An acid-tolerant yeast strain with high l-malic acid production and a construction method and application thereof

Abstract

The present application provides a genetically engineered L-malic acid producing yeast strain, wherein the strain has or has enhanced malate transporter activity and has or has enhanced NADPH-dependent malate dehydrogenase (EC 1.1.1.82) activity, optionally further has or has enhanced at least one of the following activities: (i) pyruvate carboxylase (EC 6.4.1.1) activity, (ii) phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activity, (iii) phosphoenolpyruvate carboxylase activity, and (iv) biotin transporter activity; as well as methods of making the same, methods of using the same to produce L-malic acid, and applications thereof.

Description

Technical Field

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

[0002] Malic acid, also known as 2-hydroxybutyric acid, is an intermediate metabolite in the tricarboxylic acid cycle of organisms. Due to the presence of an asymmetric carbon atom in its molecule, malic acid has two stereoisomers (L-form and D-form), but naturally occurring malic acid is always L-malic acid. L-malic acid is an important C4 bulk chemical with a wide range of applications.

[0003] Currently, malic acid production methods mainly include chemical methods, enzymatic catalysis, and bio-fermentation. Chemical synthesis primarily utilizes maleic acid or fumaric acid derived from fossil fuels to synthesize malic acid under high temperature and pressure conditions. While the chemical method is mature and cost-effective, it suffers from poor stereoselectivity, and the malic acid product is a mixture of L- and D-forms, making it difficult to obtain high-purity L-malic acid. Enzymatic catalysis mainly utilizes fumarate enzymes to catalyze the hydration of fumaric acid to form L-malic acid; although this method is clean and efficient, the cost of preparing the enzyme and substrate is high, hindering large-scale production. Currently, microbial fermentation mainly utilizes filamentous fungi, including Aspergillus oryzae (…). Aspergillus oryzae Aspergillus flavus Aspergillus flavus ), Ustilago barnyard fungus ( Utilago trichophora While filamentous fungal fermentation can achieve high L-malic acid yields, it also presents several challenges. These include slow growth, complex and time-consuming genetic modification, poor cell morphology homogeneity during submerged liquid fermentation, low tolerance to mechanical shear stress, and the tendency for hyphae to clump together, making the fermentation process difficult to control. Furthermore, some fungal strains carry mycotoxins (such as aflatoxin), posing potential safety risks. Yeast, compared to filamentous fungi, grows much faster and is easier to genetically modify, making it an ideal production strain. Currently, some yeast strains, such as *Saccharomyces cerevisiae* (Saccharomyces cerevisiae), are used in fermentation. Saccharomyces cerevisiae ), Rhubarb yeast ( Zygosaccharomycesrouxii ), black yeast ( Aureobasidium pullulans While high-yield production of L-malic acid has been achieved, the sugar-acid conversion rate is generally low, and the production cost remains high.

[0004] Furthermore, the cost of organic acid separation and purification accounts for approximately 50% of the entire fermentation process. Traditional L-malic acid production processes require calcium carbonate as a neutralizing agent to precipitate L-malic acid from the fermentation system as calcium malate. Calcium malate then needs to be further replaced with sulfuric acid to form L-malic acid, generating a large amount of calcium sulfate solid waste, which is environmentally unfriendly. However, some acid-tolerant yeast cells can grow normally in harsh environments with a pH less than 3.0. Under these conditions, malic acid mostly exists in an undissociated molecular form (malic acid pKa1 = 3.46), which can greatly simplify the downstream separation and purification process and reduce costs. Summary of the Invention

[0005] This invention uses an acid-resistant yeast, *Pichia pastoris*, isolated from the peel of wild fruits in Yunnan. Pichia kudriavzevii Starting with strain CY902 (deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 20885), metabolic engineering was performed to achieve efficient production of L-malic acid. Specifically, the modified strain can achieve efficient L-malic acid production through fermentation at pH < 3.5 with little or no addition of calcium carbonate neutralizing agent.

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

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

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

[0009] In one aspect, the present invention provides a method for producing a genetically modified malate-producing yeast strain, comprising conferring or enhancing malate transporter activity and conferring or enhancing NDAPH-dependent malate dehydrogenase activity on the strain, optionally further comprising conferring or enhancing at least one of the following activities: (i) pyruvate carboxylase activity, (ii) phosphoenolpyruvate carboxylkinase activity, (iii) phosphoenolpyruvate carboxylase activity, and (iv) biotin transporter activity. Preferably, the NDAPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, and Amaranthaceae families, or from the genera *Euglebsiella* and *Thermobacterium*, more preferably sorghum, corn, sugarcane, pea, chickpea, spinach, *Euglebsiella*, or thermoautotrophic methanotherapeutic bacteria.

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

[0011] In one aspect, the present invention provides a method for producing L-malic acid, comprising (preferably under conditions of pH < 3.5, for example, 2.0-3.5 and / or with little or no addition of a neutralizing agent) culturing the genetically modified malic acid-producing yeast strain of the present invention and / or obtaining the genetically modified malic acid-producing yeast strain by the method of the present invention for producing the genetically modified malic acid-producing yeast strain.

[0012] In one aspect, the present invention provides the use of the genetically modified malic acid-producing yeast strains described herein and / or the genetically modified malic acid-producing yeast strains obtained by the method for producing the genetically modified malic acid-producing yeast strains described herein in the production of L-malic acid (preferably in a pH range of <3.5, for example, 2.0-3.5 and / or under conditions of little or no addition of neutralizing agents). Detailed Implementation

[0013] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

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

[0015] As used herein, malic acid-producing yeast strains refer to yeasts that, under suitable conditions (e.g., through fermentation), can produce malic acid (e.g., L-malic acid) and secrete it into an extracellular medium. Preferably, the malic acid contains at least 50% L-malic acid, for example, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%. Malic acid-producing yeast strains possess proteins that can transport malic acid to the extracellular space, thus allowing the malic acid to be secreted after production. Suitable malic acid transporters for a given yeast strain are known in the art, including, but not limited to, the malic acid transporter SpMAE1 from *Schizosacchariformis* and the four-carbon dicarboxylic acid transporter C4T318 from *Aspergillus oryzae*.

[0016] Yeasts known in the art for the production of malic acid include, for example, but not limited to, the genus *Zygosaccharomyces* (…). Zygosaccharomyces ), *Gastromycium* ( Torulopsis ), Candida genus ( Candida ), Pichia pastoris ( Pichia ), Rhodotorula genus ( Rhodotroula ), Yeast ( Saccharomyces ), Yersinia ( YarrowiaIn one embodiment, the malic acid-producing yeast strain is a Pichia genus strain. In a preferred embodiment, the malic acid-producing yeast strain is Pichia kudriezwei, such as Pichia kudriezwei, which was deposited on October 14, 2020, at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 20885. Pichia kudriavzevii ).

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

[0018] As used herein, “enhanced…activity” means an increase in activity of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or more, compared to a reference strain (e.g., the initial strain or the wild-type strain) having that activity.

