Acid-tolerant yeast strain for efficient succinic acid production, method for constructing the same, and its use.

Abstract

Provided are genetically modified yeast strains for producing succinic acid, having activity or enhanced activity of NADPH-dependent malate dehydrogenase (EC 1.1.1.82), and optionally also having activity or enhanced activity of at least one of: (i) soluble fumarate reductase (EC 4.2.1.2); (ii) pyruvate carboxylase (EC 6.4.1.1); (iii) fumarase (EC 4.2.1.2); and (iv) succinate transport protein, as well as methods for preparing, producing succinic acid therewith, and uses thereof.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and more particularly to a novel acid-resistant yeast strain for the efficient production of succinic acid, a method for constructing the same, and its use. [Background technology]

[0002] Succinic acid, also known as butanediic acid, is an intermediate product in the tricarboxylic acid cycle in living organisms and is also 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 industries such as food, chemical, agricultural, and pharmaceutical industries.

[0003] Conventional succinic acid production methods are chemical processes such as paraffin oxidation, hydrogenation of maleic anhydride, and carbonylation of acrylic acid, which primarily use non-renewable petroleum resources as starting materials for synthesis. Chemical synthesis methods have drawbacks such as high energy consumption and high pollution, making it difficult to meet the requirements of green manufacturing. In contrast, biological fermentation methods for producing succinic acid have low pollution, low energy consumption, and carbon sequestration, which is more in line with national sustainable development strategies. Global production of succinic acid by biological methods is projected to 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, there are mainly two categories of microbial strains for succinic acid synthesis: one is fungal / yeast strains, mainly including Aspergillus niger, Aspergillus fumigatus, Byssochlamys nivea, Paecilomyces varioti, and Saccharomyces cerevisiae; the other is Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, and Bacteroides fragilis. This includes bacteria such as *Corynebacterium fragilis*, which can naturally produce succinic acid, and strains such as *Corynebacterium glutamicum* and *Escherichia coli*, which cannot naturally produce succinic acid (Ahn JH, Jang YS, Lee S Y. Production of succinic acid by metabolically engineered microorganisms[J]. Current Opinion in Biotechnology, 2016, 42:54-66). Filamentous fungi have problems such as slow growth, difficulty in genetic modification, poor fermentation uniformity, and a tendency for mycelium to aggregate. To maintain bacterial cell growth, a considerable amount of neutralizing agent is required for succinic acid production by bacterial fermentation, which increases the cost of isolation and purification and causes potential contamination problems. Yeast has good acid tolerance and can usually grow at pH 3.0, in which case most succinic acid exists in the form of free molecules, so the cost of subsequent isolation and purification can be greatly reduced. [Overview of the project]

[0005] In this invention, the highly efficient production of succinic acid is achieved by metabolic engineering modifications starting from the acid-resistant yeast strain Pichia cudriabuzevi CY902 (stored at the Center for Ordinary Microorganisms (CGMCC) of the China Microbial Species Depositary Administration (CGMCC) under depositary number 20885), isolated from the epidermis of wild fruits in Yunnan Province. Specifically, the modified strain can achieve highly efficient production of succinic acid at low pH through fermentation with no or minimal addition of neutralizing agents. In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain having NADPH-dependent malate dehydrogenase activity or enhanced activity, and optionally further having at least one or enhanced activity among (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase activity, (iii) fumarase activity, and (iv) succinic acid transport protein activity.

[0006] Preferably, the NDAPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae families, or from the genera Euglena or Thermobacillus, more preferably from Sorghum bicolor, Zea mays, Saccharum officinarum, Pisum sativum, Cicer arietinum, Spinacia oleracea, Euglena gracilis, or Methanothermobacter thermautotrophicus.

[0007] In one embodiment, the genetically modified succinate-producing yeast strain further comprises pyruvate decarboxylase and / or NAD-dependent glycerol 3-phosphate dehydrogenase with reduced or inactivated activity.

[0008] In a further embodiment, the present invention provides a method for constructing a genetically modified succinate-producing yeast strain, comprising conferring NADPH-dependent malate dehydrogenase activity to the strain or enhancing NADPH-dependent malate dehydrogenase activity in the strain, and optionally further comprising conferring or enhancing at least one of the following activities: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase (EC6.4.1.1) activity, (iii) fumarase (EC4.2.1.2) activity, and (iv) succinate transport protein activity. Preferably, the NDAPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae families, or from the genera Euglena or Thermobacillus, more preferably from Sorghum bicolor, Zea maize, Saccharum officinarum, Pithum sativum, Cissara aritinum, Spinasia oleracea, Euglena gracilis, or Metanothermobacter thermautotrophus.

[0009] In one embodiment, the method further comprises weakening or inactivating pyruvate decarboxylase and / or NAD-dependent glycerol 3-phosphate dehydrogenase in the strain.

[0010] In a further embodiment, the present invention provides a method for producing succinic acid, comprising culturing a genetically modified succinic acid-producing yeast strain obtained by the genetically modified succinic acid-producing yeast strain of the present invention and / or a method for constructing a genetically modified succinic acid-producing yeast strain according to the present invention (preferably at pH < 3.5, for example in the range of pH 1.5 to 3.5, and / or without or with low addition of a neutralizing agent).

[0011] In a further embodiment, the present invention provides the use of genetically modified succinic acid-producing yeast strains according to the present invention and / or genetically modified succinic acid-producing yeast strains obtained by a method for constructing genetically modified succinic acid-producing yeast strains according to the present invention in the production of succinic acid (preferably in the range of pH < 3.5, for example, pH 1.5 to 3.5, and / or with or without the addition of a neutralizing agent). Preferred embodiments of the present invention are as follows. [1] Having the activity or enhanced activity of NADPH-dependent malate dehydrogenase (EC1.1.1.82), optionally comprising (i) soluble fumarate reductase (EC1.3.1.6), optionally having the 3'-terminal glyoxisome localization peptide of the soluble fumarate reductase partially or completely cleaved, (ii) pyruvate carboxylase (EC6.4.1.1), (iii) fumarase (EC4.2.1.2), optionally having the 5'-terminal mitochondrial localization peptide of the fumarase The protein is partially or completely cleaved, and further having the activity or enhanced activity of at least one of (iv) succinate transport proteins, preferably the NADPH-dependent malate dehydrogenase is derived from plants, more preferably C4 plants, more preferably from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae families, or from the genera Euglena or Thermobacillus, more preferably Sorghum bicolor, Zea maize, Saccharum. The NADPH-dependent malate dehydrogenase is derived from *Ophiocordyceps*, *Pitsum sativum*, *Cissaea aritinum*, *Spinasia oleracea*, *Euglena gracilis*, or *Methanothermobacter thermautotrophus*, and more preferably, the NADPH-dependent malate dehydrogenase is derived from *Sorghum bicolor*, and more preferably, the soluble fumarate reductase is derived from *Saccharomyces cerevisiae*, *Trypanosoma bursei*, *Leishmania mexicana*, or *Trypanosoma creu*. A genetically modified succinate-producing yeast strain, more preferably derived from Trypanosoma bursey, wherein the succinate transport protein is selected from the group consisting of SpMAE1 protein, AnDCT-02 protein, EcDcuB protein and EcDcuC protein, more preferably SpMAE1 protein, and preferably the pyruvate carboxylase is derived from Aspergillus oryzae or Pichia cudria abuzebi, more preferably Aspergillus oryzae. [2] A genetically modified succinate-producing yeast strain as described in [1], further comprising (i) pyruvate decarboxylase (EC 4.1.1.43) with reduced activity or inactivated, and / or (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), and / or (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), and / or (iv) monocarboxylate permease, and / or (v) dicarboxylate transport protein, and / or (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and / or (vii) a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112). [3](i) an overexpressed nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, preferably the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, comprising a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or (ii) an overexpressed soluble (iii) an overexpressed nucleic acid sequence encoding the succinate transport protein, preferably the nucleic acid sequence encoding the soluble fumarate reductase, comprising a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any one of sequence 3 to 5 or a degenerate sequence thereof, and / or (iii) an overexpressed nucleic acid sequence encoding the succinate transport protein, preferably the The nucleic acid sequence encoding the succinate transport protein is the sequence of SEQ ID NO: 2 or its degenerate sequence, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with them, and comprises a nucleotide sequence encoding an amino acid sequence having succinate transport protein activity, and / or (iv) an overexpressed nucleic acid sequence encoding the pyruvate carboxylase, preferably the nucleic acid sequence encoding the pyruvate carboxylase is sequence number A nucleic acid sequence encoding fumarase, preferably the nucleic acid sequence encoding fumarase, comprising a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and having pyruvate carboxylase activity, and / or (v) overexpressed, wherein the nucleic acid sequence encoding fumarase comprises the sequence of SEQ ID NO. 8 or its degenerate sequence, or at least 75%, 80%, 85%, 90% identity thereto,An endogenous gene encoding pyruvate decarboxylase, comprising a nucleotide sequence encoding an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity and possessing fumarase activity, and / or (vi) knocked out, an endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase, and / or (viii) knocked out, an endogenous gene encoding orotidine 5'-phosphate decarboxylase, and / or The genetically modified succinic acid-producing yeast strain described in [1] or [2] has (ix) a knocked-out endogenous gene encoding monocarboxylate permease, and / or (x) a knocked-out endogenous gene encoding dicarboxylate transport protein, and / or (xi) a knocked-out endogenous gene encoding alcohol dehydrogenase 1, and / or (xii) a knocked-out endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase. [4] In the genetically modified succinate-producing yeast strain, (a) the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase and at least one of the following nucleic acid sequences: the nucleic acid sequence encoding the soluble fumarate reductase, the nucleic acid sequence encoding the succinate transport protein, the nucleic acid sequence encoding the pyruvate carboxylase, and the nucleic acid sequence encoding the fumarase are overexpressed, (b) the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase is overexpressed and the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase is knocked out, (c) the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding pyruvate carboxylase, the nucleic acid sequence encoding the soluble fumarate reductase, and the nucleic acid sequence encoding the succinate transport protein are overexpressed and the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase is knocked out, ( d) The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding pyruvate carboxylase, the nucleic acid sequence encoding soluble fumarate reductase, the nucleic acid sequence encoding fumarate, and the nucleic acid sequence encoding succinate transport protein are overexpressed, and the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out, preferably the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase is the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase of sorghum bicolor, more preferably the nucleic acid sequence containing the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, preferably the nucleic acid sequence encoding soluble fumarate reductase is the nucleic acid sequence encoding soluble fumarate reductase derived from Saccharomyces cerevisiae, Trypanosoma bursey, Leishmania mexicana, or Trypanosoma cruz, more preferably the nucleic acid sequence containing any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof, preferablyThe nucleic acid sequence encoding the succinate transport protein is a nucleic acid sequence encoding the SpMAE1 protein, more preferably a nucleic acid sequence including the sequence of SEQ ID NO: 2 or a degenerate sequence thereof, and preferably the nucleic acid sequence encoding the pyruvate carboxylase is a genetically modified succinate-producing yeast strain according to any one of [1] to [3], which encodes a pyruvate carboxylase derived from Aspergillus oryzae or Pichia cudriabuzevi, more preferably the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof. [5] The succinic acid-producing yeast strain is selected from the group consisting of the genera Pichia, Rhodotorula, Saccharomyces, Yarrowia, Zygosaccharomyces, Torlopsis and Candida, preferably selected from the group consisting of the genera Pichia, Saccharomyces and Yarrowia, more preferably Pichia cudriabzebi, Saccharomyces cerevisiae or Yarrowia liporitica, for example, Pichia cudriabzebi deposited with the Center for Ordinary Microorganisms of the Chinese Microbial Species Depositary Control Committee (CGMCC) under depositary number 20885, the genetically modified succinic acid-producing yeast strain according to any one of [1] to [4]. [6] A method for constructing a genetically modified succinate-producing yeast strain, comprising conferring or enhancing the activity of NADPH-dependent malate dehydrogenase (EC1.1.1.82) to the strain, optionally conferring or enhancing at least one of the following activities: (i) soluble fumarate reductase activity, optionally the soluble fumarate reductase being freely present in the cytoplasm, (ii) pyruvate carboxylase (EC6.4.1.1) activity, (iii) fumarase (EC4.2.1.2) activity, optionally the fumarase being freely present in the cytoplasm, and (iv) succinate transport protein activity, preferably the soluble fumarate reductase being derived from Saccharomyces cerevisiae, Trypanosoma bursey, Leishmania mexicana or Trypanosoma cruz, and preferably the succinate transport protein being SpMAE1 protein, AnD A method comprising selecting from the group consisting of CT-02 protein, EcDcuB protein, and EcDcuC protein, preferably the pyruvate carboxylase being derived from Aspergillus oryzae or Pichia cudriabzebi, preferably the NADPH-dependent malate dehydrogenase being derived from a plant, more preferably a C4 plant, more preferably from a plant of the family Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae, or from the genus Euglena or Thermobacillus, more preferably from Sorghum bicolor, Zea maize, Saccharum officinarum, Pithum sativum, Cissa aritinum, Spinacia oleracea, Euglena gracilis or Metanothermobacter thermautotrophus, and more preferably the NADPH-dependent malate dehydrogenase being Sorghum bicolor-derived NADPH-dependent malate dehydrogenase. [7] The method according to [6], further comprising attenuating or inactivating (i) pyruvate decarboxylase (EC 4.1.1.43), and / or (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), and / or (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), and / or (iv) monocarboxylate permease, and / or (v) alcohol dehydrogenase 1 (EC 1.1.1.1), and / or (vi) dicarboxylate transport protein, and / or (vii) a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112) in the strain. [8] In the succinic acid-producing yeast strain, (i) overexpress the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, preferably the sequence of SEQ ID NO: 1 or its degenerate sequence, or the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase which has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with it and which encodes an amino acid sequence having NADPH-dependent malate dehydrogenase activity. (ii) expressing and / or (ii) overexpressing the nucleic acid sequence encoding the soluble fumarate reductase, preferably one of sequence numbers 3 to 5 or its degenerate sequence, or a nucleic acid sequence encoding the soluble fumarate reductase that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with it and which encodes an amino acid sequence having soluble fumarate reductase activity, and / or (iii) Overexpressing the nucleic acid sequence encoding the succinate transport protein, preferably the sequence of SEQ ID NO: 2 or its degenerate sequence, or the nucleic acid sequence encoding the succinate transport protein which includes a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having succinate transport protein activity, and / or (iv) The pyruvate carboxylase Overexpressing a nucleic acid sequence encoding a signal, preferably the sequence of SEQ ID NO: 6 or 7 or its degenerate sequence, or a nucleic acid sequence encoding a pyruvate carboxylase that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and which encodes an amino acid sequence having pyruvate carboxylase activity, and / or (v) overexpressing a nucleic acid sequence encoding the fumarase,Preferably, overexpressing the nucleic acid sequence encoding fumarase, which includes the sequence of SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or (vi) knocking out the endogenous gene encoding pyruvate decarboxylase, and / or (vii) knocking out the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase, and / or (viii) Orochi The method according to [6] or [7], comprising knocking out an endogenous gene encoding din 5'-phosphate decarboxylase and / or (ix) an endogenous gene encoding monocarboxylate permease and / or (x) an endogenous gene encoding dicarboxylate transport protein and / or (xi) an endogenous gene encoding alcohol dehydrogenase 1 and / or (xii) an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase. [9] In the succinate-producing yeast strain, (a) a nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase and the following nucleic acid sequences: the nucleic acid sequence encoding the soluble fumarate reductase, the nucleic acid sequence encoding the succinate transport protein, the nucleic acid sequence encoding the pyruvate carboxylase, and the nucleic acid sequence encoding the fumarase, (b) overexpressing at least one of the following, (c) overexpressing the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase 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 sequence encoding NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding pyruvate carboxylase, the nucleic acid sequence encoding soluble fumarate reductase and the nucleic acid sequence encoding succinate transport protein 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 sequence encoding NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding pyruvate carboxylase, the nucleic acid sequence encoding soluble fumarate reductase, the nucleic acid sequence encoding fumarate and the nucleic acid sequence encoding succinate transport protein and knocking out the endogenous gene encoding pyruvate decarboxylase This comprises knocking out the gene and / or the endogenous gene encoding the NAD-dependent glycerol 3-phosphate dehydrogenase, preferably the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase is a nucleic acid sequence encoding NADPH-dependent malate dehydrogenase derived from Sorghum bicolor, more preferably the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, preferably the nucleic acid sequence encoding the soluble fumarate reductase is a nucleic acid sequence encoding soluble fumarate reductase derived from Saccharomyces cerevisiae, Trypanosoma burseyi, Leishmania mexicana or Trypanosoma cruz, more preferably one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof, preferably the nucleic acid sequence encoding the succinate transport protein is a nucleic acid sequence encoding the SpMAE1 protein, more preferably the sequence of SEQ ID NO: 2 or a degenerate sequence thereof, preferably the nucleic acid sequence encoding the pyruvate carboxylase is a pyruvate carboxylase derived from Aspergillus oryzae or Pichia cudriabuzevi,More preferably, the method according to any one of [6] to [8], comprising the sequence of sequence number 6 or 7 or a degenerate sequence thereof.

