An acid-tolerant yeast strain with high succinic acid yield, a construction method and application thereof

Abstract

The present invention provides a genetically engineered succinic acid producing yeast strain having or having enhanced NADPH-dependent malate dehydrogenase (EC 1.1.1.82) activity, optionally further having or having enhanced activity of at least one of (i) soluble fumarate reductase (EC 1.3.1.6), (ii) pyruvate carboxylase (EC 6.4.1.1), (iii) fumarase (EC 4.2.1.2), and (iv) succinate transporter, methods of making the same, methods of using the same to produce succinic acid, and applications thereof.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a novel acid-resistant yeast strain for efficient succinic acid production, its construction method, and its application. Background Technology

[0002] Succinic acid, also known as disuccinic acid, is an intermediate product of the tricarboxylic acid cycle in organisms and an important organic chemical raw material (Chae TU, Ahn JH, Ko YS, et al. Metabolic engineering for the production of dicarboxylic acids and diamines[J]. Metabolic Engineering, 2019). Succinic acid is widely used in the food, chemical, agricultural, and pharmaceutical industries.

[0003] The traditional production method of succinic acid is the chemical method, which mainly uses non-renewable petroleum resources as raw materials for synthesis, such as paraffin oxidation, maleic anhydride hydrogenation, and acrylic acid carbonyl synthesis. Chemical synthesis methods have disadvantages such as high energy consumption and high pollution, making it difficult to meet the requirements of green manufacturing. In contrast, the bio-fermentation method for producing succinic acid 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 output of succinic acid produced by the bio-method will reach 600,000 tons in 2020, with a market value of US$539 million (Hyohak Song, Sang Yup Lee. Production of Succinic Acid by Bacterial Fermentation[J]. Enzyme and Microbial Technology,2006, 39(3):352-361.).

[0004] Currently, the microbial strains used to synthesize succinic acid are mainly divided into two categories: the first category is fungi and yeasts, mainly including: Aspergillus niger (… Aspergillus niger Aspergillus fumigatus ( Aspergillus fumigatus ), snow-white silk clothes mold ( Byssochlamys nivea ), Penicillium wanensis ( Paecilomyces varioti ), brewer's yeast ( Saccharomyces cerevisiae The second category is bacteria, including succinate-producing actinomycetes (…). Actinobacillus succinogenes ), succinic acid-producing anaerobic spirochetes ( Anaerobiospirillum succiniciproducens ), Mansorobacterium succinate ( Mannheimia succiniciproducens Bacteroides fragilis ( Bacteroides fragilis Naturally produced strains such as *Corynebacterium glutamicum* and *Corynebacterium glutamicum* (… Corynebacterium glutamicum ) and Escherichia coli (Escherichia coli Non-natural producing strains such as succinic acid (Ahn JH, Jang YS, Lee SY. Production of succinic acid by metabolically engineered microorganisms [J]. Current Opinion in Biotechnology, 2016, 42: 54-66) were also studied. Filamentous fungi suffer from slow growth, difficulty in genetic modification, poor fermentation homogeneity, and easy mycelial clumping. Bacterial fermentation for succinic acid production requires the addition of large amounts of neutralizing agents to maintain cell growth, increasing the cost of separation and purification, and posing potential contamination problems. Yeast has good acid tolerance and can grow normally at pH below 3.0, where succinic acid is mostly in free molecular form, which can significantly reduce the cost of subsequent separation and purification. Summary of the Invention

[0005] This invention uses an acid-resistant yeast, *Pichia pastoris*, isolated from the peel of wild fruits in Yunnan. Pichia kudriavzevii Starting with strain CY902 (deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 20885, deposited on October 14, 2020), metabolic engineering was performed to achieve efficient succinic acid production. Specifically, the modified strain can achieve efficient succinic acid production under low pH conditions with little or no addition of neutralizing agents during fermentation.

[0006] In one aspect, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, and optionally also having or enhanced at least one of the following activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase activity, (iii) fumarate enzyme activity, and (iv) succinic acid transporter activity.

[0007] Preferably, the NDAPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, and Amaranthaceae families, or from Euglena or Thermobacterium genera, more preferably from sorghum ( Sorghum bicolor ),corn( Zea mays ),sugar cane( Saccharum officinarum ),pea( Pisum sativum ), chickpeas ( Cicer arietinum ),spinach( Spinacia oleracea ), small worm ( Euglena gracilis ) or thermoautotrophic methanotherapeutic bacteria ( Methanothermobacter thermautotrophicus).

[0008] In one embodiment, the genetically modified succinic acid-producing yeast strain also possesses reduced-activity or inactivated pyruvate decarboxylase and / or NAD-dependent glycerol-3-phosphate dehydrogenase.

[0009] In one aspect, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain, comprising conferring or enhancing the NADPH-dependent malate dehydrogenase activity on the strain, optionally further comprising conferring or enhancing at least one of the following activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase (EC 6.4.1.1) activity, (iii) fumarate enzyme (EC 4.2.1.2) activity, and (iv) succinic acid transporter activity. Preferably, the NADPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, and Amaranthaceae families, or from Euglena and Thermobacterium genera, more preferably sorghum, maize, sugarcane, pea, chickpea, spinach, Euglena, or thermoautotrophic methanotherapeutic bacteria.

[0010] In one embodiment, the method further includes weakening or inactivating pyruvate decarboxylase and / or NAD-dependent glycerol-3-phosphate dehydrogenase in the strain.

[0011] In one aspect, the present invention provides a method for producing succinic acid, comprising (preferably under conditions of pH < 3.5, for example, in the range of 1.5-3.5 and / or with little or no addition of a neutralizing agent) culturing the genetically modified succinic acid-producing yeast strain of the present invention and / or obtaining the genetically modified succinic acid-producing yeast strain by the method of the present invention for producing the genetically modified succinic acid-producing yeast strain.

[0012] In one aspect, the present invention provides the use of the genetically modified succinic acid-producing yeast strain described herein and / or the genetically modified succinic acid-producing yeast strain obtained by the method described herein for producing the genetically modified succinic acid-producing yeast strain in the production of succinic acid (preferably in a pH range of <3.5, for example, 1.5-3.5 and / or under conditions of little or no addition of a neutralizing agent). Detailed Implementation

[0013] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY 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).

[0014] As used herein, "genetically modified" refers to a strain that has been artificially altered through biological means, exhibiting one or more changes compared to the original strain before modification, such as gene deletion, amplification, or mutation, thereby possessing altered biological properties, such as improved production performance. As used herein, the original strain can be a natural strain to which the genetic modification is to be performed or a strain with other genetic modifications.

[0015] As used herein, succinic acid-producing yeast strains refer to yeast strains that, under suitable conditions (e.g., through fermentation), can produce succinic acid and secrete it into an extracellular medium. Succinic acid-producing yeast strains possess proteins that can transport succinic acid to the extracellular space, thus allowing the succinic acid to be secreted after production. Suitable succinic acid transporters for a given yeast strain are known in the art, for example, but not limited to, *Schizosaccharomyces cerevisiae*. Schizosaccharomyces pombe The dicarboxylic acid transporter SpMAE1 and Aspergillus niger ( Aspergillus niger AnDCT-02, a dicarboxylic acid transporter.

[0016] Yeasts known in the art for the production of succinic acid include, for example, but not limited to, the genus *Zygosaccharomyces* (…). Zygosaccharomyces ), *Gastromycium* ( Torulopsis ), Candida genus ( Candida Pichia pastoris ( ) Pichia ), Rhodotorula genus ( Rhodotroula ), Yeast ( Saccharomyces ), Yersinia ( Yarrowia In one embodiment, the succinic acid-producing yeast strain is a strain of the genus *Pichia*, *Saccharomyces*, or *Yersinia*. In a preferred embodiment, the succinic acid-producing yeast strain is *Pichia kudriozwee* (e.g., strain CICC32244), *Saccharomyces cerevisiae* (e.g., strain BY4742), or *Yersinia lipolytica* (…). Yarrowia lipolytica(e.g. strain Po1g), such as the Kudriazvichi yeast deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 20885.

[0017] As used herein, “having…activity” means having detectable activity compared to a reference strain (e.g., the initial strain or wild-type strain) that does not have that activity.

[0018] As used herein, “enhanced…activity” means 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 more, compared to a reference strain (e.g., the initial strain or the wild-type strain) having that activity.

[0019] The activity of a protein (e.g., an enzyme) can be generated or enhanced by any suitable means known in the art, such as including, but not limited to, expressing or overexpressing (e.g., via a vector such as a plasmid) the corresponding gene encoding the protein in a strain, introducing mutations that result in increased activity of the protein, etc.

[0020] In some embodiments, in the genetically modified succinic acid-producing yeast strains described in this invention, one or more copies of the target gene or its homologous gene may be integrated into the genome (e.g., through homologous recombination), optionally at any site in the genome (provided such integration does not significantly negatively impact the growth and production of the strain), for example, one copy of any gene in the genome may be replaced by one or more copies of the target gene or its homologous gene. Those skilled in the art know how to integrate transgenes and select strains that have integrated transgenes.

[0021] As used herein, “reduced or inactivated” means a reduction 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 95%, at least 99% or more, or even 100%, compared to a reference activity (e.g., the corresponding activity in the initial strain or wild-type strain).

[0022] The activity of a protein (e.g., an enzyme) can be reduced or inactivated by any suitable means known in the art, including, but not limited to, using a weakened or inactivated gene encoding the protein, introducing a mutation that causes a reduction or inactivation of the protein's activity, or using an antagonist or inhibitor of the protein (e.g., an antibody, ligand, etc.).

[0023] As used herein, a “weakened or inactivated gene” means a gene whose activity, such as expression level (when it is a protein-coding gene) or regulatory function (when it is a regulatory element), is reduced 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, compared to a reference (e.g., the corresponding gene in the initial strain or wild-type strain). In the case of a gene encoding a protein, such as an enzyme, “weakened or inactivated gene” also covers a protein whose activity level is reduced compared to the corresponding protein activity level in the initial 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%.

[0024] In this document, the reference may be a wild-type microorganism or a microorganism prior to the desired genetic manipulation (e.g., an initial microorganism used for genetic manipulation to increase gene activity). In this document, parental microorganism and initial microorganism are used interchangeably to refer to the microorganism on which the desired genetic manipulation (e.g., enhancing or weakening gene or protein activity) is performed.

[0025] As used in this article, malate dehydrogenase (EC 1.1.1.82 (NADPH-dependent)) is produced by... MDH The gene encodes malate dehydrogenase, which is involved in the conversion between oxaloacetate and malate. NADPH-dependent malate dehydrogenase is responsible for converting oxaloacetate to malate, a reaction that consumes one molecule of NADPH. Commonly used sources of NADPH-dependent malate dehydrogenase include C4 plants (e.g., grasses, sedges, asteraceae, euphorbia, chenopodiaceae, portulaca, and amaranthaceae), Euglena, and Thermobacterium species, such as sorghum, corn, sugarcane, peas, chickpeas, spinach, Euglena, or thermoautotrophic methanotherapeutic bacteria. As used herein, having or having enhanced NADPH-dependent malate dehydrogenase activity means that the strain has or has increased NADPH-dependent malate dehydrogenase activity catalyzing the conversion of oxaloacetate to malate.

[0026] As used in this article, soluble fumarate reductase (EC 1.3.1.6) was derived from... FRD The gene encodes an enzyme involved in the interconversion between fumarate and succinic acid, responsible for converting fumarate to succinic acid. Commonly used soluble fumarate reductases are derived from yeast and zoostreaks, particularly *Saccharomyces cerevisiae* and *Trypanosoma brevicornu*. Trypanosoma brucei ), Leishmania melanogaster ( Leishmania mexicana Trypanosoma cruzi (), Trypanosoma cruzi () Trypanosoma cruzi(e.g.) As used herein, having or having enhanced fumarate reductase activity means that the strain has or has increased soluble fumarate reductase activity that converts fumarate to succinic acid.

[0027] As used in this article, fumarate (EC 4.2.1.2) is derived from... FUM The gene encodes an enzyme involved in the interconversion of fumarate and malate in the cytoplasm and mitochondria, responsible for converting malate to fumarate. Commonly used sources include *Actinomyces succinate*, *Mannidaria succinate*, *Escherichia coli*, *Pichia pastoris*, and *Rhizopus oryzae*. Rhizopus oryzae (etc.) It is known that a FUM gene exists in *Pichia kudrica*. FUM1 As used herein, having or having enhanced fumarate enzyme activity means that the strain has or has increased fumarate enzyme activity that converts malic acid to fumarate.

[0028] As used in this article, succinate transporter proteins refer to proteins that can transport succinate from inside the cell to outside the cell, including, but not limited to, the SpMAE1 protein of *Schizosaccharomyces cerevisiae* (Uniprot database search number: P50537) and *Aspergillus niger* (…). Aspergillus niger Examples of dicarboxylic acid transporters include AnDCT-02 (NCBI Reference Sequence: XP_001398131.1) and EcDcuB (Gene ID: 948641) and EcDcuC (Gene ID: 945000) from *Escherichia coli*. In this article, "having or having enhanced succinic acid transporter activity" refers to strains possessing or having increased activity in transporting succinic acid to the extracellular space.

[0029] As used in this article, the SpMAE1 protein, a dicarboxylic acid transporter in *Schizosaccharide Spr.*, is derived from... SpMAE1 The SpMAE1 protein, encoded by the gene, is responsible for transporting dicarboxylic acids (DCAs) from the cell to the extracellular space. In this article, having or having enhanced SpMAE1 activity refers to strains exhibiting or having increased activity in transporting DCAs to the extracellular space.

[0030] As used in this article, pyruvate carboxylase (EC 6.4.1.1) is derived from... PYC The gene encodes pyruvate carboxylase, which participates in the interconversion between oxaloacetate and pyruvate during gluconeogenesis, responsible for converting pyruvate and carbon dioxide into oxaloacetate. Commonly used sources of pyruvate carboxylase include fungi, especially yeasts and filamentous fungi, with preferred sources being *Saccharomyces cerevisiae*, *Pichia pastoris*, *Aspergillus oryzae*, and *Kluyveromyces martensii*. Kluyveromyces marxianus ), etc. It is known that *Pichia kudrica* exists in one... PYC Genes, that is PYC1Genes. In this article, having or having enhanced pyruvate carboxylase activity means that the strain has or has increased activity in converting pyruvate to oxaloacetate.

