: 98) derived from Escherichia coli was overexpressed at the locus numbered Pk2365 of the CY902 genome.
  • the promoter and terminator are respectively the FBA1 promoter (SEQ ID NO: 17) and the INO1 terminator (SEQ ID NO: 18) of CY902 itself.
  • primers 3_EcSthA_F and 3_EcSthA_R see Table 2 to amplify the coding sequence (fragment 8) of Escherichia coli soluble pyridine nucleotide transhydrogenase EcSthA; using CY902 genomic DNA as a template, use primers 1_UP_UGA2_F and 1_UP_UGA2_R1 (see Table 2) amplify CY902 self-PkUGA2 gene upstream homology arm fragment 1 (fragment 9); use primer 2_P ENO1 _F and 2_P ENO1 _R (see Table 2) to amplify CY902 self-ENO1 promoter sequence (fragment 10 ); using the plasmid containing the AoMDH synthetic sequence as a template, amplifying the Aspergillus oryzae AoMDH coding sequence (fragment 11) with primers 3_
  • CY902 genomic DNA as a template, use primers 1_UP_UGA2_F and 1_UP_UGA2_R2 (see Table 2) to amplify CY902's own PkUGA2 gene upstream homology arm fragment 2 (fragment 14); use primers 5_DW_UGA2_F2 and 5_DW_UGA2_R (see Table 2) to amplify CY902's own PkUGA2 gene Downstream homology arm fragment 2 (fragment 15).
  • pWSPK-Cas9 plasmid uses primers sgRNA-1F and UGA2_sgRNA-1R (see Table 2) to amplify the 5' end sequence of the sgRNA (referred to as UGA2_sgRNA_1), and the 3' end of the fragment contains a special 20nt protospacer of the PkUGA2 gene sequence.
  • UGA2_sgRNA_1 The pWSPK_backbone, sgRNA_2 and UGA2_sgRNA_1 were ligated with a seamless cloning kit to finally obtain a positive clone plasmid, which was named pWSPK_UGA2.
  • the pWSPK_2365 plasmid and fragments 1, 2, 8, 4, and 5 were transferred into the CY902 ⁇ URA3 strain by yeast electrotransformation, and a positive transformant was obtained after screening, which was named SA101-3 strain (genotype: CY902 ⁇ URA3, Pk2365::P PkFBA1- ORF EcSthA -T PkINO1 ).
  • SA101-3 strain Genotype: CY902 ⁇ URA3, Pk2365::P PkFBA1- ORF EcSthA -T PkINO1
  • the pWSPK_UGA2 plasmid and fragments 14 and 15 were transformed into SA101-2 and SA101-3 strains by yeast electrotransformation, and positive transformants were obtained after screening, which were named SA102-1 strain (genotype: SA101-2, ⁇ UGA2) and SA102- 2 strains (genotype: SA101-3, ⁇ UGA2).
  • the pWSPK_UGA2 plasmid and fragments 9-13 were transformed into the SA101-3 strain by yeast electroporation, and a positive transformant was obtained after screening, which was named SA102-3 strain (genotype: SA101-3, PkUGA2::P PkENO1 -ORF AoMDH -T PkSED1 ).
  • Embodiment 3 Evaluation SA101, the succinic acid production capacity of SA102 series bacterial strain
  • strain SA101-1 SA101-2 SA101-3 SA102-1 SA102-2 SA102-3 Succinic acid g/L 0.52 0.80 0.58 0.83 0.57 0.62
  • the promoter and terminator of overexpressed FRD are the promoter (SEQ ID NO: 21) and terminator (SEQ ID NO: 22) of CY902's own alcohol dehydrogenase 1 (alcohol dehydrogenase I) ADH1, respectively.
  • the above three FRD gene sequences were synthesized by Nanjing GenScript Biotechnology Co., Ltd. and optimized according to the codon preference of CY902.
  • the specific construction method is as follows:
  • CY902 genomic DNA as a template, use primers 1_P ADH1 _F and 1_P ADH1 _R (see Table 5) to amplify the CY902 own ADH1 gene promoter sequence (upstream homology arm, fragment 16); using the plasmid containing the ScFRD synthetic sequence as a template, Use primers 2_ScFRD_F and 2_ScFRD_R (see Table 5) to amplify the ScFRD coding sequence (fragment 17-1) of Saccharomyces cerevisiae; use the plasmid containing the LmFRD synthetic sequence as a template to amplify the Mexican Leish The LmFRD coding sequence (fragment 17-2) of the glyoxylate cycle positioning peptide at the 3' end of Mannia truncated; using the plasmid containing the synthetic sequence of TbFRD as a template, the primers 2_TbFRD_F and 2_TbFRD_R (see Table
  • primers 3_T ADH1 _F2 and 3_T ADH1 _R were used to amplify CY902's own ADH1 gene terminator sequence (downstream homology arm 2, fragment 19).
  • pWSPK-Cas9 plasmid uses the primers sgRNA-1F and ADH1_sgRNA-1R (see Table 5) to amplify the 5' end sequence of the sgRNA (abbreviated as ADH1_sgRNA_1), and the 3' end of the fragment contains Kudri Azweibich Yeast ADH1 (PkADH1) gene specific 20nt protospacer sequence.
  • ADH1_sgRNA_1 The pWSPK_backbone, sgRNA_2 and ADH1_sgRNA_1 were ligated with a seamless cloning kit to finally obtain a positive clone plasmid, which was named pWSPK_ADH1.
  • the pWSPK_ADH1 plasmid and fragments 16 and 19 were transformed into the SA101-2 strain by yeast electroporation, and a positive transformant was obtained after screening, which was named SA103-1 strain (genotype: SA101-2, ⁇ ADH1).
  • the pWSPK_ADH1 plasmid and fragments 16, 17, and 18 were transformed into the SA101-2 strain by the yeast electroporation method, and positive transformants were obtained after screening, which were named SA103-2 strains (genotype: SA101-2, ADH1::ORF ScFRD ) ; SA103-3 strain (genotype: SA101-2, ADH1::ORF LmFRD ); named SA103-4 strain (genotype: SA101-2, ADH1::ORF TbFRD ).
  • CY902 genomic DNA as a template, use primers PDC1_1F and PDC1_1R, PDC1_2F and PDC1_2R (see Table 6) to amplify the upstream and downstream homology arm fragments of PDC1 gene (abbreviated as PDC1_1, PDC1_2); use primers GPD1_1F and GPD1_1R, GPD1_2F and GPD1_2R (see Table 6) Table 6) Amplification of the upstream and downstream homology arm fragments of the GPD1 gene (abbreviated as GPD1_1, GPD1_2).
  • the 5' end sequence of the sgRNA (referred to as PDC1_sgRNA_1, SEQ ID NO: 96) was amplified with primers sgRNA-1F and PDC1_sgRNA-1R (see Table 6), and the 3' end of the fragment contained a special 20nt prototype spacer sequence of the PDC1 gene.
  • the plasmid backbone pWSPK_backbone, sgRNA fragments PDC1_sgRNA_1 and sgRNA_2 were ligated with the seamless cloning kit.
  • the seamless cloning product was transferred into Trans1-T1 competent cells, and the obtained positive plasmid was named pWSPK_PDC1.
  • GPD1_sgRNA-1R Use primers sgRNA-1F and GPD1_sgRNA-1R (see Table 6) to amplify the 5' end sequence of the sgRNA (abbreviated as GPD1_sgRNA_1), and the 3' end of the fragment contains a special 20nt protospacer sequence of the GPD1 gene, pWSPK_backbone, sgRNA_2 and GPD1_sgRNA_1
  • the pWSPK_GPD1 plasmid for editing the GPD1 gene was fused by seamless cloning.
  • the fragments PDC1_1, PDC1_2 and plasmid pWSPK_PDC1 were simultaneously transferred into SA101-2.
  • the positive transformants identified using primers PDC1_1F/PDC1_2R were named SA104 strain (genotype: SA101-2, ⁇ PDC1).
  • SA104 strain wild-type: SA101-2, ⁇ PDC1
  • the pWSPK_GPD1 plasmid and the fragments GPD1_1 and GPD1_2 were electrotransformed into the SA104 strain, and the positive transformant was obtained by SD-URA medium screening, which was named SA105 strain (genotype: SA104, ⁇ GPD1).
  • the promoter and terminator used are respectively the promoter (SEQ ID NO: 19) of CY902's own 3-phosphate glyceraldehyde dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase 3) gene TDH3 and the terminator of the galactose transporter gene GAL2 (SEQ ID NO: 20).
  • the specific construction method is as follows:
  • primers 1_UP_MCH4_F and 1_UP_MCH4_R1 (see Table 7) to amplify MCH4 gene upstream homology arm fragment 1 (fragment 20); use primers 2_P TDH3 _F and 2_P TDH3 _R (see Table 7) to amplify CY902 itself TDH3 gene promoter sequence (fragment 21); with the plasmid containing the SpMAE1 synthetic sequence as a template, use primers 3_SpMAE1_F and 3_SpMAE1_R (see Table 7) to amplify the SpMAE1 coding sequence (fragment 22) of Schizosaccharomyces pombe; with CY902 genomic DNA As a template, use primers 4_T GAL2 _F and 4_T GAL2 _R (see Table 7) to amplify the CY902 self-GAL2 gene terminator sequence (fragment 23); use primers
  • primers 1_UP_MCH4_F and 1_UP_MCH4_R2 see Table 7 to amplify the MCH4 gene upstream homology arm fragment 2 (fragment 25); use primers 5_DW_MCH4_F2 and 5_DW_MCH4_R (see Table 7) to amplify the MCH4 gene downstream homology arm Fragment 2 (fragment 26).
  • pWSPK-Cas9 plasmid as a template, use the primers sgRNA-1F and MCH4_sgRNA-1R (see Table 7) to amplify the 5' end sequence of the sgRNA (abbreviated as MCH4_sgRNA_1), and the 3' end of the fragment contains Kudri Azweibich A unique 20nt protospacer sequence in the yeast PkMCH4 gene.
  • the pWSPK_backbone, sgRNA_2 and MCH4_sgRNA_1 were ligated with a seamless cloning kit to finally obtain a positive clone plasmid, which was named pWSPK_MCH4.
  • the pWSPK_MCH4 plasmid and fragments 25 and 26 were electrotransformed into the SA101-2 strain, and a positive transformant was obtained after screening, named SA106-1 (genotype: SA101-2, ⁇ PkMCH4); the pWSPK_MCH4 plasmid and fragment 20 -24 was electrotransformed into the SA101-2 strain, and a positive transformant was obtained after screening, named SA106-2 (genotype: SA101-2, PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).
  • the PYC (AoPYC) gene (Uniprot database accession number: Q2UGL1) (SEQ ID NO: 6) derived from Aspergillus oryzae was respectively overexpressed at the JEN2-1 site, and the PYC1 (PkPYC1) gene of CY902 itself was used as a control (SEQ ID NO : 7), the promoter and terminator used are respectively TDH3 promoter (SEQ ID NO: 19) and GAL2 terminator (SEQ ID NO: 20).
  • the AoPYC gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and optimized according to the codon preference of CY902 (SEQ ID NO: 6). The specific construction method is as follows:
  • primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R1 were used to amplify JEN2-1 gene upstream homology arm fragment 1 (fragment 27); use primers 3_PkPYC1_F and 3_PkPYC1_R (see Table 8) to amplify CY902's own PkPYC1 coding sequence (fragment 28-1); using the plasmid containing the AoPYC synthetic sequence as a template, use primers 3_AoPYC_F and 3_AoPYC_R (see Table 8) to amplify the AoPYC coding sequence (fragment 28-2) derived from Aspergillus oryzae; using CY902 Using genomic DNA as a template, primers 5_DW_JEN2-1_F1 and 5_DW_JEN2-1_R (see Table 8) were used to amplify JEN2-1 gene downstream homology arm
  • primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R2 see Table 8 to amplify JEN2-1 gene upstream homology arm fragment 2 (fragment 30); use primers 5_DW_JEN2-1_F2 and 5_DW_JEN2-1_R (see Table 8) Amplify JEN2-1 gene downstream homology arm fragment 2 (fragment 31).
  • pWSPK-Cas9 plasmid Using the pWSPK-Cas9 plasmid as a template, use primers sgRNA-1F and JEN2-1_sgRNA-1R (see Table 8) to amplify the 5' end sequence of the sgRNA (abbreviated as JEN2-1_sgRNA_1), and the 3' end of the fragment contains JEN2-1 Gene-specific 20nt protospacer sequence.
  • the pWSPK_backbone, sgRNA_2 and JEN2-1_sgRNA_1 in Example 1 were ligated with a seamless cloning kit to finally obtain a positive clone plasmid, which was named pWSPK_JEN2-1.
  • the pWSPK_JEN2-1 plasmid and fragments 30 and 31 were electrotransformed into the SA101-2 strain, and a positive transformant was obtained after screening, named SA107-1 (genotype: SA101-2, ⁇ PkJEN2-1); the pWSPK_JEN2-1 plasmid and fragments 27, 21, 28, 23, and 29 were electrotransformed into the SA101-2 strain, and the positive transformants were screened and named as SA107-2 (genotype: SA101-2, PkJEN2-1::P PkTDH3 -ORF PkPYC1 -T PkGAL2 ); SA107-3 (genotype: SA101-2, PkJEN2-1::P PkTDH3 -ORF AoPYC -T PkGAL2 ).
  • the fumarase gene PkFUM1 (SEQ ID NO: 8) of CY902 truncated the 5' mitochondrial localization peptide was overexpressed at the promoter site of the PDC1 gene of the SA101-2 strain.
  • the promoter and terminator used were TDH3 promoter (SEQ ID NO: 19) and GAL2 terminator (SEQ ID NO: 20), respectively.
  • the specific construction method is as follows:
  • pWSPK-Cas9 plasmid Using the pWSPK-Cas9 plasmid as a template, use primers sgRNA-1F and P PDC1_sgRNA -1R (see Table 9) to amplify the 5' end sequence of the sgRNA (abbreviated as P PDC1_sgRNA_1 ), and the 3' end of the fragment contains the P PDC1 gene Promoter-specific 20nt protospacer.
  • the pWSPK_backbone, sgRNA_2 and P PDC1 _sgRNA_1 were ligated with a seamless cloning kit to finally obtain a positive clone plasmid, which was named pWSPK_P PDC1 .
  • the pWSPK_P PDC1 plasmid and fragments 32, 21, 33, 23 and 34 were transferred into the SA101-2 strain by the yeast electroporation method, and the positive transformant was obtained by screening, which was named SA108 strain (genotype: SA101-2, P PkPDC1 :: P PkTDH3 -ORF PkFUM1 -T PkGAL2 ).
  • the 5'-orotidine phosphate decarboxylase gene PkURA3 (SEQ ID NO: 117) was complemented at the dicarboxylic acid transporter PkJEN2-2 gene site of the SA101 series strains.
  • the promoter and terminator used were the promoter (SEQ ID NO: 118) and terminator (SEQ ID NO: 119) of PkURA3 itself.
  • the specific construction method is as follows:
  • primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R1 see Table 10 to amplify CY902's own PkJEN2-2 gene upstream homology arm fragment 1 (fragment 35); use primers 2_PkURA3_F and 2_PkURA3_R (see Table 10) URA3 promoter, coding frame and terminator sequence (fragment 36) were amplified; primers 3_DW_JEN2-2_F1 and 3_DW_JEN2-2_R (see Table 10) were used to amplify CY902's own PkJEN2-2 gene downstream homology arm fragment 1 (fragment 37).
  • CY902 genomic DNA as a template, use primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R2 (see Table 10) to amplify CY902's own PkJEN2-2 gene upstream homology arm fragment 2 (fragment 38); use primers 3_DW_JEN2-2_F2 and 3_DW_JEN2-2_R( See Table 10) Amplify CY902's own PkJEN2-2 gene downstream homology arm fragment 2 (fragment 39).
  • pWSPK-Cas9 plasmid Using the pWSPK-Cas9 plasmid as a template, use primers sgRNA-1F and JEN2-2_sgRNA-1R (see Table 10) to amplify the 5' end sequence of the sgRNA (abbreviated as JEN2-2_sgRNA_1), and the 3' end of the fragment contains PkJEN2-2 Gene promoter-specific 20nt protospacer sequence.
  • the pWSPK_backbone, sgRNA_2 and JEN2-2_sgRNA_1 were ligated with a seamless cloning kit, and finally a positive clone plasmid was obtained, which was named pWSPK_JEN2-2.
  • the pWSPK_JEN2-2 plasmid and fragments 38 and 39 were transformed into SA101-1, SA101-2, and SA101-3 strains by yeast electroporation, and positive transformants were obtained by screening, which were named SA109-1 strains (genotype: SA101- 1, ⁇ PkJEN2-2), SA109-2 strain (genotype: SA101-2, ⁇ PkJEN2-2) and SA109-3 strain (genotype: SA101-3, ⁇ PkJEN2-2).
  • the pWSPK_JEN2-2 plasmid and fragments 35-37 were transformed into SA101-1, SA101-2, and SA101-3 strains by yeast electroporation method, and positive transformants were screened, which were named SA109-4 strains (genotype: SA101- 1, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ), SA109-5 strain (genotype: SA101-2, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ) and SA109-6 strain (gene Type: SA101-3, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).
  • PkURA3 was complemented at the SA102-SA108 strain PkJEN2-2 gene locus to obtain the SA110-1 strain (genotype: SA102-1, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ); SA110- 2 strains (genotype: SA102-2, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ); SA110-3 strain (genotype: SA102-3, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ); SA111-1 strain (genotype: SA103-1, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ); SA111-2 strain (genotype: SA103-2, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 );
  • the SA101-SA116 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w/v glucose, 30 g/L CaCO 3 ), fermented in shake flasks at 30°C and 250 rpm for 24 hours, and the production of succinic acid was measured by HPLC.