[10] The succinic acid-producing yeast strain is selected from the group consisting of the genera Pichia, Rhodotorula, Saccharomyces, Yarrowia, Zygosaccharomyces, Torlopsis and Candida, preferably selected from the group consisting of the genera Pichia, Saccharomyces and Yarrowia, more preferably Pichia cudriabzebi, Saccharomyces cerevisiae or Yarrowia liporitica, for example, Pichia cudriabzebi deposited with the Center for Ordinary Microorganisms of the Chinese Microbial Species Depositary Commission (CGMCC) under depositary number CGMCC number 20885, according to any one of [6] to [9].

[11] A method for producing succinic acid, comprising culturing a genetically modified succinic acid-producing yeast strain described in any of [1] to [5] or a genetically modified succinic acid-producing yeast strain prepared by any of the methods described in any of [6] to

[10] , preferably at a pH < 3.5, more preferably in the range of pH 1.5 to 3.5, and / or with no or low addition of a neutralizing agent, and optionally isolating and purifying the produced succinic acid.

[12] Use of a genetically modified succinic acid-producing yeast strain described in any of [1] to [5] or a genetically modified succinic acid-producing yeast strain prepared by any of the methods described in any of [6] to

[10] in the production of succinic acid, preferably in the range of pH < 3.5, more preferably in the range of pH 1.5 to 3.5, and / or without or with low addition of a neutralizing agent. Detailed description of the invention

[0012] Unless otherwise defined, the technical and scientific terms used herein have meanings that are generally understood by those skilled in the art, for example, by referring to Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

[0013] As used herein, the term “genetically modified” means that a strain artificially modified by biological means has one or more modifications, such as gene deletions, amplifications, or mutations, compared to the original strain before modification, and thereby has modified biological characteristics, such as improved production characteristics. As used herein, the term “original strain” may be the natural strain being genetically modified or a strain having other genetic modifications.

[0014] As used herein, the term “succinate-producing yeast strain” refers to a yeast strain that can produce succinate under suitable conditions (e.g., via fermentation) and secrete succinate into extracellular medium. Succinate-producing yeast strains possess proteins that can transport succinate extracellularly, so that succinate can be secreted extracellularly after it is produced. Suitable succinate transport proteins used in a given yeast strain are known in the art, including, but are not limited to, the dicarboxylate transport protein SpMAE1 of Schizosaccharomyces pombe and the dicarboxylate transport protein AnDCT-02 of Aspergillus niger.

[0015] Succinate-producing yeasts are known in the art and include, but are not limited to, those belonging to the genera Zygosaccharomyces, Torulopsis, Candida, Pichia, Rhodotroula, Saccharomyces, and Yarrowia. In one embodiment, the succinate-producing yeast strain is a strain derived from the genera Pichia, Saccharomyces, or Yarrowia. In a preferred embodiment, the succinic acid-producing yeast strain is Pichia cudriabuzevi (e.g., strain CICC32244), Saccharomyces cerevisiae (e.g., strain BY4742), or Yarrowia lipolytica (e.g., strain Po1g), for example, Pichia cudriabuzevi stored at the Center for Ordinary Microorganisms of the Chinese Microbial Species Depositary Commission (CGMCC) under depositary number 20885.

[0016] As used herein, “active” means having detectable activity compared to a reference that does not have such activity (e.g., an early strain or a wild-type strain).

[0017] As used herein, “having enhanced activity” means that the activity is increased 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 100%, at least 150%, at least 200%, at least 250%, at least 300%, or more, compared to a reference having such activity (e.g., an initial strain or a wild-type strain).

[0018] The activity of a protein (e.g., an enzyme) may be generated or enhanced by any suitable means known in the art, including, but not limited to, expressing or overexpressing a corresponding gene encoding the protein in a strain (e.g., via a vector such as a plasmid), or introducing a mutation that results in increased protein activity.

[0019] In some embodiments, in the genetically modified succinic acid-producing yeast strains of the present invention, one or more copies of the target gene or its homologous gene may be incorporated into the genome (e.g., via homologous recombination), and optionally, at any locus in the genome, for example, at a locus where one copy of any gene in the genome is replaced by one or more copies of the target gene or its homologous gene (provided that such incorporation does not significantly adversely affect the growth and production of the strain). Those skilled in the art know how to incorporate a transgene and how to select a strain into which the transgene has been incorporated.

[0020] As used herein, the terms “reduced activity or inactivated…” or “reduced activity or inactivated…” mean that the activity 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 100%, compared to the reference activity (e.g., the corresponding activity in the initial or wild-type strain).

[0021] The activity of a protein (e.g., an enzyme) may be reduced or inactivated by any suitable means known in the art, including, but not limited to, using a corresponding weakening or inactivation gene encoding the protein, introducing a mutation resulting in reduced or inactivation of the protein, or using a protein antagonist or inhibitor (e.g., an antibody, a ligand, etc.).

[0022] As used herein, the term “weakened or inactivated gene” means that the activity of a gene, for example, its expression level (when used as a protein-coding gene) or its regulatory capacity (when used as 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 or wild-type strain). In the case of a protein-coding gene such as an enzyme, a “weakened or inactivated gene” also includes the reduction in the activity level of the protein expressed by this gene by, for example, 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 activity level of the corresponding protein in the initial or wild-type strain.

[0023] In this specification, a reference may be a wild-type microorganism or a microorganism before the desired genetic manipulation is performed (e.g., an initial microorganism used to perform a genetic manipulation to increase gene activity). In this specification, parent microorganism and initial microorganism may be used interchangeably and refer to a microorganism on which the desired genetic manipulation (e.g., enhancing or weakening the activity of a gene or protein) is performed.

[0024] As used herein, malate dehydrogenase (EC1.1.1.82 (NADPH-dependent)) is encoded by the MDH gene and is involved in the conversion between oxaloacetate and malate. NADPH-dependent malate dehydrogenase contributes to the conversion of oxaloacetate to malate, a reaction in which one molecule of NADPH is consumed. Commonly used sources of NADPH-dependent malate dehydrogenase include C4 plants (e.g., grasses, sedges, aster, spurge, amaranthaceae, purslane, and amaranthaceae), and genera such as Euglena and Thermobacillus, e.g., Sorghum bicolor, Zea maize, Saccharum officinarum, Pithum sativum, Cissara aritinum, Spinasia oleracea, Euglena gracilis, or Metanothermobacter thermoautotrophus. As used herein, “having NADPH-dependent malate dehydrogenase activity or enhanced activity” means that the strain has NADPH-dependent malate dehydrogenase activity or increased NADPH-dependent malate dehydrogenase activity that catalyzes the conversion of oxaloacetate to malate.

[0025] As used herein, soluble fumarate reductase (EC1.3.1.6) is encoded by the FRD gene. This enzyme is involved in the interconversion between fumarate and succinate, contributing to the conversion of fumarate to succinate. Commonly used sources of soluble fumarate reductase are yeast and kinetoplastiales, particularly Saccharomyces cerevisiae, Trypanosoma brucei, Leishmania mexicana, and Trypanosoma cruzi. As used herein, "having fumarate reductase activity or enhanced activity" means that a strain has soluble fumarate reductase activity or increased soluble fumarate reductase activity that converts fumarate to succinate.

[0026] As used herein, fumarase (EC4.2.1.2) is encoded by the FUM gene. This enzyme is involved in the interconversion of fumarate and malate in the cytoplasm and mitochondria, contributing to the conversion of malate to fumarate. Commonly used sources include Actinobacillus succinogenes, Mannhemia succiniciproducens, Escherichia coli, Pichia kudoriabzebi, and Rhizopus oryzae. One FUM gene, FUM1, is known to be present in Pichia kudoriabzebi. As used herein, "having fumarase activity or enhanced activity" means that a strain has fumarase activity or increased fumarase activity that converts malate to fumarate.

[0027] As used herein, “succinate transport protein” refers to a protein capable of transporting intracellular succinate out of the cell, including, but not limited to, the SpMAE1 protein of Schizosaccharomyces pombe (Uniprot database search number: P50537), the dicarboxylate transport protein AnDCT-02 of Aspergillus niger (NCBI reference sequence: XP_001398131.1), and the dicarboxylate transport proteins EcDcuB (gene ID: 948641) and EcDcuC (gene ID: 945000) of Escherichia coli. In this specification, “having succinate transport protein activity or enhanced activity” means that the strain has activity or increased activity to transport succinate out of the cell.

[0028] As used herein, the dicarboxylate transport protein SpMAE1 of Schizosaccharomyces pombe is encoded by the SpMAE1 gene, and the SpMAE1 protein contributes to the transport of intracellular dicarboxylates to the extracellular space. In this specification, “having SpMAE1 activity or enhanced activity” means that the strain has activity or increased activity to transport dicarboxylates to the extracellular space.

[0029] As used herein, pyruvate carboxylase (EC 6.4.1.1) is encoded by the PYC gene. Pyruvate carboxylase is involved in the interconversion between oxaloacetate and pyruvate in the process of gluconeogenesis, contributing to the conversion of pyruvate and carbon dioxide to oxaloacetate. Commonly used sources of pyruvate carboxylase include fungi, particularly yeasts and filamentous fungi, preferably Saccharomyces cerevisiae, Pichia kudoriabzebi, Aspergillus oryzae, and Kluyveromyces marxianus. One PYC gene, namely the PYC1 gene, is known to be present in Pichia kudoriabzebi. In this specification, "having pyruvate carboxylase activity or enhanced activity" means that the strain has the activity or increased activity to convert pyruvate to oxaloacetate.

[0030] As used herein, pyruvate decarboxylase (EC 4.1.1.43) is encoded by the PDC gene and is involved in the decarboxylation of pyruvate in the ethanol synthesis pathway. One PDC gene present in Pichia cudriabzebi is known to be the PDC1 gene. In this specification, "deactivated or inactivated pyruvate decarboxylase" refers to a decrease or loss of the pyruvate decarboxylase activity of this enzyme.

[0031] As used herein, NAD-dependent glycerol 3-phosphate dehydrogenase (EC1.1.1.8) is encoded by the GPD gene and is involved in the interconversion between glyceron phosphate and glycerol 3-phosphate in the glycerol synthesis pathway. One GPD gene, namely the GPD1 gene, is known to be present in Pichia cudriabzebi. In this specification, "deactivated or inactivated NAD-dependent glycerol 3-phosphate dehydrogenase" refers to a decrease or loss of activity of this enzyme that catalyzes the interconversion between glyceron phosphate and glycerol 3-phosphate.

[0032] As used herein, orotidine 5'-phosphate decarboxylase (EC4.1.1.23) is encoded by the URA3 gene and is involved in the decarboxylation of orotidine 5'-phosphate in the process of pyrimidine synthesis. One URA3 gene is known to be present in Pichia cudriabzebi. In this specification, “deactivated or reduced activity of orotidine 5'-phosphate decarboxylase” means a decrease or loss of activity of this enzyme that catalyzes the orotidine 5'-phosphate decarboxylation reaction.

[0033] As used herein, alcohol dehydrogenase 1 (EC1.1.1.1) is encoded by the ADH1 gene and is involved in the interconversion between acetaldehyde and ethanol in the ethanol synthesis pathway. In this specification, “deactivated or inactivated alcohol dehydrogenase 1” refers to a decrease or loss of activity of this enzyme that catalyzes the interconversion between acetaldehyde and ethanol. In one embodiment, the ADH1 gene in a strain is knocked out, for example, by homologous recombination.

[0034] As used herein, monocarboxylate permease (NCBI reference sequence XP_029320775.1) is encoded by the MCH4 gene. In this specification, “decreased or inactivated monocarboxylate permease” refers to a decrease or loss of catalytic activity of this enzyme. In one embodiment, the MCH4 gene in a strain is knocked out, for example, by homologous recombination.

[0035] As used herein, the JEN2 gene encodes a dicarboxylate transport protein involved in the process of transporting dicarboxylate from culture medium to cells. Two JEN2 genes (JEN2-1 (encoding the polypeptide of SEQ ID NO: 14) and JEN2-2 (encoding the polypeptide of SEQ ID NO: 15)) are known to be present in Pichia cudriabzevi. In this specification, “deactivated or inactivated dicarboxylate transport protein” refers to a decrease or loss of cellular activity in transporting dicarboxylate from culture medium to cells.

[0036] As used herein, “neutralizing agent” refers to a reagent that precipitates succinic acid in the form of calcium succinate from a fermentation system. Substances that can be used as neutralizing agents include, but are not limited to, calcium carbonate, as is known in the art.

[0037] As used herein, “no or low addition of neutralizing agent” means an amount of neutralizing agent that 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%, lower than the amount of neutralizing agent known in the art to be added for the fermentation production of succinic acid. For example, the amount of neutralizing agent added in the method of the present invention may be 0 to 30 g / L.

[0038] As used herein, the terms “polypeptide,” “amino acid sequence,” “peptide,” and “protein” may be used interchangeably herein and refer to amino acid chains of any length that may contain modified amino acids and / or be interrupted by non-amino acid components. The term also encompasses amino acid chains modified by any other operation or modification, whether natural or artificial, such as the formation of disulfide bonds, glycosylation, lipidization, acetylation, phosphorylation, or conjugation with marker components.

[0039] As used herein, the terms “gene,” “nucleic acid sequence,” “polynucleotide,” and “nucleotide sequence” may be used interchangeably herein and refer to nucleotide chains including DNA and RNA. “Genes expression” or “gene expression” refers to the transcription of appropriate regulatory regions, in particular DNA regions operably linked to a promoter, into biologically active RNA and RNA that can be translated into biologically active proteins or peptides.

[0040] As used herein, “degenerate sequence” refers to a nucleotide sequence that codes for the same amino acid sequence as a particular sequence but has a different nucleotide sequence due to the degeneracy of gene codons.

[0041] Where used herein, terms such as “homology” and “sequence identity” are used interchangeably herein. Sequence identity may be detected by alignment of the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide, for example, by a standard alignment algorithm program using a default gap penalty established by each supplier. Whether two nucleic acid molecules have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% “identical” nucleotide sequences can be determined by using known computer algorithms such as BLASTN, FASTA, DNAStar, and Gap (University of Wisconsin Genetics Computer Group (UWG), Madison, Wisconsin, USA). For example, the identity percentage of nucleic acid molecules can be determined by comparing sequence information using, for example, a 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))). Briefly, the GAP program defines similarity based on the number of aligned similar symbols (i.e., nucleotides) divided by the total number of symbols in the shorter of the two sequences.

[0042] As used herein, the Pk2365 gene encodes a bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112) that is both oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase. In this specification, “bifunctional enzyme with reduced or inactivated activity of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase” means a reduction or loss of activity of this enzyme that catalyzes the decarboxylation of oxaloacetate and the aldol condensation of 3-hydroxy-3-methylglutarate.

[0043] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having NADPH-dependent malate dehydrogenase activity or enhanced activity, and optionally further having at least one or enhanced activity among (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase activity, (iii) fumarase activity, and (iv) succinate transport protein activity. The term "at least one" includes any one, two, three, or all of the activities selected therefrom.

[0044] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having NADPH-dependent malate dehydrogenase and soluble fumarate reductase activity or enhanced activity.

[0045] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having NADPH-dependent malate dehydrogenase and pyruvate carboxylase activity or enhanced activity.

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

[0047] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase and succinate transport protein.

[0048] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, soluble fumarate reductase, and pyruvate carboxylase.

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

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

[0051] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, pyruvate carboxylase, and fumarase.

[0052] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, pyruvate carboxylase, and succinate transport protein.

[0053] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, fumarase, and succinate transport protein.

[0054] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, soluble fumarate reductase, pyruvate carboxylase, and fumarase.

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

[0056] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, soluble fumarate reductase, fumarase, and succinate transport protein.

[0057] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, pyruvate carboxylase, fumarase, and succinate transport protein.