[0031] As used in this article, pyruvate decarboxylase (EC 4.1.1.1) is derived from... PDC The gene encodes pyruvate decarboxylase, which participates in the decarboxylation of pyruvate in the ethanol synthesis pathway. It is known that *Pichia kudrica* contains a... PDC Genes are PDC1 Genes. In this article, reduced or inactivated pyruvate decarboxylase refers to enzymes whose pyruvate decarboxylation activity is reduced or lost.

[0032] As used in this article, NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is derived from... GPD The gene encodes an enzyme involved in the interconversion between glycerol phosphate and glycerol 3-phosphate in the glycerol synthesis pathway. It is known that *Pichia kudrica* contains a... GPD Genes, that is GPD1 Genes. In this article, reduced or inactivated NAD-dependent glycerol-3-phosphate dehydrogenase refers to the enzyme’s reduced or lost activity in the interconversion of glycerol phosphate and glycerol 3-phosphate.

[0033] As used in this article, 5'-orhinophosphate decarboxylase (EC 4.1.1.23) is derived from... URA3 The gene encodes an enzyme involved in the decarboxylation of whey 5-phosphate during pyrimidine synthesis. It is known that *Pichia goudryanazwiica* contains one such enzyme. URA3 Genes. In this article, reduced or inactivated 5'-orotose decarboxylase refers to the enzyme's reduced or lost activity in catalyzing the decarboxylation of 5'-orotose.

[0034] As used in this article, alcohol dehydrogenase 1 (EC 1.1.1.1) is derived from... ADH1 The gene encodes an enzyme involved in the interconversion of acetaldehyde and ethanol in the ethanol synthesis pathway. In this document, reduced-activity or inactivated alcohol dehydrogenase 1 refers to a reduced or lost activity of this enzyme in catalyzing the interconversion of acetaldehyde and ethanol. In one embodiment, the strain... ADH1 Genes are knocked out, for example, through homologous recombination.

[0035] As used in this article, monocarboxylic acid permease (NCBI Reference Sequence XP_029320775.1) was obtained from... MCH4 Gene encoding. In this document, reduced or inactivated monocarboxylic acid permease refers to an enzyme whose catalytic activity is reduced or lost. In one embodiment, the strain contains... MCH4 Genes are knocked out, for example, through homologous recombination.

[0036] As used in this article, JEN2 The gene encodes a dicarboxylic acid transporter, which is involved in the transport of dicarboxylic acids from the culture medium into the cell. Two strains of *Pichia goudrya* are known to exist. JEN2 Gene( JEN2-1 (encoding the polypeptide shown in SEQ ID NO: 14) and JEN2-2 (The polypeptide represented by SEQ ID NO: 15)). In this article, deactivated or inactive dicarboxylic acid transporters are those whose activity in transporting dicarboxylic acids from the culture medium into the cell is reduced or lost.

[0037] As used herein, a neutralizing agent is a reagent 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.

[0038] As used herein, "no or minimal addition of neutralizing agent" means that the amount of neutralizing agent added is 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%, compared to the amount of neutralizing agent added in the fermentation production of succinic acid known in the art. For example, in the method of the present invention, the amount of neutralizing agent added may be 0-30 g / L.

[0039] As used herein, the terms “polypeptide,” “amino acid sequence,” “peptide,” and “protein” are used interchangeably to refer to an amino acid chain of any length that may contain modified amino acids and / or may be interrupted by non-amino acid components. The term also covers amino acid chains that have been modified by natural or artificial means; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component.

[0040] As used herein, the terms “gene,” “nucleic acid sequence,” “polynucleotide,” and “nucleotide sequence” are used interchangeably and refer to nucleotide chains, including DNA and RNA. “Gene expression” refers to the transcription of a DNA region operatively linked to appropriate regulatory regions, particularly promoters, into biologically active RNA, and the translation of RNA into biologically active proteins or peptides.

[0041] As used in this article, a degenerate sequence is a nucleotide sequence that encodes the same amino acid sequence as a specified sequence but has a different nucleotide sequence due to the degeneracy of the genetic codon.

[0042] As used herein, the terms “homology”, “sequence identity”, etc., are used interchangeably. Sequence identity can be detected by comparing the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide, for example, by using a standard alignment algorithm program with a default gap penalty specified 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). For example, the percentage of similarity between nucleic acid molecules can be determined, for instance, by comparing sequence information using the GAP computer program (e.g. Needleman et al. J. Mol. Biol. 48: 443 (1970), revised by Smith and Waterman (Adv. Appl. Math. 2: 482 (1981)). In short, the GAP program defines similarity based on the number of similarly arranged symbols (i.e., nucleotides) compared to the total number of symbols in the shorter sequence of the two sequences.

[0043] As used in this article, Pk2365 The gene encodes a bifunctional enzyme for oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation (EC 4.1.3.17 or 4.1.1.112). In this text, reduced or inactivated oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation bifunctional enzymes refer to enzymes whose activity in catalyzing oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation is reduced or lost.

[0044] In one aspect, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, and optionally also having or enhanced at least one of the following activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase activity, (iii) fumarate enzyme activity, and (iv) succinic acid transporter activity. The term "at least one" includes any one, two, three, or all four activities selected therefrom.

[0045] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity and soluble fumarate reductase activity.

[0046] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity and pyruvate carboxylase activity.

[0047] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity and fumarate enzyme activity.

[0048] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity and succinic acid transporter activity.

[0049] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, and pyruvate carboxylase activity.

[0050] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, and fumarate enzyme activity.

[0051] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, and succinic acid transporter activity.

[0052] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, pyruvate carboxylase activity, and fumarate enzyme activity.

[0053] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, pyruvate carboxylase activity, and succinic acid transporter activity.

[0054] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, fumarate enzyme activity, and succinic acid transporter activity.

[0055] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, pyruvate carboxylase activity, and fumarate enzyme activity.

[0056] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, pyruvate carboxylase activity, and succinic acid transporter activity.

[0057] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, fumarate enzyme activity, and succinic acid transporter activity.

[0058] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, pyruvate carboxylase activity, fumarate enzyme activity, and succinic acid transporter activity.

[0059] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having or enhanced NADPH-dependent malate dehydrogenase activity, soluble fumarate reductase activity, pyruvate carboxylase activity, fumarate enzyme activity, and succinic acid transporter activity.

[0060] In one embodiment, having or having enhanced activity is achieved by expressing or overexpressing the corresponding encoding gene in the strain. Thus, in one embodiment, the genetically modified succinic acid-producing yeast strain expresses or overexpresses the gene encoding NADPH-dependent malate dehydrogenase.

[0061] In one embodiment, the NADPH-dependent malate dehydrogenase is derived from plants (preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, and Amaranthaceae families), Euglena, or Thermomycium, preferably from sorghum, corn, sugarcane, peas, chickpeas, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria, more preferably from sorghum.

[0062] In one embodiment, the enzyme is expressed or overexpressed in the genetically modified succinic acid-producing yeast strain, encoding NADPH-dependent malate dehydrogenase. MDH Genes that produce or increase NADPH-dependent malate dehydrogenase activity, preferably, genes encoding said NADPH-dependent malate dehydrogenase. MDH The genes are preferably derived from plants, preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae families, or from Euglena or Thermomycium, more preferably from sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria, more preferably from sorghum.

[0063] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity.

[0064] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH Genes are incorporated into the genome of genetically modified succinic acid-producing yeasts (e.g., Pichia kudrica), for example in... Pk2365 Location of the locus. The gene encoding NADPH-dependent malate dehydrogenase... MDH Genes can be placed in appropriate promoters (e.g.) FBA1 Gene promoters (e.g., shown in SEQ ID NO: 17) and / or terminators (e.g.) INO1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 18).

[0065] In a further embodiment, the genetically modified succinic acid-producing yeast strain also possesses or has enhanced soluble fumarate reductase. In one embodiment, the soluble fumarate reductase is derived from yeasts and Zoysiata, such as, but not limited to, *Saccharomyces cerevisiae*, *Trypanosoma brevicornu*, *Leishmania megacephala*, and *Trypanosoma krusei*.

[0066] In one embodiment, soluble fumarate reductase activity is generated or increased by expressing or overexpressing a gene encoding soluble fumarate reductase in the genetically modified succinic acid-producing yeast strain. Preferably, the gene encoding the soluble fumarate reductase is derived from yeasts and Zoysiata, such as, but not limited to, Saccharomyces cerevisiae, Trypanosoma brevicornu, Leishmania mexicana, and Trypanosoma krusei.

[0067] In one embodiment, the 3'-terminal glyoxylate cycling peptide of the soluble fumarate reductase is partially or completely truncated so that it exists freely in the cytoplasm.

[0068] In one embodiment, the gene encoding soluble fumarate reductase comprises any of the sequences or degenerate sequences shown in SEQ ID NO: 3-5, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with them and encoding an amino acid sequence having soluble fumarate reductase activity.

[0069] In one embodiment, the gene encoding soluble fumarate reductase is incorporated into the genome of a genetically modified succinic acid-producing yeast (e.g., *Pichia kudrica*), for example in... ADH1 The location of the gene locus. The gene encoding soluble fumarate reductase can be placed in a suitable promoter (e.g., ADH1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) ADH1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0070] In a further embodiment, the genetically modified succinic acid-producing yeast strain also has or has enhanced succinic acid transporter activity.

[0071] In one embodiment, the succinic acid transporter is selected from SpMAE1, AnDCT-02, EcDcuB, and EcDcuC proteins. In one embodiment, succinic acid transporter activity is generated or increased by expressing or overexpressing a gene encoding a succinic acid transporter such as SpMAE1 in the genetically modified succinic acid-producing yeast strain. In one embodiment, the succinic acid transporter activity is achieved through expression or overexpression of… SpMAE1 Genes produce or enhance this.

[0072] In one implementation, the SpMAE1 The gene comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence (e.g., from *Schizosaccharomyces cerevisiae*) with succinic acid transporter activity. Optionally, the... SpMAE1 Genes are incorporated into the genome of genetically modified succinic acid-producing yeast strains (e.g., Pichia kudrica), for example in... MCH4 The location of the gene locus. (The following is a separate, unrelated sentence.) SpMAE1 Genes can be placed in appropriate promoters (e.g.) TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0073] In a further embodiment, the genetically modified succinic acid-producing yeast strain also possesses or has enhanced pyruvate carboxylase activity. This pyruvate carboxylase activity can be generated or enhanced by expressing or overexpressing a gene encoding pyruvate carboxylase. The pyruvate carboxylase can be derived from fungi, particularly yeasts and filamentous fungi, preferably *Saccharomyces cerevisiae*, *Pichia pastoris*, *Aspergillus oryzae*, *Kluyveromyces martensii*, etc. In one embodiment, the gene encoding pyruvate carboxylase can be selected from *Aspergillus oryzae*. PYC Genes and Kudria zweipichia yeast PYC1 Gene.

[0074] In one embodiment, the pyruvate carboxylase comprises an amino acid sequence encoded by the sequence shown in SEQ ID NO: 6 or 7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having pyruvate carboxylase activity.

[0075] In one embodiment, the gene encoding pyruvate carboxylase comprises a sequence as shown in SEQ ID NO: 6 or 7 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity.

[0076] Optionally, the gene encoding pyruvate carboxylase is incorporated into the genome of a genetically modified succinic acid-producing yeast (e.g., Pichia kudrica), for example in... JEN2-1 The location of the gene locus. The gene encoding pyruvate carboxylase can be placed in a suitable promoter (e.g., TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0077] In a further embodiment, the genetically modified succinic acid-producing yeast strain also possesses or has enhanced fumarate activity. This fumarate activity can be generated or enhanced by expressing or overexpressing a gene encoding fumarate. In one embodiment, the genetically modified succinic acid-producing yeast strain expresses or overexpresses a gene encoding fumarate. In one embodiment, the fumarate is derived from, for example, but not limited to, *Actinomyces succinate*, *Succinate mannheim*, *Escherichia coli*, *Pichia kudrifolia*, and *Rhizopus oryzae*.

[0078] In one embodiment, the fumarate enzyme comprises an amino acid sequence as shown in SEQ ID NO: 95 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same and having fumarate enzyme activity.

[0079] In one embodiment, the 5' mitochondrial-localizing peptide of the fumarate is partially or completely truncated so that it cannot be localized to the mitochondria and instead exists freely in the cytoplasm.

[0080] In one embodiment, the gene encoding fumarate enzyme comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and having fumarate enzyme activity. Optionally, the gene encoding fumarate enzyme is incorporated into the genome of a genetically modified succinic acid-producing yeast (e.g., Pichia kudrica), for example in... PDC1 The location of the gene locus. The gene encoding fumarate can be placed in a suitable promoter (e.g., TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0081] The gene to be expressed or overexpressed may be integrated into a suitable location in the strain genome, provided that such integration does not negatively affect the growth, reproduction, and / or production performance of the strain. For example, it may be integrated into any one or more genomic locations encoding the following proteins: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhinophosphate 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-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

[0082] In a further embodiment, the genetically modified succinic acid-producing yeast strain also has at least one of the following reduced or inactivated activities: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhinophosphate 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-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

[0083] In one embodiment, the genetically modified succinic acid-producing yeast strain has a reduced-activity or inactivated pyruvate decarboxylase. The reduced-activity or inactivated pyruvate decarboxylase can be achieved by weakening or inactivating a gene encoding pyruvate decarboxylase in the strain. Therefore, in one embodiment, the genetically modified succinic acid-producing yeast strain has a weakened or inactivated gene encoding pyruvate decarboxylase. In one embodiment, the gene encoding pyruvate decarboxylase in the genetically modified succinic acid-producing yeast strain is, for example... PDC1 The gene was knocked out.

[0084] In one embodiment, the pyruvate decarboxylase comprises an amino acid sequence as 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 of the same sequence and having pyruvate decarboxylase activity.

[0085] In one embodiment, the gene encoding pyruvate decarboxylase encodes a protein as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with pyruvate decarboxylase activity.

[0086] In one embodiment, the genetically modified succinic acid-producing yeast strain also possesses a reduced-activity or inactivated NAD-dependent glycerol-3-phosphate dehydrogenase. In one embodiment, the genetically modified succinic acid-producing yeast strain possesses a weakened or inactivated gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase. In one embodiment, the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase in the genetically modified succinic acid-producing yeast strain (e.g., Pichia kudriazine) is knocked out.

[0087] In one embodiment, the NAD-dependent glycerol-3-phosphate dehydrogenase comprises an amino acid sequence as shown in SEQ ID NO: 11 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having NAD-dependent glycerol-3-phosphate dehydrogenase activity.