  • yeast inorganic salt medium 5% w/v glucose, 30 g/L CaCO 3
  • the SA101-SA116 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w/v glucose, 0 g/L CaCO 3 ), and fermented in shake flasks at 30°C and 250 rpm for 24 hours, and the production of succinic acid was measured by HPLC.
  • yeast inorganic salt medium 5% w/v glucose, 0 g/L CaCO 3
  • Embodiment 12 Optimizing succinic acid producing bacteria
  • the SpMAE1 gene was overexpressed in SA118 to obtain SA119-1 strain (genotype: SA118, ⁇ PkMCH4); SA119-2 strain (genotype: SA118, PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).
  • the fumarase gene PkFUM1 that truncated the 5' end mitochondrial localization peptide was overexpressed in SA120-3 to obtain the SA121 strain (genotype: SA120-3, P PkPDC1 ::P PkTDH3 -ORF PkFUM1 -T PkGAL2 ).
  • the 5′-orotidine phosphate decarboxylase gene PkURA3 was complemented in the SA121 strain to obtain the SA122 strain (genotype: SA121, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).
  • the TbFRD gene was overexpressed in SA102-3 to obtain the SA123 strain (genotype: SA102-3, ADH1::ORF TbFRD ).
  • the SpMAE1 gene was overexpressed in SA125 to obtain SA126-1 strain (genotype: SA125, ⁇ PkMCH4); SA126-2 strain (genotype: SA125, PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).
  • the fumarase gene PkFUM1 that truncated the 5' end mitochondrial localization peptide was overexpressed in SA127-3 to obtain the SA128 strain (genotype: SA127-3, P PkPDC1 ::P PkTDH3 -ORF PkFUM1 -T PkGAL2 ).
  • the 5′-orotidine phosphate decarboxylase gene PkURA3 was complemented in the SA128 strain to obtain the SA129 strain (genotype: SA128, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).
  • the SA122 and SA129 strains were respectively scaled up and fermented in 5L tanks (yeast inorganic salt medium, 12% w/v glucose), and without adding any neutralizing agent, the succinic acid production reached 96.08g/L respectively in 36h and 46.02g/L, the conversion rates were 0.89g/g and 0.57g/g, respectively.
  • CICC32244 purchased from China Industrial Microorganism Culture Collection Management Center
  • orotidine 5'-phosphate decarboxylase Optidine 5'-phosphate decarboxylase
  • the pWSPK_2365 plasmid and fragments 6 and 7 were transferred into the CICC32244 ⁇ URA3 strain by yeast electroporation, and a positive transformant was obtained after screening, which was named SA130-1 strain (genotype: CICC32244 ⁇ URA3, ⁇ Pk2365); the pWSPK_2365 plasmid and fragments 1-5
  • SA130-1 strain wild-type
  • CICC32244 ⁇ URA3, ⁇ Pk2365 the pWSPK_2365 plasmid and fragments 1-5
  • the yeast was transformed into CICC32244 ⁇ URA3 strain by electroporation, and a positive transformant was obtained after screening, which was named SA130-2 strain (genotype: CICC32244 ⁇ URA3, Pk2365::P PkFBA1 -ORF SbMDH -TPkINO1 ).
  • PkURA3 was complemented at the PkJEN2-2 gene locus of the SA130 series strain to obtain the SA131-1 strain (genotype: SA130-1, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ); SA131-2 Strain (genotype: SA130-2, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).
  • the SA130 and SA131 series strains were inoculated into 30 mL of yeast mineral salt medium (5% w/v glucose, 30 g/L CaCO 3 ), and fermented in shake flasks at 30°C and 250 rpm for 24 hours, and the production of succinic acid was measured by HPLC.
  • yeast mineral salt medium 5% w/v glucose, 30 g/L CaCO 3
  • strain SA130-1 SA130-2 SA131-1 SA131-2 Succinic acid g/L 0.37 0.52 0.39 0.56
  • the SA130 and SA131 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w/v glucose, 0 g/L CaCO 3 ), and fermented in shake flasks at 30°C and 250 rpm for 24 hours, and the production of succinic acid was measured by HPLC.
  • yeast inorganic salt medium 5% w/v glucose, 0 g/L CaCO 3
  • strain SA130-1 SA130-2 SA131-1 SA131-2 Succinic acid g/L 0.37 0.53 0.40 0.55
  • Example 17 Overexpression of the SbMDH gene in Saccharomyces cerevisiae BY4742 strain
  • Sorghum-derived malate dehydrogenase SbMDH (SEQ ID NO: 1) was overexpressed at the site of Saccharomyces cerevisiae 5-phosphate orotidine decarboxylase gene ScURA3 (SEQ ID NO: 129), and the strain overexpressing the ScURA3 gene was used as a control.
  • the overexpressed SbMDH gene sequence is the same as above, optimized according to the codon preference of CY902, and the promoter and terminator used are respectively the promoter of BY4742's own 3-phosphate glyceraldehyde dehydrogenase gene ScTDH3 (SEQ ID NO: 130) and the terminator of the galactose permease gene ScGAL2 (SEQ ID NO: 131).
  • the promoter and terminator used for the overexpression of the ScURA3 gene are the promoter (SEQ ID NO: 132) of BY4742's own orotidine 5-phosphate decarboxylase gene ScURA3 and the terminator (SEQ ID NO: 133) of the cell wall glycoprotein gene ScSED1, respectively. ).
  • the specific construction method is as follows:
  • the fragments 40, 46, and 47 were mixed together and transferred into the BY4742 strain (purchased from ThermoFisher Scientific) according to the yeast electroporation method, and a positive transformant was obtained after screening, which was named SA132-1 strain (genotype: BY4742, ScURA3::P ScURA3 -ORF ScURA3 -T ScSED1t ); the fragments 40-45 were mixed together and transferred into the BY4742 strain according to the yeast electroporation method, and a positive transformant was obtained after screening, which was named SA132-2 strain (genotype: BY4742, ScURA3::P ScURA3 -ORF ScURA3 -T ScSED1t -P ScTDH3 -ORF SbMDH -T ScGAL2 ).
  • SA132 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w/v glucose, 0 g/L CaCO 3 ), and fermented in shake flasks at 30° C. and 250 rpm for 12 hours, and the production of succinic acid was measured by HPLC.
  • yeast inorganic salt medium 5% w/v glucose, 0 g/L CaCO 3
  • strain SA132-1 SA132-2 Succinic acid g/L 0.12 0.22
  • Example 19 Overexpression of the SbMDH gene in Yarrowia lipolytica Po1g strain
  • Yarrowia lipolytica has a strong ability of non-homologous end recombination
  • sorghum-derived malate dehydrogenase gene SbMDH SEQ ID NO: 1
  • selection markers were overexpressed by random insertion into the Yarrowia lipolytica genome ⁇ -isopropylmalate dehydrogenase (EC 1.1.1.85) gene YlLEU2 (SEQ ID NO: 148), and a strain overexpressing the YlLEU2 gene by random insertion was used as a control.
  • the overexpressed SbMDH gene sequence is the same as above, optimized according to the CY902 codon preference, and the promoter and terminator used are respectively the promoter of Po1g's own 3-phosphate glyceraldehyde dehydrogenase gene YlGAPDH (SEQ ID NO: 149) and the terminator of the 3-hydroxy-3-methylglutaryl-CoA reductase gene Y1HMG1 (SEQ ID NO: 150).
  • the promoter and terminator used for overexpressing the YlLEU2 gene are the promoter of Po1g's own ⁇ -isopropylmalate dehydrogenase gene YlLEU2 (SEQ ID NO: 151) and the termination of the Saccharomyces cerevisiae cytochrome c subtype 1 gene ScCYC1, respectively. Son (SEQ ID NO: 152).
  • the specific construction method is as follows:
  • Yarrowia lipolytica W29 genomic DNA (GenBank: GCA_001761485.1) as a template
  • primers 1_P YlGAPDH _F and 1_P YlGAPDH _R (see Table 17) to amplify the YlGAPDH gene promoter sequence (fragment 48); to contain the SbMDH synthetic sequence
  • the plasmid of Yarrowia lipolytica W29 was used as a template, and the SbMDH coding sequence (fragment 49) derived from sorghum was amplified with primers 2_SbMDH_F and 2_SbMDH_R (see Table 17); See Table 17) amplify the YlHMG1 gene terminator sequence (fragment 50); use primers 4_P YlLEU2_F and 4_YlLEU2_R (see Table 17) to amplify the YlLEU2 promoter and ORF sequence (fragment 51); use S288c genomic DNA as
  • fragment 48-52 uses primer 1_P YlGAPDH _F and 5_T ScCYC1 _R to amplify the expression cassette (fragment 53) overexpressing SbMDH and YlLEU2 ; Expression cassette for overexpressing Y1LEU2 (fragment 54)
  • Fragment 54 was transferred into the Po1g strain (purchased from Yisheng Biotechnology Development Co., Ltd.) according to the yeast electroporation method, and a positive transformant was obtained after screening, which was named SA133-1 strain (genotype: Polg, genome::P YlLEU2 -ORF YlLEU2 -T ScCYC1t ); fragment 53 was transferred into the Po1g strain by yeast electroporation method, and the positive transformant obtained by screening was named SA133-2 strain (genotype: Polg, genome::P YlGAPDH- SbMDH-T YlHMG1t -P YlLEU2 -ORF YlLEU2 -T ScCYC1t ).
  • SA133 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w/v glucose, 0 g/L CaCO 3 ), fermented in shake flasks at 30°C and 250 rpm for 24 hours, and the production of succinic acid was measured by HPLC.
  • yeast inorganic salt medium 5% w/v glucose, 0 g/L CaCO 3
  • strain SA133-1 SA133-2 Succinic acid g/L 1.48 2.41

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Abstract

提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶(EC 1.1.1.82)活性，任选还具有或具有增强的如下至少一种的活性：（i）可溶的富马酸还原酶（EC 4.2.1.2），（ii）丙酮酸羧化酶（EC 6.4.1.1），(iii) 富马酸酶（EC 4.2.1.2）和（iv）丁二酸转运蛋白，及其制备方法，使用其生产丁二酸的方法和应用。

Description

一种高产丁二酸的耐酸酵母菌株及其构建方法和应用 技术领域
本发明涉及生物技术领域，特别涉及一种新型的用于丁二酸高效生产的耐酸酵母菌株及其构建方法和应用。
背景技术
丁二酸又名琥珀酸，是生物体三羧酸循环的中间产物亦是一种重要的有机化工原料(Chae T U,Ahn J H,Ko Y S,et al.Metabolic engineering for the production of dicarboxylic acids and diamines[J].Metabolic Engineering,2019)。丁二酸广泛应用于食品，化学，农业，医药等行业。
丁二酸的传统生产方法为化学法，主要以不可再生的石油资源为原料进行合成，如：石蜡氧化法、顺丁烯二酸酐加氢法、丙烯酸羰基合成法等。化学合成法存在高耗能、高污染等缺点，难以达到绿色制造的要求。相比而言，生物发酵法生产丁二酸污染小、能耗低、固定二氧化碳，更加符合国家可持续发展战略。预测2020年生物法生产丁二酸的全球产量能够达到60万吨，市场价值达到5.39亿美元(Hyohak Song,Sang Yup Lee.Production of Succinic Acid by Bacterial Fermentation[J].Enzyme and Microbial Technology,2006,39(3):352-361.)。