[0058] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain having the activity or enhanced activity of NADPH-dependent malate dehydrogenase, soluble fumarate reductase, pyruvate carboxylase, fumarase, and succinate transport protein.

[0059] In one embodiment, having active or enhanced activity is achieved by expressing or overexpressing the corresponding coding gene in the strain. Thus, in one embodiment, the gene encoding NADPH-dependent malate dehydrogenase is expressed or overexpressed in a genetically modified succinate-producing yeast strain.

[0060] In one embodiment, the NADPH-dependent malate dehydrogenase is derived from a plant (preferably a C4 plant, more preferably from the grasses, sedges, daisies, euphorbias, amaranthaceae, purslane, and amaranthaceae), the genus Euglena, or the genus Thermobacillus, preferably from Sorghum bicolor, Zea maize, Saccharum officinarum, Pithum sativum, Cissara aritinum, Spinacia oleracea, Euglena gracilis, or Metanothermobacter thermautotrophus, more preferably from Sorghum bicolor.

[0061] In one embodiment, the activity of NADPH-dependent malate dehydrogenase is generated or increased by expressing or overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase in a genetically modified succinate-producing yeast strain. Preferably, the MDH gene encoding NADPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae families, or from the genera Euglena or Thermobacillus, more preferably from Sorghum bicolor, Zea maize, Saccharum officinarum, Pithum sativum, Cissara aritinum, Spinacia oleracea, Euglena gracilis or Metanothermobacter thermautotrophus, and more preferably from Sorghum bicolor.

[0062] In one embodiment, the MDH gene encoding NADPH-dependent malate dehydrogenase includes the sequence of Sequence ID No. 1 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and possessing NADPH-dependent malate dehydrogenase activity.

[0063] In one embodiment, the MDH gene encoding NADPH-dependent malate dehydrogenase is incorporated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia cudriabuzevi) at, for example, the Pk2365 locus. The MDH gene encoding NADPH-dependent malate dehydrogenase may be controlled by an appropriate promoter (e.g., the promoter of the FBA1 gene (shown in SEQ ID NO 17)) and / or terminator (e.g., the terminator of the INO1 gene (shown in SEQ ID NO 18)).

[0064] In further embodiments, the genetically modified succinate-producing yeast strain further possesses soluble fumarate reductase or has enhanced soluble fumarate reductase. In one embodiment, the soluble fumarate reductase is derived from yeast and the kinetoplastial order, for example, but not limited to Saccharomyces cerevisiae, Trypanosoma burseyi, Leishmania mexicana and Trypanosoma cruz.

[0065] In one embodiment, soluble fumarate reductase activity is generated or increased by expressing or overexpressing a gene encoding soluble fumarate reductase in a genetically modified succinic acid-producing yeast strain. Preferably, the gene encoding soluble fumarate reductase is derived from yeast and the order Kinetoplastes, for example, but not limited to Saccharomyces cerevisiae, Trypanosoma burseyi, Leishmania mexicana, and Trypanosoma cruz.

[0066] In one embodiment, the glyoxisome-localized peptide at the 3' end of soluble fumarate reductase is partially or completely cleaved and therefore freely available in the cytoplasm.

[0067] In one embodiment, the gene encoding soluble fumarate reductase includes one of sequence numbers 3 to 5 and its degenerate sequence, or a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith and possessing soluble fumarate reductase activity.

[0068] In one embodiment, the gene encoding soluble fumarate reductase is integrated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia cudriabuzevi) at, for example, the ADH1 locus. The gene encoding soluble fumarate reductase may be placed under the control of an appropriate promoter (e.g., the promoter of the ADH1 gene shown in SEQ ID NO: 21) and / or terminator (e.g., the terminator of the ADH1 gene shown in SEQ ID NO: 22).

[0069] In further embodiments, the genetically modified succinate-producing yeast strain further possesses succinate transport protein activity or enhanced succinate transport protein activity.

[0070] In one embodiment, the succinate transport protein is selected from the group consisting of SpMAE1 protein, AnDCT-02 protein, EcDcuB protein, and EcDcuC protein. In one embodiment, succinate transport protein activity is generated or increased by expressing or overexpressing a gene encoding a succinate transport protein, such as the SpMAE1 protein, in a genetically modified succinate-producing yeast strain. In one embodiment, succinate transport protein activity is generated or enhanced by expressing or overexpressing the SpMAE1 gene.

[0071] In one embodiment, the SpMAE1 gene comprises a nucleotide sequence encoding an amino acid having succinate transport protein activity (e.g., derived from Schizosaccharomyces pombe) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Optionally, the SpMAE1 gene is incorporated into the genome of a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabuzevi) at, for example, the MCH4 locus. The SpMAE1 gene may be controlled by an appropriate promoter (e.g., the promoter of the TDH3 gene (shown in SEQ ID NO: 19)) and / or a terminator (e.g., the terminator of the GAL2 gene (shown in SEQ ID NO: 20)).

[0072] In further embodiments, the genetically modified succinic acid-producing yeast strain further 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 may be derived from fungi, particularly yeasts and filamentous fungi, preferably Saccharomyces cerevisiae, Pichia kudoriabzebi, Aspergillus oryzae, Clibellomyces marsianus, and the like. In one embodiment, the gene encoding pyruvate carboxylase may be selected from the group consisting of the PYC gene of Aspergillus oryzae and the PYC1 gene of Pichia kudoriabzebi.

[0073] In one embodiment, pyruvate carboxylase comprises an amino acid sequence encoded by the sequence of 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 more identity thereto, and possessing pyruvate carboxylase activity.

[0074] In one embodiment, the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and which has pyruvate carboxylase activity.

[0075] Optionally, the gene encoding pyruvate carboxylase is incorporated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia cudriabzevi) at, for example, the JEN2-1 locus. The gene encoding pyruvate carboxylase may be placed under the control of an appropriate promoter (e.g., the promoter of the TDH3 gene (shown in SEQ ID NO: 19)) and / or terminator (e.g., the terminator of the GAL2 gene (shown in SEQ ID NO: 20)).

[0076] In further embodiments, the genetically modified succinate-producing yeast strain has further or enhanced fumarase activity. Fumarase activity can be generated or enhanced by expressing or overexpressing the gene encoding fumarase. In one embodiment, the genetically modified succinate-producing yeast strain expresses or overexpresses the gene encoding fumarase. In one embodiment, the fumarase is derived from, for example, Actinobacillus succinogenes, Mannhemia succiniciproducens, Escherichia coli, Pichia cudriabzebi, and Rhizopus oryzae.

[0077] In one embodiment, fumarase includes the amino acid sequence of SEQ ID NO: 95, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and possessing fumarase activity.

[0078] In one embodiment, the mitochondrial localization peptide at the 5' end of the fumarase is partially or completely cleaved and therefore cannot localize to mitochondria but is freely present in the cytoplasm.

[0079] In one embodiment, the gene encoding fumarase includes the sequence of Sequence ID No. 8 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and which has fumarase activity.

[0080] Optionally, the gene encoding fumarase is incorporated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia cudriabzevi) at, for example, the PDC1 locus. The gene encoding fumarase may be placed under the control of an appropriate promoter (e.g., the promoter of the TDH3 gene (shown in SEQ ID NO: 19)) and / or terminator (e.g., the terminator of the GAL2 gene (shown in SEQ ID NO: 20)).

[0081] Genes that need to be expressed or overexpressed may be incorporated at appropriate locations in the strain genome, provided that such incorporation does not adversely affect the strain's growth, proliferation, and / or productivity. For example, genes that need to be expressed or overexpressed may be incorporated at any one or more locations in the genome encoding the following proteins: (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylate permease, (v) dicarboxylate transport protein, (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) the bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112).

[0082] In further embodiments, the genetically modified succinic acid-producing yeast strain further comprises at least one reduced-activity or inactivated (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylate permease, (v) dicarboxylate transport protein, (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112).

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

[0084] In one embodiment, pyruvate decarboxylase includes the amino acid sequence of 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 more identity thereto, and possessing pyruvate decarboxylase activity.

[0085] In one embodiment, the gene encoding pyruvate decarboxylase encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the protein of Sequence ID No. 10, and having pyruvate decarboxylase activity.

[0086] In one embodiment, the genetically modified succinate-producing yeast strain further possesses a reduced-activity or inactivated NAD-dependent glycerol 3-phosphate dehydrogenase. In one embodiment, the genetically modified succinate-producing yeast strain has 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 succinate-producing yeast strain (e.g., Pichia cudriabuzevi) is knocked out.

[0087] In one embodiment, the NAD-dependent glycerol 3-phosphate dehydrogenase comprises the amino acid sequence of 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 more identity thereto, and possessing NAD-dependent glycerol 3-phosphate dehydrogenase activity.

[0088] In one embodiment, the gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase is, for example, SEQ ID NO: 1 1 The protein, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with it, and possessing NAD-dependent glycerol 3-phosphate dehydrogenase activity.

[0089] In further embodiments, the genetically modified succinate-producing yeast strain further comprises alcohol dehydrogenase 1 with reduced activity or inactivation. In one embodiment, the genetically modified succinate-producing yeast strain has a weakened or inactivated gene encoding alcohol dehydrogenase 1. In one embodiment, the gene encoding alcohol dehydrogenase 1 in the genetically modified succinate-producing yeast strain (e.g., Pichia cudriabuzevi) is knocked out.

[0090] In one embodiment, alcohol dehydrogenase 1 includes the amino acid sequence of 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 more identity thereto, and possessing alcohol dehydrogenase 1 activity.

[0091] In one embodiment, the gene encoding alcohol dehydrogenase 1 encodes, for example, the protein of sequence number 12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and possessing alcohol dehydrogenase 1 activity.

[0092] In further embodiments, the genetically modified succinic acid-producing yeast strain further comprises a reduced-activity or inactivated orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), a dicarboxylate transport protein, a bifunctional enzyme consisting of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase, and / or a monocarboxylate permease.

[0093] In further embodiments, genes encoding orotidine 5'-phosphate decarboxylase and / or dicarboxylate transport proteins (e.g., JEN2 gene, e.g., JEN2-1 gene, JEN2-2 gene) and / or monocarboxylate permease and / or genes encoding the bifunctional enzymes oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase are knocked out in genetically modified succinate-producing yeast strains.

[0094] In one embodiment, the genetically modified succinic acid-producing yeast strain further possesses a difunctional enzyme in which the activity of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase is reduced or inactivated. Specifically, the genetically modified succinic acid-producing yeast strain has a weakened or inactivated gene encoding the difunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase. Preferably, the gene encoding the difunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase in the genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi) is knocked out.

[0095] In one embodiment, the bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase includes the amino acid sequence of 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 more identity thereto, and having the activity of a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0096] In one embodiment, the gene encoding the bifunctional enzymes oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the protein of Sequence ID No. 9, or having the activity of the bifunctional enzymes oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0097] In one embodiment, the genetically modified succinate-producing yeast strain further comprises a reduced-activity or inactivated orotidine 5'-phosphate decarboxylase. Specifically, the genetically modified succinate-producing yeast strain has a weakened or inactivated gene encoding orotidine 5'-phosphate decarboxylase. Preferably, the gene encoding orotidine 5'-phosphate decarboxylase in the genetically modified succinate-producing yeast strain (e.g., Pichia cudriabuzevi) is knocked out.

[0098] In one embodiment, the orotidine 5'-phosphate decarboxylase comprises the amino acid sequence of 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 more identity thereto, and possessing orotidine 5'-phosphate decarboxylase activity.

[0099] In one embodiment, the gene encoding orotidine 5'-phosphate decarboxylase encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the protein of Sequence ID No. 13, and possessing orotidine 5'-phosphate decarboxylase activity.

[0100] In one embodiment, the genetically modified succinic acid-producing yeast strain further comprises a reduced-activity or inactivated monocarboxylate permease. Specifically, the genetically modified succinic acid-producing yeast strain has a weakened or inactivated gene encoding monocarboxylate permease. Preferably, the gene encoding monocarboxylate permease in the genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi) is knocked out.

[0101] In one embodiment, the monocarboxylate permease includes the amino acid sequence of 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 more identity thereto, and possessing monocarboxylate permease activity.

[0102] In one embodiment, the gene encoding monocarboxylate permease encodes, for example, the protein of 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 more identity thereto, and possessing monocarboxylate permease activity.

[0103] In one embodiment, the genetically modified succinic acid-producing yeast strain further has a dicarboxylate transport protein with reduced activity or inactivation. Specifically, the genetically modified succinic acid-producing yeast strain has a weakened or inactivated gene encoding a dicarboxylate transport protein. Preferably, the gene encoding the dicarboxylate transport protein in the genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi), such as the JEN2-1 or JEN2-2 gene, is knocked out.

[0104] In one embodiment, the dicarboxylate transport protein includes the amino acid sequence of 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 more identity thereto, and possessing dicarboxylate transport protein activity.

[0105] In one embodiment, the gene encoding the dicarboxylate transport protein encodes, for example, the protein of 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 more identity thereto, and possessing dicarboxylate transport protein activity.

[0106] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., strain CY902) having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, and optionally having an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase knocked out. Preferably, the MDH gene comprises the sequence of 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 cudriabuzevi, e.g., strain CY902) having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding soluble fumarate reductase, wherein an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding alcohol dehydrogenase 1 and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase is optionally knocked out. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding soluble fumarate reductase comprises any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof.

[0108] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., strain CY902) having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding succinate transport protein, e.g., SpMAE1 gene, and optionally, an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding monocarboxylate permease and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase is knocked out. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding succinate transport protein comprises the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0109] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., strain CY902) having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding fumarase, and optionally having an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or pyruvate decarboxylase and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase knocked out. Preferably, the MDH gene contains the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding fumarase contains the sequence of SEQ ID NO: 8 or a degenerate sequence thereof.

[0110] In one embodiment, the present invention provides a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., strain CY902) having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding pyruvate carboxylase, and optionally having knockout of an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding a dicarboxylate transport protein, such as the JEN2-1 or JEN2-2 gene and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase. Preferably, the MDH gene contains the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase contains the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof.

[0111] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., strain CY902) having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding pyruvate carboxylase, wherein the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out, and optionally, the endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or the endogenous gene encoding a dicarboxylate transport protein (e.g., the JEN2 gene, e.g., JEN2-1 gene, JEN2-2 gene) and / or the endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase are knocked out. Preferably, the MDH gene contains the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase contains the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof.

[0112] In one embodiment, the present invention provides a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabzebi, e.g., strain CY902) having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, a gene encoding pyruvate carboxylase, a gene encoding soluble fumarate reductase, and a gene encoding succinate transport protein, e.g., SpMAE1 gene, wherein the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out, and optionally, the endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or the endogenous gene encoding dicarboxylate transport protein (e.g., JEN2 gene, e.g., JEN2-1 gene, JEN2-2 gene) and / or the endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase and / or the endogenous gene encoding alcohol dehydrogenase 1 and / or the endogenous gene encoding monocarboxylate permease are knocked out. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof; the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof; the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof; or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0113] In one embodiment, the present invention has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, a gene encoding pyruvate carboxylase, a gene encoding soluble fumarate reductase, a gene encoding fumarase, and a succinate transport protein encoding gene, e.g., SpMAE1 gene, and the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out, and optionally an endogenous gene encoding orotidine 5'-phosphate decarboxylase, and / Alternatively, the present invention provides a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., strain CY902) in which an endogenous gene encoding a dicarboxylate transport protein (e.g., the JEN2 gene, e.g., the JEN2-1 gene, JEN2-2 gene), and / or an endogenous gene encoding a bifunctional enzyme, such as oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase, and / or an endogenous gene encoding alcohol dehydrogenase 1, and / or an endogenous gene encoding monocarboxylate permease is knocked out. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof; the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof; the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof; the gene encoding fumarase includes the sequence of SEQ ID NO: 8 or a degenerate sequence thereof; or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0114] In one embodiment, the JEN2-1 gene encodes an amino acid having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the protein of sequence number 14, and having dicarboxylate transport protein activity.

[0115] In one embodiment, the JEN2-2 gene encodes an amino acid having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the protein of sequence number 15, and having dicarboxylate transport protein activity.

[0116] In one embodiment, the present invention provides a genetically modified Pichia cudriabzevi, e.g., strain CY902, having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, and optionally having the endogenous URA3 gene and / or endogenous Pk2365 gene knocked out. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof.