[0088] In one embodiment, the gene encoding the NAD-dependent glycerol-3-phosphate dehydrogenase encodes, for example, the protein shown in SEQ ID NO: 11 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having NAD-dependent glycerol-3-phosphate dehydrogenase activity.

[0089] In a further embodiment, the genetically modified succinic acid-producing yeast strain also possesses a reduced-activity or inactivated alcohol dehydrogenase 1. In one embodiment, the genetically modified succinic acid-producing yeast strain possesses a weakened or inactivated gene encoding alcohol dehydrogenase 1. In one embodiment, the gene encoding alcohol dehydrogenase 1 in the genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*) is knocked out.

[0090] In one embodiment, the alcohol dehydrogenase 1 comprises an amino acid sequence as shown in SEQ ID NO: 12 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having alcohol dehydrogenase 1 activity.

[0091] In one embodiment, the gene encoding the alcohol dehydrogenase 1 encodes, for example, the protein shown in SEQ ID NO: 12 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having alcohol dehydrogenase 1 activity.

[0092] In a further embodiment, the genetically modified succinic acid-producing yeast strain also possesses a reduced-activity or inactivated 5'-phospho-orhodoside decarboxylase (EC 4.1.1.23), a dicarboxylic acid transporter, oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldolase bifunctional enzyme and / or monocarboxylic acid permease.

[0093] In a further embodiment, the genetically modified succinic acid-producing yeast strain contains a gene encoding 5'-orhodophosphate decarboxylase and / or a gene encoding a dicarboxylic acid transporter (e.g.,JEN2 Genes such as JEN2-1 Gene, JEN2-2 The gene encoding monocarboxylic acid permease and / or the gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes were knocked out.

[0094] In one embodiment, the genetically modified succinic acid-producing yeast strain also possesses a reduced-activity or inactivated bifunctional enzyme encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde. Specifically, the genetically modified succinic acid-producing yeast strain possesses a weakened or inactivated gene encoding the bifunctional enzyme encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde. Preferably, the gene encoding the bifunctional enzyme encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde in the genetically modified succinic acid-producing yeast strain (e.g., Pichia kudrica) is knocked out.

[0095] In one embodiment, the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme activity.

[0096] In one embodiment, the gene encoding the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme encodes a protein as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme activity.

[0097] In one embodiment, the genetically modified succinic acid-producing yeast strain also possesses a reduced-activity or inactivated 5'-orhinophosphate decarboxylase. Specifically, the genetically modified succinic acid-producing yeast strain possesses a weakened or inactivated gene encoding 5'-orhinophosphate decarboxylase. Preferably, the gene encoding 5'-orhinophosphate decarboxylase in the genetically modified succinic acid-producing yeast strain (e.g., Pichia kudrica) is knocked out.

[0098] In one embodiment, the 5'-orhodophosphoside decarboxylase comprises an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having 5'-orhodophosphoside decarboxylase activity.

[0099] In one embodiment, the gene encoding the 5'-orotose decarboxylase encodes a protein as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having 5'-orotose decarboxylase activity.

[0100] In one embodiment, the genetically modified succinic acid-producing yeast strain also possesses a reduced-activity or inactivated monocarboxylic acid permease. Specifically, the genetically modified succinic acid-producing yeast strain possesses a weakened or inactivated gene encoding the monocarboxylic acid permease. Preferably, the gene encoding the monocarboxylic acid permease in the genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*) is knocked out.

[0101] In one embodiment, the monocarboxylic acid permease comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same and having monocarboxylic acid permease activity.

[0102] In one embodiment, the gene encoding the monocarboxylic acid permease encodes, for example, the protein shown in SEQ ID NO: 16 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having monocarboxylic acid permease activity.

[0103] In one embodiment, the genetically modified succinic acid-producing yeast strain also possesses a reduced-activity or inactivated dicarboxylic acid transporter protein. Specifically, the genetically modified succinic acid-producing yeast strain possesses a weakened or inactivated gene encoding the dicarboxylic acid transporter protein. Preferably, the gene encoding the dicarboxylic acid transporter protein in the genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*) is, for example... JEN2-1 or JEN2-2 The gene was knocked out.

[0104] In one embodiment, the dicarboxylic acid transporter comprises an amino acid sequence as shown in SEQ ID NO: 14 or 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having dicarboxylic acid transporter activity.

[0105] In one embodiment, the gene encoding the dicarboxylic acid transporter encodes a protein as shown in SEQ ID NO: 14 or 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having dicarboxylic acid transporter activity.

[0106] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Optionally, the gene encoding endogenous 5'-phospho-orhodoside decarboxylase and / or the gene encoding an endogenous bifunctional enzyme of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldolase are knocked out. Preferably, the gene... MDH The gene contains the sequence shown in SEQ ID NO: 1 or a degenerate sequence thereof.

[0107] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH The gene, as well as the overexpressed gene encoding soluble fumarate reductase, optionally, the gene endogenously encoding 5'-phospho-orhodoside decarboxylase and / or the gene endogenously encoding alcohol dehydrogenase 1 and / or the gene endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme, are knocked out. Preferably, the gene... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding soluble fumarate reductase contains any of the sequences shown in SEQ ID NO: 3-5 or their degenerate sequences.

[0108] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and overexpressed genes encoding succinate transporters, such as SpMAE1The genes, optionally endogenously encoding 5'-phospholipid decarboxylase and / or endogenously encoding monocarboxylic acid permease and / or endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes, are knocked out. Preferably, the genes are... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the succinic acid transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0109] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH The gene and the overexpressed gene encoding fumarate enzyme, optionally, the gene endogenously encoding 5'-orhodophosphodiesterase and / or the gene endogenously encoding pyruvate decarboxylase and / or the gene endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldolase bifunctional enzyme are knocked out. Preferably, the gene is... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding fumarate contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence.

[0110] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and overexpressed genes encoding pyruvate carboxylase, optionally, endogenous genes encoding 5'-orhinophosphate decarboxylase and / or endogenous genes encoding dicarboxylic acid transporters, for example JEN2-1 or JEN2-2 The gene and / or the endogenous gene encoding the bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation are knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.

[0111] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDHThe gene encoding pyruvate carboxylase, as well as the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase, are knocked out, optionally the endogenous gene encoding orotidine phosphate decarboxylase and / or the endogenous gene encoding a dicarboxylic acid transporter (e.g., JEN2 Genes such as JEN2-1 Gene, JEN2-2 The gene and / or the endogenous gene encoding the bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation are knocked out. Preferably, the gene... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.

[0112] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes, such as those encoding pyruvate carboxylase, soluble fumarate reductase, and succinate transporter proteins. SpMAE1 The genes, as well as the genes endogenously encoding pyruvate decarboxylase and endogenously encoding NAD-dependent glycerol-3-phosphate dehydrogenase, are knocked out. Optionally, the genes endogenously encoding orotidine phosphate decarboxylase and / or the genes endogenously encoding dicarboxylic acid transporters (e.g., JEN2 Genes such as JEN2-1 Gene, JEN2-2 The gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes and / or the gene encoding alcohol dehydrogenase 1 and / or the gene encoding monocarboxylic acid permease are knocked out. Preferably, the gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes and / or the gene encoding alcohol dehydrogenase 1 and / or the gene encoding monocarboxylic acid permease are knocked out. MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence, the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence, or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0113] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding pyruvate carboxylase, soluble fumarate reductase, fumarate enzyme, and succinate transporter, for example...SpMAE1 The genes, as well as the genes endogenously encoding pyruvate decarboxylase and endogenously encoding NAD-dependent glycerol-3-phosphate dehydrogenase, are knocked out. Optionally, the genes endogenously encoding orotidine phosphate decarboxylase and / or the genes endogenously encoding dicarboxylic acid transporters (e.g., JEN2 Genes such as JEN2-1 Gene, JEN2-2 The gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes and / or the gene encoding alcohol dehydrogenase 1 and / or the gene encoding monocarboxylic acid permease are knocked out. Preferably, the gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes and / or the gene encoding alcohol dehydrogenase 1 and / or the gene encoding monocarboxylic acid permease are knocked out. MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence; the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence; the gene encoding fumarate enzyme contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence; or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0114] In one implementation, the JEN2-1 The gene encodes a protein as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having dicarboxylic acid transporter activity.

[0115] In one implementation, the JEN2-2 The gene encodes a protein as shown in SEQ ID NO: 15 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having dicarboxylic acid transporter activity.

[0116] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes, optional, endogenous URA3 Genes and / or endogenous Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or a degenerate sequence thereof.

[0117] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and, optionally, endogenous genes encoding soluble fumarate reductase, as well as overexpressed genes encoding soluble fumarate reductase. URA3 Genes and / or endogenous ADH1 Genes and / or endogenous Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding soluble fumarate reductase contains any of the sequences shown in SEQ ID NO: 3-5 or their degenerate sequences.

[0118] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and overexpressed genes encoding succinate transporters, such as SpMAE1 Genes, optional endogenous URA3 Genes and / or endogenous MCH4 Genes and / or endogenous Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the succinic acid transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0119] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and overexpressed genes encoding fumarate, optionally endogenous URA3 Genes and / or endogenous PDC1 Genes and / or endogenous Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding fumarate contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence.

[0120] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and overexpressed genes encoding pyruvate carboxylase, optionally endogenously URA3 Genes and / or endogenous JEN2-1 or JEN2-2Genes and / or endogenous Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.

[0121] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and genes encoding pyruvate carboxylase, as well as endogenous PDC1 Genes and endogenous GPD1 The gene is knocked out, and the endogenous source is chosen. URA3 Genes and / or endogenous JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes and / or endogenous Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.

[0122] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes, such as those encoding pyruvate carboxylase, soluble fumarate reductase, and succinate transporter proteins. SpMAE1 Genes, and endogenous PDC1 Genes and endogenous GPD1 The gene is knocked out, arbitrarily, endogenously. URA3 Genes and / or endogenous JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes and / or endogenous Pk2365 Genes and / or endogenous ADH1 Genes and / or endogenous MCH4 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence, the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence, or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0123] In one embodiment, the present invention provides a genetically modified *Pichia kudriezvichi* strain, such as CY902, which exhibits enhanced activity of sorghum NADPH-dependent malate dehydrogenase, *Aspergillus oryzae* pyruvate carboxylase, *Trypanosoma brevicornu* soluble fumarate reductase, *Pichia kudriezvichi* fumarate (with the 5' mitochondrial localization peptide truncated), and succinate transporter SpMAE1, while the activities of endogenous pyruvate decarboxylase and endogenous NAD-dependent glycerol-3-phosphate dehydrogenase are eliminated. In one embodiment, the present invention provides a genetically modified *Pichia kudriezvichi* strain, such as CY902, which has overexpressed an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding pyruvate carboxylase, soluble fumarate reductase, fumarate enzyme, and succinate transporter, for example... SpMAE1 Genes, and endogenous PDC1 Genes and endogenous GPD1 The gene is knocked out, arbitrarily, endogenously. URA3 Genes and / or endogenous JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes and / or endogenous Pk2365 Genes and / or endogenous ADH1 Genes and / or endogenous MCH4 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence; the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence; the gene encoding fumarate enzyme contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence; or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0124] In one aspect, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain, comprising conferring or enhancing the activity of NADPH-dependent malate dehydrogenase (EC 1.1.1.82) in the strain.

[0125] As used in this article, “conferring activity” means producing in a genetically modified succinic acid-producing yeast strain an activity that was not present in the initial strain before the genetic modification.

[0126] As used herein, “enhancing…activity” means increasing 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.

[0127] Various methods for conferring or enhancing the desired activity of proteins are known in the art, including, but not limited to, expressing or overexpressing protein-coding genes and mutations or other modifications that increase protein activity.

[0128] As used herein, “overexpression” means an increase in gene expression level relative to the level before genetic manipulation, for example, an increase 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. Methods for overexpressing genes are well known in the art, including, but not limited to, using strong promoters, increasing gene copy number, enhancers, etc. Increasing gene copy number can be achieved, for example, but not limited to, by introducing one or more copies of a foreign or endogenous gene, for example, through an expression vector or integration into the genome.

[0129] As used in this article, "exogenous gene" refers to a gene that comes from another cell or organism, such as a gene from the same species or a different species.

[0130] As used in this article, "endogenous genes" refers to the genes of a cell or organism itself.

[0131] The promoter can be selected from any suitable promoter known in the art, including but not limited to those encoding fructose-1,6-bisphosphate aldolase. FBA1 The promoter of the gene, encoding glyceraldehyde-3-phosphate dehydrogenase TDH3 The promoter of the gene, encoding pyruvate decarboxylase PDC1 Gene promoter, encoding alcohol dehydrogenase 1 ADH1 Gene promoter, encoding 3-phosphoglycerate kinase PGK1 Gene promoters, encoding transcription elongation factors TEF1 Gene promoter, encoding phosphoglycerate mutase GPM1 Gene promoter, encoding triose phosphate isomerase TPI1 Gene promoters and those encoding enolases ENO1 Gene promoters (e.g., shown in SEQ ID NO: 100).

[0132] In one embodiment, the NADPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, and Amaranthaceae families, or from Euglena and Thermobacterium genera, more preferably sorghum, corn, sugarcane, peas, chickpeas, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria, more preferably sorghum.

[0133] In one embodiment, the enzyme is expressed or overexpressed in the genetically modified succinic acid-producing yeast strain, encoding NADPH-dependent malate dehydrogenase. MDH Genes are used to produce or increase NADPH-dependent malate dehydrogenase activity. Preferably, the gene encoding the NADPH-dependent malate dehydrogenase... MDH The genes are preferably derived from plants, C4 plants are preferred, and plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae families are even more preferred, or from Euglena and Thermomycium genera, and sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria are even more preferred, and sorghum is even more preferred.

[0134] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity.

[0135] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH Genes are incorporated into the genome of genetically modified succinic acid-producing yeasts (e.g., Pichia kudrica), for example in... Pk2365 Location of the locus. In one embodiment, the gene encoding NADPH-dependent malate dehydrogenase... MDH Genes are incorporated into the genome of yeast (e.g., Pichia kudrica) via homologous recombination, for example in... Pk2365 Location of the locus. The gene encoding NADPH-dependent malate dehydrogenase... MDH Genes can be placed in appropriate promoters (e.g.) FBA1 Gene promoters (e.g., shown in SEQ ID NO: 17) and / or terminators (e.g.) INO1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 18).