目前，用于合成丁二酸的微生物菌株主要分为两类：第一类是真菌酵母类，主要包括：黑曲霉(Aspergillus niger)，烟曲霉(Aspergillus fumigatus)，雪白丝衣霉(Byssochlamys nivea)，宛氏拟青霉(Paecilomyces varioti)，酿酒酵母(Saccharomyces cerevisiae)等；第二类为细菌类：包括产琥珀酸放线杆菌(Actinobacillus succinogenes)、产琥珀酸厌氧螺菌(Anaerobiospirillum succiniciproducens)、产琥珀酸曼氏杆菌(Mannheimia succiniciproducens)、脆弱拟杆菌(Bacteroides fragilis)等天然生产菌株以及谷氨酸棒状杆菌(Corynebacterium glutamicum)和大肠杆菌(Escherichia coli)等非天然生产菌株(Ahn J H,Jang Y S,Lee S Y.Production of succinic acid by metabolically engineered microorganisms[J].Current Opinion in Biotechnology,2016,42:54-66)。丝状真菌存在生长慢、遗传改造困难、发酵均质性差菌丝易结团等问题；细菌发酵生产丁二酸需要添加大量的中和剂用以维持菌体生长，增加了分离纯化成本，并存在潜在的污染问题。酵母具有良好的耐酸能力，能在低于pH3.0的条件下正常生长，而此时丁二酸大多以游离的分子形式存在，可以显著降低后续分离纯化的成本。
发明内容
本发明以一株从云南野生水果表皮分离得到的耐酸酵母库德里阿兹威毕赤酵母(Pichia kudriavzevii)CY902菌株(保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号为CGMCC No.20885)出发，经过代谢工程改造使其实现丁二酸的高效生产。特别地，所述改造的菌株可以实现在低pH条件下以不添加或较少添加中和剂的发酵方式实现丁二酸的高效生产。
在一个方面，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性，任选地还具有或具有增强的至少一种如下活性：(i)可溶的富马酸还原酶活性，(ii)丙酮酸羧化酶活性，(iii)富马酸酶活性，和(iv)丁二酸转运蛋白活性。
优选地，所述NDAPH依赖型苹果酸脱氢酶来自植物，优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科和苋科植物，或来自眼虫属或热杆菌属，更优 选来自高粱(Sorghum bicolor)、玉米(Zea mays)、甘蔗(Saccharum officinarum)、豌豆(Pisum sativum)、鹰嘴豆(Cicer arietinum)、菠菜(Spinacia oleracea)、小眼虫(Euglena gracilis)或热自养甲烷热杆菌(Methanothermobacter thermautotrophicus)。
在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的丙酮酸脱羧酶和/或NAD依赖性3-磷酸甘油脱氢酶。
在一个方面，本发明提供了一种产生经遗传改造的丁二酸生产酵母菌株的方法，包括赋予所述菌株或增强所述菌株的NADPH依赖型苹果酸脱氢酶活性，任选地还包括赋予或增强至少一种如下活性：(i)可溶的富马酸还原酶活性，(ii)丙酮酸羧化酶(EC 6.4.1.1)活性，(iii)富马酸酶(EC 4.2.1.2)活性，和(iv)丁二酸转运蛋白活性。优选所述NDAPH依赖型苹果酸脱氢酶来自植物，优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科和苋科植物或来自眼虫属和热杆菌属，更优选高粱、玉米、甘蔗、豌豆、鹰嘴豆、菠菜、小眼虫或热自养甲烷热杆菌。
在一个实施方案中，所述方法还包括弱化或失活所述菌株中的丙酮酸脱羧酶和/或NAD依赖性3-磷酸甘油脱氢酶。
在一个方面，本发明提供了一种生产丁二酸的方法，包括(优选在pH<3.5例如1.5-3.5的范围内和/或不加或较少添加中和剂的条件下)培养本发明所述的经遗传改造的丁二酸生产酵母菌株和/通过本发明所述的产生经遗传改造的丁二酸生产酵母菌株的方法获得的经遗传改造的丁二酸生产酵母菌株。
在一个方面，本发明提供了本发明所述的经遗传改造的丁二酸生产酵母菌株和/通过本发明所述的产生经遗传改造的丁二酸生产酵母菌株的方法获得的经遗传改造的丁二酸生产酵母菌株在(优选在pH<3.5例如1.5-3.5的范围内和/或不加或较少添加中和剂的条件下)生产丁二酸中的应用。
具体实施方式
除非另有定义，本文所用的技术和科学术语具有本领域技术人员通常理解的含义。参见例如，Singleton et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed.,J.Wiley & Sons(New York,NY 1994)；Sambrook et al.,MOLECULAR CLONING,A LABORATORY MANUAL,Cold Springs Harbor Press(Cold Springs Harbor,NY 1989)。
如本文所用，“经遗传改造的”是指通过生物学手段人工改变的菌株，其与改造前的初始菌株相比具有一或多个改变，例如基因缺失、扩增或突变，从而具有改变的生物学性质例如改良的生产性能。如本文所用，初始菌株可以是对其要进行所述遗传改造的天然菌株或具有其它遗传改造的菌株。
如本文所用，丁二酸生产酵母菌株是指在适当条件下可以(例如经发酵)产生丁二酸以及将丁二酸分泌至胞外介质中的酵母。丁二酸生产酵母菌株具有可以将丁二酸转运至胞外的蛋白质，因此丁二酸在产生后可以分泌至细胞外。本领域已知用于给定酵母菌株的合适的丁二酸转运蛋白，例如包括但不限于粟酒裂殖酵母(Schizosaccharomyces pombe)的二羧酸转运蛋白SpMAE1和黑曲霉(Aspergillus niger)的二羧酸转运蛋白AnDCT-02。
本领域已知用于生产丁二酸的酵母，包括例如不限于鲁氏接合酵母属(Zygosaccharomyces)、球拟酵母属(Torulopsis)、假丝酵母属(Candida)、毕赤酵母属(Pichia)、红酵母属(Rhodotroula)、酵母属(Saccharomyces)、耶氏酵母属(Yarrowia)等。在一个实施方案中，所述丁二酸生产酵母菌株是毕赤酵母属、酵母属或耶氏酵母属菌株。在一个优选实施方案中，所述丁二酸生产酵母菌株是库德里阿兹威毕赤酵母(例如菌株CICC32244)、酿酒酵母(例如菌株BY4742)或解脂耶氏酵母(Yarrowia lipolytica)(例如菌株Po1g)，例如保藏在中国北京的中国微生物菌种保藏管理委员会普通微生物 中心(CGMCC)、保藏号为CGMCC No.20885的库德里阿兹威毕赤酵母。
如本文所用，“具有……活性”是指与不具有该活性的参照(例如初始菌株或野生型菌株)相比，具有可检测到的活性。
如本文所用，“具有增强的……活性”是指与具有该活性的参照(例如初始菌株或野生型菌株)相比，活性增加至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。
可以通过本领域已知的任何适当方式产生或增强蛋白(例如酶)的活性，例如包括但不限于在菌株中表达或过表达(例如通过载体如质粒)编码所述蛋白的相应基因、引入导致所述蛋白的活性增加的突变等。
在一些实施方案中，在本发明所述的经遗传改造的丁二酸生产酵母菌株中，一或多个拷贝的目的基因或其同源基因可以整合进基因组(例如通过同源重组)，任选在基因组任意位点，(只要这种整合不显著负面影响菌株的生长和生产)，例如基因组内一个拷贝的任意基因被一或多个拷贝的目的基因或其同源基因替换。本领域技术人员知道如何整合转基因以及选择整合了转基因的菌株。
如本文所用，“降低活性的或失活的”是指与参照活性(例如初始菌株或野生型菌株中的相应活性)相比，活性降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至100％。
可以通过本领域已知的任何适当方式降低或失活蛋白(例如酶)的活性，例如包括但不限于使用弱化的或失活的编码所述蛋白的相应基因、引入导致所述蛋白的活性降低或失活的突变、使用所述蛋白的拮抗剂或抑制剂(例如抗体、配体等)。
如本文所用，“弱化或失活的基因”是指与参照(例如初始菌株或野生型菌株中的相应基因)相比，基因的活性例如表达水平(作为蛋白编码基因时)或调控性能(作为调节元件时)降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是不可检测到的。在基因编码蛋白例如酶的情况下，“弱化或失活的基因”也涵盖，由该基因表达的蛋白的活性水平与初始菌株或野生型菌株中的相应蛋白的活性水平相比，是降低的，例如降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是100％。
在本文中，所述参照可以是野生型微生物或者是进行所需遗传操作前的微生物(例如用于进行遗传操作以增加基因活性的初始微生物)。在本文中，亲代微生物和初始微生物可互换使用，指对其进行所需遗传操作(例如增强或弱化基因或蛋白活性)的微生物。
如本文所用，苹果酸脱氢酶(EC 1.1.1.82(NADPH依赖型))由MDH基因编码，苹果酸脱氢酶参与草酰乙酸和苹果酸之间的转化。NADPH依赖型苹果酸脱氢酶负责将草酰乙酸转化为苹果酸，该反应消耗一分子NADPH。一般使用的NADPH依赖型苹果酸脱氢酶来源包括C4植物(例如禾本科、莎草科、菊科、大戟科、藜科、马齿苋科和苋科植物)、眼虫属和热杆菌属等，例如：高粱、玉米、甘蔗、豌豆、鹰嘴豆、菠菜、小眼虫或热自养甲烷热杆菌等。如本文所用，具有或具有增强的NDAPH依赖型苹果酸脱氢酶活性是指菌株具有或具有增加的催化草酰乙酸转化为苹果酸的NDAPH依赖型苹果酸脱氢酶活性。
如本文所用，可溶的富马酸还原酶(EC 1.3.1.6)由FRD基因编码，该酶参与富马酸与丁二酸之间的相互转化，负责将富马酸转化为丁二酸。一般使用的可溶的富马酸还原酶来源有酵母和动质体目，特别是酿酒酵母、布氏锥虫(Trypanosoma brucei)、墨西哥利什曼原虫(Leishmania mexicana)、克氏锥虫(Trypanosoma cruzi)等。如本文所用， 具有或具有增强的富马酸还原酶活性是指菌株具有或具有增加的将富马酸转化为丁二酸的可溶的富马酸还原酶活性。
如本文所用，富马酸酶(EC 4.2.1.2)由FUM基因编码，所述酶参与细胞质和线粒体中富马酸和苹果酸之间的相互转化，负责将苹果酸转化为富马酸。一般使用的来源有产琥珀酸放线杆菌、产琥珀酸曼氏杆菌、大肠杆菌、库德里阿兹威毕赤酵母、米根霉(Rhizopus oryzae)等。已知库德里阿兹威毕赤酵母中存在一个FUM基因FUM1。如本文所用，具有或具有增强的富马酸酶活性是指菌株具有或具有增加的将苹果酸转化为富马酸的富马酸酶活性。
如本文所用，丁二酸转运蛋白是指能够将细胞内的丁二酸转运至细胞外的蛋白，包括例如但不限于粟酒裂殖酵母的SpMAE1蛋白(Uniprot数据库检索号：P50537)、黑曲霉(Aspergillus niger)的二羧酸转运蛋白AnDCT-02(NCBI Reference Sequence:XP_001398131.1)和大肠杆菌的二羧酸转运蛋白EcDcuB(Gene ID:948641)和EcDcuC(Gene ID:945000)等。本文中，具有或具有增强的丁二酸转运蛋白活性是指菌株具有或具有增加的将丁二酸转运到胞外的活性。
如本文所用，粟酒裂殖酵母二羧酸转运蛋白SpMAE1蛋白由SpMAE1基因编码，SpMAE1蛋白负责将胞内的二羧酸转运到胞外。本文中，具有或具有增强的SpMAE1活性是指菌株具有或具有增加的将二羧酸转运到胞外的活性。
如本文所用，丙酮酸羧化酶(EC 6.4.1.1)由PYC基因编码，丙酮酸羧化酶参与糖异生过程中草酰乙酸和丙酮酸之间的相互转化，负责将丙酮酸和二氧化碳转化为草酰乙酸。一般使用的丙酮酸羧化酶来源包括真菌，特别是酵母和丝状真菌，优选酿酒酵母、库德里阿兹威毕赤酵母、米曲霉、马克斯克鲁维酵母(Kluyveromyces marxianus)等。已知库德里阿兹威毕赤酵母存在1个PYC基因，即PYC1基因。本文中，具有或具有增强的丙酮酸羧化酶活性是指菌株具有或具有增加的将丙酮酸转化为草酰乙酸的活性。
如本文所用，丙酮酸脱羧酶(EC 4.1.1.43)由PDC基因编码，丙酮酸脱羧酶参与合成乙醇途径中丙酮酸的脱羧过程。已知库德里阿兹威毕赤酵母存在一个PDC基因为PDC1基因。本文中，降低活性的或失活的丙酮酸脱羧酶是指该酶的丙酮酸脱羧活性降低或丧失。
如本文所用，NAD依赖性3-磷酸甘油脱氢酶(EC 1.1.1.8)由GPD基因编码，该酶参与合成甘油途径中磷酸甘油酮与3-磷酸甘油之间的相互转化。已知库德里阿兹威毕赤酵母存在一个GPD基因，即GPD1基因。本文中，降低活性的或失活的NAD依赖性3-磷酸甘油脱氢酶是指该酶的将磷酸甘油酮与3-磷酸甘油相互转化的活性降低或丧失。
如本文所用，5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)由URA3基因编码，所述酶参与嘧啶合成过程中5-磷酸乳清苷脱羧反应。已知库德里阿兹威毕赤酵母存在1个URA3基因。本文中，降低活性的或失活的5’-磷酸乳清苷脱羧酶是指该酶的催化5’-磷酸乳清苷脱羧反应的活性降低或丧失。
如本文所用，乙醇脱氢酶1(EC 1.1.1.1)由ADH1基因编码，所述酶参与合成乙醇途径中的乙醛与乙醇之间的相互转化。本文中，降低活性的或失活的乙醇脱氢酶1是指该酶的催化乙醛与乙醇之间相互转化的活性降低或丧失。在一个实施方案中，所述菌株中的ADH1基因被敲除，例如通过同源重组方式敲除。
如本文所用，单羧酸透性酶(NCBI Reference Sequence XP_029320775.1)由MCH4基因编码。本文中，降低活性的或失活的单羧酸透性酶是指该酶的催化活性降低或丧失。在一个实施方案中，所述菌株中的MCH4基因被敲除，例如通过同源重组方式敲除。
如本文所用，JEN2基因编码二羧酸转运蛋白，该转运蛋白参与将培养基中二羧酸转运到胞内的过程。已知库德里阿兹威毕赤酵母存在两个JEN2基因(JEN2-1(编码SEQ ID NO：14所示多肽)和JEN2-2(编码SEQ ID NO：15所示多肽))。本文中，降低活性的或失活的二羧酸转运蛋白是细胞将培养基中二羧酸转运到胞内的活性降低或丧失。
如本文所用，中和剂是指将丁二酸以丁二酸钙的形式从发酵体系中沉淀出来的试剂。本领域已知可以用作中和剂的物质，包括例如但不限于碳酸钙。
如本文所用，不加或较少添加中和剂是指与本领域已知的发酵生产丁二酸时添加的中和剂的量低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是100％。例如，在本发明的方法中，加入的中和剂的量可以为0-30g/L。
如本文所用，术语“多肽”、“氨基酸序列”、“肽”及“蛋白质”在本文可互换使用,指称任何长度的氨基酸链，其可能包含经修饰的氨基酸和/或可能被非氨基酸中断。该术语还涵盖经天然或人为干预修饰的氨基酸链；例如二硫键形成、糖基化、脂化、乙酰化、磷酸化或任何其他操纵或修饰，如与标记成份缀合。
如本文所用，表述“基因”、“核酸序列”、“多核苷酸”和“核苷酸序列”可互换使用，是指核苷酸链，包含DNA和RNA。“基因的表达”是指将与适当调节区特别是启动子可操作地连接的DNA区域转录成具有生物学活性的RNA以及RNA能够被翻译成生物学活性蛋白或肽。
如本文所用，简并序列是指由于遗传密码子的简并性，与指定序列编码相同氨基酸序列但是核苷酸序列不同的核苷酸序列。
如本文所用，术语“同源性”、“序列相同性”等在本文可互换使用。序列相同性可通过比对多核苷酸与参考多核苷酸之间的相同核苷酸碱基的数目而检测，例如可以通过标准排列对比算法程序使用由每个供应商制定的默认缺口罚分确定。两个核酸分子是否具有至少80％、85％、90％、95％、96％、97％、98％或99％“相同的”核苷酸序列可以使用已知的计算机算法确定，如BLASTN、FASTA、DNAStar及Gap(University of Wisconsin Genetics Computer Group(UWG),Madison WI,USA)。例如，核酸分子的相同性百分比可以例如通过使用GAP计算机程序对比序列信息而确定(例如Needleman et al.J.Mol.Biol.48:443(1970)，由Smith and Waterman(Adv.Appl.Math.2:482(1981)修订)。简而言之，GAP程序根据相似的排列对比的符号(即核苷酸)的数目除以两个序列中较短序列的符号总数而定义相似性。
如本文所用，Pk2365基因编码具有草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶(EC 4.1.3.17或4.1.1.112)。本文中，降低活性的或失活的草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶是指该酶催化草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩的活性降低或丧失。
在一个方面，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性，任选地还具有或具有增强的至少一种如下活性：(i)可溶的富马酸还原酶活性，(ii)丙酮酸羧化酶活性，(iii)富马酸酶活性，和(iv)丁二酸转运蛋白活性。所述“至少一种”包括选自其中的任意1、2、3这或全部4种的活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性以及可溶的富马酸还原酶活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性以及丙酮酸羧化酶活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性以及富马酸酶活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性以及丁二酸转运蛋白活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、可溶的富马酸还原酶活性和丙酮酸羧化酶活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、可溶的富马酸还原酶活性和富马酸酶活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、可溶的富马酸还原酶活性和丁二酸转运蛋白活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、丙酮酸羧化酶活性和富马酸酶活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、丙酮酸羧化酶活性和丁二酸转运蛋白活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、富马酸酶活性和丁二酸转运蛋白活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、可溶的富马酸还原酶活性、丙酮酸羧化酶活性和富马酸酶活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、可溶的富马酸还原酶活性、丙酮酸羧化酶活性和丁二酸转运蛋白活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、可溶的富马酸还原酶活性、富马酸酶活性和丁二酸转运蛋白活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、丙酮酸羧化酶活性、富马酸酶活性和丁二酸转运蛋白活性。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶活性、可溶的富马酸还原酶活性、丙酮酸羧化酶活性、富马酸酶活性和丁二酸转运蛋白活性。
在一个实施方案中，具有或具有增强的活性通过在所述菌株中表达或过表达相应编码基因来实现。因此，在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株表达或过表达编码NADPH依赖型苹果酸脱氢酶的基因。
在一个实施方案中，所述NADPH依赖型苹果酸脱氢酶来自植物(优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科和苋科植物)、眼虫属或热杆菌属，优选来自高粱、玉米、甘蔗、豌豆、鹰嘴豆、菠菜、小眼虫或热自养甲烷热杆菌，更优选来自高粱。
在一个实施方案中，通过在所述经遗传改造的丁二酸生产酵母菌株中表达或过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因来产生或增加NADPH依赖型苹果酸脱氢酶活性，优选地，编码所述NADPH依赖型苹果酸脱氢酶的MDH基因优选来自植物，优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科或苋科植物，或来自眼虫属或热杆菌属，更优选来自高粱、玉米、甘蔗、豌豆、鹰嘴豆、菠菜、小眼虫或热自养甲烷热杆菌，更优选来自高粱。
在一个实施方案中，所述编码NADPH依赖型苹果酸脱氢酶的MDH基因包含SEQ ID NO：1所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有NADPH依赖型苹果酸脱氢酶活性的氨基酸序列的核苷酸序列。