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

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

[0021] As used herein, “reduced or inactivated” means a reduction in activity of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even 100%, compared to a reference activity (e.g., the corresponding activity in the initial strain or wild-type strain).

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

[0023] As used herein, a “weakened or inactivated gene” means a gene whose activity, such as expression level (when it is a protein-coding gene) or regulatory function (when it is a regulatory element), is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable, compared to a reference (e.g., the corresponding gene in the initial strain or wild-type strain). In the case of a gene encoding a protein, such as an enzyme, “weakened or inactivated gene” also covers a protein whose activity level is reduced compared to the corresponding protein activity level in the initial strain or wild-type strain, for example, by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even 100%.

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

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

[0026] As used herein, malate transporters are proteins that can transport malate from inside the cell to outside the cell, including, but not limited to, *Schizosaccharomyces cerevisiae*. Schizosaccharomyces pombe The SpMAE1 protein (Uniprot database search number: P50537) of Aspergillus oryzae and the four-carbon dicarboxylic acid transporter C4T318 (Gene ID: 5992883) of Aspergillus oryzae and succinic acid-producing Actinobacillus ( Actinobacillus succinogenes The AsDct (NCBI Reference Sequence: WP_012073722.1) etc. are mentioned. In this article, having or having enhanced malate transporter activity refers to the strain having or having increased activity in transporting malate to the extracellular space.

[0027] As used in this article, the SpMAE1 protein, a dicarboxylic acid transporter in *Schizosaccharide Spr.*, is derived from... SpMAE1 The SpMAE1 protein, encoded by the gene, is responsible for transporting dicarboxylic acids (DCAs) from the cell to the extracellular space. In this article, having or having enhanced SpMAE1 activity refers to strains exhibiting or having increased activity in transporting DCAs to the extracellular space.

[0028] As used in this article, pyruvate carboxylase (EC 6.4.1.1) is derived from... PYC The gene encodes pyruvate carboxylase, which participates in the interconversion between oxaloacetate and pyruvate during gluconeogenesis, responsible for converting pyruvate and carbon dioxide into oxaloacetate. Commonly used sources of pyruvate carboxylase include fungi, especially yeasts and filamentous fungi, with *Saccharomyces cerevisiae*, *Pichia kudriezvichi*, *Aspergillus oryzae*, and *Kluyveromyces martensii* being preferred. It is known that *Pichia kudriezvichi* contains one... PYC Genes, that is PYC1 Genes. In this article, having or having enhanced pyruvate carboxylase activity means that the strain has or has increased activity in converting pyruvate to oxaloacetate.

[0029] As used in this article, phosphoenolpyruvate carboxykinase (EC 4.1.1.49) is derived from... PCK The gene encodes an enzyme involved in the interconversion between oxaloacetate and phosphoenolpyruvate during gluconeogenesis, responsible for converting phosphoenolpyruvate and carbon dioxide into oxaloacetate. Commonly used sources of phosphoenolpyruvate carboxykinase include Saccharomyces cerevisiae and Escherichia coli. Escherichia coli It is known that one *Pichia kudrica* species exists. PCK Genes, that is PCK1 Genes. In this article, having or having enhanced phosphoenolpyruvate carboxykinase refers to strains having or having increased activity in converting phosphoenolpyruvate to oxaloacetate.

[0030] Phosphoenolpyruvate carboxylase (PPC, EC 4.1.1.31) is responsible for converting phosphoenolpyruvate and carbon dioxide to oxaloacetic acid. Plant (especially diatoms and angiosperms) and bacterial (e.g., Proteobacteria) sources are commonly used, with *Escherichia coli* and *Phaeodactylum tricornutum* being preferred. Phaeodactylum tricornutum Sources include sorghum, corn, and rice. In this article, having or having enhanced phosphoenolpyruvate carboxylase activity refers to the strain having or having increased activity in converting phosphoenolpyruvate to oxaloacetate.

[0031] As used herein, a biotin (vitamin H) transporter is a protein capable of transporting biotin from the extracellular space to the intracellular space. Suitable biotin transporters for a given yeast strain are known in the art. Biotin transporters are generally derived from fungi and bacteria, particularly yeasts, Pseudomonas, and Rhizobia, with further preference given to Saccharomyces cerevisiae, Schizosoma makoysum, and Candida glabrata. Candida glabrata ), Kluyveromyces martensii, and Pseudomonas mukorossi ( Pseudomonas mutabilis ), Alfalfa rhizobia ( Sinorhizobium meliloti (e.g.) In this article, having or having enhanced biotin transporter activity refers to the strain having or having increased activity in transporting biotin from the culture medium into the cell.

[0032] In one embodiment, the biotin transporter is the *Schizosaccharomyces cerevisiae* biotin transporter SpVHT1 (Uniprot database search number: O13880), which is composed of... SpVHT1 The gene encodes the SpVHT1 transporter, responsible for transporting biotin from the culture medium into the cell. In this article, having or having enhanced biotin transporter activity means that the strain has or has increased activity in transporting biotin from the culture medium into the cell.

[0033] As used in this article, pyruvate decarboxylase (EC 4.1.1.1) is derived from... PDC The gene encodes pyruvate decarboxylase, which participates in the decarboxylation of pyruvate in the ethanol synthesis pathway. It is known that *Pichia kudrica* contains a... PDC Genes are PDC1 Genes. In this article, reduced or inactivated pyruvate decarboxylase refers to enzymes whose pyruvate decarboxylation activity is reduced or lost.

[0034] As used in this article, NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) is derived from... GPD The gene encodes an enzyme involved in the interconversion between glycerol phosphate and glycerol 3-phosphate in the glycerol synthesis pathway. It is known that *Pichia kudrica* contains a... GPD Genes, that is GPD1Genes. In this article, reduced or inactivated NAD-dependent glycerol-3-phosphate dehydrogenase refers to the enzyme’s reduced or lost activity in the interconversion of glycerol phosphate and glycerol 3-phosphate.

[0035] As used in this article, 5'-orhinophosphate decarboxylase (EC 4.1.1.23) is derived from... URA3 The gene encodes an enzyme involved in the decarboxylation of whey 5-phosphate during pyrimidine synthesis. It is known that *Pichia goudryanazwiica* contains one such enzyme. URA3 Genes. In this article, reduced or inactivated 5'-orotose decarboxylase refers to the enzyme's reduced or lost activity in catalyzing the decarboxylation of 5'-orotose.

[0036] As used in this article, monocarboxylic acid permease (NCBI Reference Sequence: XP_029320775.1) was obtained from... MCH4 Gene encoding. In this document, reduced or inactivated monocarboxylic acid permease refers to an enzyme whose catalytic activity is reduced or lost. In one embodiment, the strain contains... MCH4 Genes are knocked out, for example, through homologous recombination.

[0037] As used in this article, JEN2 The gene encodes a dicarboxylic acid transporter, which is involved in the transport of dicarboxylic acids from the culture medium into the cell. Two strains of *Pichia goudrya* are known to exist. JEN2 Gene( JEN2-1 (encoding the polypeptide shown in SEQ ID NO: 14) and JEN2-2 (The polypeptide represented by SEQ ID NO: 15)). In this article, deactivated or inactive dicarboxylic acid transporters are those whose activity in transporting dicarboxylic acids from the culture medium into the cell is reduced or lost.