[0136] In a further embodiment, the method further includes conferring or enhancing soluble fumarate reductase activity in the strain.

[0137] In one embodiment, the soluble fumarate reductase is derived from yeast and Zoysiata, such as, but not limited to, Saccharomyces cerevisiae, Trypanosoma brevicornu, Leishmania megacephala, and Trypanosoma clarithii.

[0138] In one embodiment, the 3'-terminal glyoxylate cycling peptide of the soluble fumarate reductase is partially or completely truncated so that it exists freely in the cytoplasm.

[0139] In one embodiment, soluble fumarate reductase activity is generated or increased by expressing or overexpressing a gene encoding soluble fumarate reductase in the genetically modified succinic acid-producing yeast strain. Preferably, the gene encoding the soluble fumarate reductase is derived from yeasts and Zoysiata, such as, but not limited to, Saccharomyces cerevisiae, Trypanosoma brevicornu, Leishmania mexicana, and Trypanosoma krusei.

[0140] In one embodiment, the gene encoding soluble fumarate reductase comprises any of the sequences shown in SEQ ID NO: 3-5 or their degenerate sequences, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with them and encoding an amino acid sequence having soluble fumarate reductase activity.

[0141] In one embodiment, the gene encoding soluble fumarate reductase is incorporated into the genome of a genetically modified succinic acid-producing yeast (e.g., *Pichia kudrica*), for example in... ADH1 The location of the gene locus. The gene encoding soluble fumarate reductase can be placed in a suitable promoter (e.g., ADH1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) ADH1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0142] In a further embodiment, the method further includes conferring or enhancing the activity of succinic acid transporter protein in the yeast strain.

[0143] In one embodiment, the succinic acid transporter is selected from SpMAE1 protein, AnDCT-02 protein, EcDcuB, and EcDcuC protein. In one embodiment, the succinic acid transporter is expressed or overexpressed in the genetically modified succinic acid-producing yeast strain, using a gene encoding the succinic acid transporter (e.g., ...). SpMAE1 Genes can be used to confer or enhance the activity of succinic acid transporters.

[0144] In one implementation, the SpMAE1 The gene comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and encoding an amino acid sequence with succinic acid transporter activity, optionally (e.g., via homologous recombination) incorporated into the genome of a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*), for example in... MCH4 The location of the gene locus. (The following is a description of the gene locus.) SpMAE1 Genes can be placed in appropriate promoters (e.g.) TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0145] In a further embodiment, the method further includes conferring or enhancing fumarate activity in the yeast strain.

[0146] In one embodiment, the 5' mitochondrial-localizing peptide of the fumarate is partially or completely truncated so that it cannot be localized to the mitochondria and instead exists freely in the cytoplasm.

[0147] In one embodiment, fumarate activity is conferred or enhanced by expressing or overexpressing a gene encoding fumarate in the genetically modified succinic acid-producing yeast strain. In one embodiment, the fumarate is derived from, for example, but not limited to, *Actinomyces succinate-producing*, *Succinate-producing Mannheim*, *Escherichia coli*, *Pichia kudrica*, and *Rhizopus oryzae*.

[0148] In one embodiment, the fumarate enzyme comprises an amino acid sequence such as SEQ ID NO: 95 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having fumarate enzyme activity.

[0149] In one embodiment, the gene encoding the fumarate enzyme encodes an amino acid sequence as shown in SEQ ID NO: 95 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having fumarate enzyme activity.

[0150] In one embodiment, the gene encoding the fumarate enzyme comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having fumarate enzyme activity.

[0151] Optionally, the gene encoding the fumarate enzyme (e.g., through homologous recombination) is incorporated into the genome of a genetically modified succinic acid-producing yeast (e.g., *Pichia kudrica*), for example in... PDC1 Location of the gene locus. The gene encoding the fumarate enzyme can be placed in a suitable promoter (e.g., TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0152] In a further embodiment, the method further includes conferring or enhancing pyruvate carboxylase activity in the yeast strain. Preferably, 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, particularly yeasts and filamentous fungi, preferably *Saccharomyces cerevisiae*, *Pichia pastoris*, *Aspergillus oryzae*, *Kluyveromyces martensii*, etc. In one embodiment, the gene encoding the pyruvate carboxylase can be selected from *Aspergillus oryzae*. PYC Genes and Kudria zweipichia yeast PYC1 Gene.

[0153] In one embodiment, the pyruvate carboxylase comprises an amino acid sequence encoded by the sequence shown in SEQ ID NO: 6 or 7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having pyruvate carboxylase activity.

[0154] In one embodiment, the gene encoding the pyruvate carboxylase comprises a sequence as shown in SEQ ID NO: 6 or 7 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity.

[0155] Optionally, the gene encoding pyruvate carboxylase is incorporated into the genome of a genetically modified succinic acid-producing yeast (e.g., Pichia kudrica), for example in... JEN2-1 Location of the gene locus. The gene encoding the pyruvate carboxylase can be placed in a suitable promoter (e.g., TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0156] The method may integrate genes to be expressed or overexpressed in the strain into appropriate locations in the strain genome, provided that such integration does not negatively affect the growth, reproduction, and / or production performance of the strain. For example, the method includes integrating one or more of the above-described genes into any one or more genomic locations encoding proteins such as: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhinophosphate 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-methylglutaraldehyde bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

[0157] In a further embodiment, the method further includes weakening or inactivating at least one of the following in the strain: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhinophosphate 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-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

[0158] In a further embodiment, the method further includes reducing or inactivating the pyruvate decarboxylase activity in the yeast strain.

[0159] As used herein, reducing or inactivating the activity of a protein, such as an enzyme, 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 making it undetectable. Various methods of reducing or inactivating protein activity are known in the art, including, for example, inhibiting gene expression such as knockdown (e.g., using small interfering RNA), using weak promoters (when the gene is a polypeptide-coding gene); gene knockout, deletion of part or all of a gene or polypeptide sequence; mutation of certain sites in a gene or polypeptide, such as coding sequences or active domains, to reduce gene expression or regulate activity or the activity of the expression product; and the use of antagonists or inhibitors (e.g., including but not limited to antibodies, interfering RNA, etc.).

[0160] As used herein, a weakened or inactivated gene refers to a gene whose expression level (when it is a protein-coding gene) or regulatory function (when it is a regulatory element) is reduced 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 rendered undetectable. Various methods for weakening or inactivating genes are known in the art, including, for example, suppressing gene expression such as knockdown (e.g., using small interfering RNA), using weak promoters (when the gene is a polypeptide-coding gene); gene knockout, deletion of part or all of a gene sequence; and mutation of certain sites in a gene, such as the coding sequence, to reduce gene expression or regulatory activity or the activity of the expression product.

[0161] In one implementation, reducing or inactivating pyruvate decarboxylase activity includes weakening or inactivating the gene encoding pyruvate decarboxylase.

[0162] In one embodiment, weakening or inactivating the gene encoding pyruvate decarboxylase includes knocking out the gene encoding pyruvate decarboxylase (e.g., encoding the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having pyruvate decarboxylase activity).

[0163] In a further embodiment, the method further includes reducing or inactivating the NAD-dependent glycerol-3-phosphate dehydrogenase activity in the yeast strain.

[0164] In one embodiment, reducing or inactivating NAD-dependent glycerol-3-phosphate dehydrogenase activity includes weakening or inactivating the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase. In one embodiment, weakening or inactivating the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase includes knocking out the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase (e.g., encoding the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having NAD-dependent glycerol-3-phosphate dehydrogenase activity).

[0165] In a further embodiment, the method further includes reducing or inactivating the activity of 5'-phosphoorhinoside decarboxylase and / or alcohol dehydrogenase 1 and / or monocarboxylic acid permease and / or dicarboxylic acid transporter and / or oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme in the yeast strain.

[0166] In one embodiment, the method includes weakening or inactivating the gene encoding 5'-orhinophosphate decarboxylase and the gene encoding a dicarboxylic acid transporter (e.g., ...) in the yeast strain. JEN2 Genes such as JEN2-1 Gene, JEN2-2 One or more of the following genes: gene encoding alcohol dehydrogenase 1, gene encoding monocarboxylic acid permease, and gene encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme.

[0167] In one embodiment, a gene encoding a bifunctional enzyme of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde (e.g., an amino acid sequence encoding the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having bifunctional enzyme activity of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde) is knocked out, for example by homologous recombination.

[0168] In one embodiment, the gene encoding 5'-orhodophosphoside decarboxylase (which encodes an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same and having 5'-orhodophosphoside decarboxylase activity) is knocked out, for example by homologous recombination.

[0169] In one implementation, the gene encoding the dicarboxylic acid transporter... JEN2-1 The gene (encoding, for example, the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having dicarboxylic acid transporter activity) is knocked out, for example by homologous recombination.

[0170] In one implementation, the gene encoding the dicarboxylic acid transporter... JEN2-2 The gene (encoding, for example, the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having dicarboxylic acid transporter activity) is knocked out, for example by homologous recombination.

[0171] In one embodiment, the gene encoding alcohol dehydrogenase 1 (which encodes an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same and having alcohol dehydrogenase 1 activity) is knocked out, for example by homologous recombination.

[0172] In one embodiment, the gene encoding a monocarboxylic acid permease (encoding, for example, the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same and having monocarboxylic acid permease activity) is knocked out, for example by homologous recombination.

[0173] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes, optionally including those that knock out an endogenous gene encoding orotidine phosphate decarboxylase and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde.

[0174] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding soluble fumarate reductase, optionally with the knockout of endogenous genes encoding orotidine phosphate decarboxylase and / or endogenous genes encoding alcohol dehydrogenase 1 and / or endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes.

[0175] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and genes encoding succinate transporters, for example SpMAE1 Genes, optionally, the gene encoding endogenous 5'-phospho-orhodoside decarboxylase and / or the gene encoding endogenous monocarboxylic acid permease and / or the gene encoding endogenous oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme is knocked out.

[0176] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding fumarate enzymes, and optionally, the gene encoding endogenous 5'-phospho-orhodoside decarboxylase and / or the gene encoding endogenous pyruvate decarboxylase and / or the gene encoding endogenous oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes.

[0177] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes and genes encoding pyruvate carboxylase, optionally including the knockout of endogenous genes encoding 5'-orhodophosphoside decarboxylase and / or endogenous genes encoding dicarboxylic acid transporters, for example... JEN2-1 or JEN2-2 Genes and / or endogenous genes encoding bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation.

[0178] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDHGenes encoding pyruvate carboxylase, and the knockout of endogenous genes encoding pyruvate decarboxylase and endogenous genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase, optionally knocking out endogenous genes encoding orotidine phosphate decarboxylase and / or endogenous genes encoding dicarboxylic acid transporters (e.g., JEN2 Genes such as JEN2-1 Gene, JEN2-2 (Genes) and / or endogenous genes encoding bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation.

[0179] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes, such as those encoding pyruvate carboxylase, soluble fumarate reductase, and succinate transporter proteins. SpMAE1 Genes, and knockout of the gene encoding endogenous pyruvate decarboxylase and the gene encoding endogenous NAD-dependent glycerol-3-phosphate dehydrogenase, optionally knockout of the gene encoding endogenous 5'-orhodophosphoside decarboxylase and / or the gene encoding endogenous dicarboxylic acid transporter (e.g. JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation bifunctional enzymes and / or genes encoding alcohol dehydrogenase 1 and / or genes encoding monocarboxylic acid permeases. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence; the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence; the gene encoding fumarate enzyme contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence; or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0180] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902), comprising overexpressing in the genetically modified succinic acid-producing yeast strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding pyruvate carboxylase, soluble fumarate reductase, fumarate enzyme, and succinate transporter, for example... SpMAE1Genes, including the knockout of genes encoding endogenous pyruvate decarboxylase and endogenous NAD-dependent glycerol-3-phosphate dehydrogenase, optionally, the knockout of genes encoding endogenous 5'-orhodophosphoside decarboxylase and / or endogenous dicarboxylic acid transporters (e.g., JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation bifunctional enzymes and / or genes encoding alcohol dehydrogenase 1 and / or genes encoding monocarboxylic acid permeases. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence; the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence; the gene encoding fumarate enzyme contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence; or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0181] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDH Genes, optionally, knock out endogenous URA3 Genes and / or endogenous Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or a degenerate sequence thereof.

[0182] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDH The gene and the gene encoding soluble fumarate reductase, optionally, are knocked out endogenously. URA3 Genes and / or endogenous ADH1 Genes and / or endogenous Pk2365 Gene. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding soluble fumarate reductase contains any of the sequences shown in SEQ ID NO: 3-5 or their degenerate sequences.

[0183] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDHGenes and genes encoding succinate transporters, for example SpMAE1 Genes, optional knockout of endogenous genes URA3 Genes and / or endogenous MCH4 Genes and / or endogenous Pk2365 Gene. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the succinic acid transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0184] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDH The gene and the gene encoding fumarate, optionally, are knocked out endogenously. URA3 Genes and / or endogenous PDC1 Genes and / or endogenous Pk2365 Gene. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding fumarate contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence.

[0185] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDH The gene encoding pyruvate carboxylase and, optionally, the endogenous gene is knocked out. URA3 Genes and / or endogenous JEN2-1 or JEN2-2 Genes and / or endogenous Pk2365 Gene. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.

[0186] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDH Genes and genes encoding pyruvate carboxylase, as well as knocking out endogenous genes. PDC1 Genes and endogenous GPD1 Genes, optional knockout of endogenous genes URA3 Genes and / or endogenous JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes and / or endogenous Pk2365 Gene. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence.