在一个实施方案中，所述编码NADPH依赖型苹果酸脱氢酶的MDH基因掺入经遗传 改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在Pk2365基因座的位置。所述编码NADPH依赖型苹果酸脱氢酶的MDH基因可以置于合适的启动子(例如FBA1基因的启动子(例如SEQ ID NO：17所示))和/或终止子(例如INO1基因的终止子(例如SEQ ID NO：18所示))的控制之下。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有或具有增强的可溶的富马酸还原酶。在一个实施方案中，所述可溶的富马酸还原酶来自酵母和动质体目，例如但不限于酿酒酵母、布氏锥虫、墨西哥利什曼原虫、克氏锥虫。
在一个实施方案中，通过在所述经遗传改造的丁二酸生产酵母菌株中表达或过表达编码可溶的富马酸还原酶的基因来产生或增加可溶的富马酸还原酶活性，优选地，编码所述可溶的富马酸还原酶的基因优选来自来自酵母和动质体目，例如但不限于酿酒酵母、布氏锥虫、墨西哥利什曼原虫、克氏锥虫。
在一个实施方案中，所述可溶的富马酸还原酶的3’端乙醛酸循环体定位肽被部分或全部截短，使其是游离存在细胞质中的。
在一个实施方案中，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有可溶的富马酸还原酶活性的氨基酸序列的核苷酸序列。
在一个实施方案中，所述编码可溶的富马酸还原酶的基因掺入经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在ADH1基因座的位置。所述编码可溶的富马酸还原酶的基因可以置于合适的启动子(例如ADH1基因的启动子(例如SEQ ID NO：21所示))和/或终止子(例如ADH1基因的终止子(例如SEQ ID NO：22所示))的控制之下。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有或具有增强的丁二酸转运蛋白活性。
在一个实施方案中，所述丁二酸转运蛋白选自SpMAE1蛋白、AnDCT-02蛋白、EcDcuB和EcDcuC蛋白。在一个实施方案中，通过在所述经遗传改造的丁二酸生产酵母菌株中表达或过表达编码丁二酸转运蛋白例如SpMAE1蛋白的基因来产生或增加丁二酸转运蛋白活性。在一个实施方案中，所述丁二酸转运蛋白活性通过表达或过表达SpMAE1基因而产生或增强。
在一个实施方案中，所述SpMAE1基因包含SEQ ID NO：2所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丁二酸转运蛋白活性的氨基酸序列(例如来自粟酒裂殖酵母)的核苷酸序列。任选地，所述SpMAE1基因掺入经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)的基因组中，例如在MCH4基因座的位置。所述SpMAE1基因可以置于合适的启动子(例如TDH3基因的启动子(例如SEQ ID NO：19所示))和/或终止子(例如GAL2基因的终止子(例如SEQ ID NO：20所示))的控制之下。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有或具有增强的丙酮酸羧化酶活性。所述丙酮酸羧化酶活性可以通过表达或过表达编码丙酮酸羧化酶的基因而产生或增强。所述丙酮酸羧化酶可以来自真菌，特别是酵母和丝状真菌，优选酿酒酵母、库德里阿兹威毕赤酵母、米曲霉、马克斯克鲁维酵母等。在一个实施方案中，所述编码丙酮酸羧化酶的基因可以选自米曲霉的PYC基因和库德里阿兹威毕赤酵母的PYC1基因。
在一个实施方案中，所述丙酮酸羧化酶包含如SEQ ID NO：6或7所示序列编码的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有丙酮酸羧化酶活性的氨基酸序列。
在一个实施方案中，所述编码丙酮酸羧化酶的基因包含如SEQ ID NO：6或7所示序 列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丙酮酸羧化酶活性的氨基酸序列的核苷酸序列。
任选地，编码丙酮酸羧化酶的基因掺入经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在JEN2-1基因座的位置。所述编码丙酮酸羧化酶的基因可以置于合适的启动子(例如TDH3基因的启动子(例如SEQ ID NO：19所示))和/或终止子(例如GAL2基因的终止子(例如SEQ ID NO：20所示))的控制之下。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有或具有增强的富马酸酶活性。所述富马酸酶活性可以通过表达或过表达编码富马酸酶的基因而产生或增强。在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株表达或过表达编码富马酸酶的基因。在一个实施方案中，所述富马酸酶来自例如但不限于产琥珀酸放线杆菌、曼海姆产琥珀酸杆菌、大肠杆菌、库德里阿兹威毕赤酵母、米根霉。
在一个实施方案中，所述富马酸酶包含如SEQ ID NO：95所示的或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有富马酸酶活性的氨基酸序列。
在一个实施方案中，所述富马酸酶的5’端线粒体定位肽被部分或全部截短，使其不能定位于线粒体，而是游离存在细胞质中的。
在一个实施方案中，所述编码富马酸酶的基因包含如SEQ ID NO：8所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有富马酸酶活性的氨基酸序列。任选地，所述编码富马酸酶的基因掺入经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在PDC1基因座的位置。所述编码富马酸酶的基因可以置于合适的启动子(例如TDH3基因的启动子(例如SEQ ID NO：19所示))和/或终止子(例如GAL2基因的终止子(例如SEQ ID NO：20所示))的控制之下。
所述需要被表达或过表达的基因可以整合在菌株基因组中的合适位置，只要这种整合不负面影响菌株的生长、繁殖和/或生产性能。例如，其可以整合在编码如下蛋白的任一或多个基因组位置：(i)丙酮酸脱羧酶(EC 4.1.1.43)，(ii)NAD依赖性3-磷酸甘油脱氢酶(EC 1.1.1.8)，(iii)5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)，(iv)单羧酸透性酶，(v)二羧酸转运蛋白，(vi)乙醇脱氢酶1(EC 1.1.1.1)，和(vii)草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶(EC 4.1.3.17或4.1.1.112)。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的以下至少之一：(i)丙酮酸脱羧酶(EC 4.1.1.43)，(ii)NAD依赖性3-磷酸甘油脱氢酶(EC 1.1.1.8)，(iii)5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)，(iv)单羧酸透性酶，(v)二羧酸转运蛋白，(vi)乙醇脱氢酶1(EC 1.1.1.1)，和(vii)草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶(EC 4.1.3.17或4.1.1.112)。
在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株具有降低活性的或失活的丙酮酸脱羧酶。降低活性或失活的丙酮酸脱羧酶可以通过在所述菌株中弱化或失活的编码丙酮酸脱羧酶的基因来实现。因此，在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株具有弱化或失活的编码丙酮酸脱羧酶的基因。在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株中的编码丙酮酸脱羧酶的基因例如PDC1基因被敲除。
在一个实施方案中，所述丙酮酸脱羧酶包含如SEQ ID NO：10所示的或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有丙酮酸脱羧酶活性的氨基酸序列。
在一个实施方案中，所述编码丙酮酸脱羧酶的基因编码如SEQ ID NO：10所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有丙酮酸脱羧酶活性的氨基酸序列。
在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的NAD依赖性3-磷酸甘油脱氢酶。在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株具有弱化或失活的编码NAD依赖性3-磷酸甘油脱氢酶的基因。在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)中的编码NAD依赖性3-磷酸甘油脱氢酶的基因被敲除。
在一个实施方案中，所述NAD依赖性3-磷酸甘油脱氢酶包含如SEQ ID NO：11所示的或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有NAD依赖性3-磷酸甘油脱氢酶活性的氨基酸序列。
在一个实施方案中，编码所述NAD依赖性3-磷酸甘油脱氢酶的基因编码例如SEQ ID NO：10所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有NAD依赖性3-磷酸甘油脱氢酶活性的氨基酸序列。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的乙醇脱氢酶1。在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株具有弱化或失活的编码乙醇脱氢酶1的基因。在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)中的编码乙醇脱氢酶1的基因被敲除。
在一个实施方案中，所述乙醇脱氢酶1包含如SEQ ID NO：12所示的或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有乙醇脱氢酶1活性的氨基酸序列。
在一个实施方案中，编码所述乙醇脱氢酶1的基因编码例如SEQ ID NO：12所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有乙醇脱氢酶1活性的氨基酸序列。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)、二羧酸转运蛋白、草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶和/或单羧酸透性酶。
在进一步的实施方案中，所述经遗传改造的丁二酸生产酵母菌株中的编码5’-磷酸乳清苷脱羧酶的基因和/或编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)和/或编码单羧酸透性酶的基因和/或编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。
在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶。特别地，所述经遗传改造的丁二酸生产酵母菌株具有弱化或失活的编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。优选地，所述经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)中的编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因被敲除。
在一个实施方案中，所述草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶包含如SEQ ID NO：9所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶活性的氨基酸序列。
在一个实施方案中，编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因编码如SEQ ID NO：9所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶活性的氨基酸序列。
在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的5’-磷酸乳清苷脱羧酶。特别地，所述经遗传改造的丁二酸生产酵母菌株具有弱化或失活的编码5’-磷酸乳清苷脱羧酶的基因。优选地，所述经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)中的编码5’-磷酸乳清苷脱羧酶的基因被敲除。
在一个实施方案中，所述5’-磷酸乳清苷脱羧酶包含如SEQ ID NO：13所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有5’-磷酸乳清苷脱羧酶活性的氨基酸序列。
在一个实施方案中，编码所述5’-磷酸乳清苷脱羧酶的基因编码如SEQ ID NO：13所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有5’-磷酸乳清苷脱羧酶活性的氨基酸序列。
在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的单羧酸透性酶。特别地，所述经遗传改造的丁二酸生产酵母菌株具有弱化或失活的编码单羧酸透性酶的基因。优选地，所述经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)中的编码单羧酸透性酶的基因被敲除。
在一个实施方案中，所述单羧酸透性酶包含如SEQ ID NO：16所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有单羧酸透性酶活性的氨基酸序列。
在一个实施方案中，编码所述单羧酸透性酶的基因编码例如SEQ ID NO：16所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有单羧酸透性酶活性的氨基酸序列。
在一个实施方案中，所述经遗传改造的丁二酸生产酵母菌株还具有降低活性的或失活的二羧酸转运蛋白。特别地，所述经遗传改造的丁二酸生产酵母菌株具有弱化或失活的编码二羧酸转运蛋白的基因。优选地，所述经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)中的编码二羧酸转运蛋白的基因例如JEN2-1或JEN2-2基因被敲除。
在一个实施方案中，所述二羧酸转运蛋白包含如SEQ ID NO：14或15所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有二羧酸转运蛋白活性的氨基酸序列。
在一个实施方案中，编码所述二羧酸转运蛋白的基因编码如SEQ ID NO：14或15所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有二羧酸转运蛋白活性的氨基酸序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因，任选地，内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因以及过表达的编码可溶的富马酸还原酶的基因，任选地，内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码乙醇脱氢酶1的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和过表达的编码丁二酸转运蛋白的基因例如SpMAE1基因，任选内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码单羧酸透性酶的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和过表达的编码富马酸酶的基因，任选地，内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码丙酮酸脱羧酶的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和过表达的编码丙酮酸羧化酶的基因，任选地，内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码二羧酸转运蛋白的基因例如JEN2-1或JEN2-2基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和编码丙酮酸羧化酶的基因，以及内源编码丙酮酸脱羧酶的基因和内源编码NAD依赖性3-磷酸甘油脱氢酶的基因是被敲除的，任选内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及内源编码丙酮酸脱羧酶的基因和内源编码NAD依赖性3-磷酸甘油脱氢酶的基因是被敲除的，任选地，内源编码5’-磷酸乳清苷脱羧酶基因和/或内源编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因和/或内源编码乙醇脱氢酶1的基因和/或内源编码单羧酸透性酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因、编码富马酸酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及内源编码丙酮酸脱羧酶的基因和内源编码NAD依赖性3-磷酸甘油脱氢酶的基因是被敲除的，任选地，内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因和/或内源编码乙醇脱氢酶1的基因和/或内源编码单羧酸透性酶的基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列，或所述编码丁二酸转运蛋白的 基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，所述JEN2-1基因编码如SEQ ID NO：14所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有二羧酸转运蛋白活性的氨基酸序列。
在一个实施方案中，所述JEN2-2基因编码如SEQ ID NO：15所示的蛋白或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有二羧酸转运蛋白活性的氨基酸序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因，任选地，内源URA3基因和/或内源Pk2365基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因以及过表达的编码可溶的富马酸还原酶基因，任选地，内源URA3基因和/或内源ADH1基因和/或内源Pk2365基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和过表达的编码丁二酸转运蛋白的基因例如SpMAE1基因，任选内源URA3基因和/或内源MCH4基因和/或内源Pk2365基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和过表达的编码富马酸酶的基因，任选地，内源URA3基因和/或内源PDC1基因和/或内源Pk2365基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和过表达的编码丙酮酸羧化酶的基因，任选地，内源URA3基因和/或内源JEN2-1或JEN2-2基因和/或内源Pk2365基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因和编码丙酮酸羧化酶的基因，以及内源PDC1基因和内源GPD1基因是被敲除的，任选内源URA3基因和/或内源JEN2基因如JEN2-1基因、JEN2-2基因和/或内源Pk2365基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及内源PDC1基因和内源GPD1基因是被敲除的，任选地，内源URA3基因和/或内源JEN2基因如JEN2-1基因、JEN2-2基因和/或内源Pk2365基因和/或内源ADH1基因和/或内源MCH4基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1 所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有增强活性的高粱NADPH依赖型苹果酸脱氢酶、米曲霉丙酮酸羧化酶、布氏锥虫可溶的富马酸还原酶、库德里阿兹威毕赤酵母富马酸酶(5’端线粒体定位肽被截掉)和丁二酸转运蛋白SpMAE1，以及内源丙酮酸脱羧酶和内源NAD依赖性3-磷酸甘油脱氢酶活性是被消除的。在一个实施方案中，本发明提供了一种经遗传改造的库德里阿兹威毕赤酵母如CY902菌株，其具有过表达的编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因、编码富马酸酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及内源PDC1基因和内源GPD1基因是被敲除的，任选地，内源URA3基因和/或内源JEN2基因如JEN2-1基因、JEN2-2基因和/或内源Pk2365基因和/或内源ADH1基因和/或内源MCH4基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个方面，本发明提供了一种产生经遗传改造的丁二酸生产酵母菌株的方法，包括赋予所述菌株或增强所述菌株中NADPH依赖型苹果酸脱氢酶(EC 1.1.1.82)活性。
如本文所用，“赋予……活性”是指在经遗传改造的丁二酸生产酵母菌株中产生在进行遗传改造之前的初始菌株中不存在的活性。