[0117] In one embodiment, the present invention provides a genetically modified Pichia cudriabzevi, such as the CY902 strain, having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding soluble fumarate reductase, and optionally having the endogenous URA3 gene and / or endogenous ADH1 gene and / or endogenous Pk2365 gene knocked out. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding soluble fumarate reductase comprises any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof.

[0118] In one embodiment, the present invention provides a genetically modified Pichia cudriabzevi, such as the CY902 strain, having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding succinate transport protein, such as the SpMAE1 gene, and optionally having the endogenous URA3 gene and / or endogenous MCH4 gene and / or endogenous Pk2365 gene knocked out. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding succinate transport protein comprises the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0119] In one embodiment, the present invention provides a genetically modified Pichia cudriabzevi, such as the CY902 strain, having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding fumarase, and optionally having the endogenous URA3 gene and / or endogenous PDC1 gene and / or endogenous Pk2365 gene knocked out. Preferably, the MDH gene contains the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding fumarase contains the sequence of SEQ ID NO: 8 or a degenerate sequence thereof.

[0120] In one embodiment, the present invention provides a genetically modified Pichia cudriabuzevi strain, e.g., CY902, having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding pyruvate carboxylase, and optionally having the endogenous URA3 gene and / or the endogenous JEN2-1 or JEN2-2 gene and / or the endogenous Pk2365 gene knocked out. Preferably, the MDH gene contains the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase contains the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof.

[0121] In one embodiment, the present invention provides a genetically modified Pichia cudriabuzevi strain, e.g., CY902, having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase and an overexpressed gene encoding pyruvate carboxylase, wherein the endogenous PDC1 gene and endogenous GPD1 gene are knocked out, and optionally, the endogenous URA3 gene and / or endogenous JEN2 genes, e.g., JEN2-1 gene, JEN2-2 gene, and / or endogenous Pk2365 gene are knocked out. Preferably, the MDH gene contains the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase contains the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof.

[0122] In one embodiment, the present invention provides a genetically modified Pichia cudriabzebi strain, such as the CY902 strain, which has an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, an overexpressed gene encoding pyruvate carboxylase, a gene encoding soluble fumarate reductase, and a gene encoding succinate transport protein, such as the SpMAE1 gene, and in which the endogenous PDC1 gene and endogenous GPD1 gene are knocked out, and optionally the endogenous URA3 gene and / or endogenous JEN2 gene, such as the JEN2-1 gene, JEN2-2 gene and / or the endogenous Pk2365 gene and / or the endogenous ADH1 gene and / or the endogenous MCH4 gene are knocked out. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof; the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof; the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof; or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0123] In one embodiment, the present invention provides a genetically modified Pichia kudriabzebi strain, such as the CY902 strain, which has enhanced activity of NADPH-dependent malate dehydrogenase from Sorghum bicolor, pyruvate carboxylase from Aspergillus oryzae, soluble fumarate reductase from Trypanosoma burseyi, fumarase from Pichia kudriabzebi (with its mitochondrial localization peptide at the 5' end cleaved), and succinate transport protein SpMAE1, and in which endogenous pyruvate decarboxylase and endogenous NAD-dependent glycerol 3-phosphate dehydrogenase activity are eliminated. In one embodiment, the present invention provides a genetically modified Pichia cudriabzebi strain, such as the CY902 strain, having an overexpressed MDH gene encoding NADPH-dependent malate dehydrogenase, a gene encoding pyruvate carboxylase, a gene encoding soluble fumarate reductase, a gene encoding fumarase, and a gene encoding succinate transport protein, such as the SpMAE1 gene, wherein the endogenous PDC1 gene and endogenous GPD1 gene are knocked out, and optionally the endogenous URA3 gene and / or endogenous JEN2 genes, such as the JEN2-1 gene, JEN2-2 gene, and / or the endogenous Pk2365 gene and / or the endogenous ADH1 gene and / or the endogenous MCH4 gene are knocked out. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof, the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof, the gene encoding fumarase includes the sequence of SEQ ID NO: 8 or a degenerate sequence thereof, or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0124] In a further embodiment, the present invention provides a method for constructing a genetically modified succinic acid-producing yeast strain, comprising conferring NADPH-dependent malate dehydrogenase (EC1.1.1.82) activity to the strain or enhancing NADPH-dependent malate dehydrogenase (EC1.1.1.82) activity in the strain.

[0125] As used herein, "...conferring activity" refers to the production of activity in a genetically modified succinic acid-producing yeast strain that was not present in the original strain before genetic modification.

[0126] As used herein, "enhance the activity of..." means, for example, increasing the activity 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 more.

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

[0128] As used herein, “overexpression” means that the level of gene expression is increased compared to the level before genetic manipulation, 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 more. Methods for overexpressing a gene are well known in the art and include, but are not limited to, the use of strong promoters, gene copy number increases, enhancers, etc. Gene copy number increases can be achieved, for example, by introducing one or more copies of an exogenous or endogenous gene, for example, by integration into an expression vector or genome.

[0129] As used herein, “exogenous gene” means a gene of origin from another cell or organism, for example, a gene of the same species or a different species.

[0130] As used herein, “endogenous gene” refers to a gene of cell or organismal origin.

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

[0132] In one embodiment, the NADPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae families, or from the genera Euglena or Thermobacillus, more preferably from Sorghum bicolor, Zea maize, Saccharum officinarum, Pithum sativum, Cissara aritinum, Spinasia oleracea, Euglena gracilis or Metanothermobacter thermautotrophus, more preferably from Sorghum bicolor.

[0133] In one embodiment, NADPH-dependent malate dehydrogenase activity is generated or increased by expressing or overexpressing an MDH gene encoding NADPH-dependent malate dehydrogenase in a genetically modified succinate-producing yeast strain. Preferably, the MDH gene encoding NADPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae families, or from the genera Euglena and Thermobacillus, more preferably from Sorghum bicolor, Zea maize, Saccharum officinarum, Pithum sativum, Cissara aritinum, Spinasia oleracea, Euglena gracilis or Metanothermobacter thermautotrophus, more preferably from Sorghum bicolor.

[0134] In one embodiment, the MDH gene encoding NADPH-dependent malate dehydrogenase includes the sequence of Sequence ID No. 1 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and possessing NADPH-dependent malate dehydrogenase activity.

[0135] In one embodiment, the MDH gene encoding NADPH-dependent malate dehydrogenase is incorporated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia kudria abuzevi) at, for example, the Pk2365 locus. In one embodiment, the MDH gene encoding NADPH-dependent malate dehydrogenase is incorporated into the genome of a yeast (e.g., Pichia kudria abuzevi) at, for example, the Pk2365 locus via homologous recombination. The MDH gene encoding NADPH-dependent malate dehydrogenase may be placed under the control of an appropriate promoter (e.g., the promoter of the FBA1 gene (shown in SEQ ID NO 17)) and / or terminator (e.g., the terminator of the INO1 gene (shown in SEQ ID NO 18)).

[0136] In further embodiments, the method further comprises conferring soluble fumarate reductase activity to a strain or enhancing soluble fumarate reductase activity in a strain.

[0137] In one embodiment, the soluble fumarate reductase is derived from yeast and kinetoplastiales, for example, but not limited to, Saccharomyces cerevisiae, Trypanosoma burseyi, Leishmania mexicana, and Trypanosoma cruz.

[0138] In one embodiment, the glyoxisome-localized peptide at the 3' end of soluble fumarate reductase is partially or completely cleaved and therefore freely available 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 a genetically modified succinate-producing yeast strain. Preferably, the gene encoding soluble fumarate reductase is derived from yeast and the order Kinetoplastes (e.g., Saccharomyces cerevisiae, Trypanosoma burseyi, Leishmania mexicana, and Trypanosoma cruz).

[0140] In one embodiment, the gene encoding soluble fumarate reductase includes one of sequence numbers 3 to 5 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith and possessing fumarate reductase activity.

[0141] In one embodiment, the gene encoding soluble fumarate reductase is integrated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia cudriabuzevi) at, for example, the ADH1 locus. The gene encoding soluble fumarate reductase may be placed under the control of an appropriate promoter (e.g., the promoter of the ADH1 gene shown in SEQ ID NO: 21) and / or terminator (e.g., the terminator of the ADH1 gene shown in SEQ ID NO: 22).

[0142] In further embodiments, the method further comprises conferring or enhancing succinate transport protein activity in a yeast strain.

[0143] In one embodiment, the succinate transport protein is selected from the group consisting of SpMAE1 protein, AnDCT-02 protein, EcDcuB protein, and EcDcuC protein. In one embodiment, succinate transport protein activity is conferred or enhanced by expressing or overexpressing a gene encoding a succinate transport protein (e.g., the SpMAE1 gene) in a genetically modified succinate-producing yeast strain.

[0144] In one embodiment, the SpMAE1 gene comprises a nucleotide sequence encoding an amino acid sequence having succinate transport protein activity, which is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and optionally incorporated into the genome of a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabzevi) for example at the MCH4 locus (e.g., via homologous recombination). The SpMAE1 gene may be controlled by an appropriate promoter (e.g., the promoter of the TDH3 gene (shown in SEQ ID NO: 19)) and / or a terminator (e.g., the terminator of the GAL2 gene (shown in SEQ ID NO: 20)).

[0145] In further embodiments, the method further comprises conferring or enhancing fumarase activity in a yeast strain.

[0146] In one embodiment, the mitochondrial localization peptide at the 5' end of the fumarase is partially or completely cleaved and therefore cannot localize to mitochondria but is freely present in the cytoplasm.

[0147] In one embodiment, fumarase activity is conferred or enhanced by expressing or overexpressing a gene encoding fumarase in a genetically modified succinic acid-producing yeast strain. In one embodiment, the fumarase is derived from, but is not limited to, Actinobacillus succinogenes, Mannhemia succiniciproducens, Escherichia coli, Pichia cudriabzebi, or Rhizopus oryzae.

[0148] In one embodiment, fumarase contains amino acids having fumarase activity and having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.

[0149] In one embodiment, the gene encoding fumarase encodes an amino acid sequence having fumarase activity, which is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto.

[0150] In one embodiment, the gene encoding fumarase includes the sequence of Sequence ID No. 8 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and which has fumarase activity.

[0151] Optionally, the gene encoding fumarase is incorporated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia cudriabzevi) at, for example, the PDC1 locus (e.g., via homologous recombination). The gene encoding fumarase may be placed under the control of an appropriate promoter (e.g., the promoter of the TDH3 gene (shown in SEQ ID NO: 19)) and / or terminator (e.g., the terminator of the GAL2 gene (shown in SEQ ID NO: 20)).

[0152] In further embodiments, the method further comprises conferring or enhancing pyruvate carboxylase activity in a yeast strain. Preferably, conferring or enhancing pyruvate carboxylase activity is achieved by expressing or overexpressing a gene encoding pyruvate carboxylase in the strain. Pyruvate carboxylase may be derived from fungi, particularly yeasts and filamentous fungi, preferably Saccharomyces cerevisiae, Pichia kudoriabzebi, Aspergillus oryzae, Clibellomyces marsianus, and the like. In one embodiment, the gene encoding pyruvate carboxylase may be selected from the group consisting of the PYC gene of Aspergillus oryzae and the PYC1 gene of Pichia kudoriabzebi.

[0153] In one embodiment, the pyruvate carboxylase includes an amino acid sequence encoded by the sequence of 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 more identity thereto, and possessing pyruvate carboxylase activity.

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

[0155] Optionally, the gene encoding pyruvate carboxylase is incorporated into the genome of a genetically modified succinate-producing yeast (e.g., Pichia cudriabzevi) at, for example, the JEN2-1 locus. The gene encoding pyruvate carboxylase may be placed under the control of an appropriate promoter (e.g., the promoter of the TDH3 gene (shown in SEQ ID NO: 19)) and / or terminator (e.g., the terminator of the GAL2 gene (shown in SEQ ID NO: 20)).

[0156] By this method, genes expressed or overexpressed in a strain can be incorporated at appropriate locations in the strain genome, provided that such incorporation does not adversely affect the strain's growth, proliferation, and / or productivity. For example, this method involves incorporating one or more of the aforementioned genes at any one or more locations in the genome encoding the following proteins: (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylate permease, (v) dicarboxylate transport protein, (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112).

[0157] In further embodiments, the method further comprises weakening or inactivating at least one of the following in a strain: (i) pyruvate decarboxylase (EC 4.1.1.43), (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylate permease, (v) dicarboxylate transport protein, (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and (vii) a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112).

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

[0159] As used herein, reducing or inactivating the activity of a protein, such as an enzyme, means that the activity of the protein 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. There are various means in the art for reducing and inactivating, including, for example, gene knockout and deletion of a partial or complete sequence of a gene or polypeptide, such as inhibition of gene expression, e.g., knockdown (e.g., using small interfering RNA), use of a weak promoter (if the gene is a polypeptide coding gene), mutation of a specific locus, such as a coding sequence or active domain in a gene or polypeptide, to reduce gene expression or modulate activity or express the activity of a product, and the use of antagonists or inhibitors (including, but not limited to, antibodies, interfering RNA, etc.).

[0160] As used herein, attenuating or inactivating a gene means that the gene's expression level (when used as a protein-coding gene) or regulatory capacity (when used as 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. There are various means in the art for attenuating or inactivating a gene, including, for example, inhibition of gene expression, e.g., knockdown (e.g., using small interfering RNA), use of a weak promoter (when the gene is a polypeptide-coding gene), partial or complete gene knockout and deletion of gene sequences, mutations in specific loci such as the coding sequence in a gene to reduce gene expression, modulate activity, or express the activity of a product.

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

[0162] In one embodiment, weakening or inactivating a gene encoding pyruvate decarboxylase includes knocking out a gene encoding pyruvate decarboxylase (for example, the amino acid sequence of 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 more identity and having pyruvate decarboxylase activity).

[0163] In further embodiments, the method further comprises reducing or inactivating NAD-dependent glycerol 3-phosphate dehydrogenase activity in a 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 (for example, the amino acid sequence of 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 more identity thereto and possessing NAD-dependent glycerol 3-phosphate dehydrogenase activity).

[0165] In further embodiments, the method further comprises reducing or inactivating the activity of the bifunctional enzymes orotidine 5'-phosphate decarboxylase and / or alcohol dehydrogenase 1 and / or monocarboxylate permease and / or dicarboxylate transport protein and / or oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase in a yeast strain.

[0166] In one embodiment, the method involves weakening or inactivating one or more genes encoding an orthidine 5'-phosphate decarboxylase, a dicarboxylate transport protein (e.g., the JEN2 gene, e.g., the JEN2-1 gene, the JEN2-2 gene), an alcohol dehydrogenase 1, a monocarboxylate permease, and a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase in a yeast strain.

[0167] In one embodiment, a gene encoding the bifunctional enzymes oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (for example, the amino acid sequence of 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 more identity thereto, and encoding the activity of the bifunctional enzymes oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase) is knocked out, for example, by homologous recombination.

[0168] In one embodiment, a gene encoding orotidine 5'-phosphate decarboxylase (encoding the amino acid sequence of 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 more identity thereto, and having orotidine 5'-phosphate decarboxylase activity) is knocked out, for example, by homologous recombination.

[0169] In one embodiment, the JEN2-1 gene encoding a dicarboxylate transport protein (for example, the amino acid sequence of 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 more identity thereto, and having dicarboxylate transport protein activity) is knocked out, for example, by homologous recombination.

[0170] In one embodiment, the JEN2-2 gene, which encodes a dicarboxylate transport protein (for example, the amino acid sequence of 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 more identity thereto, and which has dicarboxylate transport protein activity), is knocked out, for example, by homologous recombination.

[0171] In one embodiment, a gene encoding alcohol dehydrogenase 1 (encoding the amino acid sequence of 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 more identity thereto, and having alcohol dehydrogenase 1 activity) is knocked out, for example, by homologous recombination.