[0038] As used herein, a neutralizing agent is a reagent that precipitates L-malic acid from a fermentation system in the form of calcium malate. Substances known in the art that can be used as neutralizing agents include, for example, but not limited to, calcium carbonate.

[0039] As used herein, "no or minimal addition of neutralizing agent" means an amount of neutralizing agent added 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%, compared to the amount of neutralizing agent added in the fermentation production of L-malic acid known in the art. For example, in the method of the present invention, the amount of neutralizing agent added can be 0-60 g / L.

[0040] As used herein, the terms “polypeptide,” “amino acid sequence,” “peptide,” and “protein” are used interchangeably to refer to an amino acid chain of any length that may contain modified amino acids and / or may be interrupted by non-amino acid components. The term also covers amino acid chains that have been modified by natural or artificial means; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component.

[0041] As used herein, the terms “gene,” “nucleic acid sequence,” “polynucleotide,” and “nucleotide sequence” are used interchangeably and refer to nucleotide chains, including DNA and RNA. “Gene expression” refers to the transcription of a DNA region operatively linked to appropriate regulatory regions, particularly promoters, into biologically active RNA, and the translation of RNA into biologically active proteins or peptides.

[0042] As used in this article, a degenerate sequence is a nucleotide sequence that encodes the same amino acid sequence as a specified sequence but has a different nucleotide sequence due to the degeneracy of the genetic codon.

[0043] As used herein, the terms “homology”, “sequence identity”, etc., are used interchangeably. Sequence identity can be detected by comparing the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide, for example, by using a standard alignment algorithm program with a default gap penalty specified by each vendor. Whether two nucleic acid molecules have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% “identical” nucleotide sequences can be determined using known computer algorithms such as BLASTN, FASTA, DNAStar, and Gap (University of Wisconsin Genetics Computer Group (UWG), Madison WI, USA). For example, the percentage of similarity between nucleic acid molecules can be determined, for instance, by comparing sequence information using the GAP computer program (e.g. Needleman et al. J. Mol. Biol. 48: 443 (1970), revised by Smith and Waterman (Adv. Appl. Math. 2: 482 (1981)). In short, the GAP program defines similarity based on the number of similarly arranged symbols (i.e., nucleotides) compared to the total number of symbols in the shorter sequence of the two sequences.

[0044] As used in this article, malic acid oxidase (EC 1.1.1.38) is derived from... MAE1 The gene encodes this enzyme, which is responsible for the oxidative decarboxylation of malic acid to pyruvate. In this article, reduced activity or inactivated malic enzyme refers to the enzyme's reduced or lost activity in oxidative decarboxylation of malic acid to pyruvate.

[0045] As used in this article, Pk2365 The gene encodes a bifunctional enzyme that performs oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation (EC 4.1.3.17 or 4.1.1.112). In this article, reduced or inactivated oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation bifunctional enzyme refers to an enzyme whose activity in catalyzing the decarboxylation of oxaloacetate and the condensation of 3-hydroxy-3-methylglutaraldehyde is reduced or lost.

[0046] In one aspect, the present invention provides a genetically modified malate-producing yeast strain having or enhanced malate transporter activity, such as SpMAE1 protein activity, and having or enhanced NADPH-dependent malate dehydrogenase activity, optionally also having or enhanced activity of at least one of the following: (i) pyruvate carboxylase (EC 6.4.1.1), (ii) phosphoenolpyruvate carboxylkinase (EC 4.1.1.49), (iii) phosphoenolpyruvate carboxylase (EC 4.1.1.31), preferably *Escherichia coli* phosphoenolpyruvate carboxylase, and (iv) biotin transporter. The term "at least one" includes any one, two, three, or all four activities selected therefrom.

[0047] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, and pyruvate carboxylase activity.

[0048] In one embodiment, the present invention provides a genetically modified malate-producing yeast strain having or enhanced malate transporter activity, such as SpMAE1 protein activity, NADPH-dependent malate dehydrogenase activity, and phosphoenolpyruvate carboxykinase activity.

[0049] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activity, such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, and phosphoenolpyruvate carboxylase activity, preferably Escherichia coli phosphoenolpyruvate carboxylase activity.

[0050] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, and biotin transporter activity.

[0051] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, pyruvate carboxylase activity, and phosphoenolpyruvate carboxylkinase activity.

[0052] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activity, such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, pyruvate carboxylase activity, and phosphoenolpyruvate carboxylase activity, preferably Escherichia coli phosphoenolpyruvate carboxylase activity.

[0053] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, pyruvate carboxylase activity, and biotin transporter activity.

[0054] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activity, such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, phosphoenolpyruvate carboxylkinase activity, and phosphoenolpyruvate carboxylase activity, preferably Escherichia coli phosphoenolpyruvate carboxylase activity.

[0055] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, phosphoenolpyruvate carboxykinase activity, and biotin transporter activity.

[0056] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activity, such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, phosphoenolpyruvate carboxylase activity (preferably Escherichia coli phosphoenolpyruvate carboxylase activity), and biotin transporter activity.

[0057] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activity, such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, pyruvate carboxylase activity, phosphoenolpyruvate carboxylkinase activity, and preferably Escherichia coli phosphoenolpyruvate carboxylase activity.

[0058] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, pyruvate carboxylase activity, phosphoenolpyruvate carboxylkinase activity, and biotin transporter activity.

[0059] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, pyruvate carboxylase activity, phosphoenolpyruvate carboxylase activity, preferably Escherichia coli phosphoenolpyruvate carboxylase activity and biotin transporter activity.

[0060] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activity, such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, phosphoenolpyruvate carboxylkinase activity, phosphoenolpyruvate carboxylase activity, preferably Escherichia coli phosphoenolpyruvate carboxylase activity and biotin transporter activity.

[0061] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain having or enhanced malic acid transporter activities such as SpMAE1 protein activity, NADPH-dependent malic acid dehydrogenase activity, pyruvate carboxylase activity, phosphoenolpyruvate carboxylkinase activity, phosphoenolpyruvate carboxylase activity (preferably Escherichia coli phosphoenolpyruvate carboxylase activity and biotin transporter activity).

[0062] In one embodiment, having or having enhanced activity is achieved by enhancing or overexpressing the corresponding encoding gene in the strain. Thus, in one embodiment, the genetically modified malic acid-producing yeast strain expresses or overexpresses a gene encoding a malic acid transporter protein, for example... SpMAE1 Genes and / or NADPH-dependent malate dehydrogenase genes.

[0063] In one embodiment, the NADPH-dependent malate dehydrogenase is derived from plants (preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, and Amaranthaceae families), Euglena, or Thermomycium, preferably from sorghum, corn, sugarcane, peas, chickpeas, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria, more preferably from sorghum.