如本文所用，“增强……活性”是指增加活性，例如至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。
本领域已知多种用于赋予或增强所需蛋白活性的方法，例如包括但不限于表达或过表达蛋白编码基因以及增加蛋白活性的突变或其他修饰。
如本文所用，“过表达”是指相对于遗传操作前的水平，基因的表达水平是升高的，例如升高至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少150％、至少200％、至少250％、至少300％或更高。过表达基因的方法是本领域熟知的，例如包括但不限于使用强启动子、增加基因拷贝数、增强子等。增加基因拷贝数可以例如但不限于通过引入一或多个拷贝的外源基因或内源基因实现，例如通过表达载体或整合进基因组中。
如本文所用，“外源基因”是指来自另一细胞或生物体的基因，例如来自相同物种或不同物种的基因。
如本文所用，“内源基因”是指细胞或生物体自身的基因。
所述启动子可以选自本领域已知的任何合适的启动子，例如包括但不限于编码果糖1,6-二磷酸醛缩酶的FBA1基因的启动子、编码3-磷酸甘油醛脱氢酶的TDH3基因的启动子、编码丙酮酸脱羧酶的PDC1基因启动子、编码乙醇脱氢酶1的ADH1基因启动子、编码3-磷酸甘油酸激酶的PGK1基因启动子、编码转录延伸因子的TEF1基因启动子、编码磷酸甘油酸变位酶GPM1基因启动子、编码磷酸丙糖异构酶TPI1基因启动子和编码烯醇化酶的ENO1基因的启动子(例如SEQ ID NO：100所示)。
在一个实施方案中，所述NADPH依赖型苹果酸脱氢酶来自植物，优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科和苋科植物，或来自眼虫属和热杆菌属，更优选高粱、玉米、甘蔗、豌豆、鹰嘴豆、菠菜、小眼虫或热自养甲烷热杆菌， 更优选高粱。
在一个实施方案中，通过在所述经遗传改造的丁二酸生产酵母菌株中表达或过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因来产生或增加NADPH依赖型苹果酸脱氢酶活性。优选地，编码所述NADPH依赖型苹果酸脱氢酶的MDH基因优选来自植物，优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科和苋科植物，或来自眼虫属和热杆菌属，更优选高粱、玉米、甘蔗、豌豆、鹰嘴豆、菠菜、小眼虫或热自养甲烷热杆菌，更优选高粱。
在一个实施方案中，所述编码NADPH依赖型苹果酸脱氢酶的MDH基因包含SEQ ID NO：1所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有NADPH依赖型苹果酸脱氢酶活性的氨基酸序列的核苷酸序列的序列。
在一个实施方案中，所述编码NADPH依赖型苹果酸脱氢酶的MDH基因掺入经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在Pk2365基因座的位置。在一个实施方案中，所述编码NADPH依赖型苹果酸脱氢酶的MDH基因经同源重组掺入酵母(例如库德里阿兹威毕赤酵母)基因组中，例如在Pk2365基因座的位置。所述编码NADPH依赖型苹果酸脱氢酶的MDH基因可以置于合适的启动子(例如FBA1基因的启动子(例如SEQ ID NO：17所示))和/或终止子(例如INO1基因的终止子(例如SEQ ID NO：18所示))的控制之下。
在进一步的实施方案中，所述方法还包括赋予所述菌株或增强所述菌株中可溶的富马酸还原酶活性。
在一个实施方案中，所述可溶的富马酸还原酶来自酵母和动质体目，例如但不限于酿酒酵母、布氏锥虫、墨西哥利什曼原虫、克氏锥虫。
在一个实施方案中，所述可溶的富马酸还原酶的3’端乙醛酸循环体定位肽被部分或全部截短，使其是游离存在细胞质中的。
在一个实施方案中，通过在所述经遗传改造的丁二酸生产酵母菌株中表达或过表达编码可溶的富马酸还原酶的基因来产生或增加可溶的富马酸还原酶活性，优选地，编码所述可溶的富马酸还原酶的基因优选来自来自酵母和动质体目，例如但不限于酿酒酵母、布氏锥虫、墨西哥利什曼原虫、克氏锥虫。
在一个实施方案中，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有可溶的富马酸还原酶活性的氨基酸序列的核苷酸序列。
在一个实施方案中，所述编码可溶的富马酸还原酶的基因掺入经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在ADH1基因座的位置。所述编码可溶的富马酸还原酶的基因可以置于合适的启动子(例如ADH1基因的启动子(例如SEQ ID NO：21所示))和/或终止子(例如ADH1基因的终止子(例如SEQ ID NO：22所示))的控制之下。
在进一步的实施方案中，所述方法还包括赋予或增强所述酵母菌株中的丁二酸转运蛋白活性。
在一个实施方案中，所述丁二酸转运蛋白选自SpMAE1蛋白、AnDCT-02蛋白、EcDcuB和EcDcuC蛋白。在一个实施方案中，通过在所述经遗传改造的丁二酸生产酵母菌株中表达或过表达编码丁二酸转运蛋白的基因(例如SpMAE1基因)来赋予或增强丁二酸转运蛋白活性。
在一个实施方案中，所述SpMAE1基因包含如SEQ ID NO：2所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丁二酸转运蛋白活性的氨基酸序列的核苷酸序列， 任选地，(例如经同源重组)掺入经遗传改造的丁二酸生产酵母菌株(例如库德里阿兹威毕赤酵母)的基因组中，例如在MCH4基因座的位置。所述SpMAE1基因可以置于合适的启动子(例如TDH3基因的启动子(例如SEQ ID NO：19所示))和/或终止子(例如GAL2基因的终止子(例如SEQ ID NO：20所示))的控制之下。
在进一步的实施方案中，所述方法还包括赋予或增强所述酵母菌株中的富马酸酶活性。
在一个实施方案中，所述富马酸酶的5’端线粒体定位肽被部分或全部截短，使其不能定位于线粒体，而是游离存在细胞质中的。
在一个实施方案中，通过在所述经遗传改造的丁二酸生产酵母菌株中表达或过表达编码富马酸酶的基因来赋予或增强富马酸酶活性。在一个实施方案中，所述富马酸酶来自例如但不限于产琥珀酸放线杆菌、曼海姆产琥珀酸杆菌、大肠杆菌、库德里阿兹威毕赤酵母、米根霉。
在一个实施方案中，所述富马酸酶包含如SEQ ID NO：95或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有富马酸酶活性的氨基酸序列。
在一个实施方案中，编码所述富马酸酶的基因编码如SEQ ID NO：95所示或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有富马酸酶活性的氨基酸序列。
在一个实施方案中，编码所述富马酸酶的基因包含如SEQ ID NO：8所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有富马酸酶活性的氨基酸序列的核苷酸序列。
任选地，编码所述富马酸酶的基因(例如经同源重组)掺入经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在PDC1基因座的位置。编码所述富马酸酶的基因可以置于合适的启动子(例如TDH3基因的启动子(例如SEQ ID NO：19所示))和/或终止子(例如GAL2基因的终止子(例如SEQ ID NO：20所示))的控制之下。
在进一步的实施方案中，所述方法还包括赋予或增强所述酵母菌株中的丙酮酸羧化酶活性。优选地，赋予或增强丙酮酸羧化酶活性通过在菌株中表达或过表达编码丙酮酸羧化酶的基因实现。所述丙酮酸羧化酶可以来自真菌，特别是酵母和丝状真菌，优选酿酒酵母、库德里阿兹威毕赤酵母、米曲霉、马克斯克鲁维酵母等。在一个实施方案中，编码所述丙酮酸羧化酶的基因可以选自米曲霉的PYC基因和库德里阿兹威毕赤酵母的PYC1基因。
在一个实施方案中，所述丙酮酸羧化酶包含如SEQ ID NO：6或7所示序列编码的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有丙酮酸羧化酶活性的氨基酸序列。
在一个实施方案中，编码所述丙酮酸羧化酶的基因包含如SEQ ID NO：6或7所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丙酮酸羧化酶活性的氨基酸序列的核苷酸序列。
任选地，编码丙酮酸羧化酶的基因掺入经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母)的基因组中，例如在JEN2-1基因座的位置。编码所述丙酮酸羧化酶的基因可以置于合适的启动子(例如TDH3基因的启动子(例如SEQ ID NO：19所示))和/或终止子(例如GAL2基因的终止子(例如SEQ ID NO：20所示))的控制之下。
所述方法可以将要在菌株中表达或过表达的基因整合在菌株基因组中的合适位置，只要这种整合不负面影响菌株的生长、繁殖和/或生产性能。例如，所述方法包括将上述的一或多个基因整合在编码如下蛋白的任一或多个基因组位置：(i)丙酮酸脱羧酶(EC  4.1.1.43)，(ii)NAD依赖性3-磷酸甘油脱氢酶(EC 1.1.1.8)，(iii)5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)，(iv)单羧酸透性酶，(v)二羧酸转运蛋白，(vi)乙醇脱氢酶1(EC 1.1.1.1)，和(vii)草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶(EC 4.1.3.17或4.1.1.112)。
在进一步的实施方案中，所述方法还包括弱化或失活所述菌株中的以下中的至少之一：(i)丙酮酸脱羧酶(EC 4.1.1.43)，(ii)NAD依赖性3-磷酸甘油脱氢酶(EC 1.1.1.8)，(iii)5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)，(iv)单羧酸透性酶，(v)二羧酸转运蛋白，(vi)乙醇脱氢酶1(EC 1.1.1.1)，和(vii)草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶(EC 4.1.3.17或4.1.1.112)。
在进一步的实施方案中，所述方法还包括降低或失活所述酵母菌株中的丙酮酸脱羧酶活性。
如本文所用，降低或失活蛋白例如酶的活性是指使得蛋白的活性降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是不可检测到。本领域已知多种降低或失活的手段，包括例如抑制基因表达如敲低(knockdown)(例如使用小干扰RNA)、使用弱启动子(基因是多肽编码基因时)等；基因敲除、缺失部分或全部基因或多肽序列；突变基因或多肽中某些位点例如编码序列或活性结构域以降低基因表达或调控活性或表达产物的活性；以及使用拮抗剂或抑制剂(例如包括但不限于抗体、干扰RNA等)。
如本文所用，弱化或失活基因是指使得基因的表达水平(作为蛋白编码基因时)或调控性能(作为调节元件时)降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或更多，或甚至是不可检测到。本领域已知多种弱化或失活基因的手段，包括例如抑制基因表达如敲低(例如使用小干扰RNA)、使用弱启动子(基因是多肽编码基因时)等；基因敲除、缺失部分或全部基因序列；突变基因中某些位点例如编码序列以降低基因表达或调控活性或表达产物的活性等。
在一个实施方案中，降低或失活丙酮酸脱羧酶活性包括弱化或失活编码丙酮酸脱羧酶的基因。
在一个实施方案中，弱化或失活编码丙酮酸脱羧酶的基因包括敲除编码丙酮酸脱羧酶的基因(例如编码SEQ ID NO：10所示氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有丙酮酸脱羧酶活性的氨基酸序列)。
在进一步的实施方案中，所述方法还包括降低或失活所述酵母菌株中的NAD依赖性3-磷酸甘油脱氢酶活性。
在一个实施方案中，降低或失活NAD依赖性3-磷酸甘油脱氢酶活性包括弱化或失活编码NAD依赖性3-磷酸甘油脱氢酶的基因。在一个实施方案中，弱化或失活编码NAD依赖性3-磷酸甘油脱氢酶的基因包括敲除编码NAD依赖性3-磷酸甘油脱氢酶的基因(例如编码SEQ ID NO：11所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有NAD依赖性3-磷酸甘油脱氢酶活性的氨基酸序列)。
在进一步的实施方案中，所述方法还包括降低或失活所述酵母菌株中的5’-磷酸乳清苷脱羧酶和/或乙醇脱氢酶1和/或单羧酸透性酶和/或二羧酸转运蛋白和/或草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的活性。
在一个实施方案中，所述方法包括弱化或失活所述酵母菌株中的编码5’-磷酸乳清苷脱羧酶的基因、编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)、编码乙醇脱氢酶1的基因、编码单羧酸透性酶的基因、编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因中的一或多种。
在一个实施方案中，编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因(例如编码如SEQ ID NO：9所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶活性的氨基酸序列)被敲除，例如通过同源重组方式敲除。
在一个实施方案中，编码5’-磷酸乳清苷脱羧酶的基因(其编码如SEQ ID NO：13所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有5’-磷酸乳清苷脱羧酶活性的氨基酸序列)被敲除，例如通过同源重组方式敲除。
在一个实施方案中，编码二羧酸转运蛋白的基因JEN2-1基因(编码例如SEQ ID NO：14所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有二羧酸转运蛋白活性的氨基酸序列)被敲除，例如通过同源重组方式敲除。
在一个实施方案中，编码二羧酸转运蛋白的基因JEN2-2基因(编码例如SEQ ID NO：15所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有二羧酸转运蛋白活性的氨基酸序列)被敲除，例如通过同源重组方式敲除。
在一个实施方案中，编码乙醇脱氢酶1的基因(其编码如SEQ ID NO：12所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有乙醇脱氢酶1活性的氨基酸序列)被敲除，例如通过同源重组方式敲除。
在一个实施方案中，编码单羧酸透性酶的基因(编码例如SEQ ID NO：16所示的氨基酸序列或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且具有单羧酸透性酶活性的氨基酸序列)被敲除，例如通过同源重组方式敲除。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因，任选地包括敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因以及编码可溶的富马酸还原酶的基因，任选敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码乙醇脱氢酶1的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因以及编码丁二酸转运蛋白的基因例如SpMAE1基因，任选敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码单羧酸透性酶的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因以及编码富马酸酶的基因，任选敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码丙酮酸脱羧酶的基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因以及编码丙酮酸羧化酶的基因，任选敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码二羧酸转运蛋白的基因例如JEN2-1或JEN2-2基因和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因以及编码丙酮酸羧化酶的基因，以及敲除内源编码丙酮酸脱羧酶的基因和内源编码NAD依赖性3-磷酸甘油脱氢酶的基因，任选敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及敲除内源编码丙酮酸脱羧酶的基因和内源编码NAD依赖性3-磷酸甘油脱氢酶的基因，任选敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因和/或内源编码乙醇脱氢酶1的基因和/或内源编码单羧酸透性酶的基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的丁二酸生产酵母(例如库德里阿兹威毕赤酵母，如CY902菌株)菌株的方法，包括在所述经遗传改造的丁二酸生产酵母菌株中过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因、编码富马酸酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及敲除内源编码丙酮酸脱羧酶的基因和内源编码NAD依赖性3-磷酸甘油脱氢酶的基因，任选地，敲除内源编码5’-磷酸乳清苷脱羧酶的基因和/或内源编码二羧酸转运蛋白的基因(例如JEN2基因如JEN2-1基因、JEN2-2基因)和/或内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因和/或内源编码乙醇脱氢酶1的基因和/或内源编码单羧酸透性酶的基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因，任选地，敲除内源URA3基因和/或内源Pk2365基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因以及编码可溶的富马酸还原酶的基因，任选地，敲除内源URA3基因和/或内源ADH1基因和/或内 源Pk2365基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，任选敲除内源URA3基因和/或内源MCH4基因和/或内源Pk2365基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因和编码富马酸酶的基因，任选地，敲除内源URA3基因和/或内源PDC1基因和/或内源Pk2365基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因和编码丙酮酸羧化酶的基因，任选地，敲除内源URA3基因和/或内源JEN2-1或JEN2-2基因和/或内源Pk2365基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因和编码丙酮酸羧化酶的基因，以及敲除内源PDC1基因和内源GPD1基因，任选敲除内源URA3基因和/或内源JEN2基因如JEN2-1基因、JEN2-2基因和/或内源Pk2365基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，或所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及敲除内源PDC1基因和内源GPD1基因，任选地，敲除内源URA3基因和/或内源JEN2基因如JEN2-1基因、JEN2-2基因和/或内源Pk2365基因和/或内源ADH1基因和/或内源MCH4基因。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，本发明提供了一种产生经遗传改造的库德里阿兹威毕赤酵母如CY902菌株的方法，包括过表达编码NADPH依赖型苹果酸脱氢酶的MDH基因、编码丙酮酸羧化酶的基因、编码可溶的富马酸还原酶的基因、编码富马酸酶的基因和编码丁二酸转运蛋白的基因例如SpMAE1基因，以及敲除内源PDC1基因和内源GPD1基因，任选地，敲除内源URA3基因和/或内源JEN2基因如JEN2-1基因、JEN2-2基因和/或内源Pk2365基因和/或内源ADH1基因和/或内源MCH4基因是被敲除的。优选地，所述MDH基因包含SEQ ID NO：1所示序列或其简并序列，所述编码丙酮酸羧化酶的基因包含SEQ ID NO：6或7所示序列或其简并序列，所述编码可溶的富马酸还原酶的基因包含SEQ ID NO：3-5任一所示序列或其简并序列，所述编码富马酸酶的基因包含SEQ ID NO：8所示序列或其简并序列，或所述编码丁二酸转运蛋白的基因包含SEQ ID NO：2所示序列或其简并序列。
在一个实施方案中，用于进行遗传改造的丁二酸生产酵母包括例如但不限于假丝酵母属、毕赤酵母属、红酵母属、酵母属、耶氏酵母属、鲁氏接合酵母属、球拟酵母属。在一个实施方案中，用于进行遗传改造的丁二酸生产酵母选自毕赤酵母属、酵母属或耶 氏酵母属菌株。在一个优选实施方案中，用于进行遗传改造的丁二酸生产酵母是库德里阿兹威毕赤酵母、酿酒酵母或解脂耶氏酵母，例如保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)、保藏号为CGMCC No.20885的库德里阿兹威毕赤酵母。
在一个方面，本发明提供了一种生产丁二酸的方法，包括在适于发酵生产丁二酸的条件下培养本发明所述的经遗传改造的丁二酸生产酵母菌株或者根据本发明所述的产生经遗传改造的丁二酸生产酵母菌株的方法制备的经遗传改造的丁二酸生产酵母菌株，任选包括分离纯化产生的丁二酸。