[0172] In one embodiment, a gene encoding a monocarboxylate permease (for example, the amino acid sequence of 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 more identity thereto, and having monocarboxylate 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 cudriabuzevi, e.g., CY902), comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase in the genetically modified succinic acid-producing yeast strain, and optionally knocking out an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0174] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., CY902), comprising: overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding soluble fumarate reductase in the genetically modified succinic acid-producing yeast strain; and optionally knocking out the endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or the endogenous gene encoding alcohol dehydrogenase 1 and / or the endogenous gene encoding the bifunctional enzyme oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0175] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., CY902), comprising: overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding succinic acid transport protein, e.g., the SpMAE1 gene, in the genetically modified succinic acid-producing yeast strain; and optionally knocking out an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding monocarboxylate permease and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0176] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., CY902), comprising: overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding fumarase in the genetically modified succinic acid-producing yeast strain; and optionally knocking out the endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding the bifunctional enzyme oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0177] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., CY902), comprising: overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding pyruvate carboxylase in the genetically modified succinic acid-producing yeast strain; and optionally knocking out an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding a dicarboxylate transport protein, e.g., the JEN2-1 or JEN2-2 gene and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0178] In one embodiment, the present invention provides a method for producing a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabuzevi, e.g., CY902), comprising: overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and the gene encoding pyruvate carboxylase in the genetically modified succinic acid-producing yeast strain; knocking out the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase; and optionally knocking out the endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or the endogenous gene encoding a dicarboxylate transport protein (e.g., the JEN2 gene, e.g., the JEN2-1 gene, the JEN2-2 gene) and / or the endogenous gene encoding the bifunctional enzyme oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase.

[0179] In one embodiment, the present invention relates to a method for producing a genetically modified succinic acid-producing yeast strain (e.g., Pichia cudriabzebi, e.g., CY902), wherein the genetically modified succinic acid-producing yeast strain is overexpressed with the MDH gene encoding NADPH-dependent malate dehydrogenase, the gene encoding pyruvate carboxylase, the gene encoding soluble fumarate reductase, and the gene encoding succinate transport protein, e.g., the SpMAE1 gene, and the endogenous gene encoding pyruvate decarboxylase and the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out. The present invention provides a method comprising knocking out an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding a dicarboxylate transport protein (e.g., the JEN2 gene, e.g., the JEN2-1 gene, the JEN2-2 gene) and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase and / or an endogenous gene encoding alcohol dehydrogenase 1 and / or an endogenous gene encoding monocarboxylate permease. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof, the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof, the gene encoding fumarase includes the sequence of SEQ ID NO: 8 or a degenerate sequence thereof, or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0180] In one embodiment, the present invention relates to a method for producing a genetically modified succinate-producing yeast strain (e.g., Pichia cudriabzevi, e.g., CY902), wherein the genetically modified succinate-producing yeast strain is overexpressed with the following genes: an MDH gene encoding NADPH-dependent malate dehydrogenase, a gene encoding pyruvate carboxylase, a gene encoding soluble fumarate reductase, a gene encoding fumarase, and a gene encoding succinate transport protein, e.g., the SpMAE1 gene; and an endogenous gene encoding pyruvate decarboxylase and an endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase. The present invention provides a method comprising knocking out a gene and, optionally, knocking out an endogenous gene encoding orotidine 5'-phosphate decarboxylase and / or an endogenous gene encoding a dicarboxylate transport protein (e.g., the JEN2 gene, e.g., the JEN2-1 gene, the JEN2-2 gene) and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase and / or an endogenous gene encoding alcohol dehydrogenase 1 and / or an endogenous gene encoding monocarboxylate permease. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof; the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof; the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof; the gene encoding fumarase includes the sequence of SEQ ID NO: 8 or a degenerate sequence thereof; or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0181] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzevi, for example, the CY902 strain, comprising overexpressing an MDH gene encoding NADPH-dependent malate dehydrogenase and optionally knocking out an endogenous URA3 gene and / or an endogenous Pk2365 gene. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof.

[0182] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzebi, for example, the CY902 strain, comprising overexpressing an MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding soluble fumarate reductase, and optionally knocking out an endogenous URA3 gene and / or an endogenous ADH1 gene and / or an endogenous Pk2365 gene. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding soluble fumarate reductase comprises any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof.

[0183] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzebi, for example, the CY902 strain, comprising overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding succinate transport protein, for example, the SpMAE1 gene, and optionally knocking out the endogenous URA3 gene and / or the endogenous MCH4 gene and / or the endogenous Pk2365 gene. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding succinate transport protein comprises the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0184] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzevi, for example, the CY902 strain, comprising overexpressing an MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding fumarase, and optionally knocking out an endogenous URA3 gene and / or an endogenous PDC1 gene and / or an endogenous Pk2365 gene. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding fumarase comprises the sequence of SEQ ID NO: 8 or a degenerate sequence thereof.

[0185] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzevi, for example, the CY902 strain, comprising overexpressing an MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding pyruvate carboxylase, and optionally knocking out an endogenous URA3 gene and / or an endogenous JEN2-1 or JEN2-2 gene and / or an endogenous Pk2365 gene. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase comprises the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof.

[0186] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzevi, for example, the CY902 strain, comprising: overexpressing an MDH gene encoding NADPH-dependent malate dehydrogenase and a gene encoding pyruvate carboxylase; knocking out the endogenous PDC1 gene and the endogenous GPD1 gene; and optionally knocking out the endogenous URA3 gene and / or the endogenous JEN2 gene, for example, the JEN2-1 gene, the JEN2-2 gene, and / or the endogenous Pk2365 gene. Preferably, the MDH gene comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase comprises the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof.

[0187] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzevi, for example, the CY902 strain, comprising: overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase, a gene encoding pyruvate carboxylase, a gene encoding soluble fumarate reductase, and a gene encoding succinate transport protein, for example, the SpMAE1 gene; knocking out the endogenous PDC1 gene and the endogenous GPD1 gene; and optionally knocking out the endogenous URA3 gene and / or the endogenous JEN2 gene, for example, the JEN2-1 gene, the JEN2-2 gene, and / or the endogenous Pk2365 gene and / or the endogenous ADH1 gene and / or the endogenous MCH4 gene. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof, the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof, or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0188] In one embodiment, the present invention provides a method for producing a genetically modified Pichia cudriabzevi, for example, the CY902 strain, comprising: overexpressing the MDH gene encoding NADPH-dependent malate dehydrogenase, a gene encoding pyruvate carboxylase, a gene encoding soluble fumarate reductase, a gene encoding fumarase, and a gene encoding succinate transport protein, for example, the SpMAE1 gene; knocking out the endogenous PDC1 gene and the endogenous GPD1 gene; and optionally knocking out the endogenous URA3 gene and / or the endogenous JEN2 gene, for example, the JEN2-1 gene, the JEN2-2 gene, and / or the endogenous Pk2365 gene and / or the endogenous ADH1 gene and / or the endogenous MCH4 gene. Preferably, the MDH gene includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or the gene encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof, the gene encoding soluble fumarate reductase includes any one of the sequences of SEQ ID NOs: 3 to 5 or a degenerate sequence thereof, or the gene encoding fumarase includes the sequence of SEQ ID NO: 8 or a degenerate sequence thereof, or the gene encoding succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof.

[0189] In one embodiment, the succinic acid-producing yeast strain to be genetically modified includes, but is not limited to, the genera Candida, Pichia, Rhodotorula, Saccharomyces, Yarrowia, Zygosaccharomyces, and Torlopsis. In one embodiment, the succinic acid-producing yeast strain used for genetic modification is selected from the group consisting of Pichia, Saccharomyces, or Yarrowia species. In a preferred embodiment, the succinic acid-producing yeast strain used for genetic modification is Pichia cudriabzebi, Saccharomyces cerevisiae, or Yarrowia liporitica, for example, Pichia cudriabzebi, which is stored at the Center for Ordinary Microorganisms of the Chinese Microbial Species Depositary Administration (CGMCC) under depositary number 20885.

[0190] In one embodiment, the present invention provides a method for producing succinic acid, comprising culturing a genetically modified succinic acid-producing yeast strain prepared by the present invention or a method for constructing a genetically modified succinic acid-producing yeast strain according to the present invention under conditions suitable for the fermentation production of succinic acid, and optionally isolating and purifying the produced succinic acid.

[0191] It is well known in the art that the conditions for succinic acid fermentation production, in which succinic acid-producing yeast strains are cultured by fermentation, include, but are not limited to, pH, temperature, culture medium components, and fermentation time.

[0192] It is known in the art that culture media for succinic acid fermentation production by succinic acid-producing yeast strains include, but are not limited to, inorganic salt culture media (containing approximately 5-12% w / v glucose and optionally 30 g / L CaCO3).

[0193] The temperature for fermentation production of succinic acid by succinic acid-producing yeast strains is known to be, for example, about 25-37°C, such as about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. In one embodiment, succinic acid is produced by fermenting the succinic acid-producing yeast strain of the present invention at 30°C.

[0194] The succinic acid-producing yeast strain of the present invention is suitable for pH levels known in the art, for example, 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, about 1.0 to 7.0, 1.0 to 6.0, 1.0 to 5.5, 1.0 to 5.0, 1.0 to 4.5, 1.0 to 4.0, 1.0 to 3.5, Fermentation may be carried out at pH values ​​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, and 4.0-4.5. In one embodiment, succinic acid is produced by fermenting the succinic acid-producing yeast strain of the present invention at a pH of less than approximately 3.0.

[0195] To produce succinic acid, the succinic acid-producing yeast strain of the present invention may be fermented for an appropriate amount of time, for example, about 12 to 96 hours, for example, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or about 96 hours. In one embodiment, to produce succinic acid, the succinic acid-producing yeast strain of the present invention may be fermented for about 24 to 72 hours, for example, about 30 hours.

[0196] The succinic acid-producing yeast strain of the present invention may be fermented under shaking conditions (for example, about 100-300 rpm, for example, about 150, about 200, or about 250 rpm) to produce succinic acid.

[0197] The succinic acid content in the fermentation broth can be determined by any known method in the art, such as high-performance liquid chromatography (HPLC).

[0198] In one embodiment, the present invention relates to a method for producing succinic acid, wherein a genetically modified succinic acid-producing yeast strain is cultured in an inorganic salt culture medium (for example, containing about 5% w / v glucose and about 30 g / L of CaCO3, or containing about 12% w / v glucose) at an acidic pH (less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, less than 1.0, for example, about 1.0 to 7.0, 1.0 to 6.0, 1.0 to 5.5, 1.0 to 5.0). The present invention provides a method comprising culturing under the following conditions: 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 optionally isolating and purifying the produced succinic acid.

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

[0200] In one embodiment, the present invention relates to the production of succinic acid, particularly at acidic pH (less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, less than 1.0, for example, about 1.0 to 7.0, 1.0 to 6.0, 1.0 to 5.5, 1.0 to 5.0, 1.0 to 4.5, 1.0 to 4.0, 1.0 to 3.5, 1.0 to 3.0, 2.0 to 7.0, 2.0 to 6.0, 2.0 to 5.5, 2.0 to 5.0, 2.0 to 4.5, 2.0 to 4. The present invention provides the use of the 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 constructing the genetically modified succinic acid-producing yeast strain of the present invention in the production of succinic acid under the conditions of 0, 2.0~3.5, 2.0~3.0, 3.0~7.0, 3.0~5.5, 3.0~5.0, 3.0~4.5, 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 the occurrence or non-occurrence of the event or situation described therein, and such description includes the occurrence and non-occurrence of the event or situation described therein. For example, “optionally included step” means the presence or non-existence of that step.

[0202] As used herein, the term “approximately” refers to a range of numerical values ​​that include a particular value which can be reasonably considered by those skilled in the art to be similar to that particular value. In some embodiments, the term “approximately” refers to a range within the standard error using measurements commonly accepted in the art. In some embodiments, the term “approximately” refers to + / - 10% of a particular value.

[0203] The scope disclosed herein should also be considered to specifically disclose all possible subranges and various values ​​within such a range. For example, a description of the range 1–6 should be considered to explicitly disclose subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, and 3–6, as well as single numbers within this range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0204] The present invention will be further illustrated by the following non-limiting embodiments. As is well known to those skilled in the art, various modifications are possible without departing from the spirit of this application, and such modifications are also included within the scope of the present invention.

[0205] Unless otherwise specified, the following experimental methods are conventional, and the experimental materials used can be readily obtained from commercial companies.

[0206] Example 1: Overexpression of the SbMDH gene in Pichia cudriabzebi CY902ΔURA3 strain The ΔURA3 mutant was obtained by knocking out the URA3 gene encoding orotidine 5'-phosphate decarboxylase (at the PK2075 locus of CY902) via homologous recombination (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 succinate production. Microb Biotechnol 2021.0(0),1-18.doi:10.1111 / 1751-7915.13781). Subsequently, a CRISPR / Cas9 plasmid pWSPK-Cas9 (GenBank search number: MW296878.1) suitable for this strain was constructed. This plasmid contained a URA3 screening marker, which allowed for the acquisition of positive transformants by nutrient requirement screening.

[0207] Using CY902ΔURA3 as the starting strain, the sorghum bicolor-derived malate dehydrogenase SbMDH (Uniprot database search number: P17606) (SEQ ID NO: 1) was overexpressed at locus Pk2365 of the CY902 genome. The promoter and terminator used were the promoter of the FBA1 gene encoding CY902's own fructose 1,6-diphosphate aldolase (SEQ ID NO: 17) and the terminator of the inositol-3-phosphate synthase gene INO1 (SEQ ID NO: 18), respectively. The SbMDH gene was synthesized by Nanjing GenScript Biotech Co., Ltd. and optimized according to CY902 codon selection. The specific construction method was as follows.

[0208] 1. Construction of donor DNA fragments for homologous recombination Using CY902 genomic DNA as a template, the upstream homologous arm fragment 1 (fragment 1) of the Pk2365 gene of CY902 itself was amplified with primers 1_UP_2365_F and 1_UP_2365_R1 (see Table 1). The amplification system and procedure were based on the product specifications of TAKARA PrimeSTAR® HS DNA polymerase. The FBA1 promoter sequence (fragment 2) of CY902 itself was amplified with primer 2_P FBA1 _F and 2_P FBA1 Amplification was performed with _R (see Table 1). Using a plasmid containing the SbMDH synthetic sequence as a template, the SbMDH coding sequence of sorghum bicolor (fragment 3) was amplified with primers 3_SbMDH_F and 3_SbMDH_R (see Table 1). Using CY902 genomic DNA as a template, the INO1 gene terminator sequence of CY902 itself (fragment 4) was amplified with primer 4_T INO1 _F and 4_T INO1The Pk2365 gene downstream homologous arm fragment 1 (fragment 5) of CY902 itself was amplified with primers 5_DW_2365_F1 and 5_DW_2365_R (see Table 1). Using CY902 genomic DNA as a template, the Pk2365 gene upstream homologous arm fragment 2 (fragment 6) of CY902 itself was amplified with primers 1_UP_2365_F and 1_UP_2365_R2 (see Table 1). The Pk2365 gene downstream homologous arm fragment 2 (fragment 7) of CY902 itself was amplified with primers 5_DW_2365_F2 and 5_DW_2365_R (see Table 1).

[0209] 2. Construction of a plasmid for editing the Pk2365 gene locus Using the pWSPK-Cas9 plasmid (GenBank search number: MW296878.1) as a template, a 9052 bp plasmid skeleton (abbreviated as pWSPK_skeleton, SEQ ID NO: 23) that did not contain an sgRNA sequence was amplified with primers pWSPK-F and pWSPK-R (see Table 1). When the 5' terminal sequence of sgRNA (GenBank search number: MW296878.1) (abbreviated as 2365_sgRNA_1) was amplified with primers sgRNA-1F and 2365_sgRNA-1R (see Table 1), the 3' end of the fragment contained a 20 nt protospacer specific to the Pk2365 gene. Using the pWSPK-Cas9 plasmid as a template, a 500 bp 3' sequence of sgRNA (abbreviated as sgRNA_2, SEQ ID NO: 97) was amplified with primers sgRNA-2F and sgRNA-2R (see Table 1). The plasmid skeleton pWSPK_skeleton and 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 transferred into Trans1-T1 competent cells (TransGen Biotech Co., Ltd., Beijing, product number: CD501-02), and the resulting positive plasmid was named pWSPK_2365.

[0210] 3. Construction of a strain that overexpresses the SbMDH gene. The pWSPK_2365 plasmid and fragments 6 and 7 were transferred into the CY902ΔURA3 strain by yeast electrotransmission (doi:10.1111 / 1751-7915.13781), and positive transformants were screened and obtained, named strain SA101-1 (genotype: CY902ΔURA3, ΔPk2365). The pWSPK_2365 plasmid and fragments 1-5 were transferred into the CY902ΔURA3 strain by yeast electrotransmission, and positive transformants were screened and obtained, named strain SA101-2 (genotype: CY902ΔURA3, Pk2365::P PkFBA1 -ORF SbMDH -T PkINO1 ) was named as such.