[0064] In one embodiment, the enzyme is expressed or overexpressed in the genetically modified malate-producing yeast strain, encoding an NADPH-dependent malate dehydrogenase. MDHGenes that produce or increase NADPH-dependent malate dehydrogenase activity, preferably, genes encoding said NADPH-dependent malate dehydrogenase. MDH The genes are preferably derived from plants, preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae or Amaranthaceae families, or from Euglena or Thermomycium, more preferably from sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria, more preferably from sorghum.

[0065] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity.

[0066] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH Genes are incorporated into the genome of genetically modified malic acid-producing yeasts (e.g., Pichia kudrica), for example, in the corresponding... Pk2365 Location of the locus. The gene encoding NADPH-dependent malate dehydrogenase... MDH Genes can be placed in appropriate promoters (e.g.) FBA1 Gene promoters (e.g., shown in SEQ ID NO: 17) and / or terminators (e.g.) INO1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 18).

[0067] In one embodiment, the malate transporter is selected from SpMAE1 protein, C4T318 protein, and AsDct protein. In one embodiment, malate transporter activity is generated or increased by expressing or overexpressing a gene encoding a malate transporter such as SpMAE1 protein in the genetically modified malate-producing yeast strain. In one embodiment, the malate transporter activity is achieved through expression or overexpression of... SpMAE1 Genes produce or enhance this.

[0068] In one implementation, the SpMAE1The gene contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having malic acid transporter activity (e.g., from *Schizosaccharomyces cerevisiae*), optionally incorporated into the genome of a genetically modified malic acid-producing yeast strain (e.g., *Pichia kudrica*), for example in... MCH4 The location of the gene locus. (The following is a description of the gene locus.) SpMAE1 Genes can be placed in appropriate promoters (e.g.) TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0069] In a further embodiment, the genetically modified malic acid-producing yeast strain also possesses or has enhanced biotin transporter activity, such as SpVHT1 activity. Biotin transporters are generally derived from fungi and bacteria, particularly yeasts, Pseudomonas, and Rhizobia, with further preferred species being Saccharomyces cerevisiae, Schizosoma sacchari, Candida glabrata, Kluyveromyces martensii, Pseudomonas mukorossi, and Rhizobia sinensis.

[0070] Biotin transporter activity can be generated or increased by expressing or overexpressing a gene encoding a biotin transporter protein, such as SpVHT1, in the genetically modified malic acid-producing yeast strain. Therefore, in one embodiment, the biotin transporter activity is achieved by expressing or overexpressing a gene encoding a biotin transporter protein, such as SpVHT1. SpVHT1 Genes produce or enhance this.

[0071] In one embodiment, the gene encoding the biotin transporter comprises the sequence shown in SEQ ID NO: 3 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding a polypeptide (e.g., from *Schizosaccharomyces cerevisiae*) having the biotin transporter activity. Optionally, it is incorporated into the genome of a genetically modified malate-producing yeast (e.g., *Pichia kudrica*), for example in... PkMAE1 The location of the gene locus. (The following is a description of the gene locus.) SpVHT1 Genes can be placed in appropriate promoters (e.g.) TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0072] In a further embodiment, the genetically modified malic acid-producing yeast strain also possesses or has enhanced pyruvate carboxylase activity. This pyruvate carboxylase activity can be generated or enhanced by expressing or overexpressing a gene encoding pyruvate carboxylase. The pyruvate carboxylase can be derived from fungi, particularly yeasts and filamentous fungi, preferably *Saccharomyces cerevisiae*, *Pichia pastoris*, *Aspergillus oryzae*, *Kluyveromyces martensii*, etc. In one embodiment, the gene encoding pyruvate carboxylase can be selected from *Aspergillus oryzae*. PYC Genes and Kudria zweipichia yeast PYC1 Gene.

[0073] In one embodiment, the pyruvate carboxylase comprises an amino acid sequence as shown in SEQ ID NO: 5 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having the pyruvate carboxylase activity.

[0074] In one embodiment, the pyruvate carboxylase comprises an amino acid sequence encoded by the sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having pyruvate carboxylase activity.

[0075] In one embodiment, the gene encoding the pyruvate carboxylase encodes an amino acid sequence as shown in SEQ ID NO: 5 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having the pyruvate carboxylase activity.

[0076] In one embodiment, the gene encoding the pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity.

[0077] Optionally, the gene encoding pyruvate carboxylase is incorporated into the genome of a genetically modified malate-producing yeast (e.g., Pichia kudrica), for example in... JEN2-1 Location of the gene locus. The gene encoding the pyruvate carboxylase can be placed in a suitable promoter (e.g., TDH3Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0078] In a further embodiment, the genetically modified malic acid-producing yeast strain also possesses or has enhanced phosphoenolpyruvate carboxykinase activity. This phosphoenolpyruvate carboxykinase activity can be generated or enhanced by expressing or overexpressing a gene encoding phosphoenolpyruvate carboxykinase. In one embodiment, the genetically modified malic acid-producing yeast strain expresses or overexpresses a gene encoding phosphoenolpyruvate carboxykinase, for example... PCK Gene. In one embodiment, the phosphoenolpyruvate carboxykinase is derived from Saccharomyces cerevisiae, Pichia kudrica, or Escherichia coli.

[0079] In one embodiment, the phosphoenolpyruvate carboxykinase comprises an amino acid sequence such as SEQ ID NO: 6 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with and having phosphoenolpyruvate carboxykinase activity.

[0080] In one embodiment, the phosphoenolpyruvate carboxykinase comprises an amino acid sequence encoded by the sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having phosphoenolpyruvate carboxykinase activity.

[0081] In one embodiment, the gene encoding the phosphoenolpyruvate carboxylkinase comprises the sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having pyruvate carboxylase activity.

[0082] In one embodiment, the gene encoding the phosphoenolpyruvate carboxykinase comprises the sequence shown in SEQ ID NO: 7 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxykinase activity.

[0083] Optionally, the gene encoding the phosphoenolpyruvate carboxykinase is incorporated into the genome of a genetically modified malate-producing yeast (e.g., Pichia kudrica), for example in... JEN2-2 Location of the gene locus. The gene encoding the phosphoenolpyruvate carboxykinase can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0084] In a further embodiment, the genetically modified malic acid-producing yeast strain also possesses or has enhanced activity of at least one of the following enzymes: (I) Escherichia coli phosphoenolpyruvate carboxylkinase (… PCK (i) phosphoenolpyruvate carboxylase (e.g., *Escherichia coli* phosphoenolpyruvate carboxylase, Uniprot database search number: P00864) and (iii) *Pichia kudriezvichi* (e.g., *Pichia kudriezvichi* strain CY902 with accession number CGMCC No. 20885) phosphoenolpyruvate carboxylkinase (locus number PK0402). The enzyme activity can be produced or enhanced by expressing or overexpressing the enzyme-encoding gene.