本领域已知发酵培养丁二酸生产酵母菌株用于发酵生产丁二酸的条件，包括例如但不限于pH、温度、培养基成分、发酵时间等。
本领域已知用于丁二酸生产酵母菌株发酵生产丁二酸的培养基，包括例如但不限于无机盐培养基(约5-12％w/v葡萄糖，任选含有约30g/L CaCO 3)。
本领域已知用于丁二酸生产酵母菌株发酵生产丁二酸的温度，例如约25-37℃，例如约25℃、约26℃、约27℃、约28℃、约29℃、约30℃、约31℃、约32℃、约33℃、约34℃、约35℃、约36℃、约37℃。在一个实施方案中，本发明所述丁二酸生产酵母菌株在30℃进行发酵以生产丁二酸。
本发明所述丁二酸生产酵母菌株可以在本领域已知的合适的pH值进行发酵，例如小于约7.0、、小于约6.5、小于约6.0、小于约5.5、小于约5.0、小于约4.5、小于约4.0、小于约3.5、小于约3.0、小于约2.5、小于约2.0、小于约1.5、小于约1.0的pH值(例如pH约1.0-7.0、1.0-6.0、1.0-5.5、1.0-5.0、1.0-4.5、1.0-4.0、1.0-3.5、1.0-3.0、2.0-7.0、2.0-6.0、2.0-5.5、2.0-5.0、2.0-4.5、2.0-4.0、2.0-3.5、2.0-3.0、3.0-7.0、3.0-6.0、3.0-5.5、3.0-5.0、3.0-4.5、3.0-4.0、3.0-3.5、4.0-7.0、4.0-6.0、4.0-5.5、4.0-5.0、4.0-4.5)。在一个实施方案中，本发明所述丁二酸生产酵母菌株在pH<约3.0进行发酵以生产丁二酸。
为生产丁二酸，本发明所述丁二酸生产酵母菌株可以发酵合适的时间，例如约12-96小时，如约12小时、约24小时、约36小时、约48小时、约60小时、约72小时、约96小时。在一个实施方案中，为生产丁二酸，本发明所述丁二酸生产酵母菌株发酵约24-72小时，例如约30小时。
本发明所述丁二酸生产酵母菌株可以在摇动的条件下(例如约100-300rpm，如约150、约200、约250rpm)进行发酵以生产丁二酸。
发酵液中的丁二酸含量可以通过本领域已知的合适的方法测定，例如HPLC。
在一个实施方案中，本发明提供了一种生产丁二酸的方法，包括在酸性pH(小于约7.0、小于约6.5、小于约6.0、小于约5.5、小于约5.0、小于约4.5、小于约4.0、小于约3.5、小于约3.0、小于约2.5、小于约2.0、小于约1.5、小于约1.0，例如pH约1.0-7.0、1.0-6.0、1.0-5.5、1.0-5.0、1.0-4.5、1.0-4.0、1.0-3.5、1.0-3.0、2.0-7.0、2.0-6.0、2.0-5.5、2.0-5.0、2.0-4.5、2.0-4.0、2.0-3.5、2.0-3.0、3.0-7.0、3.0-6.0、3.0-5.5、3.0-5.0、3.0-4.5、3.0-4.0、3.0-3.5、4.0-7.0、4.0-6.0、4.0-5.5、4.0-5.0、4.0-4.5)条件下，在无机盐培养基(如包含约5％w/v葡萄糖和约30g/L CaCO 3，或者约12％w/v葡萄糖)中培养经遗传改造的丁二酸生产酵母菌株，任选包括分离纯化产生的丁二酸。
在一个实施方案中，本发明所述的生产丁二酸的方法不需要添加中和剂。
在一个方面，本发明提供了本发明所述的经遗传改造的丁二酸生产酵母菌株或者根据本发明所述的产生经遗传改造的丁二酸生产酵母菌株的方法制备的经遗传改造的丁二酸生产酵母菌株在生产丁二酸中的应用，特别是在酸性pH(小于约7.0、小于约6.5、小于约6.0、小于约5.5、小于约5.0、小于约4.5、小于约4.0、小于约3.5、小于约3.0、小于约2.5、小于约2.0、小于约1.5、小于约1.0，例如pH约1.0-7.0、1.0-6.0、1.0-5.5、1.0-5.0、1.0-4.5、1.0-4.0、1.0-3.5、1.0-3.0、2.0-7.0、2.0-6.0、2.0-5.5、2.0-5.0、2.0-4.5、2.0-4.0、2.0-3.5、2.0-3.0、3.0-7.0、3.0-6.0、3.0-5.5、3.0-5.0、3.0-4.5、3.0-4.0、3.0-3.5、4.0-7.0、4.0-6.0、4.0-5.5、4.0-5.0、4.0-4.5)和/或不添加中和剂的条件下生产丁二酸中的应用。
如本文所用，“任选”或“任选地”是指随后描述的事件或情况发生或不发生，该描述包括其中所述事件或情况发生及不发生的情况。例如，任选包括的步骤是指该步骤存在或不存在。
如本文所用，术语“约”是指包括具体数值的数值范围，本领域技术人员可以合理认为其类似于具体数值。在一些实施方案中，术语“约”是指在使用本领域通常接受的测量的标准误差内。在一些实施方案中，约是指到具体数值的+/-10％。
本文公开的范围应该认为也具体公开了所有可能的子范围以及该范围内的各个数值。例如，对范围从1到6的描述应视为已明确公开了从1到3，从1到4，从1到5，从2到4，从2到6，从3至6等的子范围，以及该范围内的单个数字，例如1、2、3、4、5和6。无论范围的广度均适用这点。
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。
实施例1：在库德里阿兹威毕赤酵母CY902 ΔURA3菌株过表达SbMDH基因
通过同源重组方式对其乳清酸核苷-5'-磷酸脱羧酶(Orotidine 5'-phosphate decarboxylase)编码基因URA3(CY902基因座编号PK2075)进行敲除，获得了ΔURA3突变体(Xi,Y.；Zhan,T.；Xu,H.；Chen,J.；Bi,C.；Fan,F.；Zhang,X.,Characterization of JEN family carboxylate transporters from the acid-tolerant yeast Pichia kudriavzevii and their applications in succinic acid production.Microb Biotechnol 2021.0(0),1–18.doi:10.1111/1751-7915.13781)。随后我们构建了适用于该菌的CRISPR/Cas9质粒pWSPK-Cas9(GenBank检索号：MW296878.1)，该质粒含有URA3筛选标记，可以通过营养缺陷型筛选获取阳性转化子。
以CY902 ΔURA3为出发菌，在CY902基因组的基因座编号为Pk2365的位点过表达高粱来源苹果酸脱氢酶SbMDH(Uniprot数据库检索号：P17606)(SEQ ID NO：1)，所用启动子和终止子分别是CY902自身的果糖1,6-二磷酸醛缩酶基因FBA1的启动子(SEQ ID NO：17)和肌醇-3-磷酸合成酶基因INO1的终止子(SEQ ID NO：18)。SbMDH基因由南京金斯瑞生物科技有限公司合成，根据CY902密码子偏好性进行优化。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以CY902基因组DNA为模板，用引物1_UP_2365_F和1_UP_2365_R1(见表1)扩增CY902自身Pk2365基因上游同源臂片段1(片段1)；扩增体系和程序参照TAKARA
Figure PCTCN2022133973-appb-000001
DNA聚合酶的产品说明书；用引物2_P FBA1_F和2_P FBA1_R(见表1)扩增CY902自身FBA1启动子序列(片段2)；以含有SbMDH合成序列的质粒为模板，用引物3_SbMDH_F和3_SbMDH_R(见表1)扩增高粱的SbMDH编码序列(片段3)；以CY902基因组DNA为模板，用引物4_T INO1_F和4_T INO1_R(见表1)扩增CY902自身INO1基因终止子序列(片段4)；用引物5_DW_2365_F1和5_DW_2365_R(见表1)扩增CY902自身Pk2365基因下游同源臂片段1(片段5)。以CY902基因组DNA为模板，用引物1_UP_2365_F和1_UP_2365_R2(见表1)扩增CY902自身Pk2365基因上游同源臂片段2(片段6)；用引物5_DW_2365_F2和5_DW_2365_R(见表1)扩增CY902自身Pk2365基因下游同源臂片段2(片段7)。
2.构建用于编辑Pk2365基因位点的质粒
以pWSPK-Cas9质粒(GenBank检索号：MW296878.1)为模板，用引物pWSPK-F和 pWSPK-R(见表1)扩增9052bp的不含sgRNA序列的质粒骨架(简称为pWSPK_backbone，SEQ ID NO：23)；用引物sgRNA-1F和2365_sgRNA-1R(见表1)扩增sgRNA(GenBank检索号：MW296878.1)的5’端序列(简称为2365_sgRNA_1)，该片段的3’末端含有Pk2365基因特殊的20nt原型间隔序列。以pWSPK-Cas9质粒为模板，用引物sgRNA-2F和sgRNA-2R(见表1)扩增500bp的sgRNA的3’端序列(简称为sgRNA_2，SEQ ID NO：97)。将上述质粒骨架pWSPK_backbone，sgRNA片段2365_sgRNA_1和sgRNA_2用无缝克隆试剂盒(碧云天生物技术有限公司，上海，商品编号：D7010S)进行连接处理。将无缝克隆连接产物，转入Trans1-T1感受态细胞(全式金生物技术有限公司，北京，商品编号：CD501-02)，所获得的阳性质粒命名为pWSPK_2365。
3.构建过表达SbMDH基因菌株
将pWSPK_2365质粒以及片段6和7按酵母电转化法(doi:10.1111/1751-7915.13781)转入CY902 ΔURA3菌株中，筛选获得阳性转化子，命名为SA101-1菌株(基因型：CY902ΔURA3，ΔPk2365)；将pWSPK_2365质粒以及片段1-5按酵母电转化转入CY902 ΔURA3菌株中，筛选获得阳性转化子，命名为SA101-2菌株(基因型：CY902 ΔURA3，Pk2365::P PkFBA1-ORF SbMDH-T PkINO1)。
表1.过表达SbMDH基因和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000002
实施例2：在库德里阿兹威毕赤酵母CY902 ΔURA3中过表达EcSthA和AoMDH基因
以CY902 ΔURA3为出发菌，在CY902基因组的基因座编号为Pk2365的位点过表达大肠杆菌来源的可溶性吡啶核苷酸转氢酶EcSthA(EC 1.6.1.1)(SEQ ID NO：98)， 所用启动子和终止子分别是CY902自身的FBA1启动子(SEQ ID NO：17)和INO1终止子(SEQ ID NO：18)。在丁二酸半醛脱氢酶(EC 1.2.1.16)基因PkUGA2位点过表达米曲霉来源的苹果酸脱氢酶AoMDH(EC 1.1.1.37；Uniprot数据库检索号：I8U0T6)(SEQ ID NO：99)，所用启动子和终止子分别是CY902自身的烯醇化酶基因ENO1的启动子(SEQ ID NO：100)和细胞壁糖蛋白(GPI-cell wall glycoprotein)基因SED1的终止子(SEQ ID NO：101)。EcSthA和AoMDH基因由南京金斯瑞生物科技有限公司合成，根据CY902密码子偏好性进行优化。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以含有EcSthA合成序列的质粒为模板，用引物3_EcSthA_F和3_EcSthA_R(见表2)扩增大肠杆菌可溶性吡啶核苷酸转氢酶EcSthA的编码序列(片段8)；以CY902基因组DNA为模板，用引物1_UP_UGA2_F和1_UP_UGA2_R1(见表2)扩增CY902自身PkUGA2基因上游同源臂片段1(片段9)；用引物2_P ENO1_F和2_P ENO1_R(见表2)扩增CY902自身ENO1启动子序列(片段10)；以含有AoMDH合成序列的质粒为模板，用引物3_AoMDH_F和3_AoMDH_R(见表2)扩增米曲霉AoMDH编码序列(片段11)；以CY902基因组DNA为模板，用引物4_T SED1_F和4_T SED1_R(见表1)扩增CY902自身SED1基因终止子序列(片段12)；用引物5_DW_UGA2_F1和5_DW_UGA2_R(见表2)扩增CY902自身PkUGA2基因下游同源臂片段1(片段13)。以CY902基因组DNA为模板，用引物1_UP_UGA2_F和1_UP_UGA2_R2(见表2)扩增CY902自身PkUGA2基因上游同源臂片段2(片段14)；用引物5_DW_UGA2_F2和5_DW_UGA2_R(见表2)扩增CY902自身PkUGA2基因下游同源臂片段2(片段15)。
2.构建用于编辑PkUGA2基因位点的质粒
以pWSPK-Cas9质粒为模板，用引物sgRNA-1F和UGA2_sgRNA-1R(见表2)扩增sgRNA的5’端序列(简称为UGA2_sgRNA_1)，该片段的3’末端含有PkUGA2基因特殊的20nt原型间隔序列。将pWSPK_backbone，sgRNA_2与UGA2_sgRNA_1用无缝克隆试剂盒进行连接处理，最终获得阳性克隆质粒，将其命名为pWSPK_UGA2。
3.构建过表达EcSthA和AoMDH基因菌株
将pWSPK_2365质粒以及片段1、2、8、4、5按酵母电转化转入CY902 ΔURA3菌株中，筛选获得阳性转化子，命名为SA101-3菌株(基因型：CY902 ΔURA3，Pk2365::P PkFBA1-ORF EcSthA-T PkINO1)。将pWSPK_UGA2质粒以及片段14、15按酵母电转化转入SA101-2和SA101-3菌株中，筛选获得阳性转化子，分别命名为SA102-1菌株(基因型：SA101-2，ΔUGA2)和SA102-2菌株(基因型：SA101-3，ΔUGA2)。将pWSPK_UGA2质粒以及片段9-13按酵母电转化转入SA101-3菌株中,筛选获得阳性转化子，命名为SA102-3菌株(基因型：SA101-3，PkUGA2::P PkENO1-ORF AoMDH-T PkSED1)。
表2.过表达EcSthA及AoMDH基因和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000003
Figure PCTCN2022133973-appb-000004
实施例3：评价SA101，SA102系列菌株的丁二酸生产能力
1.有中和剂发酵
将SA101，SA102系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，30g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵24h，通过HPLC测量丁二酸产量如下表3所示。
表3.SA101，SA102系列菌株丁二酸摇瓶发酵产量
菌株 SA101-1 SA101-2 SA101-3 SA102-1 SA102-2 SA102-3
丁二酸g/L 0.52 0.80 0.58 0.83 0.57 0.62
2.无中和剂发酵
将SA101，SA102系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，0g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵24h，通过HPLC测量丁二酸产量如下表4所示。
表4.SA101，SA102系列菌株丁二酸摇瓶发酵产量
菌株 SA101-1 SA101-2 SA101-3 SA102-1 SA102-2 SA102-3
丁二酸g/L 0.31 0.52 0.38 0.54 0.40 0.42
实施例4：在SA101-2菌株中过表达FRD基因
以SA101-2为出发菌，在ADH1基因位点分别过表达三种来源的富马酸还原酶，包括(I)酿酒酵母的FRD(ScFRD，Uniprot数据库检索号：P32614)(SEQ ID NO：3)；(II)墨西哥利什曼原虫的FRD(LmFRD，SEQ ID NO：4)；(III)布氏锥虫的FRD(TbFRD，SEQ ID NO：5)。过表达FRD的启动子和终止子分别是CY902自身的乙醇脱氢酶1(alcohol dehydrogenase I)ADH1的启动子(SEQ ID NO：21)和终止子(SEQ ID NO：22)。以上三种FRD基因序列由南京金斯瑞生物科技有限公司合成，根据CY902密码子偏好性进行优化。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以CY902基因组DNA为模板，用引物1_P ADH1_F和1_P ADH1_R(见表5)扩增CY902自身ADH1基因启动子序列(上游同源臂，片段16)；以含有ScFRD合成序列的质粒为模板，用引物2_ScFRD_F和2_ScFRD_R(见表5)扩增酿酒酵母的ScFRD编码序列(片段17-1)；以含有LmFRD合成序列的质粒为模板，用引物2_LmFRD_F和2_LmFRD_R(见表5)扩增墨西哥利什曼原虫截掉3’端乙醛酸循环体定位肽的的LmFRD编码序列(片段17-2)；以含有TbFRD合成序列的质粒为模板，用引物2_TbFRD_F和2_TbFRD_R(见表5)扩增布氏锥虫截掉3’端乙醛酸循环体定位肽的TbFRD编码序列(片段17-3)；以CY902基因组DNA为模板，用引物3_T ADH1_F1和3_T ADH1_R(见表5)扩增CY902 自身ADH1基因终止子序列(下游同源臂1，片段18)。以CY902基因组DNA为模板，用引物3_T ADH1_F2和3_T ADH1_R(见表5)扩增CY902自身ADH1基因终止子序列(下游同源臂2，片段19)。
2.构建用于编辑ADH1基因位点的质粒
以pWSPK-Cas9质粒为模板，用引物sgRNA-1F和ADH1_sgRNA-1R(见表5)扩增sgRNA的5’端序列(简称为ADH1_sgRNA_1)，该片段的3’末端含有库德里阿兹威毕赤酵母ADH1(PkADH1)基因特殊的20nt原型间隔序列。将pWSPK_backbone，sgRNA_2与ADH1_sgRNA_1用无缝克隆试剂盒进行连接处理，最终获得阳性克隆质粒，将其命名为pWSPK_ADH1。
3.构建过表达FRD基因菌株
将pWSPK_ADH1质粒以及片段16、19按酵母电转化法转入SA101-2菌株中，筛选获得阳性转化子，命名为SA103-1菌株(基因型：SA101-2，ΔADH1)。将pWSPK_ADH1质粒以及片段16、17、18按酵母电转化法转入SA101-2菌株中，筛选获得阳性转化子，分别命名为SA103-2菌株(基因型：SA101-2，ADH1::ORF ScFRD)；SA103-3菌株(基因型：SA101-2，ADH1::ORF LmFRD)；命名为SA103-4菌株(基因型：SA101-2，ADH1::ORF TbFRD)。
表5.过表达FRD基因和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000005
实施例5：在SA101-2菌株中敲除PDC1，GPD1基因
1.构建用于敲除PDC1，GPD1的DNA片段
以CY902基因组DNA为模板，用引物PDC1_1F和PDC1_1R，PDC1_2F和PDC1_2R(见表6)扩增PDC1基因上下游同源臂片段(简称为PDC1_1，PDC1_2)；用引物GPD1_1F和GPD1_1R，GPD1_2F和GPD1_2R(见表6)扩增GPD1基因上下游同源臂片段(简称为GPD1_1，GPD1_2)。
2.构建用于编辑PDC1，GPD1基因的CRISPR/Cas9质粒
用引物sgRNA-1F和PDC1_sgRNA-1R(见表6)扩增sgRNA的5’端序列(简称为PDC1_sgRNA_1，SEQ ID NO：96)，该片段的3’末端含有PDC1基因特殊的20nt原型间隔序列。将质粒骨架pWSPK_backbone，sgRNA片段PDC1_sgRNA_1和sgRNA_2用无缝克隆试剂盒进行连接处理。将无缝克隆连接产物，转入Trans1-T1感受态细胞，所获得的阳性质粒命名为pWSPK_PDC1。用引物sgRNA-1F和GPD1_sgRNA-1R(见表6)扩增sgRNA的5’端序列(简称为GPD1_sgRNA_1)，该片段的3’末端含有GPD1基因特殊的20nt原型间隔序列，将pWSPK_backbone，sgRNA_2与GPD1_sgRNA_1通过无缝克隆融合为用于编辑GPD1基因的pWSPK_GPD1质粒。
表6.PDC1，GPD1基因敲除和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000006
3.构建PDC1，GPD1基因敲除菌株
将片段PDC1_1，PDC1_2和质粒pWSPK_PDC1同时转入SA101-2。使用引物PDC1_1F/PDC1_2R鉴定出阳性转化子命名为SA104菌株(基因型：SA101-2，ΔPDC1)。将pWSPK_GPD1质粒和片段GPD1_1，GPD1_2电转化入SA104菌株中，经SD-URA培养基筛选获得阳性转化子，命名为SA105菌株(基因型：SA104，ΔGPD1)。
实施例6：在SA101-2菌株中过表达SpMAE1基因
在CY902基因组的MCH4基因位点表达了粟酒裂殖酵母来源的SpMAE1转运蛋白(Uniprot数据库检索号：P50537)，SpMAE1基因由南京金斯瑞生物科技有限公司合成(SEQ ID NO：2)，根据CY902密码子偏好性进行优化。所用启动子和终止子分别是CY902自身的3-磷酸甘油醛脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase 3)基因TDH3的启动子(SEQ ID NO：19)和半乳糖转运蛋白基因GAL2的终止子(SEQ ID NO：20)。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以CY902基因组DNA为模板，用引物1_UP_MCH4_F和1_UP_MCH4_R1(见表7)扩增MCH4基因上游同源臂片段1(片段20)；用引物2_P TDH3_F和2_P TDH3_R(见表7)扩增CY902自身TDH3基因启动子序列(片段21)；以含有SpMAE1合成序列的质粒为模板，用引物3_SpMAE1_F和3_SpMAE1_R(见表7)扩增粟酒裂殖酵母的 SpMAE1编码序列(片段22)；以CY902基因组DNA为模板，用引物4_T GAL2_F和4_T GAL2_R(见表7)扩增CY902自身GAL2基因终止子序列(片段23)；用引物5_DW_MCH4_F1和5_DW_MCH4_R(见表7)扩增MCH4基因下游同源臂片段1(片段24)。以CY902基因组DNA为模板，用引物1_UP_MCH4_F和1_UP_MCH4_R2(见表7)扩增MCH4基因上游同源臂片段2(片段25)；用引物5_DW_MCH4_F2和5_DW_MCH4_R(见表7)扩增MCH4基因下游同源臂片段2(片段26)。