[0187] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDH Genes, such as those encoding pyruvate carboxylase, soluble fumarate reductase, and succinate transporter proteins. SpMAE1 Genes, and knocking out endogenous genes PDC1 Genes and endogenous GPD1 Genes, optionally, knock out endogenous URA3 Genes and / or endogenous JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes and / or endogenous Pk2365 Genes and / or endogenous ADH1 Genes and / or endogenous MCH4 Gene. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence, the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence, or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0188] In one embodiment, the present invention provides a method for producing genetically modified Pichia kudriezvichia strains such as CY902, comprising overexpressing a gene encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding pyruvate carboxylase, soluble fumarate reductase, fumarate enzyme, and succinate transporter, for example... SpMAE1 Genes, and knocking out endogenous genes PDC1 Genes and endogenous GPD1 Genes, optionally, knock out endogenous URA3 Genes and / or endogenous JEN2 Genes such as JEN2-1 Gene, JEN2-2 Genes and / or endogenous Pk2365 Genes and / or endogenous ADH1 Genes and / or endogenous MCH4 The gene is knocked out. Preferably, the... MDHThe gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 6 or 7 or its degenerate sequence; the gene encoding soluble fumarate reductase contains any one of the sequences shown in SEQ ID NO: 3-5 or its degenerate sequence; the gene encoding fumarate enzyme contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence; or the gene encoding succinate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0189] In one embodiment, the succinic acid-producing yeast used for genetic modification includes, but is not limited to, *Candida*, *Pichia*, *Rhodotorula*, *Saccharomyces*, *Yersinia*, *Zygosaccharomyces rouxii*, and *Globosa*. In one embodiment, the succinic acid-producing yeast used for genetic modification is selected from strains of *Pichia*, *Saccharomyces*, or *Yersinia*. In a preferred embodiment, the succinic acid-producing yeast used for genetic modification is *Pichia kudriezwei*, *Saccharomyces cerevisiae*, or *Yersinia lipolytica*, such as *Pichia kudriezwei* deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20885.

[0190] In one aspect, the present invention provides a method for producing succinic acid, comprising culturing a genetically modified succinic acid-producing yeast strain of the present invention, or a genetically modified succinic acid-producing yeast strain prepared by the method for producing a genetically modified succinic acid-producing yeast strain of the present invention, under conditions suitable for fermentation production of succinic acid, optionally including the isolated and purified succinic acid produced.

[0191] Conditions known in the art for fermenting succinic acid-producing yeast strains for succinic acid production include, for example, but not limited to, pH, temperature, culture medium composition, fermentation time, etc.

[0192] Culture media known in the art for the fermentation of succinic acid by yeast strains are, for example, but not limited to, inorganic salt media (about 5-12% w / v glucose, optionally containing about 30 g / L CaCO3).

[0193] Temperatures known in the art for the fermentation of succinic acid-producing yeast strains to produce succinic acid include, for example, about 25-37°C, such as about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, and about 37°C. In one embodiment, the succinic acid-producing yeast strain of the present invention is fermented at 30°C to produce succinic acid.

[0194] The succinic acid-producing yeast strain described in this invention can ferment at suitable pH values ​​known in the art, such as pH values ​​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, and less than about 1.0 (e.g., pH values ​​of 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). In one embodiment, the succinic acid-producing yeast strain of the present invention is fermented at a pH < about 3.0 to produce succinic acid.

[0195] To produce succinic acid, the succinic acid-producing yeast strain of the present invention can ferment for a suitable time, such as about 12-96 hours, including about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, and about 96 hours. In one embodiment, to produce succinic acid, the succinic acid-producing yeast strain of the present invention ferments for about 24-72 hours, for example, about 30 hours.

[0196] The succinic acid producing yeast strain described in this invention can be fermented to produce succinic acid under shaking conditions (e.g., about 100-300 rpm, such as about 150, about 200, or about 250 rpm).

[0197] The succinic acid content in the fermentation broth can be determined by suitable methods known in the art, such as HPLC.

[0198] In one embodiment, the present invention provides a method for producing succinic acid, comprising: at acidic pH values ​​(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 values ​​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), ... Genetically modified succinic acid-producing yeast strains were cultured in an inorganic salt medium (such as one containing approximately 5% w / v glucose and approximately 30 g / L CaCO3, or approximately 12% w / v glucose) under conditions of 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), optionally including isolated and purified succinic acid.

[0199] In one embodiment, the method for producing succinic acid according to the present invention does not require the addition of a neutralizing agent.

[0200] In one aspect, the present invention provides the application of the genetically modified succinic acid-producing yeast strain described in this invention, or the genetically modified succinic acid-producing yeast strain prepared according to the method for producing the genetically modified succinic acid-producing yeast strain described in this invention, in the production of succinic acid, particularly at acidic pH values ​​(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 values ​​of about 1.0-7.0, 1.0-6.0, 1.0-5.5, 1... Applications in the production of succinic acid under conditions of 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) and / or without the addition of a neutralizing agent.

[0201] As used herein, "optional" or "optionally" means that the event or situation subsequently described occurs or does not occur, including both the occurrence and non-occurrence of said event or situation. For example, a step optionally included means that the step is present or absent.

[0202] As used herein, the term "about" refers to a range of values ​​that includes a specific value and that would be reasonably regarded by those skilled in the art as similar to that specific value. In some embodiments, the term "about" refers to within the standard error of measurements generally accepted in the art. In some embodiments, "about" refers to + / - 10% of a specific value.

[0203] The scope disclosed herein should be considered to specifically disclose all possible subranges and the individual values ​​within those ranges. For example, a description of the range from 1 to 6 should be considered as explicitly disclosing subranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0204] The present invention will be further illustrated below through the following non-limiting embodiments. As will be known to those skilled in the art, many modifications can be made to the present invention without departing from the spirit of the invention, and such modifications also fall within the scope of the present invention.

[0205] Unless otherwise specified, the experimental methods described below are standard methods, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.

[0206] Example 1: In Pichia pastoris CY902 ΔURA3 strain overexpression SbMDH Gene The gene encoding orotidine 5'-phosphate decarboxylase was synthesized via homologous recombination. URA3 Knockout of (CY902 locus number PK2075) yielded ΔURA3 Mutants (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 Biotechnol2021. 0(0), 1–18. doi:10.1111 / 1751-7915.13781). We then constructed the CRISPR / Cas9 plasmid pWSPK-Cas9 (GenBank accession number: MW296878.1) suitable for this bacterium, which contains... URA3 Screening markers can be used to obtain positive transformants through auxotrophic screening.

[0207] With CY902 ΔURA3 The originating bacterium is located at locus number [number missing] in the CY902 genome. Pk2365 The site overexpressed sorghum-derived malate dehydrogenase SbMDH (Uniprot database search number: P17606) (SEQ ID NO: 1), using the promoter and terminator of CY902's own fructose 1,6-bisphosphate aldolase gene. FBA1 The promoter (SEQ ID NO: 17) and the inositol-3-phosphate synthase gene INO1 The terminator (SEQ ID NO: 18). SbMDH The gene was synthesized by Nanjing Genscript Biotech Co., Ltd., and optimized based on the codon bias of CY902. The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_2365_F and 1_UP_2365_R1 (see Table 1). Pk2365 Upstream homologous arm fragment 1 (fragment 1); amplification system and procedure were performed according to the TAKARA PrimeSTAR® HS DNA polymerase product instructions; primer 2_P was used. FBA1 _F and 2_P FBA1 _R (see Table 1) amplification of CY902 itself FBA1 Promoter sequence (segment 2); containing SbMDH Using the plasmid containing the synthesized sequence as a template, the SbMDH coding sequence (fragment 3) of sorghum was amplified using primers 3_SbMDH_F and 3_SbMDH_R (see Table 1); using CY902 genomic DNA as a template, primer 4_T INO1 _F and 4_T INO1 _R (see Table 1) amplification of CY902 itself INO1 Gene terminator sequence (fragment 4); CY902 itself was amplified using primers 5_DW_2365_F1 and 5_DW_2365_R (see Table 1). Pk2365Downstream homologous arm fragment 1 (fragment 5). Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_2365_F and 1_UP_2365_R2 (see Table 1). Pk2365 Upstream homologous arm fragment 2 (fragment 6); CY902 itself was amplified using primers 5_DW_2365_F2 and 5_DW_2365_R (see Table 1). Pk2365 Downstream homologous arm fragment 2 (fragment 7).

[0208] 2. Build for editing Pk2365 plasmids of gene loci Using the pWSPK-Cas9 plasmid (GenBank accession number: MW296878.1) as a template, a 9052 bp plasmid backbone (referred to as pWSPK_backbone, SEQ ID NO: 23) without the sgRNA sequence was amplified using primers pWSPK-F and pWSPK-R (see Table 1). The 5' end sequence of the sgRNA (GenBank accession number: MW296878.1) (referred to as 2365_sgRNA_1) was amplified using primers sgRNA-1F and 2365_sgRNA-1R (see Table 1). This fragment contains […]. Pk2365 The gene contains a unique 20nt prototype spacer sequence. Using the pWSPK-Cas9 plasmid as a template, the 3' end sequence of a 500bp sgRNA (abbreviated as sgRNA_2, SEQ ID NO: 97) was amplified using primers sgRNA-2F and sgRNA-2R (see Table 1). The plasmid backbone pWSPK_backbone, sgRNA fragments 2365_sgRNA_1 and sgRNA_2 were ligated using a seamless cloning kit (Beyotime Biotechnology Co., Ltd., Shanghai, product number: D7010S). The seamless cloning ligation product was transformed into Trans1-T1 competent cells (TransGen Biotech Co., Ltd., Beijing, product number: CD501-02), and the resulting positive plasmid was named pWSPK_2365.

[0209] 3. Construct overexpression SbMDH Gene strains The pWSPK_2365 plasmid and fragments 6 and 7 were transformed into CY902 using yeast electroporation (doi:10.1111 / 1751-7915.13781). ΔURA3 Positive transformants were obtained from the strain and named strain SA101-1 (genotype: CY902). ΔURA3 , ΔPk2365 The pWSPK_2365 plasmid and fragments 1-5 were transformed into CY902 via yeast electroporation. ΔURA3Positive transformants were obtained from the strain and named strain SA101-2 (genotype: CY902). ΔURA3 , Pk2365::P PkFBA1 - ORF SbMDH -T PkINO1 ).

[0210] Table 1. Overexpression SbMDH Primers required for gene and CRISPR / Cas9 plasmid construction

[0211] Example 2: In Pichia pastoris CY902 ΔURA3 overexpression EcSthA and AoMDH Gene With CY902 ΔURA3 The originating bacterium is located at locus number [number missing] in the CY902 genome. Pk2365 The site was used to overexpress the soluble pyridine nucleotide transhydrogenase EcSthA (EC 1.6.1.1) (SEQ ID NO: 98) derived from E. coli, using the promoter and terminator of CY902 itself. FBA1 Promoter (SEQ ID NO: 17) and INO1 Terminator (SEQ ID NO: 18). In the succinate semialdehyde dehydrogenase (EC 1.2.1.16) gene. PkUGA2 The site overexpressed the malate dehydrogenase AoMDH (EC 1.1.1.37; Uniprot database search number: I8U0T6) (SEQ ID NO: 99) derived from Aspergillus oryzae, using the promoter and terminator of the CY902's own enolase gene. ENO1 The promoter (SEQ ID NO: 100) and cell wall glycoprotein (GPI) gene SED1 The terminator (SEQ ID NO: 101). EcSthA and AoMDH The gene was synthesized by Nanjing Genscript Biotech Co., Ltd., and optimized based on the codon bias of CY902. The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. With EcSthA Using the plasmid containing the synthesized sequence as a template, the coding sequence (fragment 8) of E. coli soluble pyridine nucleotide transhydrogenase EcSthA was amplified using primers 3_EcSthA_F and 3_EcSthA_R (see Table 2); using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_UGA2_F and 1_UP_UGA2_R1 (see Table 2). PkUGA2 Upstream homologous arm fragment 1 (fragment 9); using primer 2_P ENO1 _F and 2_P ENO1 _R (see Table 2) amplification of CY902 itself ENO1 Promoter sequence (segment 10); containing AoMDH Using the plasmid containing the synthesized sequence as a template, the Aspergillus oryzae AoMDH coding sequence (fragment 11) was amplified using primers 3_AoMDH_F and 3_AoMDH_R (see Table 2); using CY902 genomic DNA as a template, primer 4_T... SED1 _F and 4_T SED1 _R (see Table 1) amplification of CY902 itself SED1 Gene terminator sequence (fragment 12); CY902 itself was amplified using primers 5_DW_UGA2_F1 and 5_DW_UGA2_R (see Table 2). PkUGA2 Downstream homologous arm fragment 1 (fragment 13). Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_UGA2_F and 1_UP_UGA2_R2 (see Table 2). PkUGA2 Upstream homologous arm fragment 2 (fragment 14); CY902 itself was amplified using primers 5_DW_UGA2_F2 and 5_DW_UGA2_R (see Table 2). PkUGA2 Downstream homologous arm fragment 2 (fragment 15).

[0212] 2. Build for editing PkUGA2 plasmids of gene loci Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (abbreviated as UGA2_sgRNA_1) was amplified using primers sgRNA-1F and UGA2_sgRNA-1R (see Table 2). The 3' end of this fragment contains PkUGA2 The gene has a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2, and UGA2_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_UGA2.

[0213] 3. Construct overexpression EcSthA and AoMDH Gene strains The pWSPK_2365 plasmid and fragments 1, 2, 8, 4, and 5 were transferred into CY902 via yeast electroporation. ΔURA3 Positive transformants were obtained from the strains and named strain SA101-3 (genotype: CY902). ΔURA3 , Pk2365::P PkFBA1 -ORF EcSthA - T PkINO1 The pWSPK_UGA2 plasmid and fragments 14 and 15 were transformed into yeast strains SA101-2 and SA101-3 via yeast electroporation. Positive transformants were obtained and named strains SA102-1 (genotype: SA101-2). ΔUGA2 ) and SA102-2 strain (genotype: SA101-3, ΔUGA2 The pWSPK_UGA2 plasmid and fragments 9-13 were transformed into yeast strain SA101-3 via electroporation. Positive transformants were obtained and named strain SA102-3 (genotype: SA101-3). PkUGA2::P PkENO1 -ORF AoMDH - T PkSED1 ).

[0214] Table 2. Overexpression EcSthA and AoMDH Primers required for gene and CRISPR / Cas9 plasmid construction

[0215] Example 3: Evaluation of succinic acid production capacity of SA101 and SA102 series strains 1. Fermentation with neutralizing agent SA101 and SA102 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 30 g / L CaCO3) and fermented in shake flasks at 30 °C and 250 rpm for 24 h. The yield of succinic acid was measured by HPLC and is shown in Table 3 below.

[0216] Table 3. Succinic acid yield from shake-flask fermentation of SA101 and SA102 series strains

[0217] 2. Fermentation without neutralizer SA101 and SA102 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30℃ and 250 rpm for 24 h. The yield of succinic acid was measured by HPLC and is shown in Table 4 below.