[0085] In one embodiment, the gene encoding the *E. coli* phosphoenolpyruvate carboxykinase comprises the sequence shown in SEQ ID NO: 7 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxykinase activity, optionally incorporated into the genome of a genetically modified malate-producing yeast (e.g., *Pichia kudrica*), for example in... JEN2-2 Location of the gene locus. The gene encoding the carboxykinase can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0086] In one embodiment, the phosphoenolpyruvate carboxylase is derived from plants (especially diatoms and angiosperms) and bacteria (e.g., Proteobacteria), preferably *Escherichia coli* or *Phaeodactylum tricornutum*. Phaeodactylum tricornutum ), sorghum, corn, and rice.

[0087] In one embodiment, the gene encoding the *E. coli* phosphoenolpyruvate carboxylase comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxylase activity, optionally incorporated into the genome of a genetically modified malate-producing yeast (e.g., *Pichia kudrica*), for example in... JEN2-2 The location of the gene locus. The gene encoding the carboxylase can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0088] In one embodiment, the gene encoding the phosphoenolpyruvate carboxykinase of the *Pichia kudriezvichi* yeast encodes an amino acid sequence as shown in SEQ ID NO: 6 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having phosphoenolpyruvate carboxykinase activity, optionally, is incorporated into the genome of a genetically modified malate-producing yeast (e.g., *Pichia kudriezvichi*), for example in... JEN2-2 Location of the gene locus. The gene encoding the phosphoenolpyruvate carboxykinase can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0089] The gene to be expressed or overexpressed may be integrated into a suitable location in the strain genome, provided that such integration does not negatively affect the growth, reproduction, and / or production performance of the strain. For example, it may be integrated into any one or more genomic locations encoding the following proteins: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhinophosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) dicarboxylic acid transporter, (vi) malatease (EC 1.1.1.38), and (vii) oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldolase bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

[0090] In a further embodiment, the genetically modified malic acid-producing yeast strain also possesses at least one of the following reduced-activity or inactivated: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhodophosphoside decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) dicarboxylic acid transporter, (vi) malatease (EC 1.1.1.38), and (vii) oxaloacetate decarboxylase and 3-hydroxy-3-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112). In one embodiment, the genetically modified malic acid-producing yeast strain possesses a reduced-activity or inactivated pyruvate decarboxylase. The reduced-activity or inactivated pyruvate decarboxylase can be achieved by weakening or inactivating the gene encoding the pyruvate decarboxylase in the strain. Therefore, in one embodiment, the genetically modified malic acid-producing yeast strain has a weakened or inactivated gene encoding pyruvate decarboxylase. In one embodiment, the gene encoding pyruvate decarboxylase in the genetically modified malic acid-producing yeast strain is knocked out.

[0091] In one embodiment, the pyruvate decarboxylase comprises an amino acid sequence as shown in SEQ ID NO: 10 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having pyruvate decarboxylase activity.

[0092] In one embodiment, the gene encoding the pyruvate decarboxylase encodes a protein as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having pyruvate decarboxylase activity.

[0093] In a further embodiment, the genetically modified malate-producing yeast strain also possesses a reduced-activity or inactivated NAD-dependent glycerol-3-phosphate dehydrogenase. In one embodiment, the genetically modified malate-producing yeast strain possesses a weakened or inactivated gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase. In one embodiment, the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase in the genetically modified malate-producing yeast strain (e.g., Pichia kudriazine) is knocked out.

[0094] In one embodiment, the NAD-dependent glycerol-3-phosphate dehydrogenase comprises an amino acid sequence as shown in SEQ ID NO: 11 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having NAD-dependent glycerol-3-phosphate dehydrogenase activity.

[0095] In one embodiment, the gene encoding the NAD-dependent glycerol-3-phosphate dehydrogenase encodes, for example, the protein shown in SEQ ID NO: 11 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having NAD-dependent glycerol-3-phosphate dehydrogenase activity.

[0096] In a further embodiment, the genetically modified malic acid-producing yeast strain also possesses reduced or inactivated 5'-orhodophosphoside decarboxylase, dicarboxylic acid transporter, malatease, oxaloacetate decarboxylase, and 3-hydroxy-3-methylglutarate aldolase bifunctional enzyme and / or monocarboxylic acid permease. In a further embodiment, the genetically modified malic acid-producing yeast strain contains genes encoding 5'-orhodophosphoside decarboxylase and / or genes encoding dicarboxylic acid transporters (e.g., ...). JEN2 Genes such as JEN2-1 Gene, JEN2-2 The gene encoding monocarboxylic acid permease and / or malate enzyme and / or oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme was knocked out.

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

[0098] In one embodiment, the malic enzyme comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having malic enzyme activity.

[0099] In one embodiment, the gene encoding the malicase encodes a protein as shown in SEQ ID NO: 12 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having malicase activity.

[0100] In one embodiment, the genetically modified malic acid-producing yeast strain also possesses a reduced-activity or inactivated bifunctional enzyme encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde. Specifically, the genetically modified malic acid-producing yeast strain possesses a weakened or inactivated gene encoding the bifunctional enzyme. Preferably, the gene encoding the bifunctional enzyme in the genetically modified malic acid-producing yeast strain (e.g., Pichia kudrica) is knocked out.

[0101] In one embodiment, the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme activity.

[0102] In one embodiment, the gene encoding the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme encodes a protein as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme activity.

[0103] In one embodiment, the genetically modified malic acid-producing yeast strain also possesses a reduced-activity or inactivated 5'-orhinophosphate decarboxylase. Specifically, the genetically modified malic acid-producing yeast strain possesses a weakened or inactivated gene encoding 5'-orhinophosphate decarboxylase. Preferably, the gene encoding 5'-orhinophosphate decarboxylase in the genetically modified malic acid-producing yeast strain (e.g., *Pichia kudrica*) is knocked out.

[0104] In one embodiment, the 5'-orhodophosphoside decarboxylase comprises an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having 5'-orhodophosphoside decarboxylase activity.

[0105] In one embodiment, the gene encoding the 5'-orotose decarboxylase encodes a protein as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having 5'-orotose decarboxylase activity.

[0106] In one embodiment, the genetically modified malic acid-producing yeast strain also possesses a reduced-activity or inactivated monocarboxylic acid permease. Specifically, the genetically modified malic acid-producing yeast strain possesses a weakened or inactivated gene encoding the monocarboxylic acid permease. Preferably, the gene encoding the monocarboxylic acid permease in the genetically modified malic acid-producing yeast strain (e.g., *Pichia kudrica*) is knocked out.

[0107] In one embodiment, the monocarboxylic acid permease comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same and having monocarboxylic acid permease activity.

[0108] In one embodiment, the gene encoding the monocarboxylic acid permease encodes, for example, the protein shown in SEQ ID NO: 16 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having monocarboxylic acid permease activity.

[0109] In one embodiment, the genetically modified malic acid-producing yeast strain also possesses a reduced-activity or inactivated dicarboxylic acid transporter protein. Specifically, the genetically modified malic acid-producing yeast strain possesses a weakened or inactivated gene encoding the dicarboxylic acid transporter protein. Preferably, the gene encoding the dicarboxylic acid transporter protein in the genetically modified malic acid-producing yeast strain (e.g., *Pichia kudrica*) is, for example... JEN2-1FORJEN2-2 The gene was knocked out.