[0211] [Table 1]

[0212] Example 2: Overexpression of EcSthA and AoMDH genes in Pichia kudriabzevi CY902ΔURA3 Using CY902ΔURA3 as the starting strain, the Escherichia coli-derived soluble pyridine nucleotide transhydrogenase EcSthA (EC1.6.1.1) (SEQ ID NO: 98) was overexpressed at the locus Pk2365 of the CY902 genome. The promoter and terminator used were the FBA1 promoter (SEQ ID NO: 17) and INO1 terminator (SEQ ID NO: 18) of CY902 itself, respectively. The malate dehydrogenase AoMDH (EC1.1.1.37, Uniprot database search number: I8U0T6) (SEQ ID NO: 99) derived from Aspergillus oryzae was overexpressed at the locus of the succinic semialdehyde dehydrogenase (EC1.2.1.16) gene PkUGA2. The promoter and terminator used were the promoter of the enolase gene ENO1 (SEQ ID NO: 100) and the terminator of the gene SED1 encoding the GPI cell wall glycoprotein (SEQ ID NO: 101) of CY902 itself, respectively. The EcSthA and AoMDH genes were synthesized by Nanjing GenScript Biotech Co., Ltd and optimized according to the codon selection of CY902. The specific construction method was as follows.

[0213] 1. Construction of donor DNA fragments for homologous recombination Using the plasmid containing the EcSthA synthetic sequence as a template, the sequence encoding Escherichia coli soluble pyridine nucleotide transhydrogenase EcSthA (fragment 8) was amplified with primers 3_EcSthA_F and 3_EcSthA_R (see Table 2). Using CY902 genomic DNA as a template, the upstream homologous arm fragment 1 (fragment 9) of the PkUGA2 gene of CY902 itself was amplified with primers 1_UP_UGA2_F and 1_UP_UGA2_R1 (see Table 2). The ENO1 promoter sequence (fragment 10) of CY902 itself was amplified with primers 2_P ENO1 _F and 2_P ENO1Amplification was performed with _R (see Table 2). Using a plasmid containing the AoMDH synthesis sequence as a template, the Aspergillus oryzae AoMDH coding sequence (fragment 11) was amplified with primers 3_AoMDH_F and 3_AoMDH_R (see Table 2). Using CY902 genomic DNA as a template, the SED1 gene terminator sequence of CY902 itself (fragment 12) was amplified with primer 4_T SED1 _F and 4_T SED1 The PkUGA2 gene downstream homologous arm fragment 1 (fragment 13) of CY902 itself was amplified with primers 5_DW_UGA2_F1 and 5_DW_UGA2_R (see Table 2). Using CY902 genomic DNA as a template, the PkUGA2 gene upstream homologous arm fragment 2 (fragment 14) of CY902 itself was amplified with primers 1_UP_UGA2_F and 1_UP_UGA2_R2 (see Table 2). The PkUGA2 gene downstream homologous arm fragment 2 (fragment 15) of CY902 itself was amplified with primers 5_DW_UGA2_F2 and 5_DW_UGA2_R (see Table 2).

[0214] 2. Construction of a plasmid for editing the PkUGA2 gene locus Using the pWSPK-Cas9 plasmid as a template, the 5' terminal sequence of the sgRNA (abbreviated as UGA2_sgRNA_1) was amplified with primers sgRNA-1F and UGA2_sgRNA-1R (see Table 2). The 3' end of the fragment contained a 20nt protospacer specific to the PkUGA2 gene. The pWSPK_backbone, sgRNA_2, and UGA2_sgRNA_1 were ligated using a seamless cloning kit to ultimately obtain a positive cloning plasmid named pWSPK_UGA2.

[0215] 3. Construction of strains that overexpress the EcSthA and AoMDH genes. The pWSPK_2365 plasmid and fragments 1, 2, 8, 4, and 5 were transferred into the CY902ΔURA3 strain by yeast electrotransformation, and positive transformants were screened to obtain strain SA101-3 (genotype: CY902ΔURA3, Pk2365::P PkFBA1 -ORF EcSthA -T PkINO1 The pWSPK_UGA2 plasmid and fragments 14 and 15 were transferred into SA101-2 and SA101-3 strains by yeast electrotransformation, and positive transformants were screened and named SA102-1 strain (genotype: SA101-2, ΔUGA2) and SA102-2 strain (SA101-3, ΔUGA2), respectively. The pWSPK_UGA2 plasmid and fragments 9-13 were transferred into SA101-3 strain by yeast electrotransformation, and positive transformants were screened and named SA102-3 strain (genotype: SA101-3, PkUGA2::P PkENO1 -ORF AoMDH -T PkSED1 ) was named as such.

[0216] [Table 2]

[0217] Example 3: Evaluation of succinic acid production ability of SA101 and SA102 series strains 1. Fermentation using a neutralizing agent SA101 and SA102 series strains were inoculated into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 30 g / L CaCO3) and fermented in a flask with shaking at 30°C and 250 rpm for 24 hours. The succinic acid titer was measured by HPLC and is shown in Table 3 below.

[0218] [Table 3]

[0219] 2. Fermentation without the use of neutralizing agents The strains of the SA101 and SA102 series were inoculated into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 0 g / L CaCO3), and after flask shaking fermentation at 30 °C and 250 rpm for 24 hours, the titer of succinic acid was measured by HPLC and shown in Table 4 below.

[0220]

Table 4

[0221] Example 4: Overexpression of the FRD gene in the SA101-2 strain Using SA101-2 as the starting strain, at the locus of the ADH1 gene, (I) fumarate reductase from three sources including FRD of Saccharomyces cerevisiae (ScFRD, Uniprot database search number: P32614) (SEQ ID NO: 3), (II) FRD of Leishmania mexicana (LmFRD, SEQ ID NO: 4), (III) FRD of Trypanosoma brucei (TbFRD, SEQ ID NO: 5) were each overexpressed. The promoter and terminator for overexpressing FRD were respectively the promoter (SEQ ID NO: 21) and terminator (SEQ ID NO: 22) of the alcohol dehydrogenase 1 ADH1 of CY902 itself. The above three FRD gene sequences were synthesized by Nanjing GenScript Biotech Co., Ltd and optimized according to the codon selection of CY902. The specific construction method was as follows.

[0222] 1. Construction of the donor DNA fragment for homologous recombination Using CY902 genomic DNA as a template, the ADH1 gene promoter sequence (upstream homologous arm, fragment 16) of CY902 itself was amplified with primer 1_P ADH1 _F and 1_P ADH1The sequences were amplified using primers 2_ScFRD_F and 2_ScFRD_R (see Table 5). Using a plasmid containing the ScFRD synthesis 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). Using a plasmid containing the LmFRD synthesis sequence as a template, the LmFRD coding sequence (fragment 17-2) of Leishmania mexicana, with the 3' glyoxisome localization peptide cleaved, was amplified with primers 2_LmFRD_F and 2_LmFRD_R (see Table 5). Using a plasmid containing the TbFRD synthesis sequence as a template, the TbFRD coding sequence (fragment 17-3) of Trypanosoma bursey, with the 3' glyoxisome localization peptide cleaved, was amplified with primers 2_TbFRD_F and 2_TbFRD_R (see Table 5). Using CY902 genomic DNA as a template, the ADH1 gene terminator sequence of CY902 itself (downstream homologous arm 1, fragment 18) is applied to primer 3_T. ADH1 _F1 and 3_T ADH1 Amplification was performed using _R (see Table 5). Using CY902 genomic DNA as a template, the ADH1 gene terminator sequence of CY902 itself (downstream homologous arm 2, fragment 19) was amplified with primer 3_T. ADH1 _F2 and 3_T ADH1 The signal was amplified using _R (see Table 5).

[0223] 2. Construction of a plasmid for editing the ADH1 gene locus Using the pWSPK-Cas9 plasmid as a template, the 5' end sequence of the sgRNA (abbreviated as ADH1_sgRNA_1) was amplified with primers sgRNA-1F and ADH1_sgRNA-1R (see Table 5). The 3' end of this fragment contained a 20nt protospacer specific to the Pichia kudriabzevi ADH1 (PkADH1) gene. The pWSPK_backbone, sgRNA_2, and ADH1_sgRNA_1 were ligated using a seamless cloning kit to ultimately obtain a positive cloning plasmid named pWSPK_ADH1.

[0224] 3. Construction of a strain that overexpresses the FRD gene. The pWSPK_ADH1 plasmid and fragments 16 and 19 were transferred into the SA101-2 strain via yeast electrotransformation, and positive cloning transformants were screened and named SA103-1 strain (genotype: SA101-2, ΔADH1). The pWSPK_ADH1 plasmid and fragments 16, 17, and 18 were each transferred into the SA101-2 strain via yeast electrotransformation, and positive transformants were screened and named SA103-2 strain (genotype: SA101-2, ADH1::ORF ScFRD ), SA103-3 strain (genotype: SA101-2, ADH1::ORF LmFRD ), SA103-4 strain (genotype: SA101-2, ADH1::ORF TbFRD ) was named as such.

[0225] [Table 5]

[0226] Example 5: Knockout of PDC1 and GPD1 genes in SA101-2 strain 1. Construction of DNA fragments for knocking out the PDC1 and GPD1 genes. Using CY902 genomic DNA as a template, the upstream and downstream homologous arm fragments of the PDC1 gene (abbreviated as PDC1_1 and PDC1_2) were amplified with primers PDC1_1F and PDC1_1R, and PDC1_2F and PDC1_2R (see Table 6). The upstream and downstream homologous arm fragments of the GPD1 gene (abbreviated as GPD1_1 and GPD1_2) were amplified with primers GPD1_1F and GPD1_1R, and GPD1_2F and GPD1_2R (see Table 6).

[0227] 2. Construction of CRISPR / Cas9 plasmids for editing the PDC1 and GPD1 genes The 5' terminal sequence of the 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 contained a 20nt protospacer specific to the PDC1 gene. The plasmid backbone, pWSPK_backbone, and the sgRNA fragments, PDC1_sgRNA_1 and sgRNA_2, were ligated using a seamless cloning kit. The seamless cloning ligation product was transferred into Trans1-T1 competent cells, and the resulting positive plasmid was named pWSPK_PDC1. The 5' terminal sequence of the sgRNA (abbreviated as GPD1_sgRNA_1) was amplified using primers sgRNA-1F and GPD1_sgRNA-1R (see Table 6). The 3' end of this fragment contained a 20nt protospacer specific to the GPD1 gene. The pWSPK_GPD1 plasmid for editing the GPD1 gene was formed by seamlessly cloning and fusing the pWSPK_skeleton, sgRNA_2, and GPD1_sgRNA_1.

[0228] [Table 6]

[0229] 3. Construction of strains with knockout of the PDC1 and GPD1 genes. Fragments PDC1_1 and PDC1_2 and plasmid pWSPK_PDC1 were simultaneously transferred into SA101-2. Positive transformants were identified using primers PDC1_1F / PDC1_2R and named SA104 strain (genotype: SA101-2, ΔPDC1). After electrotransforming SA104 strain with the pWSPK_GPD1 plasmid and fragments GPD1_1 and GPD1_2, positive transformants were obtained by screening in SD-URA culture medium and named SA105 strain (genotype: SA104, ΔGPD1).

[0230] Example 6: Overexpression of the SpMAE1 gene in SA101-2 strain The SpMAE1 transport protein derived from Schizosaccharomyces pombe (Uniprot database search number: P50537) was expressed at the MCH4 locus of the CY902 genome. The SpMAE1 gene was synthesized by Nanjing GenScript Biotech Co., Ltd. (SEQ ID NO: 2) and optimized according to the codon selection of CY902. The promoter and terminator used were the promoter of the glyceraldehyde-3-phosphate dehydrogenase 3 gene TDH3 (SEQ ID NO: 19) and the terminator of the gene GAL2 encoding the galactose transport protein (SEQ ID NO: 20), respectively. The specific construction method was as follows.

[0231] 1. Construction of donor DNA fragments for homologous recombination Using CY902 genomic DNA as a template, the upstream homologous arm fragment 1 (fragment 20) of the MCH4 gene was amplified with primers 1_UP_MCH4_F and 1_UP_MCH4_R1 (see Table 7). The TDH3 gene promoter sequence of CY902 itself (fragment 21) was amplified with primer 2_P TDH3 _F and 2_P TDH3 Amplification was performed with _R (see Table 7). Using a plasmid containing the SpMAE1 synthetic sequence as a template, the SpMAE1 coding sequence (fragment 22) of Schizosaccharomyces pombe was amplified with primers 3_SpMAE1_F and 3_SpMAE1_R (see Table 7). Using CY902 genomic DNA as a template, the GAL2 gene terminator sequence of CY902 itself (fragment 23) was amplified with primer 4_T GAL2 _F and 4_T GAL2It was amplified with _R (see Table 7). The downstream homologous arm fragment 1 (fragment 24) of the MCH4 gene was amplified with primers 5_DW_MCH4_F1 and 5_DW_MCH4_R (see Table 7). Using CY902 genomic DNA as a template, the upstream homologous arm fragment 2 (fragment 25) of the MCH4 gene was amplified with primers 1_UP_MCH4_F and 1_UP_MCH4_R2 (see Table 7). The downstream homologous arm fragment 2 (fragment 26) of the MCH4 gene was amplified with primers 5_DW_MCH4_F2 and 5_DW_MCH4_R (see Table 7).

[0232] 2. Construction of a plasmid for editing MCH4 of CY902 itself Using the pWSPK-Cas9 plasmid as a template, when the 5' end sequence of the sgRNA (abbreviated as MCH4_sgRNA_1) was amplified with primers sgRNA-1F and MCH4_sgRNA-1R (see Table 7), a 20-nt protospacer specific to the Pichia kudriavzevii PkMCH4 gene was contained at the 3' end of this fragment. pWSPK_skeleton, sgRNA_2 and MCH4_sgRNA_1 were ligated using a seamless cloning kit, and finally a positive cloning plasmid named pWSPK_MCH4 was obtained.

[0233] [Table 7]

[0234] 3. Construction of a strain overexpressing the SpMAE1 gene The pWSPK_MCH4 plasmid and fragments 25 and 26 were transferred into the SA101-2 strain according to the yeast electrotransformation method, and positive transformants were screened to obtain SA106-1 (genotype: SA101-2, ΔPkMCH4), named. The pWSPK_MCH4 plasmid and fragments 20 - 24 were electrotransformed into the SA101-2 strain, and positive transformants were screened to obtain SA106-2 (genotype: SA101-2, PkMCH4::P PkTDH3 -ORF SpMAE1-T PkGAL2 ) was named as such.

[0235] Example 7: Overexpression of the PYC gene in SA101-2 strain The Aspergillus oryzae-derived PYC (AoPYC) gene (Uniprot database search number: Q2UGL1) (SEQ ID NO: 6) was overexpressed at the JEN2-1 locus, and the CY902's own PYC1 (PkPYC1) gene was used as a control (SEQ ID NO: 7). The promoter and terminator used were the TDH3 promoter (SEQ ID NO: 19) and the GAL2 terminator (SEQ ID NO: 20), respectively. The AoPYC gene was synthesized by Nanjing GenScript Biotech Co., Ltd. and optimized according to the codon selection of CY902 (SEQ ID NO: 6). The specific construction method was as follows.

[0236] 1. Construction of donor DNA fragments for homologous recombination Using CY902 genomic DNA as a template, the upstream homologous arm fragment 1 (fragment 27) of the JEN2-1 gene was amplified with primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R1 (see Table 8). The PkPYC1 coding sequence of CY902 itself (fragment 28-1) was amplified with primers 3_PkPYC1_F and 3_PkPYC1_R (see Table 8). Using a plasmid containing the AoPYC synthesis sequence as a template, the AoPYC coding sequence from Aspergillus oryzae (fragment 28-2) was amplified with primers 3_AoPYC_F and 3_AoPYC_R (see Table 8). Using CY902 genomic DNA as a template, downstream homologous arm fragment 1 (fragment 29) of the JEN2-1 gene was amplified with primers 5_DW_JEN2-1_F1 and 5_DW_JEN2-1_R (see Table 8). Using CY902 genomic DNA as a template, upstream homologous arm fragment 2 (fragment 30) of the JEN2-1 gene was amplified with primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R2 (see Table 8). Downstream homologous arm fragment 2 (fragment 31) of the JEN2-1 gene was amplified with primers 5_DW_JEN2-1_F2 and 5_DW_JEN2-1_R (see Table 8).