[0218] Table 4. Succinic acid yield from shake-flask fermentation of SA101 and SA102 series strains

[0219] Example 4: Overexpression in SA101-2 strain FRD Gene Using SA101-2 as the starting strain, in ADH1 The gene loci were overexpressed with fumarate reductases from three different sources, including (I) FRD from *Saccharomyces cerevisiae* (ScFRD, Uniprot database search number: P32614) (SEQ ID NO: 3); (II) FRD from *Leishmania megacephala* (LmFRD, SEQ ID NO: 4); and (III) FRD from *T. bryophyte* (TbFRD, SEQ ID NO: 5). Overexpression FRD The promoter and terminator are CY902's own alcohol dehydrogenase I. ADH1 The promoter (SEQ ID NO: 21) and terminator (SEQ ID NO: 22). The above three... FRD The gene sequence was synthesized by Nanjing Genscript Biotech Co., Ltd., and optimized based on the codon bias of CY902. The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using CY902 genomic DNA as a template, primer 1_P ADH1 _F and 1_P ADH1 _R (see Table 5) amplification of CY902 itself ADH1 Gene promoter sequence (upstream homologous arm, fragment 16); containing ScFRD Using the plasmid containing the synthesized sequence as a template, the ScFRD coding sequence (fragment 17-1) of *Saccharomyces cerevisiae* was amplified with primers 2_ScFRD_F and 2_ScFRD_R (see Table 5); LmFRD Using the plasmid containing the synthesized sequence as a template, the LmFRD coding sequence (fragment 17-2) of *Leishmania melanogaster* with the 3' end glyoxylate cycling body localization peptide was amplified using primers 2_LmFRD_F and 2_LmFRD_R (see Table 5); TbFRD Using the plasmid containing the synthesized sequence as a template, primers 2_TbFRD_F and 2_TbFRD_R (see Table 5) were used to amplify the TbFRD coding sequence (fragment 17-3) of Trypanosoma brevicornuate with the 3' end glyoxylate cycling peptide removed; using CY902 genomic DNA as a template, primer 3_T ADH1 _F1 and 3_T ADH1 _R (see Table 5) amplification of CY902 itself ADH1 Gene terminator sequence (downstream homologous arm 1, fragment 18). Using CY902 genomic DNA as a template, primer 3_T was used. ADH1 _F2 and 3_T ADH1 _R (see Table 5) amplification of CY902 itself ADH1 Gene terminator sequence (downstream homologous arm 2, fragment 19).

[0220] 2. Build for editing ADH1 plasmids of gene loci Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (abbreviated as ADH1_sgRNA_1) was amplified using primers sgRNA-1F and ADH1_sgRNA-1R (see Table 5). The 3' end of this fragment contains *Pichia pastoris*. ADH1 ( PkADH1 The gene contains a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2, and ADH1_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_ADH1.

[0221] 3. Construct overexpression FRD Gene strains The pWSPK_ADH1 plasmid and fragments 16 and 19 were transformed into strain SA101-2 using yeast electroporation. Positive transformants were obtained and named strain SA103-1 (genotype: SA101-2). ΔADH1 The pWSPK_ADH1 plasmid and fragments 16, 17, and 18 were transformed into strain SA101-2 using yeast electroporation. Positive transformants were screened and named strains SA103-2 (genotype: SA101-2). ADH1:: ORF ScFRD SA103-3 strain (genotype: SA101-2, ADH1:: ORF LmFRD ); named strain SA103-4 (genotype: SA101-2, ADH1:: ORF TbFRD ).

[0222] Table 5. Overexpression FRD Primers required for gene and CRISPR / Cas9 plasmid construction

[0223] Example 5: Knockout in SA101-2 strain PDC1 , GPD1 Gene 1. Build a tool for knocking out PDC1 , GPD1 DNA fragments Using CY902 genomic DNA as a template, amplification was performed using primers PDC1_1F and PDC1_1R, PDC1_2F and PDC1_2R (see Table 6). PDC1 Upstream and downstream homologous arm fragments of the gene (abbreviated as PDC1_1, PDC1_2); amplified using primers GPD1_1F and GPD1_1R, GPD1_2F and GPD1_2R (see Table 6). GPD1 Upstream and downstream homologous arm fragments of the gene (abbreviated as GPD1_1, GPD1_2).

[0224] 2. Build for editing PDC1 , GPD1 CRISPR / Cas9 plasmids for genes The 5' end sequence of sgRNA (abbreviated as PDC1_sgRNA_1, SEQ ID NO: 96) was amplified using primers sgRNA-1F and PDC1_sgRNA-1R (see Table 6). The 3' end of this fragment contains... PDC1 The gene contains a unique 20nt prototype spacer sequence. The plasmid backbone pWSPK_backbone, sgRNA fragments PDC1_sgRNA_1, and sgRNA_2 were ligated using a seamless cloning kit. The seamless cloning ligation product was transformed into Trans1-T1 competent cells, and the resulting positive plasmid was named pWSPK_PDC1. The 5' end sequence of the sgRNA (referred to as GPD1_sgRNA_1) was amplified using primers sgRNA-1F and GPD1_sgRNA-1R (see Table 6). This fragment contains a unique 20nt prototype spacer sequence at its 3' end. GPD1 The gene's unique 20nt prototype spacer sequence allows for seamless cloning and fusion of pWSPK_backbone, sgRNA_2, and GPD1_sgRNA_1 into a single gene for editing. GPD1 The pWSPK_GPD1 plasmid of the gene.

[0225] Table 6. PDC1 , GPD1 Primers required for gene knockout and CRISPR / Cas9 plasmid construction

[0226] 3. Construction PDC1 , GPD1 Gene knockout strains Fragments PDC1_1, PDC1_2, and plasmid pWSPK_PDC1 were simultaneously transformed into SA101-2. Positive transformants were identified using primers PDC1_1F / PDC1_2R and named strain SA104 (genotype: SA101-2). ΔPDC1 The pWSPK_GPD1 plasmid and fragments GPD1_1 and GPD1_2 were electroporated into strain SA104. Positive transformants were obtained by screening on SD-URA medium and named strain SA105 (genotype: SA104). ΔGPD1 ).

[0227] Example 6: Overexpression in SA101-2 strain SpMAE1 Gene In the CY902 genome MCH4 The gene locus expressed the SpMAE1 transporter protein derived from Schizosaccharomyces cerevisiae (Uniprot database search number: P50537). SpMAE1 The gene was synthesized by Nanjing Genscript Biotech Co., Ltd. (SEQ ID NO: 2) and optimized according to the codon preference of CY902. The promoter and terminator used are those of CY902's own glyceraldehyde-3-phosphate dehydrogenase 3 gene. TDH3 The promoter (SEQ ID NO: 19) and galactose transporter gene GAL2 The terminator (SEQ ID NO: 20). The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_MCH4_F and 1_UP_MCH4_R1 (see Table 7). MCH4 Upstream homologous arm fragment 1 (fragment 20); using primer 2_P TDH3 _F and 2_P TDH3 _R (see Table 7) amplifies CY902 itself TDH3 Gene promoter sequence (fragment 21); containing SpMAE1 Using the plasmid containing the synthesized sequence as a template, the SpMAE1 coding sequence (fragment 22) of *Schizosaccharomyces cerevisiae* was amplified using primers 3_SpMAE1_F and 3_SpMAE1_R (see Table 7); using CY902 genomic DNA as a template, primer 4_T... GAL2 _F and 4_T GAL2 _R (see Table 7) amplifies CY902 itself GAL2 Gene terminator sequence (fragment 23); amplified using primers 5_DW_MCH4_F1 and 5_DW_MCH4_R (see Table 7). MCH4 Downstream homologous arm fragment 1 (fragment 24). Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_MCH4_F and 1_UP_MCH4_R2 (see Table 7). MCH4 Upstream homologous arm fragment 2 (fragment 25); amplified using primers 5_DW_MCH4_F2 and 5_DW_MCH4_R (see Table 7). MCH4 Downstream homologous arm fragment 2 (fragment 26).

[0228] 2. Construct a tool for editing CY902 itself. MCH4 plasmid Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (referred to as MCH4_sgRNA_1) was amplified using primers sgRNA-1F and MCH4_sgRNA-1R (see Table 7). The 3' end of this fragment contains *Pichia pastoris*. PkMCH4 The gene contains a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2, and MCH4_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_MCH4.

[0229] Table 7. Overexpression SpMAE1 Primers required for gene and CRISPR / Cas9 plasmid construction

[0230] 3. Construct overexpression SpMAE1 Gene strains The pWSPK_MCH4 plasmid and fragments 25 and 26 were electroporated into strain SA101-2 using the yeast electroporation method. Positive transformants were obtained by screening and named SA106-1 (genotype: SA101-2). ΔPkMCH4 The pWSPK_MCH4 plasmid and fragments 20-24 were electroporated into strain SA101-2, and positive transformants were obtained by screening and named SA106-2 (genotype: SA101-2). PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).

[0231] Example 7: Overexpression in SA101-2 strain PYC Gene exist JEN2-1 The sites were overexpressed with Aspergillus oryzae-derived [products / products]. PYC ( AoPYC ) gene (Uniprot database search number: Q2UGL1) (SEQ ID NO: 6), CY902 itself PYC1 ( PkPYC1 The gene was used as a control (SEQ ID NO: 7), and the promoter and terminator used were respectively... TDH3 Promoter (SEQ ID NO: 19) and GAL2 Terminator (SEQ ID NO: 20). AoPYC The gene was synthesized by Nanjing Genscript Biotech Co., Ltd., and optimized based on the codon bias of CY902 (SEQ ID NO: 6). The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R1 (see Table 8). JEN2-1 Upstream homologous arm fragment 1 (fragment 27); amplified the CY902 self-PkPYC1 coding sequence (fragment 28-1) using primers 3_PkPYC1_F and 3_PkPYC1_R (see Table 8); with... AoPYC Using the plasmid containing the synthesized sequence as a template, the AoPYC coding sequence (fragment 28-2) from Aspergillus oryzae was amplified using primers 3_AoPYC_F and 3_AoPYC_R (see Table 8); using CY902 genomic DNA as a template, the sequence was amplified using primers 5_DW_JEN2-1_F1 and 5_DW_JEN2-1_R (see Table 8). JEN2-1 Downstream homologous arm fragment 1 (fragment 29). Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R2 (see Table 8). JEN2-1 Upstream homologous arm fragment 2 (fragment 30); amplified using primers 5_DW_JEN2-1_F2 and 5_DW_JEN2-1_R (see Table 8). JEN2-1 Downstream homologous arm fragment 2 (fragment 31).

[0232] Table 8. Overexpression PYC Primers required for gene and CRISPR / Cas9 plasmid construction

[0233] 2. Build for editing JEN2-1 CRISPR / Cas9 plasmids for genes Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (referred to as JEN2-1_sgRNA_1) was amplified using primers sgRNA-1F and JEN2-1_sgRNA-1R (see Table 8). The 3' end of this fragment contains JEN2-1 The gene has a unique 20nt prototype spacer sequence. The pWSPK_backbone, sgRNA_2, and JEN2-1_sgRNA_1 from Example 1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_JEN2-1.

[0234] 3. Construct strains overexpressing the PYC gene The pWSPK_JEN2-1 plasmid and fragments 30 and 31 were electroporated into strain SA101-2, and positive transformants were obtained by screening and named SA107-1 (genotype: SA101-2). ΔPkJEN2-1 The pWSPK_JEN2-1 plasmid and fragments 27, 21, 28, 23, and 29 were electroporated into strain SA101-2, and positive transformants were obtained by screening and named 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 ).

[0235] Example 8: Overexpression in SA101-2 strain PkFUM1 Gene In strain SA101-2 PDC1 The gene promoter site overexpresses the fumarate enzyme gene of CY902 with the 5' mitochondrial localization peptide truncated. PkFUM1 (SEQ ID NO: 8). The promoter and terminator used are respectively TDH3 Promoter (SEQ ID NO: 19) and GAL2 Terminator (SEQ ID NO: 20). The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using CY902 genomic DNA as a template, primer 1_UP_P PDC1 _F and 1_UP_P PDC1 _R (see Table 9) amplification of CY902 itself PDC1 The upstream homologous arm fragment of the gene promoter (fragment 32); the 5' mitochondrial localization peptide was amplified using primers 3_PkFUM1_F and 3_PkFUM1_R (see Table 9). FUM1 Encoding sequence (segment 33); using primer 5_DW_P PDC1 _F and 5_DW_P PDC1 _R (see Table 9) amplification of CY902 itself PDC1 Downstream homologous arm fragment of the gene promoter (fragment 34).

[0236] 2. Build for editing PDC1 plasmids for gene promoter sites Using pWSPK-Cas9 plasmid as a template, primers sgRNA-1F and P were used. PDC1 _sgRNA-1R (see Table 9) amplifies the 5' end sequence of sgRNA (abbreviated as P). PDC1 _sgRNA_1), the 3' end of this fragment contains P PDC1 The gene promoter has a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2, and P... PDC1 _sgRNA_1 was ligated using a seamless cloning kit, and a positive clone plasmid was obtained, which was named pWSPK_P. PDC1 .

[0237] Table 9. Overexpression PkFUM1 Primers required for gene and CRISPR / Cas9 plasmid construction

[0238] 3. Construct overexpression PkFUM1 Gene strains pWSPK_P PDC1 Plasmids and fragments 32, 21, 33, 23, and 34 were transformed into strain SA101-2 using yeast electroporation. Positive transformants were obtained through screening and named strain SA108 (genotype: SA101-2). P PkPDC1 :: P PkTDH3 -ORF PkFUM1 - T PkGAL2 ).

[0239] Example 9: Replacement in SA101 series strains PkURA3 Gene dicarboxylic acid transporter in SA101 series strains PkJEN2-2 The 5'-phospho-orhinoside decarboxylase gene was reintroduced at the gene locus. PkURA3 (SEQ ID NO: 117). The promoter and terminator used are: PkURA3 Its own promoter (SEQ ID NO: 118) and terminator (SEQ ID NO: 119). The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R1 (see Table 10). PkJEN2-2 Upstream homologous arm fragment 1 (fragment 35); amplified using primers 2_PkURA3_F and 2_PkURA3_R (see Table 10). URA3 Promoter, coding frame, and terminator sequences (fragment 36); CY902 itself was amplified using primers 3_DW_JEN2-2_F1 and 3_DW_JEN2-2_R (see Table 10). PkJEN2-2 Downstream homologous arm fragment 1 (fragment 37). Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R2 (see Table 10). PkJEN2-2 Upstream homologous arm fragment 2 (fragment 38); CY902 itself was amplified using primers 3_DW_JEN2-2_F2 and 3_DW_JEN2-2_R (see Table 10). PkJEN2-2 Downstream homologous arm fragment 2 (fragment 39).