[0110] In one embodiment, the dicarboxylic acid transporter comprises an amino acid sequence as shown in SEQ ID NO: 14 or 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having dicarboxylic acid transporter activity.

[0111] In one embodiment, the gene encoding the dicarboxylic acid transporter encodes a protein as shown in SEQ ID NO: 14 or 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having dicarboxylic acid transporter activity.

[0112] In one embodiment, the present invention provides a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) having an overexpressed gene encoding a malate transporter protein, for example... SpMAE1 Genes and overexpressed genes encoding NADPH-dependent malate dehydrogenase MDH Optionally, genes endogenously encoding 5'-orhodophosphoglycerate decarboxylase and / or genes endogenously encoding monocarboxylic acid permease and / or genes endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes are knocked out. Preferably, the genes are... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

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

[0114] In one embodiment, the present invention provides a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding an NADPH-dependent malate dehydrogenase. MDH Genes and genes encoding malate transport proteins, for example SpMAE1 The gene, as well as the gene endogenously encoding malic acid oxidase, is knocked out; optionally, the gene endogenously encoding orotidine phosphate decarboxylase and / or the gene endogenously encoding monocarboxylic acid permease and / or the gene endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme is knocked out. Preferably, the gene... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0115] In one embodiment, the present invention provides a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as strain CY902) that overexpresses an enzyme encoding an NADPH-dependent malate dehydrogenase. MDH Genes and genes encoding malate transport proteins, for example SpMAE1 The genes, including those endogenously encoding pyruvate decarboxylase, NAD-dependent glycerol-3-phosphate dehydrogenase, and malate enzyme, are knocked out. Optionally, the genes endogenously encoding orotidine phosphate decarboxylase and / or monocarboxylic acid permease and / or oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldolase bifunctional enzymes are knocked out. Preferably, the genes are... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0116] In one embodiment, the present invention provides a genetically modified malic acid-producing yeast strain (e.g., Pichia kudricazvichia) that overexpresses the following gene:

[0117] Encoding NADPH-dependent malate dehydrogenase MDH Gene,

[0118] Genes encoding malate transport proteins, for example SpMAE1 Genes, and

[0119] At least one of the following genes: a gene encoding a biotin transporter, for example... SpVHT1Genes encoding *Escherichia coli* phosphoenolpyruvate carboxylkinase, genes encoding phosphoenolpyruvate carboxylase (preferably *Escherichia coli* phosphoenolpyruvate carboxylase), genes encoding *Pichia pastoris* phosphoenolpyruvate carboxylkinase, and genes encoding pyruvate carboxylase.

[0120] Optionally, the genes endogenously encoding pyruvate decarboxylase and endogenously encoding NAD-dependent glycerol-3-phosphate dehydrogenase in the strain are knocked out.

[0121] Optionally, genes endogenously encoding 5'-orhinophosphate decarboxylase and / or genes endogenously encoding monocarboxylic acid permease and / or genes endogenously encoding dicarboxylic acid transporters, for example... JEN2-1 or JEN2-2 The gene and / or the gene encoding the bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation and / or the gene encoding malate enzyme were knocked out.

[0122] In one embodiment, the present invention provides a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*) that overexpresses an enzyme encoding an NADPH-dependent malate dehydrogenase. MDH Genes, genes encoding malate transport proteins, for example SpMAE1 Genes and genes encoding biotin transport proteins, for example SpVHT1 Optionally, the genes endogenously encoding 5'-orhodophosphoglycerate decarboxylase and / or endogenously encoding monocarboxylic acid permease and / or endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme and / or endogenously encoding malate enzyme are knocked out. Preferably, the genes are... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or the gene encoding the biotin transporter contains the sequence shown in SEQ ID NO: 3 or its degenerate sequence.

[0123] In one embodiment, the present invention provides a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as CY902) that overexpresses an enzyme encoding an NADPH-dependent malate dehydrogenase. MDH Genes, genes encoding malate transport proteins, for example SpMAE1 Genes and genes encoding biotin transport proteins, for example SpVHT1 Genes, and endogenous genes encoding malic acid enzymes, such as PkMAE1 The gene is knocked out, optionally, the gene endogenously encoding 5'-orhinophosphate decarboxylase, for example... URA3 Genes and / or endogenous genes encoding monocarboxylic acid permeases, such as MCH4 Genes and / or endogenous genes encoding bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde, such as Pk2365 The gene is knocked out. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or the gene encoding the biotin transporter contains the sequence shown in SEQ ID NO: 3 or its degenerate sequence.

[0124] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain (e.g., CY902 strain) that overexpresses an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding malate transport proteins, and overexpressing at least one of the following genes:

[0125] The genes encoding biotin transporter, the gene encoding *Escherichia coli* phosphoenolpyruvate carboxylkinase, and the gene encoding phosphoenolpyruvate carboxylase are preferably encoded by the gene encoding *Escherichia coli* phosphoenolpyruvate carboxylase, the gene encoding *Pichia pastoris* phosphoenolpyruvate carboxylkinase, and the gene encoding pyruvate carboxylase.

[0126] Optional, endogenous PDC1 Genes and endogenous GPD1 The gene was knocked out.

[0127] Optionally, the endogenous in the strain URA3 Genes and / or endogenous MCH4 Genes and / or endogenous JEN2-1 and / or JEN2-2 Genes and / or endogenous PkMAE The gene was knocked out.

[0128] Preferably, the MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the gene encoding malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence; the gene encoding biotin transporter contains the sequence shown in SEQ ID NO: 3 or its degenerate sequence; the gene encoding *Escherichia coli* phosphoenolpyruvate carboxylkinase contains the sequence shown in SEQ ID NO: 7 or its degenerate sequence; the gene encoding phosphoenolpyruvate carboxylase contains the sequence shown in SEQ ID NO: 8 or its degenerate sequence; the gene encoding *Pichia pastoris* phosphoenolpyruvate carboxylkinase encodes the sequence shown in SEQ ID NO: 6; or the gene encoding pyruvate carboxylase contains the sequence shown in SEQ ID NO: 4 or its degenerate sequence.

[0129] In one embodiment, the present invention provides a genetically modified *Pichia gondii* strain (e.g., CY902 strain) that overexpresses: encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding malate transporter; genes encoding *Escherichia coli* phosphoenolpyruvate carboxylase; genes encoding biotin transporter; and genes encoding *Aspergillus oryzae* pyruvate carboxylase, as well as endogenous genes encoding pyruvate decarboxylase and / or endogenous genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase, are knocked out. Optionally, endogenous genes in the strains are also knocked out. URA3 Genes and / or endogenous MCH4 Genes and / or endogenous JEN2-1 and / or JEN2-2 Genes and / or endogenous PkMAE1 Genes and / or endogenous Pk2365 The gene was knocked out.