2.构建用于编辑CY902自身MCH4的质粒
以pWSPK-Cas9质粒为模板，用引物sgRNA-1F和MCH4_sgRNA-1R(见表7)扩增sgRNA的5’端序列(简称为MCH4_sgRNA_1)，该片段的3’末端含有库德里阿兹威毕赤酵母PkMCH4基因特殊的20nt原型间隔序列。将pWSPK_backbone，sgRNA_2与MCH4_sgRNA_1用无缝克隆试剂盒进行连接处理，最终获得阳性克隆质粒，将其命名为pWSPK_MCH4。
表7.过表达SpMAE1基因和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000007
3.构建过表达SpMAE1基因菌株
按照酵母电转化方法将pWSPK_MCH4质粒以及片段25和26电转化入SA101-2菌株中，筛选获得阳性转化子，命名为SA106-1(基因型：SA101-2，ΔPkMCH4)；将pWSPK_MCH4质粒以及片段20-24电转化入SA101-2菌株中，筛选获得阳性转化子，命名为SA106-2(基因型：SA101-2，PkMCH4::P PkTDH3-ORF SpMAE1-T PkGAL2)。
实施例7：在SA101-2菌株中过表达PYC基因
在JEN2-1位点分别过表达了米曲霉来源的PYC(AoPYC)基因(Uniprot数据库检索号：Q2UGL1)(SEQ ID NO：6)，CY902自身的PYC1(PkPYC1)基因用作对照(SEQ ID NO：7)，所用启动子和终止子分别是TDH3启动子(SEQ ID NO：19)和GAL2终止子(SEQ ID NO：20)。AoPYC基因由南京金斯瑞生物科技有限公司合成，根据CY902密码子偏好性进行优化(SEQ ID NO：6)。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以CY902基因组DNA为模板，用引物1_UP_JEN2-1_F和1_UP_JEN2-1_R1(见表8)扩增JEN2-1基因上游同源臂片段1(片段27)；用引物3_PkPYC1_F和3_PkPYC1_R(见表8)扩增CY902自身PkPYC1编码序列(片段28-1)；以含有AoPYC合成序列的质粒为模板，用引物3_AoPYC_F和3_AoPYC_R(见表8)扩增米曲霉来源的AoPYC编码序列(片段28-2)；以CY902基因组DNA为模板，用引物5_DW_JEN2-1_F1和5_DW_JEN2-1_R(见表8)扩增JEN2-1基因下游同源臂片段1(片段29)。以CY902基因组DNA为模板，用引物1_UP_JEN2-1_F和1_UP_JEN2-1_R2(见表8)扩增JEN2-1基因上游同源臂片段2(片段30)；用引物5_DW_JEN2-1_F2和5_DW_JEN2-1_R(见表8)扩增JEN2-1基因下游同源臂片段2(片段31)。
表8.过表达PYC基因和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000008
2.构建用于编辑JEN2-1基因的CRISPR/Cas9质粒
以pWSPK-Cas9质粒为模板，用引物sgRNA-1F和JEN2-1_sgRNA-1R(见表8)扩增sgRNA的5’端序列(简称为JEN2-1_sgRNA_1)，该片段的3’末端含有JEN2-1基因特殊的20nt原型间隔序列。将实施例1中的pWSPK_backbone，sgRNA_2与JEN2-1_sgRNA_1用无缝克隆试剂盒进行连接处理，最终获得阳性克隆质粒，将其命名为pWSPK_JEN2-1。
3.构建过表达PYC基因菌株
将pWSPK_JEN2-1质粒以及片段30和31电转化入SA101-2菌株中，筛选获得阳性转化子，命名为SA107-1(基因型：SA101-2，ΔPkJEN2-1)；将pWSPK_JEN2-1质粒以及片段27、21、28、23、29电转化入SA101-2菌株中，筛选获得阳性转化子，分别命名为SA107-2(基因型：SA101-2，PkJEN2-1::P PkTDH3-ORF PkPYC1-T PkGAL2)；SA107-3(基因型：SA101-2，PkJEN2-1::P PkTDH3-ORF AoPYC-T PkGAL2)。
实施例8：在SA101-2菌株中过表达PkFUM1基因
在SA101-2菌株PDC1基因启动子位点过表达了CY902截掉5’端线粒体定位肽的富马酸酶基因PkFUM1(SEQ ID NO：8)。所用启动子和终止子分别为TDH3启动子(SEQ ID NO：19)和GAL2终止子(SEQ ID NO：20)。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以CY902基因组DNA为模板，用引物1_UP_P PDC1_F和1_UP_P PDC1_R(见表9)扩增CY902自身PDC1基因启动子上游同源臂片段(片段32)；用引物3_PkFUM1_F和3_PkFUM1_R(见表9)扩增截掉5’端线粒体定位肽的FUM1编码序列(片段33)；用引物5_DW_P PDC1_F和5_DW_P PDC1_R(见表9)扩增CY902自身PDC1基因启动子下游同源臂片段(片段34)。
2.构建用于编辑PDC1基因启动子位点的质粒
以pWSPK-Cas9质粒为模板，用引物sgRNA-1F和P PDC1_sgRNA-1R(见表9)扩增sgRNA的5’端序列(简称为P PDC1_sgRNA_1)，该片段的3’末端含有P PDC1基因启动子特殊的20nt原型间隔序列。将pWSPK_backbone，sgRNA_2与P PDC1_sgRNA_1用无缝克隆试剂盒进行连接处理，最终获得阳性克隆质粒，将其命名为pWSPK_P PDC1。
表9.过表达PkFUM1基因和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000009
3.构建过表达PkFUM1基因菌株
将pWSPK_P PDC1质粒以及片段32、21、33、23和34按酵母电转化法转入SA101-2菌株中，筛选获得阳性转化子，命名为SA108菌株(基因型：SA101-2，P PkPDC1::P PkTDH3-ORF PkFUM1-T PkGAL2)。
实施例9：在SA101系列菌株中回补PkURA3基因
在SA101系列菌株二羧酸转运蛋白PkJEN2-2基因位点回补了5’-磷酸乳清苷脱羧酶基因PkURA3(SEQ ID NO：117)。所用启动子和终止子为PkURA3自身的启动子(SEQ ID NO：118)和终止子(SEQ ID NO：119)。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以CY902基因组DNA为模板，用引物1_UP_JEN2-2_F和1_UP_JEN2-2_R1(见表10)扩增CY902自身PkJEN2-2基因上游同源臂片段1(片段35)；用引物2_PkURA3_F和2_PkURA3_R(见表10)扩增URA3启动子、编码框及终止子序列(片段36)；用引物3_DW_JEN2-2_F1和3_DW_JEN2-2_R(见表10)扩增CY902自身PkJEN2-2基因下游同源臂片段1(片段37)。以CY902基因组DNA为模板，用引物1_UP_JEN2-2_F和1_UP_JEN2-2_R2(见表10)扩增CY902自身PkJEN2-2基因上游同源臂片段2(片段38)；用引物3_DW_JEN2-2_F2和3_DW_JEN2-2_R(见表10)扩增CY902自身 PkJEN2-2基因下游同源臂片段2(片段39)。
2.构建用于编辑PkJEN2-2基因位点的质粒
以pWSPK-Cas9质粒为模板，用引物sgRNA-1F和JEN2-2_sgRNA-1R(见表10)扩增sgRNA的5’端序列(简称为JEN2-2_sgRNA_1)，该片段的3’末端含有PkJEN2-2基因启动子特殊的20nt原型间隔序列。将pWSPK_backbone，sgRNA_2与JEN2-2_sgRNA_1用无缝克隆试剂盒进行连接处理，最终获得阳性克隆质粒，将其命名为pWSPK_JEN2-2。
表10.回补PkURA3基因和CRISPR/Cas9质粒构建所需引物
Figure PCTCN2022133973-appb-000010
3.构建回补PkURA3基因菌株
将pWSPK_JEN2-2质粒以及片段38、39按酵母电转化法转入SA101-1、SA101-2、SA101-3菌株中，筛选获得阳性转化子，分别命名为SA109-1菌株(基因型：SA101-1，ΔPkJEN2-2)、SA109-2菌株(基因型：SA101-2，ΔPkJEN2-2)和SA109-3菌株(基因型：SA101-3，ΔPkJEN2-2)。将pWSPK_JEN2-2质粒以及片段35-37按酵母电转化法转入SA101-1、SA101-2、SA101-3菌株中，筛选获得阳性转化子，分别命名为SA109-4菌株(基因型：SA101-1，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)、SA109-5菌株(基因型：SA101-2，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)和SA109-6菌株(基因型：SA101-3，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)。
实施例10：在SA102—SA108系列菌株中回补PkURA3基因
参照实施例9，在SA102—SA108菌株PkJEN2-2基因位点回补PkURA3，得到SA110-1菌株(基因型：SA102-1，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA110-2菌株(基因型：SA102-2，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA110-3菌株(基因型：SA102-3，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA111-1菌株(基因型：SA103-1，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA111-2菌株(基因型：SA103-2，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA111-3菌株(基因型：SA103-3，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA111-4菌株(基因型：SA103-4，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA112菌株(基因型：SA104，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA113菌株(基因型：SA105，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA114-1菌株(基因型：SA106-1，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA114-2菌株(基因型：SA106-2，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA115-1菌株(基因型：SA107-1， PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA115-2菌株(基因型：SA107-2，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA115-3菌株(基因型：SA107-3，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA116菌株(基因型：SA108，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)。
实施例11：评价SA101—SA116系列菌株丁二酸生产能力
1.有中和剂发酵
将SA101—SA116系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，30g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵24h，通过HPLC测量丁二酸产量。
表11.SA101—SA116系列菌株丁二酸摇瓶发酵产量
菌株 SA101-1 SA101-2 SA101-3 SA102-1 SA102-2 SA102-3
丁二酸g/L 0.52 0.80 0.58 0.83 0.57 0.62
菌株 SA103-1 SA103-2 SA103-3 SA103-4 SA104 SA105
丁二酸g/L 0.97 1.02 2.84 9.93 1.13 1.25
菌株 SA106-1 SA106-2 SA107-1 SA107-2 SA107-3 SA108
丁二酸g/L 0.82 6.07 0.80 1.04 1.12 1.57
菌株 SA109-1 SA109-2 SA109-3 SA109-4 SA109-5 SA109-6
丁二酸g/L 0.53 0.78 0.57 0.54 0.83 0.60
菌株 SA110-1 SA110-2 SA110-3 SA111-1 SA111-2 SA111-3
丁二酸g/L 0.88 0.59 0.68 1.08 1.17 3.12
菌株 SA111-4 SA112 SA113 SA114-1 SA114-2 SA115-1
丁二酸g/L 10.74 1.24 1.37 0.81 6.23 0.86
菌株 SA115-2 SA115-3 SA116      
丁二酸g/L 1.11 1.21 1.73      
2.无中和剂发酵
将SA101—SA116系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，0g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵24h，通过HPLC测量丁二酸产量。
表12.SA101—SA116系列菌株丁二酸摇瓶发酵产量
菌株 SA101-1 SA101-2 SA101-3 SA102-1 SA102-2 SA102-3
丁二酸g/L 0.31 0.52 0.38 0.54 0.40 0.42
菌株 SA103-1 SA103-2 SA103-3 SA103-4 SA104 SA105
丁二酸g/L 0.59 0.62 3.28 10.63 0.93 1.11
菌株 SA106-1 SA106-2 SA107-1 SA107-2 SA107-3 SA108
丁二酸g/L 0.52 5.31 0.51 0.77 0.86 1.21
菌株 SA109-1 SA109-2 SA109-3 SA109-4 SA109-5 SA109-6
丁二酸g/L 0.29 0.50 0.39 0.33 0.55 0.40
菌株 SA110-1 SA110-2 SA110-3 SA111-1 SA111-2 SA111-3
丁二酸g/L 0.53 0.42 0.45 0.63 0.66 3.73
菌株 SA111-4 SA112 SA113 SA114-1 SA114-2 SA115-1
丁二酸g/L 11.27 1.01 1.18 0.54 5.47 0.55
菌株 SA115-2 SA115-3 SA116      
丁二酸g/L 0.81 0.93 1.42      
实施例12：优化丁二酸生产菌
参照实施例5，在SA103-4中敲除PDC1和GPD1基因，得到SA117菌株(基因型：SA103-4，ΔPDC1)；SA118菌株(基因型：SA117，ΔGPD1)。
参照实施例6，在SA118中过表达SpMAE1基因，得到SA119-1菌株(基因型：SA118，ΔPkMCH4)；SA119-2菌株(基因型：SA118，PkMCH4::P PkTDH3-ORF SpMAE1-T PkGAL2)。
参照实施例7，在SA119-2菌株中过表达菌株中过表达两种来源的PYC，得到SA120-1菌株(基因型：SA119-2，ΔPkJEN2-1)；得到SA120-2菌株(基因型：SA119-2，PkJEN2-1::P PkTDH3-ORF PkPYC1-T PkGAL2)；SA120-3菌株(基因型：SA119-2，PkJEN2-1::P PkTDH3-ORF AoPYC-T PkGAL2)。
参照实施例8，在SA120-3中过表达截掉5’端线粒体定位肽的富马酸酶基因PkFUM1，得到SA121菌株(基因型：SA120-3，P PkPDC1::P PkTDH3-ORF PkFUM1-T PkGAL2)。
参照实施例9，在SA121菌株中回补5’-磷酸乳清苷脱羧酶基因PkURA3，得到SA122菌株(基因型：SA121，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)。
参照实施例4，在SA102-3中过表达TbFRD基因，得到SA123菌株(基因型：SA102-3，ADH1::ORF TbFRD)。
参照实施例5，在SA123中敲除PDC1和GPD1基因，得到SA124菌株(基因型：SA123，ΔPDC1)；SA125菌株(基因型：SA124，ΔGPD1)。
参照实施例6，在SA125中过表达SpMAE1基因，得到SA126-1菌株(基因型：SA125，ΔPkMCH4)；SA126-2菌株(基因型：SA125，PkMCH4::P PkTDH3-ORF SpMAE1-T PkGAL2)。
参照实施例7，在SA126-2菌株中过表达菌株中过表达两种来源的PYC，得到SA127-1菌株(基因型：SA126-2，ΔPkJEN2-1)；得到SA127-2菌株(基因型：SA126-2，PkJEN2-1::P PkTDH3-ORF PkPYC1-T PkGAL2)；SA127-3菌株(基因型：SA126-2，PkJEN2-1::P PkTDH3-ORF AoPYC-T PkGAL2)。
参照实施例8，在SA127-3中过表达截掉5’端线粒体定位肽的富马酸酶基因PkFUM1，得到SA128菌株(基因型：SA127-3，P PkPDC1::P PkTDH3-ORF PkFUM1-T PkGAL2)。
参照实施例9，在SA128菌株中回补5’-磷酸乳清苷脱羧酶基因PkURA3，得到SA129菌株(基因型：SA128，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)。
实施例13：评价SA122和SA129菌株丁二酸生产能力
将SA122和SA129菌株在5L罐中分别进行放大发酵(酵母无机盐培养基，12％w/v葡萄糖)，在不添加任何中和剂的条件下，36h丁二酸产量分别达到96.08g/L和46.02g/L，转化率分别是0.89g/g和0.57g/g。
实施例14：在库德里阿兹威毕赤酵母CICC32244 ΔURA3菌株过表达SbMDH基因
参考实施例1，通过同源重组方式对CICC32244(购于中国工业微生物菌种保藏管理中心)乳清酸核苷-5'-磷酸脱羧酶(Orotidine 5'-phosphate decarboxylase)编码基因URA3进行敲除，获得了CICC32244 ΔURA3突变体。
将pWSPK_2365质粒以及片段6和7按酵母电转化法转入CICC32244 ΔURA3菌株中，筛选获得阳性转化子，命名为SA130-1菌株(基因型：CICC32244 ΔURA3，ΔPk2365)；将pWSPK_2365质粒以及片段1-5按酵母电转化转入CICC32244 ΔURA3菌株中，筛选获得阳性转化子，命名为SA130-2菌株(基因型：CICC32244 ΔURA3，Pk2365::P PkFBA1-ORF SbMDH-T PkINO1)。
实施例15：在SA130系列菌株中回补PkURA3基因
参照实施例9，在SA130系列菌株PkJEN2-2基因位点回补PkURA3，得到SA131-1菌株(基因型：SA130-1，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)；SA131-2菌株(基因型：SA130-2，PkJEN2-2::P PkURA3-ORF PkURA3-T PkURA3)。
实施例16：评价SA130和SA131系列菌株的丁二酸生产能力
1.有中和剂发酵
将SA130和SA131系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，30g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵24h，通过HPLC测量丁二酸产量。
表13.SA130和SA131系列菌株丁二酸摇瓶发酵产量
菌株 SA130-1 SA130-2 SA131-1 SA131-2
丁二酸g/L 0.37 0.52 0.39 0.56
2.无中和剂发酵
将SA130和SA131系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，0g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵24h，通过HPLC测量丁二酸产量。
表14.SA130和SA131系列菌株丁二酸摇瓶发酵产量
菌株 SA130-1 SA130-2 SA131-1 SA131-2
丁二酸g/L 0.37 0.53 0.40 0.55