[0237] [Table 8]

[0238] 2. Construction of a CRISPR / Cas9 plasmid for editing the JEN2-1 gene Using the pWSPK-Cas9 plasmid as a template, the 5' terminal sequence of the sgRNA (abbreviated as JEN2-1_sgRNA_1) was amplified with primers sgRNA-1F and JEN2-1_sgRNA-1R (see Table 8). The 3' end of this fragment contained a 20nt protospacer specific to the JEN2-1 gene. The pWSPK_backbone and sgRNA_2 from Example 1, along with JEN2-1_sgRNA_1, were ligated using a seamless cloning kit to obtain a positive cloning plasmid named pWSPK_JEN2-1.

[0239] 3. Construction of a strain that overexpresses the PYC gene. The pWSPK_JEN2-1 plasmid and fragments 30 and 31 were electrotransformed into the SA101-2 strain, and positive transformants were screened and named SA107-1 (genotype: SA101-2, ΔPkJEN2-1). The pWSPK_JEN2-1 plasmid and fragments 27, 21, 28, 23 and 29 were electrotransformed into the SA101-2 strain, and positive transformants were screened 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 ) was named as such.

[0240] Example 8: Overexpression of the PkFUM1 gene in SA101-2 strain The cytofumarase gene PkFUM1 (SEQ ID NO: 8), from which the 5'-terminal mitochondrial localization peptide was cleaved, was overexpressed at the PDC1 gene promoter locus of the SA101-2 strain. The promoter and terminator used were the TDH3 promoter (SEQ ID NO: 19) and the GAL2 terminator (SEQ ID NO: 20). The specific construction method was as follows.

[0241] 1. Construction of donor DNA fragments for homologous recombination Using CY902 genomic DNA as a template, the upstream homologous arm fragment (fragment 32) of the PDC1 gene promoter of CY902 itself is applied to primer 1_UP_P PDC1 _F and 1_UP_P PDC1The FUM1 coding sequence with the 5'-terminus mitochondrial localization peptide cleaved (fragment 33) was amplified with primers 3_PkFUM1_F and 3_PkFUM1_R (see Table 9). The downstream homologous arm fragment of the PDC1 gene promoter of CY902 itself (fragment 34) was amplified with primer 5_DW_P PDC1 _F and 5_DW_P PDC1 The signal was amplified using _R (see Table 9).

[0242] 2. Construction of a plasmid for editing the PDC1 gene promoter locus. Using the pWSPK-Cas9 plasmid as a template, the 5' end sequence of the sgRNA (P PDC1 (abbreviated as _sgRNA_1) with primers sgRNA-1F and P PDC1 When amplified with _sgRNA-1R (see Table 9), the 3' end of this fragment contained P PDC1 It contained a 20nt protospacer specific to the gene promoter. pWSPK_backbone, sgRNA_2 and P PDC1 _sgRNA_1 was ligated using a seamless cloning kit, and pWSPK_P PDC1 We ultimately obtained a positive cloning plasmid, which was named [name of plasmid].

[0243] [Table 9]

[0244] 3. Construction of a strain that overexpresses the PkFUM1 gene. pWSPK_P PDC1 Plasmids and fragments 32, 21, 33, 23, and 34 were transferred into the SA101-2 strain according to yeast electrotransformation, and positive transformants were screened to obtain the SA108 strain (genotype: SA101-2, P PkPDC1 ::P PkTDH3 -ORF PkFUM1 -T PkGAL2 ) was named as such.

[0245] Example 9: Complementary expression of the PkURA3 gene in SA101 series strains The orotidine 5'-phosphate decarboxylase gene PkURA3 (SEQ ID NO: 117) was complemented at the PkJEN2-2 locus of the dicarboxylate transport protein in the SA101 series strain. The promoter and terminator used were the promoter (SEQ ID NO: 118) and terminator (SEQ ID NO: 119) of PkURA3 itself. The specific construction method was as follows.

[0246] 1. Construction of donor DNA fragments for homologous recombination Using CY902 genomic DNA as a template, the upstream homologous arm fragment 1 (fragment 35) of the PkJEN2-2 gene of CY902 itself was amplified with primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R1 (see Table 10). The URA3 promoter, open reading frame, and terminator sequence (fragment 36) were amplified with primers 2_PkURA3_F and 2_PkURA3_R (see Table 10). The downstream homologous arm fragment 1 (fragment 37) of the PkJEN2-2 gene of CY902 itself was amplified with primers 3_DW_JEN2-2_F1 and 3_DW_JEN2-2_R (see Table 10). Using CY902 genomic DNA as a template, the upstream homologous arm fragment 2 (fragment 38) of the PkJEN2-2 gene of CY902 itself was amplified with primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R2 (see Table 10). The downstream homologous arm fragment 2 (fragment 39) of the PkJEN2-2 gene of CY902 itself was amplified with primers 3_DW_JEN2-2_F2 and 3_DW_JEN2-2_R (see Table 10).

[0247] 2. Construction of a plasmid for editing the PkJEN2-2 gene site. When the 5' end sequence of the sgRNA (abbreviated as JEN2-2_sgRNA_1) was amplified using the pWSPK-Cas9 plasmid as a template with primers sgRNA-1F and JEN2-2_sgRNA-1R (see Table 10), the 3' end of this fragment contained the PkJEN2-2 gene. to child A specific 20nt protospacer was present. The pWSPK_backbone, sgRNA_2, and JEN2-2_sgRNA_1 were ligated using a seamless cloning kit to ultimately obtain a positive cloning plasmid named pWSPK_JEN2-2.

[0248] [Table 10]

[0249] 3. Construction of a strain possessing the complementary PkURA3 gene. The pWSPK_JEN2-2 plasmid and fragments 38 and 39 were transferred into yeast strains SA101-1, SA101-2, and SA101-3 according to electrotransmission, and positive transformants were screened and named strain SA109-1 (genotype: SA101-1, ΔPkJEN2-2), strain SA109-2 (genotype: SA101-2, ΔPkJEN2-2), and strain SA109-3 (genotype: SA101-3, ΔPkJEN2-2), respectively. The pWSPK_JEN2-2 plasmid and fragments 35-37 were transferred into yeast strains SA101-1, SA101-2, and SA101-3 according to electrotransmission, and positive transformants were screened and named strain 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 ) was named as such.

[0250] Example 10: Complementary Expression of the PkURA3 Gene in SA102 - SA108 Series Strains Referring to Example 9, the PkURA3 was made complementary at the PkJEN2 - 2 locus of SA102 - SA108 strains to obtain SA110 - 1 strain (genotype: SA102 - 1, 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[[ID=​​​​​​​​​​​​​​​​​​​​​​) SA115-1 strain (genotype: SA107-1, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ) 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 ) and SA116 strain (genotype: SA108, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ) were obtained.

[0251] Example 11: Evaluation of Succinic Acid Production Capacity of SA101 - SA116 Series Strains 1. Fermentation with Neutralizing Agent After inoculating the SA101 - SA116 series of strains into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 30 g / L CaCO3), flask shaking fermentation was carried out at 30 °C and 250 rpm for 24 hours. The titer of succinic acid was measured by HPLC.

[0252] [Table 11]

[0253] 2. Fermentation without Neutralizing Agent After inoculating the SA101 - SA116 series of strains into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 0 g / L CaCO3), flask shaking fermentation was carried out at 30 °C and 250 rpm for 24 hours. The titer of succinic acid was measured by HPLC.

[0254] [Table 12]

[0255] Example 12: Optimization of Succinic Acid - Producing Strains Referring to Example 5, the PDC1 and GPD1 genes were knocked out in SA103-4 to obtain strains SA117 (genotype: SA103-4, ΔPDC1) and SA118 (genotype: SA117, ΔGPD1).

[0256] Referring to Example 6, the SpMAE1 gene was overexpressed in SA118 to produce SA119-1 strain (genotype: SA118, ΔPkMCH4) and SA119-2 strain (genotype: SA118, PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ) was obtained.

[0257] Referring to Example 7, PYC from two sources was overexpressed in the SA119-2 strain, resulting in the SA120-1 strain (genotype: SA119-2, ΔPkJEN2-1) and the SA120-2 strain (genotype: SA119-2, PkJEN2-1::P PkTDH3 -ORF PkPYC1 -T PkGAL2 ), and SA120-3 strain (genotype: SA119-2, PkJEN2-1::P PkTDH3 -ORF AoPYC -T PkGAL2 ) was obtained.

[0258] Referring to Example 8, the fumarase gene PkFUM1, in which the 5' terminal mitochondrial localization peptide has been cleaved, was overexpressed in SA120-3 to obtain the SA121 strain (genotype: SA120-3, P PkPDC1 ::P PkTDH3 -ORF PkFUM1 -T PkGAL2 ) was obtained.

[0259] Referring to Example 9, the orotidine 5'-phosphate decarboxylase gene PkURA3 was complemented to the SA121 strain to create the SA122 strain (genotype: SA121, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ) was obtained.

[0260] Referring to Example 4, the TbFRD gene was overexpressed in SA102-3 to create the SA123 strain (genotype: SA102-3, ADH1::ORF TbFRD ) was obtained.

[0261] Referring to Example 5, the PDC1 and GPD1 genes were knocked out in SA123 to obtain the SA124 strain (genotype: SA123, ΔPDC1) and the SA125 strain (genotype: SA124, ΔGPD1).

[0262] Referring to Example 6, the SpMAE1 gene was overexpressed in SA125 to produce SA126-1 strain (genotype: SA125, ΔPkMCH4) and SA126-2 strain (genotype: SA125, PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ) was obtained.

[0263] Referring to Example 7, PYC from two sources was overexpressed in the SA126-2 strain, resulting in the SA127-1 strain (genotype: SA126-2, ΔPkJEN2-1) and the SA127-2 strain (genotype: SA126-2, PkJEN2-1::P PkTDH3 -ORF PkPYC1 -T PkGAL2 ), and SA127-3 strain (genotype: SA126-2, PkJEN2-1::P PkTDH3 -ORF AoPYC -T PkGAL2 ) was obtained.

[0264] Referring to Example 8, the fumarase gene PkFUM1, in which the 5' terminal mitochondrial localization peptide has been cleaved, was overexpressed in SA127-3 to obtain the SA128 strain (genotype: SA127-3, P PkPDC1 ::P PkTDH3 -ORF PkFUM1 -T PkGAL2 ) was obtained.

[0265] Referring to Example 9, the orotidine 5'-phosphate decarboxylase gene PkURA3 was complemented to the SA128 strain to create the SA129 strain (genotype: SA128, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ) was obtained.

[0266] Example 13: Evaluation of succinic acid production ability of SA122 and SA129 strains Strains SA122 and SA129 were grown and fermented in 5L tanks (yeast inorganic salt culture medium, 12% w / v glucose). Without a neutralizing agent, the 36-hour titers of succinic acid reached 96.08 g / L and 46.02 g / L, respectively, with yields of 0.89 g / g and 0.57 g / g, respectively.

[0267] Example 14: Overexpression of the SbMDH gene in Pichia cudriabzebi strain CICC32244ΔURA3 Referring to Example 1, the URA3 gene encoding orotidine 5'-phosphate decarboxylase was knocked out to obtain the CICC32244ΔURA3 mutant.

[0268] The pWSPK_2365 plasmid and fragments 6 and 7 were transferred into the CICC32244ΔURA3 strain via yeast electrotransmission, and positive transformants were screened and obtained, named strain SA130-1 (genotype: CICC32244ΔURA3, ΔPk2365). The pWSPK_2365 plasmid and fragments 1-5 were transferred into the CICC32244ΔURA3 strain via yeast electrotransmission, and positive transformants were screened and obtained, named strain SA130-2 (genotype: CICC32244ΔURA3, Pk2365::P PkFBA1 -ORF SbMDH -T PkINO1 ) was named as such.

[0269] Example 15: Complementary expression of the PkURA3 gene in SA130 series strains Referring to Example 9, PkURA3 was complemented at the PkJEN2-2 locus of the SA130 series strain to create the SA131-1 strain (genotype: SA130-1, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ), and SA131-2 strain (SA130-2, PkJEN2-2::P PkURA3 -ORF PkURA3 -T PkURA3 ) was obtained.

[0270] Example 16: Evaluation of succinic acid production ability of SA130 and SA131 series strains 1. Fermentation using a neutralizing agent SA130 and SA131 series strains were inoculated into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 30 g / L CaCO3) and fermented in a flask with shaking at 30°C and 250 rpm for 24 hours. The succinic acid titer was measured by HPLC.

[0271] [Table 13] 2. Fermentation without the use of neutralizing agents SA130 and SA131 series strains were inoculated into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 0 g / L CaCO3) and fermented in a flask with shaking at 30°C and 250 rpm for 24 hours. The succinic acid titer was measured by HPLC.

[0272] [Table 14] Example 17: Overexpression of the SbMDH gene in Saccharomyces cerevisiae BY4742 strain Sorghum bicolor-derived malate dehydrogenase SbMDH (SEQ ID NO: 1) was overexpressed at the ScURA3 (SEQ ID NO: 129) locus of the orotidine 5'-phosphate decarboxylase gene in Saccharomyces cerevisiae, and strains overexpressing the ScURA3 gene were used as a control. The overexpression of the SbMDH gene sequence was the same as described above and was optimized according to the codon selection of CY902. The promoter and terminator used were the promoter of the ScTDH3 gene encoding glyceraldehyde 3-phosphate dehydrogenase in BY4742 itself (SEQ ID NO: 130) and the terminator of the galactose permyase gene ScGAL2 (SEQ ID NO: 131), respectively. The promoter and terminator used for overexpression of the ScURA3 gene were the promoter of the orotidine 5'-phosphate decarboxylase gene ScURA3 (SEQ ID NO: 132) and the terminator of the gene ScSED1 encoding the GPI cell wall glycoprotein (SEQ ID NO: 133), respectively, within BY4742. The specific construction method was as follows.

[0273] 1. Construction of donor DNA fragments for homologous recombination Using genomic DNA from Saccharomyces cerevisiae S288c (purchased from ThermoFisher Scientific) as a template, the ScURA3 upstream homologous arm and open reading frame (ORF) sequence (fragment 40) were amplified with primers 1_UP_ScURA3_F and 1_ScURA3_R (see Table 15). The ScSED1 terminator sequence 1 (fragment 41) was amplified with primer 2_T ScSED1 _F and 2_T ScSED1 The ScTDH3 gene promoter sequence (fragment 42) was amplified with primer 3_P. ScTDH3 _F and 3_P ScTDH3Amplification was performed with _R (see Table 15). Using a plasmid containing the SbMDH synthetic sequence as a template, the SbMDH coding sequence (fragment 43) derived from sorghum bicolor was amplified with primers 4_SbMDH_F and 4_SbMDH_R (see Table 15). Using S288c genomic DNA as a template, the ScGAL2 gene terminator sequence (fragment 44) was amplified with primer 5_T ScGAL2 _F and 5_T ScGAL2 The ScURA3 gene downstream homologous arm fragment 1 (fragment 45) was amplified with primers 6_DW_ScURA3_F1 and 6_DW_ScURA3_R (see Table 15). Using S288c genomic DNA as a template, the ScSED1 terminator sequence 2 (fragment 46) was amplified with primer 2_T ScSED1 _F and 2_T ScSED1 The gene was amplified with _R2 (see Table 15). Downstream homologous arm fragment 2 (fragment 47) of the ScURA3 gene was amplified with primers 6_DW_ScURA3_F2 and 6_DW_ScURA3_R (see Table 15).

[0274] 2. Construction of a Saccharomyces cerevisiae strain that overexpresses SbMDH. Fragments 40, 46, and 47 were mixed together and transferred to the BY4742 strain (purchased from ThermoFisher Scientific) according to yeast electrotransformation, and positive transformants were screened to obtain the SA132-1 strain (genotype: BY4742, ScURA3::P ScURA3 -ORF ScURA3 -T ScSED1t ) was named. Fragments 40-45 were mixed together and transferred to the BY4742 strain according to yeast electrotransformation, and positive transformants were screened to obtain strain SA132-2 (genotype: BY4742, ScURA3::P ScURA3 -ORF ScURA3 -T ScSED1t -P ScTDH3 -ORF SbMDH -T ScGAL2 ) was named as such.

[0275] [Table 15]

[0276] Example 18: Evaluation of succinic acid production ability of SA132 series strains 1. Fermentation without the use of neutralizing agents SA132 series strains were inoculated into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 0 g / L CaCO3) and fermented in a flask with shaking at 30°C and 250 rpm for 12 hours. The succinic acid titer was measured by HPLC.