[0240] 2. Build for editing PkJEN2-2 plasmids of gene loci Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (abbreviated as JEN2-2_sgRNA_1) was amplified using primers sgRNA-1F and JEN2-2_sgRNA-1R (see Table 10). The 3' end of this fragment contains PkJEN2-2 The gene has a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2 and JEN2-2_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_JEN2-2.

[0241] Table 10. Replenishment PkURA3 Primers required for gene and CRISPR / Cas9 plasmid construction

[0242] 3. Construct replenishment PkURA3 Gene strains The pWSPK_JEN2-2 plasmid and fragments 38 and 39 were transformed into strains SA101-1, SA101-2, and SA101-3 using yeast electroporation. Positive transformants were screened and named strains SA109-1 (genotype: SA101-1). Δ PkJEN2-2 SA109-2 strain (genotype: SA101-2, ΔPkJEN2-2 ) and SA109-3 strain (genotype: SA101-3, ΔPkJEN2-2 The pWSPK_JEN2-2 plasmid and fragments 35-37 were transformed into strains SA101-1, SA101-2, and SA101-3 using yeast electroporation. Positive transformants were screened and named strains SA109-4 (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 (genotype: SA101-3, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).

[0243] Example 10: Replacement in SA102-SA108 series strains PkURA3 Gene Referring to Example 9, in strains SA102-SA108 PkJEN2-2 Gene locus replacement PkURA3 The strain SA110-1 (genotype: SA102-1) was obtained. PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA110-2 strain (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 SA111-3 strain (genotype: SA103-3, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA111-4 strain (genotype: SA103-4, PkJEN2-2:: P PkURA3 -ORF PkURA3 -T PkURA3 SA112 strain (genotype: SA104, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA113 strain (genotype: SA105, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA114-1 strain (genotype: SA106-1, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA114-2 strain (genotype: SA106-2, PkJEN2-2:: P PkURA3 -ORF PkURA3 -T PkURA3 SA115-1 strain (genotype: SA107-1, PkJEN2-2::P PkURA3 -ORF PkURA3 - TPkURA3 SA115-2 strain (genotype: SA107-2, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA115-3 strain (genotype: SA107-3, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA116 strain (genotype: SA108, PkJEN2- 2::P PkURA3 -ORF PkURA3 -T PkURA3 ).

[0244] Example 11: Evaluation of succinic acid production capacity of SA101-SA116 series strains 1. Fermentation with neutralizing agent The SA101-SA116 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 30 g / L CaCO3) and fermented in shake flasks at 30℃ and 250 rpm for 24 h. The yield of succinic acid was measured by HPLC.

[0245] Table 11. Succinic acid yield from shake-flask fermentation of SA101–SA116 series strains

[0246] 2. Fermentation without neutralizer The SA101-SA116 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30℃ and 250 rpm for 24 h. The yield of succinic acid was measured by HPLC.

[0247] Table 12. Succinic acid yield from shake-flask fermentation of SA101–SA116 series strains

[0248] Example 12: Optimization of succinic acid producing bacteria Referring to Example 5, knockout was performed in SA103-4. PDC1 and GPD1 Genes were used to obtain strain SA117 (genotype: SA103-4). ΔPDC1 SA118 strain (genotype: SA117, ΔGPD1 ).

[0249] Referring to Example 6, overexpression in SA118 SpMAE1 Genes were used to obtain strain SA119-1 (genotype: SA118, Δ PkMCH4 SA119-2 strain (genotype: SA118, PkMCH4:: P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).

[0250] Referring to Example 7, the SA119-2 strain overexpressed two sources of [a specific ingredient / factor]. PYC The strain SA120-1 (genotype: SA119-2) was obtained. ΔPkJEN2-1 ); strain SA120-2 (genotype: SA119-2, PkJEN2-1:: P PkTDH3 -ORF PkPYC1 -T PkGAL2 SA120-3 strain (genotype: SA119-2, PkJEN2-1::P PkTDH3 -ORF AoPYC - T PkGAL2 ).

[0251] Referring to Example 8, the fumarate gene with the 5' mitochondrial localization peptide truncated was overexpressed in SA120-3. PkFUM1 The SA121 strain (genotype: SA120-3) was obtained. P PkPDC1 :: P PkTDH3 -ORF PkFUM1 -T PkGAL2 ).

[0252] Referring to Example 9, the 5'-phospho-orhodoside decarboxylase gene was reintroduced into strain SA121. PkURA3 The SA122 strain (genotype: SA121) was obtained. PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).

[0253] Referring to Example 4, overexpression in SA102-3 TbFRD Genes were used to obtain strain SA123 (genotype: SA102-3). ADH1:: ORF TbFRD ).

[0254] Referring to Example 5, knockout in SA123 PDC1 and GPD1 Genes were used to obtain strain SA124 (genotype: SA123). Δ PDC1 SA125 strain (genotype: SA124, ΔGPD1 ).

[0255] Referring to Example 6, overexpression in SA125 SpMAE1 Genes were used to obtain strain SA126-1 (genotype: SA125, Δ PkMCH4 SA126-2 strain (genotype: SA125, PkMCH4:: P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).

[0256] Referring to Example 7, the SA126-2 strain overexpressed two sources of [unclear - possibly a specific ingredient or strain]. PYC The strain SA127-1 (genotype: SA126-2) was obtained. ΔPkJEN2-1 ); strain SA127-2 (genotype: SA126-2, PkJEN2-1:: P PkTDH3 -ORF PkPYC1 -T PkGAL2 SA127-3 strain (genotype: SA126-2, PkJEN2-1::P PkTDH3 -ORF AoPYC - T PkGAL2 ).

[0257] Referring to Example 8, the fumarate gene with the 5' mitochondrial localization peptide truncated was overexpressed in SA127-3. PkFUM1 The SA128 strain (genotype: SA127-3) was obtained. P PkPDC1 :: P PkTDH3 -ORF PkFUM1 -T PkGAL2 ).

[0258] Referring to Example 9, the 5'-phospho-orhodoside decarboxylase gene was reintroduced into strain SA128. PkURA3 The SA129 strain (genotype: SA128) was obtained. PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).

[0259] Example 13: Evaluation of succinic acid production capacity of strains SA122 and SA129 SA122 and SA129 strains were scaled up and fermented in 5L tanks (yeast inorganic salt medium, 12% w / v glucose). Under the condition of no addition of any neutralizing agent, the succinic acid yields reached 96.08 g / L and 46.02 g / L, respectively, after 36 h, with conversion rates of 0.89 g / g and 0.57 g / g, respectively.

[0260] Example 14: In Pichia pastoris CICC32244 ΔURA3 strain overexpression SbMDH Gene Referring to Example 1, the orotidine 5'-phosphate decarboxylase encoding gene of CICC32244 (purchased from China Industrial Microbial Culture Collection Center) was synthesized via homologous recombination. URA3 Knockout was performed, resulting in CICC32244. ΔURA3 Mutant.

[0261] pWSPK_2365 plasmid and fragments 6 and 7 were transformed into CICC32244 using yeast electroporation. ΔURA3 Positive transformants were obtained from the strain and named strain SA130-1 (genotype: CICC32244). ΔURA3 , ΔPk2365 The pWSPK_2365 plasmid and fragments 1-5 were transformed into CICC32244 via yeast electroporation. ΔURA3 Positive transformants were obtained from the strain and named strain SA130-2 (genotype: CICC32244). ΔURA3 , Pk2365::P PkFBA1 -ORF SbMDH -T PkINO1 ).

[0262] Example 15: Replacement in SA130 series strains PkURA3 Gene Referring to Example 9, in the SA130 series strains PkJEN2-2 Gene locus replacement PkURA3 The strain SA131-1 (genotype: SA130-1) was obtained. PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 SA131-2 strain (genotype: SA130-2, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ).

[0263] Example 16: Evaluation of succinic acid production capacity of SA130 and SA131 series strains 1. Fermentation with neutralizing agent SA130 and SA131 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 30 g / L CaCO3) and fermented in shake flasks at 30 °C and 250 rpm for 24 h. The yield of succinic acid was measured by HPLC.

[0264] Table 13. Succinic acid yield from shake-flask fermentation of SA130 and SA131 series strains

[0265] 2. Fermentation without neutralizer SA130 and SA131 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30 °C and 250 rpm for 24 h. The yield of succinic acid was measured by HPLC.

[0266] Table 14. Succinic acid yield from shake-flask fermentation of SA130 and SA131 series strains

[0267] Example 17: Overexpression in Saccharomyces cerevisiae strain BY4742 SbMDH Gene In the 5-phosphate whey glycoside decarboxylase gene of Saccharomyces cerevisiae ScURA3 (SEQ ID NO: 129) Overexpression of sorghum-derived malate dehydrogenase SbMDH (SEQ ID NO: 1), overexpression ScURA3 Gene-producing strains were used as controls. Overexpression SbMDH The gene sequence is the same as above, optimized according to the codon preference of CY902. The promoter and terminator used are the BY4742's own glyceraldehyde-3-phosphate dehydrogenase gene. ScTDH3 The promoter (SEQ ID NO: 130) and galactose permease gene ScGAL2 Terminator (SEQ ID NO: 131). Overexpression ScURA3 The promoter and terminator used in this gene are the BY4742's own whey 5-phosphate decarboxylase gene. ScURA3 The promoter (SEQ ID NO: 132) and cell wall glycoprotein gene ScSED1 The terminator (SEQ ID NO: 133). The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using Saccharomyces cerevisiae S288c (purchased from ThermoFisher Scientific) genomic DNA as a template, amplification was performed using primers 1_UP_ScURA3_F and 1_ScURA3_R (see Table 15). ScURA3 Upstream homologous arm and ORF sequence (fragment 40); using primer 2_T ScSED1 _F and 2_T ScSED1 _R1 (see Table 15) amplification ScSED1 Termination subsequence 1 (fragment 41); using primer 3_P ScTDH3 _F and 3_P ScTDH3 _R (see Table 15) amplification ScTDH3 Gene promoter sequence (fragment 42); containing SbMDH Using the plasmid containing the synthesized sequence as a template, primers 4_SbMDH_F and 4_SbMDH_R (see Table 15) were used to amplify sorghum-derived sequences. SbMDH Coding sequence (fragment 43); using S288c genomic DNA as a template, primer 5_T ScGAL2 _F and 5_T ScGAL2 _R (see Table 15) amplification ScGAL2 Gene terminator sequence (fragment 44); amplified using primers 6_DW_ScURA3_F1 and 6_DW_ScURA3_R (see Table 15). ScURA3 Downstream homologous arm fragment 1 (fragment 45). Using S288c genomic DNA as a template, primer 2_T was used. ScSED1 _F and 2_T ScSED1 _R2 (see Table 15) amplification ScSED1 Termination subsequence 2 (fragment 46); amplified using primers 6_DW_ScURA3_F2 and 6_DW_ScURA3_R (see Table 15). ScURA3 Downstream homologous arm fragment 2 (fragment 47).

[0268] 2. Constructing an overexpression mechanism in Saccharomyces cerevisiae SbMDH strains Fragments 40, 46, and 47 were mixed together and transformed into yeast strain BY4742 (purchased from ThermoFisher Scientific) using yeast electroporation. Positive transformants were obtained through screening and named strain SA132-1 (genotype: BY4742). ScURA3:: P ScURA3 -ORF ScURA3 -T ScSED1t Fragments 40-45 were mixed together and transformed into strain BY4742 using yeast electroporation. Positive transformants were obtained through screening and named strain SA132-2 (genotype: BY4742). ScURA3:: P ScURA3 -ORF ScURA3 -T ScSED1t -P ScTDH3 -ORF SbMDH -T ScGAL2 ).

[0269] Table 15. Overexpression in Saccharomyces cerevisiae SbMDH Primers required for gene generation

[0270] Example 18: Evaluation of succinic acid production capacity of SA132 series strains 1. Fermentation without neutralizer The SA132 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30 °C and 250 rpm for 12 h. The yield of succinic acid was measured by HPLC.

[0271] Table 16. Succinic acid yield from shake-flask fermentation of SA132 series strains

[0272] Example 19: Overexpression in Yersinia lipophila strain Po1g SbMDH Gene Because *Yersinia lipolyticis* has a very strong ability for non-homologous end recombination, the sorghum-derived malate dehydrogenase gene was overexpressed by randomly inserting it into the *Yersinia lipolyticis* genome. SbMDH (SEQ ID NO: 1) and the selection marker β-isopropylmalate dehydrogenase (EC 1.1.1.85) gene YlLEU2 (SEQ ID NO: 148), and with random insertion overexpression YlLEU2 Gene-producing strains were used as controls. Overexpression SbMDH The gene sequence is the same as above, optimized according to the codon preference of CY902. The promoter and terminator used are Po1g's own glyceraldehyde-3-phosphate dehydrogenase gene. YlGAPDH The promoter (SEQ ID NO: 149) and the 3-hydroxy-3-methylglutaryl-CoA reductase gene YlHMG1 Terminator (SEQ ID NO: 150). Overexpression YlLEU2 The promoter and terminator used in the gene are Po1g's own β-isopropylmalate dehydrogenase gene. YlLEU2 The promoter (SEQ ID NO: 151) and the Saccharomyces cerevisiae cytochrome c subtype 1 gene ScCYC1 The terminator (SEQ ID NO: 152). The specific construction method is as follows: 1. Constructing donor DNA fragments for homologous recombination. Using Yersinia lipolyticis W29 genomic DNA (GenBank: GCA_001761485.1) as a template, primer 1_P was used. YlGAPDH _F and 1_P YlGAPDH _R (see Table 17) amplification YlGAPDH Gene promoter sequence (fragment 48); containing SbMDH Using the plasmid containing the synthesized sequence as a template, primers 2_SbMDH_F and 2_SbMDH_R (see Table 17) were used to amplify sorghum-derived sequences. SbMDH Coding sequence (fragment 49); using Yersinia lipophila W29 genomic DNA as a template, primer 3_T YlHMG1 _F and 3_T YlHMG1 _R (see Table 17) amplification YlHMG1 Gene terminator sequence (fragment 50); using primer 4_P YlLEU2 Amplification of _F and 4_YlLEU2_R (see Table 17) YlLEU2 Promoter and ORF sequence (fragment 51); using S288c genomic DNA as a template, primer 5_T ScCYC1 _F and 5_T ScCYC1 _R (see Table 17) amplification ScCYC1 Termination subsequence (fragment 52). Using fragments 48-52 as templates, primer 1_P... YlGAPDH _F and 5_T ScCYC1 _R amplification overexpression SbMDH and YlLEU2 The expression box (fragment 53); using fragments 51 and 52 as templates, primer 4_P YlLEU2 _F and 5_T ScCYC1 _R amplification overexpression YlLEU2 The expression box (segment 54) 2. Construction of overexpression in Yersinia lipophila SbMDH strains Fragment 54 was transformed into strain Po1g (purchased from Yisheng Biotechnology Development Co., Ltd.) using yeast electroporation. Positive transformants were obtained through screening and named strain SA133-1 (genotype: Po1g). genome:: P YlLEU2 -ORF YlLEU2 - T ScCYC1t Fragment 53 was transformed into the Po1g strain using yeast electroporation, and positive transformants were obtained through screening and named strain SA133-2 (genotype: Po1g). genome:: P YlGAPDH -SbMDH-T YlHMG1t -P YlLEU2 -ORF YlLEU2 -T ScCYC1t ).