[0130] In one aspect, the present invention provides a method for producing a genetically modified malate-producing yeast strain, comprising conferring or enhancing malate transporter activity, such as SpMAE1 protein activity, in the strain, and conferring or enhancing NADPH-dependent malate dehydrogenase (EC 1.1.1.82) activity in the strain, optionally further comprising conferring or enhancing at least one of the following activities: (i) pyruvate carboxylase (EC 6.4.1.1) activity, (ii) phosphoenolpyruvate carboxylkinase (EC 4.1.1.49) activity, (iii) phosphoenolpyruvate carboxylase activity, preferably Escherichia coli phosphoenolpyruvate carboxylase activity, and (iv) biotin transporter activity.

[0131] As used in this article, “conferring activity” means producing in a genetically modified malic acid-producing yeast strain an activity that was not present in the initial strain before the genetic modification.

[0132] As used herein, “enhancing…activity” means increasing activity, for example, by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or higher.

[0133] Various methods for conferring or enhancing the desired activity of proteins are known in the art, including, but not limited to, expressing or overexpressing protein-coding genes and mutations or other modifications that increase protein activity.

[0134] As used herein, “overexpression” means an increase in gene expression level relative to the level before genetic manipulation, for example, an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, or higher. Methods for overexpressing genes are well known in the art, including, but not limited to, using strong promoters, increasing gene copy number, enhancers, etc. Increasing gene copy number can be achieved, for example, but not limited to, by introducing one or more copies of a foreign or endogenous gene, for example, through an expression vector or integration into the genome.

[0135] As used in this article, "exogenous gene" refers to a gene that comes from another cell or organism, such as a gene from the same species or a different species.

[0136] As used in this article, "endogenous genes" refers to the genes of a cell or organism itself.

[0137] The promoter can be selected from any suitable promoter known in the art, including but not limited to those encoding fructose-1,6-bisphosphate aldolase. FBA1 The promoter of the gene, encoding glyceraldehyde-3-phosphate dehydrogenase TDH3 The promoter of the gene, encoding pyruvate decarboxylase PDC1 Gene promoter, encoding alcohol dehydrogenase ADH1 Gene promoter, encoding 3-phosphoglycerate kinase PGK1 Gene promoters, encoding transcription elongation factors TEF1 Gene promoter, encoding phosphoglycerate mutase GPM1 Gene promoter, encoding triose phosphate isomerase TPI1 Gene promoters and those encoding enolases ENO1 Gene promoters.

[0138] In one embodiment, the NADPH-dependent malate dehydrogenase is derived from plants, preferably C4 plants, more preferably plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, and Amaranthaceae families, or from Euglena and Thermobacterium genera, more preferably sorghum, corn, sugarcane, peas, chickpeas, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria.

[0139] In one embodiment, the enzyme is expressed or overexpressed in the genetically modified malate-producing yeast strain, encoding an NADPH-dependent malate dehydrogenase. MDH Genes are used to produce or increase NADPH-dependent malate dehydrogenase activity. Preferably, the gene encoding the NADPH-dependent malate dehydrogenase... MDHThe genes are preferably derived from plants, C4 plants are preferred, and plants from the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae and Amaranthaceae families are even more preferred, or from Euglena and Thermomycium genera, and sorghum, corn, sugarcane, pea, chickpea, spinach, Euglena or thermoautotrophic methanotherapeutic bacteria are even more preferred, and sorghum is even more preferred.

[0140] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity.

[0141] In one implementation, the enzyme encoding NADPH-dependent malate dehydrogenase... MDH Genes are incorporated into the genome of genetically modified malic acid-producing yeasts (e.g., Pichia kudrica), for example in... Pk2365 Location of the locus. In one embodiment, the gene encoding NADPH-dependent malate dehydrogenase... MDH Genes are incorporated into the genome of yeast (e.g., Pichia kudrica) via homologous recombination, for example in... MDH2 Location of the locus. The gene encoding NADPH-dependent malate dehydrogenase... MDH Genes can be placed in appropriate promoters (e.g.) FBA1 Gene promoters (e.g., shown in SEQ ID NO: 17) and / or terminators (e.g.) INO1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 18).

[0142] In one embodiment, the malate transporter is selected from SpMAE1 protein, C4T318 protein, and AsDct protein. In one embodiment, the malate transporter is expressed or overexpressed in the genetically modified malate-producing yeast strain, using a gene encoding a malate transporter, such as SpMAE1 protein (e.g., ...). SpMAE1 Genes can be used to confer or enhance the activity of malic acid transport proteins.

[0143] In one implementation, the SpMAE1The gene comprises the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and encoding an amino acid sequence having malic acid transporter activity, optionally (e.g., via homologous recombination) incorporated into the genome of a genetically modified malic acid-producing yeast strain (e.g., *Pichia kudrica*), for example in... MCH4 The location of the gene locus. (The following is a description of the gene locus.) SpMAE1 Genes can be placed in appropriate promoters (e.g.) TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0144] In one embodiment, the method further includes conferring or enhancing biotin transporter activity in the strain. Biotin transporters are generally derived from fungi and bacteria, particularly yeasts, Pseudomonas, and Rhizobia, with further preferred species being *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, *Candida glabrata*, *Kluyveromyces martensii*, *Pseudomonas hibiscus*, and *Rhizobia sinensis*.

[0145] In one embodiment, by expressing or overexpressing a gene encoding a biotin transporter protein such as SpVHT1 in the genetically modified malic acid-producing yeast strain (e.g., SpVHT1 Genes are used to confer or enhance biotin transporter activity. Therefore, in one embodiment, the biotin transporter activity is achieved by expressing or overexpressing... SpVHT1 Genes produce or enhance this.

[0146] In one embodiment, the gene encoding the biotin transporter protein comprises the sequence shown in SEQ ID NO: 3 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding a polypeptide having the biotin transporter protein activity, optionally incorporated into the genome of a genetically modified malate-producing yeast (e.g., Pichia kudrica), for example in... PkMAE1 The location of the gene locus. The gene can be placed in a suitable promoter (e.g., ...). TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0147] In a further embodiment, the method further includes conferring or enhancing pyruvate carboxylase activity in the yeast strain. Preferably, conferring or enhancing pyruvate carboxylase activity is done by expressing or overexpressing a gene encoding pyruvate carboxylase in the strain, for example... PYC Genetic implementation. The pyruvate carboxylase can be derived from fungi, particularly yeasts and filamentous fungi, preferably *Saccharomyces cerevisiae*, *Pichia pastoris*, *Aspergillus oryzae*, and *Kluyveromyces martensii*. Kluyveromyces marxianus In one embodiment, the gene encoding the pyruvate carboxylase may be selected from Aspergillus oryzae. PYC Genes and Kudria zweipichia yeast PYC1 Gene.

[0148] In one embodiment, the pyruvate carboxylase comprises an amino acid sequence as shown in SEQ ID NO: 5 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having the pyruvate carboxylase activity.

[0149] In one embodiment, the gene encoding the pyruvate carboxylase comprises the sequence shown in SEQ ID NO: 4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity.