[0277] [Table 16]

[0278] Example 19: Overexpression of the SbMDH gene in Yarouia liporitica Po1g strain Because *Yarrowia liporitica* possesses very strong non-homologous end joining ability, random insertions into the *Yarrowia liporitica* genome were used to overexpress the malate dehydrogenase gene SbMDH (SEQ ID NO: 1) derived from *Sorghum bicolor* and the screening marker β-isopropylmalate dehydrogenase (EC1.1.1.85) gene YlLEU2 (SEQ ID NO: 148). A *Yarrowia liporitica* strain with randomly inserted overexpressed YlLEU2 genes was used as a control. Overexpression of the SbMDH gene sequence was the same as described above and was optimized according to CY902 codon selection. The promoters and terminators used were the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene YlGAPDH (SEQ ID NO: 149) and the terminator of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene YlHMG1 (SEQ ID NO: 150), respectively, from *Po1g* itself. The promoter and terminator used for overexpression of the YlLEU2 gene were the promoter of the β-isopropylmalate dehydrogenase gene YlLEU2 (SEQ ID NO: 151) and the terminator of the Saccharomyces cerevisiae cytochrome c subtype 1 gene ScCYC1 (SEQ ID NO: 152), respectively. The specific construction method was as follows.

[0279] 1. Construction of donor DNA fragments for homologous recombination Using Yarouia liporitica W29 genomic DNA (GenBank: GCA_001761485.1) as a template, the YlGAPDH gene promoter sequence (fragment 48) was applied to primer 1_P YlGAPDH _F and 1_P YlGAPDH Amplification was performed with _R (see Table 17). Using a plasmid containing the SbMDH synthetic sequence as a template, the SbMDH coding sequence (fragment 49) derived from Sorghum bicolor was amplified with primers 2_SbMDH_F and 2_SbMDH_R (see Table 17). Using Yarouia liporitica W29 genomic DNA as a template, the YlHMG1 gene terminal sequence (fragment 50) was amplified with primer 3_T YlHMG1 _F and 3_T YlHMG1 The signal was amplified using _R (see Table 17). The YlLEU2 promoter and ORF sequence (fragment 51) were primed with primer 4_P YlLEU2 Amplification was performed using _F and 4_YlLEU2_R (see Table 17). Using S288c genomic DNA as a template, the ScCYC1 terminator sequence (fragment 52) ​​was amplified with primer 5_T ScCYC1 _F and 5_T ScCYC1 The expression was amplified with _R (see Table 17). Using fragments 48-52 as templates, an expression cassette (fragment 53) overexpressing SbMDH and YlLEU2 was applied to primer 1_P YlGAPDH _F and 5_T ScCYC1 The expression cassette (fragment 54) overexpressing YlLEU2 was amplified with _R. Fragments 51 and 52 were used as templates, and primer 4_P YlLEU2 _F and 5_T ScCYC1 It was amplified using _R.

[0280] 2. Construction of a Yaroia liporitica strain that overexpresses SbMDH Fragment 54 was transferred to the Po1g strain (purchased from Yeastern Biotechnology Development Co., Ltd.) according to yeast electrotransformation, and positive transformants were screened to obtain strain SA133-1 (genotype: Polg, genome::P).YlLEU2 -ORF YlLEU2 -T ScCYC1t It was named SA133-2 (genotype: Polg, genome::P). Fragment 53 was transferred to the Po1g strain according to yeast electrotransformation, and positive transformants were screened and obtained. YlGAPDH -SbMDH-T YlHMG1t -P YlLEU2 -ORF YlLEU2 -T ScCYC1t ) was named as such.

[0281] [Table 17]

[0282] Example 20: Evaluation of succinic acid production ability of SA133 series strains 1. Fermentation without the use of neutralizing agents SA133 series strains were inoculated into 30 mL of yeast inorganic salt culture medium (5% w / v glucose, 0 g / L CaCO3) and fermented in a flask with shaking at 30°C and 250 rpm for 24 hours. The succinic acid titer was measured by HPLC.

[0283] [Table 18]

Claims

1. A genetically modified succinate-producing yeast strain having NADPH-dependent malate dehydrogenase (EC1.1.1.82) activity or enhanced activity, The aforementioned succinic acid-producing yeast strain is a genetically modified succinic acid-producing yeast strain, which was modified from Pichia cudriabzebi, deposited with the China Microbial Species Depositary Administration Center for Ordinary Microorganisms (CGMCC) under CGMCC number 20885.

2. (i) having the activity or 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) fumarase (EC 4.2.1.2), and (iv) succinate transport protein, and / or A genetically modified succinic acid-producing yeast strain according to claim 1, further comprising (i) pyruvate decarboxylase (EC 4.1.1.43) with reduced activity or inactivated activity, and / or (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), and / or (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), and / or (iv) monocarboxylate permease, and / or (v) dicarboxylate transport protein, and / or (vi) alcohol dehydrogenase 1 (EC 1.1.1.1), and / or (vii) a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112).

3. The 3'-terminal glyoxisome localization peptide of the soluble fumarate reductase is partially or completely cleaved, and / or The 5'-terminal mitochondrial localization peptide of the fumarase is partially or completely cleaved, and / or The NADPH-dependent malate dehydrogenase is derived from plants, and / or The soluble fumarate reductase is derived from Saccharomyces cerevisiae, Trypanosoma brusey, Leishmania mexicana, or Trypanosoma cruz, and / or The succinate transport protein is selected from the group consisting of SpMAE1 protein, AnDCT-02 protein, EcDcuB protein and EcDcuC protein, and / or The genetically modified succinate-producing yeast strain according to claim 2, wherein the pyruvate carboxylase is derived from Aspergillus oryzae or Pichia cudriabzebi.

4. The genetically modified succinic acid-producing yeast strain according to claim 1, wherein the NADPH-dependent malate dehydrogenase is derived from a plant of the family Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, or Amaranthaceae, or from a plant of the genus Euglena or Thermobacillus.

5. (i) an overexpressed nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, wherein the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase comprises the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or a nucleotide sequence having at least 90% identity thereto and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity, and / or (ii) A nucleic acid sequence that is overexpressed and encodes soluble fumarate reductase, wherein the nucleic acid sequence encoding soluble fumarate reductase comprises any one of sequence numbers 3 to 5 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 90% identity thereto and having soluble fumarate reductase activity, and / or (iii) an overexpressed nucleic acid sequence encoding a succinate transport protein, wherein the nucleic acid sequence encoding the succinate transport protein comprises a nucleotide sequence encoding the sequence of SEQ ID NO: 2 or a degenerate sequence thereof, or an amino acid sequence having at least 90% identity thereto and possessing succinate transport protein activity, and / or (iv) an overexpressed nucleic acid sequence encoding pyruvate carboxylase, wherein the nucleic acid sequence encoding pyruvate carboxylase comprises a nucleotide sequence encoding the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof, or an amino acid sequence having at least 90% identity thereto and possessing pyruvate carboxylase activity, and / or (v) an overexpressed nucleic acid sequence encoding fumarase, wherein the nucleic acid sequence encoding fumarase comprises the sequence of Sequence ID No. 8 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 90% identity thereto and possessing fumarase activity, and / or (vi) a knockout of an endogenous gene encoding pyruvate decarboxylase, and / or (vii) A knockout of an endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase, and / or (viiii) The knockout of the endogenous gene encoding orotidine 5'-phosphate decarboxylase, and / or (ix) a knockout of the endogenous gene encoding monocarboxylate permease, and / or (x) a knockout of an endogenous gene encoding a dicarboxylate transport protein, and / or (xi) The knockout of the endogenous gene encoding alcohol dehydrogenase 1, and / or (xi) Endogenous gene encoding a bifunctional enzyme, oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase, which has been knocked out. A genetically modified succinic acid-producing yeast strain according to any one of claims 1 to 4, having the above characteristics.

6. In the aforementioned genetically modified succinate-producing yeast strain, (a) The nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase and the following nucleic acid sequence: The nucleic acid sequence encoding the soluble fumarate reductase, The nucleic acid sequence encoding the succinate transport protein, The nucleic acid sequence encoding the pyruvate carboxylase, and The nucleic acid sequence encoding the aforementioned fumarose, At least one of the following is overexpressed, (b) The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase is overexpressed, and the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase is knocked out, (c) The nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding the pyruvate carboxylase, the nucleic acid sequence encoding the soluble fumarate reductase, and the nucleic acid sequence encoding the succinate transport protein are overexpressed, and the endogenous gene encoding the pyruvate decarboxylase and / or the endogenous gene encoding the NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out, or (d) The nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding the pyruvate carboxylase, the nucleic acid sequence encoding the soluble fumarate reductase, the nucleic acid sequence encoding the fumarase, and the nucleic acid sequence encoding the succinate transport protein are overexpressed, and the endogenous gene encoding the pyruvate decarboxylase and / or the endogenous gene encoding the NAD-dependent glycerol 3-phosphate dehydrogenase are knocked out, and The nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase is a nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase of sorghum bicolor, or a nucleic acid sequence containing the sequence of Sequence ID No. 1 or a degenerate sequence thereof, and / or The nucleic acid sequence encoding the soluble fumarate reductase is a nucleic acid sequence encoding a soluble fumarate reductase derived from Saccharomyces cerevisiae, Trypanosoma burseyi, Leishmania mexicana, or Trypanosoma cruz, or a nucleic acid sequence containing any one of Sequence IDs 3-5 or a degenerate sequence thereof, and / or The nucleic acid sequence encoding the succinate transport protein is a nucleic acid sequence encoding the SpMAE1 protein, or a nucleic acid sequence containing the sequence of Sequence ID No. 2 or a degenerate sequence thereof, and / or The nucleic acid sequence encoding the pyruvate carboxylase encodes a pyruvate carboxylase derived from Aspergillus oryzae or Pichia cudriabzebi, or includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof. The genetically modified succinic acid-producing yeast strain according to claim 2 or 3.

7. A method for constructing a genetically modified succinic acid-producing yeast strain, comprising conferring or enhancing the activity of NADPH-dependent malate dehydrogenase (EC1.1.1.82) to the succinic acid-producing yeast strain, wherein the genetically modified succinic acid-producing yeast strain is modified from Pichia cudriabzebi, which is deposited with the China Microbial Species Depositary Administration Center for Ordinary Microorganisms (CGMCC) under CGMCC number 20885.

8. The following activities are further comprising conferring or enhancing at least one of the following: (i) soluble fumarate reductase activity, (ii) pyruvate carboxylase (EC 6.4.1.1) activity, (iii) fumarase (EC 4.2.1.2) activity, and (iv) succinate transport protein activity, and / or The method according to claim 7, further comprising attenuating or inactivating (i) pyruvate decarboxylase (EC 4.1.1.43), and / or (ii) NAD-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), and / or (iii) orotidine 5'-phosphate decarboxylase (EC 4.1.1.23), and / or (iv) monocarboxylate permease, and / or (v) alcohol dehydrogenase 1 (EC 1.1.1.1), and / or (vi) dicarboxylate transport protein, and / or (vii) a bifunctional enzyme of oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase (EC 4.1.3.17 or 4.1.1.112) in the strain.

9. The soluble fumarate reductase is freely present in the cytoplasm, and / or The fumarase is freely present in the cytoplasm, and / or The soluble fumarate reductase is derived from Saccharomyces cerevisiae, Trypanosoma brusey, Leishmania mexicana, or Trypanosoma cruz, and / or The succinate transport protein is selected from the group consisting of SpMAE1 protein, AnDCT-02 protein, EcDcuB protein and EcDcuC protein, and / or The pyruvate carboxylase is derived from Aspergillus oryzae or Pichia cudriabzebi, and / or The method according to claim 8, wherein the NADPH-dependent malate dehydrogenase is derived from a plant.

10. The method according to claim 7, wherein the NADPH-dependent malate dehydrogenase is derived from a plant of the family Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae, or from a plant of the genus Euglena or Thermobacillus.

11. In the succinic acid-producing yeast strain mentioned above, (i) Overexpressing the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, wherein the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase includes the sequence of SEQ ID NO: 1 or a degenerate sequence thereof, or a nucleotide sequence having at least 90% identity thereto and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity, and / or (ii) Overexpressing a nucleic acid sequence encoding soluble fumarate reductase, wherein the nucleic acid sequence encoding soluble fumarate reductase includes any one sequence of SEQ ID NOs. 3 to 5 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 90% identity thereto and possessing soluble fumarate reductase activity, and / or (iii) Overexpressing a nucleic acid sequence encoding a succinate transport protein, wherein the nucleic acid sequence encoding the succinate transport protein includes the sequence of SEQ ID NO: 2 or a degenerate sequence thereof, or a nucleotide sequence having at least 90% identity thereto and encoding an amino acid sequence having succinate transport protein activity, and / or (iv) Overexpressing a nucleic acid sequence encoding pyruvate carboxylase, wherein the nucleic acid sequence encoding pyruvate carboxylase includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 90% identity thereto and possessing pyruvate carboxylase activity, and / or (v) Overexpressing a nucleic acid sequence encoding fumarase, wherein the nucleic acid sequence encoding fumarase includes the sequence of SEQ ID NO: 8 or a degenerate sequence thereof, or a nucleotide sequence encoding an amino acid sequence having at least 90% identity thereto and possessing fumarase activity, and / or (vi) Knocking out the endogenous gene encoding pyruvate decarboxylase, and / or (vii) Knocking out the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase, and / or (viiii) Knocking out the endogenous gene encoding orotidine 5'-phosphate decarboxylase, and / or (ix) Knocking out the endogenous gene encoding monocarboxylate permease, and / or (x) Knocking out an endogenous gene encoding a dicarboxylate transport protein, and / or (xi) Knocking out the endogenous gene encoding alcohol dehydrogenase 1, and / or (xi) Knock out the endogenous gene encoding the bifunctional enzymes oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldolase. The method according to any one of claims 7 to 10, including the method described in any one of claims 7 to 10.

12. In the succinic acid-producing yeast strain mentioned above, (a) The nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase and the following nucleic acid sequence: The nucleic acid sequence encoding the soluble fumarate reductase, The nucleic acid sequence encoding the succinate transport protein, The nucleic acid sequence encoding the pyruvate carboxylase, and The nucleic acid sequence encoding the aforementioned fumarose, Overexpression of at least one of the following, or (b) Overexpressing the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase and knocking out the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase, or (c) Overexpressing the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding pyruvate carboxylase, the nucleic acid sequence encoding soluble fumarate reductase, and the nucleic acid sequence encoding succinate transport protein, and knocking out the endogenous gene encoding pyruvate decarboxylase and / or the endogenous gene encoding NAD-dependent glycerol 3-phosphate dehydrogenase, or (d) Overexpressing the nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase, the nucleic acid sequence encoding the pyruvate carboxylase, the nucleic acid sequence encoding the soluble fumarate reductase, the nucleic acid sequence encoding the fumarase, and the nucleic acid sequence encoding the succinate transport protein, and knocking out the endogenous gene encoding the pyruvate decarboxylase and / or the endogenous gene encoding the NAD-dependent glycerol 3-phosphate dehydrogenase, The nucleic acid sequence encoding the NADPH-dependent malate dehydrogenase is a nucleic acid sequence encoding NADPH-dependent malate dehydrogenase derived from sorghum bicolor, or includes the sequence of Sequence ID No. 1 or a degenerate sequence thereof, and / or The nucleic acid sequence encoding the soluble fumarate reductase is a nucleic acid sequence encoding a soluble fumarate reductase derived from Saccharomyces cerevisiae, Trypanosoma bursey, Leishmania mexicana, or Trypanosoma cruz, or includes any one of the sequences of SEQ ID NOs. 3 to 5 or a degenerate sequence thereof, and / or The nucleic acid sequence encoding the succinate transport protein is the nucleic acid sequence encoding the SpMAE1 protein, or includes the sequence of Sequence ID No. 2 or a degenerate sequence thereof, and / or The nucleic acid sequence encoding the pyruvate carboxylase encodes a pyruvate carboxylase derived from Aspergillus oryzae or Pichia cudriabzebi, or includes the sequence of SEQ ID NO: 6 or 7 or a degenerate sequence thereof. The method according to claim 8 or 9.

13. A method for producing succinic acid, comprising culturing a genetically modified succinic acid-producing yeast strain described in any one of claims 1 to 4 or a genetically modified succinic acid-producing yeast strain prepared by the method described in any one of claims 7 to 10.

14. The genetically modified succinic acid-producing yeast strain is cultured at pH < 3.5 and / or with no or minimal addition of a neutralizing agent, and / or The method according to claim 13, comprising isolating and purifying the succinic acid produced.

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