[0273] Table 17. Overexpression in Yersinia lipophila SbMDH Primers required for gene generation

[0274] Example 20: Evaluation of succinic acid production capacity of SA133 series strains 1. Fermentation without neutralizer SA133 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30 °C and 250 rpm for 24 h. The yield of succinic acid was measured by HPLC.

[0275] Table 18. Succinic acid yield from shake-flask fermentation of SA133 series strains

Claims

1. A genetically modified succinic acid-producing yeast strain that possesses or has enhanced NADPH-dependent malate dehydrogenase (EC50) 1.1.1.82) activity, wherein the NADPH-dependent malate dehydrogenase is derived from sorghum, and the succinic acid-producing yeast strain is *Kudriazwibich*, preserved at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20885.

2. The genetically modified succinic acid-producing yeast strain of claim 1, further having or having enhanced activity of at least one of the following: (i) soluble fumarate reductase (EC 1.3.1.6), (ii) pyruvate carboxylase (EC 6.4.1.1), (iii) fumarate enzyme (EC 4.2.1.2), and (iv) succinic acid transporter.

3. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the 3'-terminal glyoxylate cycling peptide of the soluble fumarate reductase is partially or completely truncated.

4. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the 5' mitochondrial-localizing peptide of the fumarate enzyme is partially or completely truncated.

5. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the soluble fumarate reductase is derived from Saccharomyces cerevisiae, Trypanosoma brevicornu, Leishmania mexicanum, or Trypanosoma krusei.

6. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the soluble fumarate reductase is derived from Trypanosoma brevicornu.

7. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the succinic acid transporter protein is selected from SpMAE1 protein and AnDCT-02 protein.

8. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the succinic acid transporter protein is selected from SpMAE1 protein.

9. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the pyruvate carboxylase is derived from Aspergillus oryzae or Pichia kudrica.

10. The genetically modified succinic acid-producing yeast strain of claim 2, wherein the pyruvate carboxylase is derived from Aspergillus oryzae.

11. The genetically modified succinic acid-producing yeast strain of claim 1, further comprising the following reduced or inactivated characteristics: (i) pyruvate decarboxylase (EC 4.1.1.1), and / or (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), and / or (iii) 5'-phospho-orhinoside decarboxylase (EC 4.1.1.23), and / or (iv) Monocarboxylic acid permeases, and / or (v) dicarboxylic acid transporters, and / or (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and / or (vii) Oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

12. The genetically modified succinic acid-producing yeast strain according to any one of claims 1 to 11, comprising: (i) the overexpressed nucleic acid sequence encoding NADPH-dependent malate dehydrogenase, and / or (ii) the overexpressed nucleic acid sequence encoding a soluble fumarate reductase, and / or (iii) The overexpressed nucleic acid sequence encoding the succinate transporter, and / or (iv) Overexpressed nucleic acid sequence encoding pyruvate carboxylase, and / or (v) Overexpressed nucleic acid sequences encoding fumarate, and / or (vi) The endogenous gene encoding pyruvate decarboxylase is knocked out, and / or (vii) The gene encoding the endogenous NAD-dependent glycerol-3-phosphate dehydrogenase is knocked out, and / or (viii) The gene encoding endogenous 5'-orhinophosphate decarboxylase is knocked out, and / or (ix) The endogenous gene encoding monocarboxylic acid permease is knocked out, and / or (x) The gene encoding the endogenous dicarboxylic acid transporter is knocked out, and / or (xi) The endogenous gene encoding alcohol dehydrogenase 1 is knocked out, and / or (xii) The gene encoding the bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation was knocked out.

13. The genetically modified succinic acid-producing yeast strain of claim 12, which has, The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase includes the sequence shown in SEQ ID NO: 1, and / or The nucleic acid sequence encoding soluble fumarate reductase comprises any of the sequences shown in SEQ ID NO: 3-5, and / or The nucleic acid sequence encoding the succinic acid transporter contains the sequence shown in SEQ ID NO: 2, and / or The nucleic acid sequence encoding pyruvate carboxylase includes the sequence shown in SEQ ID NO: 6 or 7, and / or The nucleic acid sequence encoding fumarate enzyme contains the sequence shown in SEQ ID NO:

8.

14. The genetically modified succinic acid-producing yeast strain according to any one of claims 1 to 11, wherein, In the genetically modified succinic acid-producing yeast strain, (a) The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase and at least one of the following nucleic acid sequences are overexpressed: The nucleic acid sequence encoding soluble fumarate reductase; Nucleic acid sequence encoding succinic acid transporter protein; The nucleic acid sequence encoding pyruvate carboxylase; and The nucleic acid sequence encoding fumarate enzyme, (b) The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase is overexpressed, and the gene encoding endogenous pyruvate decarboxylase and / or the gene encoding endogenous NAD-dependent glycerol-3-phosphate dehydrogenase is knocked out. (c) The nucleic acid sequences encoding NADPH-dependent malate dehydrogenase, pyruvate carboxylase, soluble fumarate reductase and succinate transporter are overexpressed, and the endogenous genes encoding pyruvate decarboxylase and / or endogenous genes encoding NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out. (d) The nucleic acid sequences encoding NADPH-dependent malate dehydrogenase, pyruvate carboxylase, soluble fumarate reductase, fumarate enzyme, and succinate transporter are overexpressed, and the genes encoding endogenous pyruvate decarboxylase and / or endogenous NAD-dependent glycerol-3-phosphate dehydrogenase are knocked out.

15. The genetically modified succinic acid-producing yeast strain of claim 14, wherein the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase comprises the sequence shown in SEQ ID NO:

1.

16. The genetically modified succinic acid-producing yeast strain of claim 14, wherein the nucleic acid sequence encoding soluble fumarate reductase is a nucleic acid sequence encoding soluble fumarate reductase of Saccharomyces cerevisiae, Trypanosoma brevicornu, Leishmania megacephala, or Trypanosoma krusei.

17. The genetically modified succinic acid-producing yeast strain of claim 14, wherein the nucleic acid sequence encoding soluble fumarate reductase comprises any one of the sequences shown in SEQ ID NO: 3-5.

18. The genetically modified succinic acid-producing yeast strain of claim 14, wherein the nucleic acid sequence encoding the succinic acid transporter protein is a nucleic acid sequence encoding the SpMAE1 protein.

19. The genetically modified succinic acid-producing yeast strain of claim 14, wherein the nucleic acid sequence encoding the succinic acid transporter protein comprises the sequence shown in SEQ ID NO:

2.

20. The genetically modified succinic acid-producing yeast strain of claim 14, wherein the nucleic acid sequence encoding pyruvate carboxylase encodes pyruvate carboxylase from Aspergillus oryzae or Pichia kudrica.

21. The genetically modified succinic acid-producing yeast strain of claim 14, wherein the nucleic acid sequence encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7.

22. A method for producing a genetically modified succinic acid-producing yeast strain, comprising conferring or enhancing NADPH-dependent malate dehydrogenase (EC 1.1.1.82) activity in the strain, wherein the NADPH-dependent malate dehydrogenase is derived from sorghum, and wherein the succinic acid-producing yeast strain is *Pichia kudriezvichi*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20885.

23. The method of claim 22 further comprises conferring or enhancing at least one of the following activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase (EC 6.4.1.1) activity, (iii) fumarate enzyme (EC 4.2.1.2) activity, and (iv) succinate transporter activity.

24. The method of claim 23, wherein the soluble fumarate reductase is present in the cytoplasm.

25. The method of claim 23, wherein the fumarate enzyme is present in the cytoplasm in a free state.

26. The method of claim 23, wherein the soluble fumarate reductase is derived from Saccharomyces cerevisiae, Trypanosoma brevicornu, Leishmania megacephala, or Trypanosoma krusei.

27. The method of claim 23, wherein the succinic acid transporter is selected from SpMAE1 protein and AnDCT-02 protein.

28. The method of claim 23, wherein the pyruvate carboxylase is derived from Aspergillus oryzae or Pichia kudrica.

29. The method of any one of claims 22 to 28, further comprising weakening or inactivating the following in the strain: (i) pyruvate decarboxylase (EC 4.1.1.1), and / or (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), and / or (iii) 5'-phospho-orhinoside decarboxylase (EC 4.1.1.23), and / or (iv) Monocarboxylic acid permeases, and / or (v) alcohol dehydrogenase 1 (EC 1.1.1.1), and / or (vi) dicarboxylic acid transporters, and / or (vii) Oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

30. The method of any one of claims 22 to 28, comprising: In the succinic acid-producing yeast strain. (i) Overexpression of a nucleic acid sequence encoding NADPH-dependent malate dehydrogenase, and / or (ii) Overexpression of a nucleic acid sequence encoding a soluble fumarate reductase, and / or (iii) Overexpression of a nucleic acid sequence encoding a succinic acid transporter, and / or (iv) Overexpression of a nucleic acid sequence encoding pyruvate carboxylase, and / or (v) Overexpression of a nucleic acid sequence encoding fumarate, and / or (vi) Knock out the gene encoding endogenous pyruvate decarboxylase, and / or (vii) Knock out the gene encoding endogenous NAD-dependent glycerol-3-phosphate dehydrogenase, and / or (viii) Knock out the gene encoding endogenous 5'-phospho-orhinoside decarboxylase, and / or (ix) Knock out the gene encoding an endogenous monocarboxylic acid permease, and / or (x) Knockout of the gene encoding the endogenous dicarboxylic acid transporter, and / or (xi) Knock out the gene encoding endogenous alcohol dehydrogenase 1, and / or (xii) Knock out the endogenous gene encoding the bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate condensation.

31. The method of claim 30, wherein, The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase includes the sequence shown in SEQ ID NO: 1, and / or The nucleic acid sequence encoding soluble fumarate reductase comprises any of the sequences shown in SEQ ID NO: 3-5, and / or The nucleic acid sequence encoding the succinic acid transporter contains the sequence shown in SEQ ID NO: 2, and / or The nucleic acid sequence encoding pyruvate carboxylase includes the sequence shown in SEQ ID NO: 6 or 7, and / or The nucleic acid sequence encoding fumarate enzyme contains the sequence shown in SEQ ID NO:

8.

32. The method of any one of claims 22 to 28, comprising: In the succinic acid-producing yeast strain. (a) Overexpression of a nucleic acid sequence encoding NADPH-dependent malate dehydrogenase and at least one of the following nucleic acid sequences: The nucleic acid sequence encoding soluble fumarate reductase; Nucleic acid sequence encoding succinic acid transporter protein; The nucleic acid sequence encoding pyruvate carboxylase; and The nucleic acid sequence encoding fumarate enzyme, (b) Overexpression of the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase, and knockout of the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase. (c) Overexpressing the nucleic acid sequences encoding NADPH-dependent malate dehydrogenase, pyruvate carboxylase, soluble fumarate reductase, and succinate transporter, and knocking out the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase. (d) Overexpressing the nucleic acid sequences encoding NADPH-dependent malate dehydrogenase, pyruvate carboxylase, soluble fumarate reductase, fumarate enzyme, and succinate transporter, and knocking out the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase.

33. The method of claim 32, wherein the nucleic acid sequence of the NADPH-dependent malate dehydrogenase comprises the sequence shown in SEQ ID NO:

1.

34. The method of claim 32, wherein the nucleic acid sequence encoding the soluble fumarate reductase is a nucleic acid sequence encoding the soluble fumarate reductase of *Saccharomyces cerevisiae*, *Trypanosoma brevicornu*, *Leishmania megacephala*, or *Trypanosoma krusei*.

35. The method of claim 32, wherein the nucleic acid sequence encoding soluble fumarate reductase comprises any of the sequences shown in SEQ ID NO: 3-5.

36. The method of claim 32, wherein the nucleic acid sequence encoding the succinic acid transporter is a nucleic acid sequence encoding the SpMAE1 protein.

37. The method of claim 32, wherein the nucleic acid sequence encoding the succinic acid transporter comprises the sequence shown in SEQ ID NO:

2.

38. The method of claim 32, wherein the nucleic acid sequence encoding pyruvate carboxylase encodes pyruvate carboxylase from Aspergillus oryzae or Pichia kudria.

39. The method of claim 32, wherein the nucleic acid sequence encoding pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 6 or 7.

40. A method for producing succinic acid, comprising culturing a genetically modified succinic acid-producing yeast strain according to any one of claims 1 to 21 or a genetically modified succinic acid-producing yeast strain prepared according to any one of claims 22 to 39.

41. The method of claim 40, wherein the culture is carried out in a pH range of <3.5 and / or with little or no addition of a neutralizing agent.

42. The method of claim 40, wherein the culture is carried out under conditions of pH ranging from 1.5 to 3.5 and / or with little or no addition of a neutralizing agent.

43. The method of any one of claims 40 to 42, comprising separating and purifying the succinic acid produced.

44. The use of the genetically modified succinic acid-producing yeast strain of any one of claims 1 to 21, or the genetically modified succinic acid-producing yeast strain prepared by the method of any one of claims 22 to 39, in the production of succinic acid.

45. The application of claim 44, wherein succinic acid is produced under conditions of pH < 3.5 and / or with little or no addition of a neutralizing agent.

46. ​​The application of claim 44, wherein succinic acid is produced under conditions of pH ranging from 1.5 to 3.5 and / or with little or no addition of a neutralizing agent.

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