实施例17：在酿酒酵母BY4742菌株中过表达SbMDH基因
在酿酒酵母5-磷酸乳清苷脱羧酶基因ScURA3(SEQ ID NO：129)位点过表达高粱来源苹果酸脱氢酶SbMDH(SEQ ID NO：1)，过表达ScURA3基因菌株用作对照。过表达SbMDH基因序列与上文相同，根据CY902密码子偏好性进行优化，所用启动子和终止子分别是BY4742自身的3-磷酸甘油醛脱氢酶基因ScTDH3的启动子(SEQ ID NO：130)和半乳糖透性酶基因ScGAL2的终止子(SEQ ID NO：131)。过表达ScURA3基因所用启动子和终止子分别是BY4742自身的5-磷酸乳清苷脱羧酶基因ScURA3的启动子(SEQ ID NO：132)和细胞壁糖蛋白基因ScSED1的终止子(SEQ ID NO：133)。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以酿酒酵母S288c(购自ThermoFisher Scientific)基因组DNA为模板，用引物1_UP_ScURA3_F和1_ScURA3_R(见表15)扩增ScURA3上游同源臂和ORF序列(片段40)；用引物2_T ScSED1_F和2_T ScSED1_R1(见表15)扩增ScSED1终止子序列1(片段41)；用引物3_P ScTDH3_F和3_P ScTDH3_R(见表15)扩增ScTDH3基因启动子序列(片段42)；以含有SbMDH合成序列的质粒为模板，用引物4_SbMDH_F和4_SbMDH_R(见表15)扩增高粱来源的SbMDH编码序列(片段43)；以S288c基因组DNA为模板，用引物5_T ScGAL2_F和5_T ScGAL2_R(见表15)扩增ScGAL2基因终止子序列(片段44)；用引物6_DW_ScURA3_F1和6_DW_ScURA3_R(见表15)扩增ScURA3基因下游同源臂片段1(片段45)。以S288c基因组DNA为模板，用引物2_T ScSED1_F和2_T ScSED1_R2(见表15)扩增ScSED1终止子序列2(片段46)；用引物6_DW_ScURA3_F2和6_DW_ScURA3_R(见表15)扩增ScURA3基因下游同源臂片段2(片段47)。
2.构建在酿酒酵母中过表达SbMDH菌株
将片段40、46、47混合在一起按酵母电转化方法转入BY4742菌株(购自ThermoFisher Scientific)中，筛选获得阳性转化子，命名为SA132-1菌株(基因型：BY4742，ScURA3::P ScURA3-ORF ScURA3-T ScSED1t)；将片段40-45混合在一起按酵母电转化方法转入BY4742菌株中，筛选获得阳性转化子，命名为SA132-2菌株(基因型：BY4742，ScURA3::P ScURA3-ORF ScURA3-T ScSED1t-P ScTDH3-ORF SbMDH-T ScGAL2)。
表15.在酿酒酵母中过表达SbMDH基因所需引物
Figure PCTCN2022133973-appb-000011
Figure PCTCN2022133973-appb-000012
实施例18：评价SA132系列菌株的丁二酸生产能力
1.无中和剂发酵
将SA132系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，0g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵12h，通过HPLC测量丁二酸产量。
表16.SA132系列菌株丁二酸摇瓶发酵产量
菌株 SA132-1 SA132-2
丁二酸g/L 0.12 0.22
实施例19：在解脂耶氏酵母Po1g菌株中过表达SbMDH基因
因为解脂耶氏酵母有极强的非同源末端重组能力，所以采用随机插入解脂耶氏酵母基因组的方式过表达高粱来源苹果酸脱氢酶基因SbMDH(SEQ ID NO：1)和筛选标记β-异丙基苹果酸脱氢酶(EC 1.1.1.85)基因YlLEU2(SEQ ID NO：148)，并以随机插入过表达YlLEU2基因菌株用作对照。过表达SbMDH基因序列与上文相同，根据CY902密码子偏好性进行优化，所用启动子和终止子分别是Po1g自身的3-磷酸甘油醛脱氢酶基因YlGAPDH的启动子(SEQ ID NO：149)和3-羟基-3-甲基戊二酰辅酶A还原酶基因YlHMG1的终止子(SEQ ID NO：150)。过表达YlLEU2基因所用启动子和终止子分别是Po1g自身的β-异丙基苹果酸脱氢酶基因YlLEU2的启动子(SEQ ID NO：151)和酿酒酵母细胞色素c亚型1基因ScCYC1的终止子(SEQ ID NO：152)。具体构建方法如下：
1.构建用于同源重组的供体DNA(Donor DNA)片段
以解脂耶氏酵母W29基因组DNA(GenBank：GCA_001761485.1)为模板，用引物1_P YlGAPDH_F和1_P YlGAPDH_R(见表17)扩增YlGAPDH基因启动子序列(片段48)；以含有SbMDH合成序列的质粒为模板，用引物2_SbMDH_F和2_SbMDH_R(见表17)扩增高粱来源的SbMDH编码序列(片段49)；以解脂耶氏酵母W29基因组DNA为模板，用引物3_T YlHMG1_F和3_T YlHMG1_R(见表17)扩增YlHMG1基因终止子序列(片段50)；用引物4_P YlLEU2_F和4_YlLEU2_R(见表17)扩增YlLEU2启动子和ORF序列(片段51)；以S288c基因组DNA为模板，用引物5_T ScCYC1_F和5_T ScCYC1_R(见表17)扩增ScCYC1终止子序 列(片段52)。以片段48-52为模板，用引物1_P YlGAPDH_F和5_T ScCYC1_R扩增过表达SbMDH和YlLEU2的表达盒(片段53)；以片段51、52为模板，用引物4_P YlLEU2_F和5_T ScCYC1_R扩增过表达YlLEU2的表达盒(片段54)
2.构建在解脂耶氏酵母中过表达SbMDH菌株
将片段54按酵母电转化方法转入Po1g菌株(购自益生生技开发股份有限公司)中，筛选获得阳性转化子，命名为SA133-1菌株(基因型：Polg，genome::P YlLEU2-ORF YlLEU2-T ScCYC1t)；将片段53按酵母电转化方法转入Po1g菌株中，筛选获得阳性转化子，命名为SA133-2菌株(基因型：Polg，genome::P YlGAPDH-SbMDH-T YlHMG1t-P YlLEU2-ORF YlLEU2-T ScCYC1t)。
表17.在解脂耶氏酵母中过表达SbMDH基因所需引物
Figure PCTCN2022133973-appb-000013
实施例20：评价SA133系列菌株的丁二酸生产能力
1.无中和剂发酵
将SA133系列菌株接种30mL酵母无机盐培养基(5％w/v葡萄糖，0g/L CaCO 3)，在30℃，250rpm条件下摇瓶发酵24h，通过HPLC测量丁二酸产量。
表18.SA133系列菌株丁二酸摇瓶发酵产量
菌株 SA133-1 SA133-2
丁二酸g/L 1.48 2.41

Claims (12)

  1. 一种经遗传改造的丁二酸生产酵母菌株，其具有或具有增强的NADPH依赖型苹果酸脱氢酶(EC 1.1.1.82)活性，
    任选地，还具有或具有增强的如下至少一种的活性：(i)可溶的富马酸还原酶(EC 1.3.1.6)，任选地，所述可溶的富马酸还原酶的3’端乙醛酸循环体定位肽被部分或全部截短，(ii)丙酮酸羧化酶(EC 6.4.1.1)，(iii)富马酸酶(EC 4.2.1.2)，任选地，所述富马酸酶的5’端线粒体定位肽被部分或全部截短，和(iv)丁二酸转运蛋白，
    优选地，所述NADPH依赖型苹果酸脱氢酶来自植物，更优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科或苋科植物、或来自眼虫属、或热杆菌属，更优选高粱(Sorghum bicolor)、玉米(Zea mays)、甘蔗(Saccharum officinarum)、豌豆(Pisum sativum)、鹰嘴豆(Cicer arietinum)、菠菜(Spinacia oleracea)、小眼虫(Euglena gracilis)或热自养甲烷热杆菌(Methanothermobacter thermautotrophicus)，
    更优选地，所述NADPH依赖型苹果酸脱氢酶是来自高粱的NADPH依赖型苹果酸脱氢酶，
    优选地，所述可溶的富马酸还原酶来自酿酒酵母、布氏锥虫、墨西哥利什曼原虫或克氏锥虫，更优选布氏锥虫；
    优选地，所述丁二酸转运蛋白选自SpMAE1蛋白、AnDCT-02蛋白、EcDcuB和EcDcuC蛋白，更优选SpMAE1蛋白；
    优选地，所述丙酮酸羧化酶来自米曲霉或库德里阿兹威毕赤酵母，更优选米曲霉。
  2. 权利要求1的经遗传改造的丁二酸生产酵母菌株，其还具有降低活性的或失活的：
    (i)丙酮酸脱羧酶(EC 4.1.1.43)，和/或
    (ii)NAD依赖性3-磷酸甘油脱氢酶(EC 1.1.1.8)，和/或
    (iii)5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)，和/或
    (iv)单羧酸透性酶，和/或
    (v)二羧酸转运蛋白，和/或
    (vi)乙醇脱氢酶1(EC 1.1.1.1)，和/或
    (vii)草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶(EC 4.1.3.17或4.1.1.112)。
  3. 权利要求1或2的经遗传改造的丁二酸生产酵母菌株，其具有：
    (i)过表达的编码NADPH依赖型苹果酸脱氢酶的核酸序列，优选地，所述编码NADPH依赖型苹果酸脱氢酶的核酸序列包含SEQ ID NO：1所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有NADPH依赖型苹果酸脱氢酶活性的氨基酸序列的核苷酸序列，和/或
    (ii)过表达的编码可溶的富马酸还原酶的核酸序列，优选地，所述编码可溶的富马酸还原酶的核酸序列包含SEQ ID NO：3-5任一所示序列或其简并序列，或与其具有 至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有可溶的富马酸还原酶活性的氨基酸序列的核苷酸序列，和/或
    (iii)过表达的编码丁二酸转运蛋白的核酸序列，优选地，所述编码丁二酸转运蛋白的核酸序列包含SEQ ID NO：2所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丁二酸转运蛋白活性的氨基酸序列的核苷酸序列，和/或
    (iv)过表达的编码丙酮酸羧化酶的核酸序列，优选地，所述编码丙酮酸羧化酶的核酸序列包含如SEQ ID NO：6或7所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丙酮酸羧化酶活性的氨基酸序列的核苷酸序列，和/或
    (v)过表达的编码富马酸酶的核酸序列，优选地，所述编码富马酸酶的核酸序列包含如SEQ ID NO：8所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有磷酸烯醇式丙酮酸羧化酶活性的氨基酸序列的核苷酸序列，和/或
    (vi)内源编码丙酮酸脱羧酶的基因是被敲除的，和/或
    (vii)内源编码NAD依赖性3-磷酸甘油脱氢酶的基因是被敲除的，和/或
    (viii)内源编码5’-磷酸乳清苷脱羧酶的基因是被敲除的，和/或
    (ix)内源编码单羧酸透性酶的基因是被敲除的，和/或
    (x)内源编码二羧酸转运蛋白的基因是被敲除的，和/或
    (xi)内源编码乙醇脱氢酶1的基因是被敲除的，和/或
    (xii)内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因是被敲除的。
  4. 权利要求1-3任一项的经遗传改造的丁二酸生产酵母菌株，其中，在所述经遗传改造的丁二酸生产酵母菌株中，
    (a)编码NADPH依赖型苹果酸脱氢酶的核酸序列以及至少一种如下核酸序列是过表达的：
    编码可溶的富马酸还原酶的核酸序列；
    编码丁二酸转运蛋白的核酸序列；
    编码丙酮酸羧化酶的核酸序列；和
    编码富马酸酶的核酸序列，
    (b)编码NADPH依赖型苹果酸脱氢酶的核酸序列是过表达的，以及内源编码丙酮酸脱羧酶的基因和/或内源编码NAD依赖性3-磷酸甘油脱氢酶的基因是被敲除的，
    (c)编码NADPH依赖型苹果酸脱氢酶的核酸序列、编码丙酮酸羧化酶的核酸序列、编码可溶的富马酸还原酶的核酸序列和编码丁二酸转运蛋白的核酸序列是过表达的，以及内源编码丙酮酸脱羧酶的基因和/或内源编码NAD依赖性3-磷酸甘油脱氢酶的基因是被敲除的；
    (d)编码NADPH依赖型苹果酸脱氢酶的核酸序列、编码丙酮酸羧化酶的核酸序列、编码可溶的富马酸还原酶的核酸序列、编码富马酸酶的核酸序列和编码丁二酸转运蛋白的核酸序列是过表达的，以及内源编码丙酮酸脱羧酶的基因和/或内源编码NAD依赖性3-磷酸甘油脱氢酶的基因是被敲除的，
    优选地，所述编码NADPH依赖型苹果酸脱氢酶的核酸序列是编码高粱NADPH依赖 型苹果酸脱氢酶的核酸序列，更优选包含SEQ ID NO：1所示序列或其简并序列，
    优选地，所述编码可溶的富马酸还原酶的核酸序列是编码酿酒酵母、布氏锥虫、墨西哥利什曼原虫或克氏锥虫的可溶的富马酸还原酶的核酸序列，更优选包含SEQ ID NO：3-5任一所示序列或其简并序列，
    优选地，所述编码丁二酸转运蛋白的核酸序列是编码SpMAE1蛋白的核酸序列，更优选包含SEQ ID NO：2所示序列或其简并序列；
    优选地，所述编码丙酮酸羧化酶的核酸序列编码来自米曲霉或库德里阿兹威毕赤酵母的丙酮酸羧化酶，更优选地，包含如SEQ ID NO：6或7所示序列或其简并序列。
  5. 权利要求1-4任一项的经遗传改造的丁二酸生产酵母菌株，其中所述丁二酸生产酵母菌株选自毕赤酵母属(Pichia)、红酵母属(Rhodotroula)、酵母属(Saccharomyces)、耶氏酵母属(Yarrowia)、鲁氏接合酵母属(Zygosaccharomyces)、球拟酵母属(Torulopsis)、假丝酵母属(Candida)，优选选自毕赤酵母属、酵母属和耶氏酵母属，更优选为库德里阿兹威毕赤酵母(Pichia kudriavzevii)、酿酒酵母(Saccharomyces cerevisiae)或解脂耶氏酵母(Yarrowia lipolytica)，例如保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)、保藏号为CGMCC No.20885的库德里阿兹威毕赤酵母。
  6. 一种产生经遗传改造的丁二酸生产酵母菌株的方法，包括赋予所述菌株或增强所述菌株中的NADPH依赖型苹果酸脱氢酶(EC 1.1.1.82)活性，任选地还包括赋予或增强至少一种如下活性：(i)可溶的富马酸还原酶活性，任选地，所述可溶的富马酸还原酶游离存在细胞质中，(ii)丙酮酸羧化酶(EC 6.4.1.1)活性，(iii)富马酸酶(EC 4.2.1.2)活性，任选地，所述富马酸酶游离存在细胞质中，和(iv)丁二酸转运蛋白活性，
    优选地，所述可溶的富马酸还原酶来自酿酒酵母、布氏锥虫、墨西哥利什曼原虫或克氏锥虫；
    优选地，所述丁二酸转运蛋白选自SpMAE1蛋白、AnDCT-02蛋白、EcDcuB和EcDcuC蛋白；
    优选地，所述丙酮酸羧化酶来自米曲霉或库德里阿兹威毕赤酵母，
    优选地，所述NADPH依赖型苹果酸脱氢酶来自植物，更优选C4植物，更优选禾本科、莎草科、菊科、大戟科、藜科、马齿苋科或苋科植物、或来自眼虫属、或热杆菌属，更优选高粱、玉米、甘蔗、豌豆、鹰嘴豆、菠菜、小眼虫或热自养甲烷热杆菌，
    更优选地，所述NADPH依赖型苹果酸脱氢酶是来自高粱的NADPH依赖型苹果酸脱氢酶。
  7. 权利要求6的方法，还包括弱化或失活所述菌株中的：
    (i)丙酮酸脱羧酶(EC 4.1.1.43)，和/或
    (ii)NAD依赖性3-磷酸甘油脱氢酶(EC 1.1.1.8)，和/或
    (iii)5’-磷酸乳清苷脱羧酶(EC 4.1.1.23)，和/或
    (iv)单羧酸透性酶，和/或
    (v)乙醇脱氢酶1(EC 1.1.1.1)，和/或
    (vi)二羧酸转运蛋白，和/或
    (vii)草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶(EC 4.1.3.17或4.1.1.112)。
  8. 权利要求6或7的方法，包括：在所述丁二酸生产酵母菌株中，
    (i)过表达编码NADPH依赖型苹果酸脱氢酶的核酸序列，优选地，所述编码NADPH依赖型苹果酸脱氢酶的核酸序列包含SEQ ID NO：1所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有NADPH依赖型苹果酸脱氢酶活性的氨基酸序列的核苷酸序列，和/或
    (ii)过表达编码可溶的富马酸还原酶的核酸序列，优选地，所述编码可溶的富马酸还原酶的核酸序列包含SEQ ID NO：3-5任一所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有可溶的富马酸还原酶活性的氨基酸序列的核苷酸序列，和/或
    (iii)过表达编码丁二酸转运蛋白的核酸序列，优选地，所述编码丁二酸转运蛋白的核酸序列包含SEQ ID NO：2所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丁二酸转运蛋白活性的氨基酸序列的核苷酸序列，和/或
    (iv)过表达编码丙酮酸羧化酶的核酸序列，优选地，所述编码丙酮酸羧化酶的核酸序列包含如SEQ ID NO：6或7所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有丙酮酸羧化酶活性的氨基酸序列的核苷酸序列，和/或
    (v)过表达编码富马酸酶的核酸序列，优选地，所述编码富马酸酶的核酸序列包含如SEQ ID NO：8所示序列或其简并序列，或与其具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高相同性且编码具有磷酸烯醇式丙酮酸羧化酶活性的氨基酸序列的核苷酸序列，和/或
    (vi)敲除内源编码丙酮酸脱羧酶的基因，和/或
    (vii)敲除内源编码NAD依赖性3-磷酸甘油脱氢酶的基因，和/或
    (viii)敲除内源编码5’-磷酸乳清苷脱羧酶的基因，和/或
    (ix)敲除内源编码单羧酸透性酶的基因，和/或
    (x)敲除内源编码二羧酸转运蛋白的基因，和/或
    (xi)敲除内源编码乙醇脱氢酶1的基因，和/或
    (xii)敲除内源编码草酰乙酸脱羧和3-羟基-3-甲基戊二酸醛缩双功能酶的基因。
  9. 权利要求6-8任一项的方法，包括：在所述丁二酸生产酵母菌株中，
    (a)过表达编码NADPH依赖型苹果酸脱氢酶的核酸序列以及至少一种如下核酸序列：
    编码可溶的富马酸还原酶的核酸序列；
    编码丁二酸转运蛋白的核酸序列；
    编码丙酮酸羧化酶的核酸序列；和
    编码富马酸酶的核酸序列，
    (b)过表达编码NADPH依赖型苹果酸脱氢酶的核酸序列，以及敲除内源编码丙酮酸脱羧酶的基因和/或内源编码NAD依赖性3-磷酸甘油脱氢酶的基因，
    (c)过表达编码NADPH依赖型苹果酸脱氢酶的核酸序列、编码丙酮酸羧化酶的核酸序列、编码可溶的富马酸还原酶的核酸序列和编码丁二酸转运蛋白的核酸序列，以及敲除内源编码丙酮酸脱羧酶的基因和/或内源编码NAD依赖性3-磷酸甘油脱氢酶的基因；
    (d)过表达编码NADPH依赖型苹果酸脱氢酶的核酸序列、编码丙酮酸羧化酶的核酸序列、编码可溶的富马酸还原酶的核酸序列、编码富马酸酶的核酸序列和编码丁二酸转运蛋白的核酸序列，以及敲除内源编码丙酮酸脱羧酶的基因和/或内源编码NAD依赖性3-磷酸甘油脱氢酶的基因，
    优选地，所述NADPH依赖型苹果酸脱氢酶的核酸序列是编码高粱NADPH依赖型苹果酸脱氢酶的核酸序列，更优选包含SEQ ID NO：1所示序列或其简并序列，
    优选地，所述编码可溶的富马酸还原酶的核酸序列是编码酿酒酵母、布氏锥虫、墨西哥利什曼原虫或克氏锥虫的可溶的富马酸还原酶的核酸序列，更优选包含SEQ ID NO：3-5任一所示序列或其简并序列，
    优选地，所述编码丁二酸转运蛋白的核酸序列是编码SpMAE1蛋白的核酸序列，更优选包含SEQ ID NO：2所示序列或其简并序列；
    优选地，所述编码丙酮酸羧化酶的核酸序列编码来自米曲霉或库德里阿兹威毕赤酵母的丙酮酸羧化酶，更优选地，包含如SEQ ID NO：6或7所示序列或其简并序列。
  10. 权利要求6-9任一项的方法，其中所述丁二酸生产酵母菌株选自毕赤酵母属、红酵母属、酵母属、耶氏酵母属、鲁氏接合酵母属、球拟酵母属、假丝酵母属，优选选自毕赤酵母属、酵母属和耶氏酵母属，更优选为库德里阿兹威毕赤酵母、酿酒酵母或解脂耶氏酵母，例如保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)、保藏号为CGMCC No.20885的库德里阿兹威毕赤酵母。
  11. 一种生产丁二酸的方法，包括培养权利要求1-5任一项的经遗传改造的丁二酸生产酵母菌株或者根据权利要求6-10任一项的方法制备的经遗传改造的丁二酸生产酵母菌株，优选在pH<3.5更优选1.5-3.5的范围内和/或不加或较少添加中和剂的条件下进行培养，任选包括分离纯化产生的丁二酸。
  12. 权利要求1-5任一项的经遗传改造的丁二酸生产酵母菌株或者根据权利要求6-10任一项的方法制备的经遗传改造的丁二酸生产酵母菌株在生产丁二酸、优选在pH<3.5更优选1.5-3.5的范围内和/或不加或较少添加中和剂的条件下生产丁二酸中的应用。
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