[0150] Optionally, the gene encoding pyruvate carboxylase is incorporated into the genome of a genetically modified malate-producing yeast (e.g., Pichia kudrica), for example in... JEN2-1 Location of the gene locus. Genes encoding pyruvate carboxylase can be placed in suitable promoters (e.g., TDH3 Gene promoters (e.g., shown in SEQ ID NO: 19) and / or terminators (e.g.) GAL2 Under the control of gene terminators (e.g., shown in SEQ ID NO: 20).

[0151] In a further embodiment, the method further includes conferring or enhancing phosphoenolpyruvate carboxykinase activity. In one embodiment, the phosphoenolpyruvate carboxykinase is derived from *Saccharomyces cerevisiae*, *Pichia pastoris*, or *Escherichia coli*. In one embodiment, the method includes expressing or overexpressing a gene encoding phosphoenolpyruvate carboxykinase. In a further embodiment, the method includes overexpressing a gene encoding phosphoenolpyruvate carboxykinase.

[0152] In one embodiment, the phosphoenolpyruvate carboxykinase comprises an amino acid sequence such as SEQ ID NO: 6 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with and having phosphoenolpyruvate carboxykinase activity.

[0153] In one embodiment, the gene encoding the phosphoenolpyruvate carboxykinase comprises the sequence shown in SEQ ID NO: 7 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxykinase activity.

[0154] Optionally, the gene encoding the phosphoenolpyruvate carboxykinase is incorporated into the genome of a genetically modified malate-producing yeast (e.g., Pichia kudrica), for example in... JEN2-2 Location of the gene locus. The gene encoding the phosphoenolpyruvate carboxykinase can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0155] In a further embodiment, the method further includes conferring or enhancing the activity of one or more of the following enzymes: (I) *Escherichia coli* phosphoenolpyruvate carboxykinase (… PCK (i) phosphoenolpyruvate carboxylase (e.g., *Escherichia coli* phosphoenolpyruvate carboxylase, Uniprot database search number: P22259); (ii) phosphoenolpyruvate carboxylase (e.g., *Escherichia coli* phosphoenolpyruvate carboxylase, Uniprot database search number: P00864); and (iii) *Pichia kudriezvichi* (e.g., *Pichia kudriezvichi* strain CY902 with accession number CGMCC No. 20885) phosphoenolpyruvate carboxylkinase (locus number PK0402). In one embodiment, the method comprises expressing or overexpressing the gene encoding the enzyme to confer or enhance enzyme activity.

[0156] In one embodiment, the gene encoding the *E. coli* phosphoenolpyruvate carboxykinase comprises the sequence shown in SEQ ID NO: 7 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having the activity of the phosphoenolpyruvate carboxykinase, optionally incorporated into the genome of a genetically modified malate-producing yeast (e.g., *Pichia kudrica*), for example in... JEN2-2 The location of the gene locus. The gene encoding the enzyme can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0157] The phosphoenolpyruvate carboxylase can be derived from plants (especially diatoms and angiosperms) and bacteria (e.g., Proteobacteria), preferably *Escherichia coli* and *Phaeodactylum tricornutum*. Phaeodactylum tricornutum ), sorghum, corn, and rice.

[0158] In one embodiment, the gene encoding the *E. coli* phosphoenolpyruvate carboxylase comprises the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxylase activity, optionally incorporated into the genome of a genetically modified malate-producing yeast (e.g., *Pichia kudrica*), for example in... JEN2-2 The location of the gene locus. The gene encoding the carboxylase can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0159] In one embodiment, the gene encoding the phosphoenolpyruvate carboxykinase of the *Pichia kudriezvichi* yeast encodes an amino acid sequence as shown in SEQ ID NO: 6 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having phosphoenolpyruvate carboxykinase activity, optionally, is incorporated into the genome of a genetically modified malate-producing yeast (e.g., *Pichia kudriezvichi*), for example in... JEN2-2 The location of the gene locus. The gene encoding the enzyme can be placed in a suitable promoter (e.g., ENO1 Gene promoters (e.g., shown in SEQ ID NO: 21) and / or terminators (e.g.) SED1 Under the control of gene terminators (e.g., shown in SEQ ID NO: 22).

[0160] The method may integrate genes to be expressed or overexpressed in the strain into appropriate locations in the strain genome, provided that such integration does not negatively affect the growth, reproduction, and / or production performance of the strain. For example, the method includes integrating one or more of the above-described genes into any one or more genomic locations encoding proteins such as: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhinophosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) dicarboxylic acid transporter, (vi) malatease (EC 1.1.1.38), and (vii) oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

[0161] In a further embodiment, the method further includes weakening or inactivating at least one of the following in the strain: (i) pyruvate decarboxylase (EC 4.1.1.1), (ii) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), (iii) 5'-orhinophosphate decarboxylase (EC 4.1.1.23), (iv) monocarboxylic acid permease, (v) dicarboxylic acid transporter, (vi) malatease (EC 1.1.1.38), and (vii) oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldolase bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

[0162] In one embodiment, the method further includes reducing or inactivating pyruvate decarboxylase activity in the yeast strain.

[0163] As used herein, reducing or inactivating the activity of a protein, such as an enzyme, means reducing the activity of the protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even making it undetectable. Various methods of reducing or inactivating protein activity are known in the art, including, for example, inhibiting gene expression such as knockdown (e.g., using small interfering RNA), using weak promoters (when the gene is a polypeptide-coding gene); gene knockout, deletion of part or all of a gene or polypeptide sequence; mutation of certain sites in a gene or polypeptide, such as coding sequences or active domains, to reduce gene expression or regulate activity or the activity of the expression product; and the use of antagonists or inhibitors (e.g., including but not limited to antibodies, interfering RNA, etc.).

[0164] As used herein, a weakened or inactivated gene refers to a gene whose expression level (when it is a protein-coding gene) or regulatory function (when it is a regulatory element) is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even rendered undetectable. Various methods for weakening or inactivating genes are known in the art, including, for example, suppressing gene expression such as knockdown (e.g., using small interfering RNA), using weak promoters (when the gene is a polypeptide-coding gene), gene knockout, deletion of part or all of a gene sequence, and mutation of certain sites in a gene, such as the coding sequence, to reduce gene expression or regulatory activity or the activity of the expression product.

[0165] In one implementation, reducing or inactivating the gene encoding pyruvate decarboxylase activity includes weakening or inactivating the gene encoding pyruvate decarboxylase.

[0166] In one embodiment, weakening or inactivating the gene encoding pyruvate decarboxylase includes knocking out the gene encoding pyruvate decarboxylase (e.g., encoding the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having pyruvate decarboxylase activity).

[0167] In a further embodiment, the method further includes reducing or inactivating the NAD-dependent glycerol-3-phosphate dehydrogenase activity in the yeast strain.

[0168] In one embodiment, reducing or inactivating NAD-dependent glycerol-3-phosphate dehydrogenase activity includes weakening or inactivating the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase. In one embodiment, weakening or inactivating the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase includes knocking out the gene encoding NAD-dependent glycerol-3-phosphate dehydrogenase (e.g., encoding the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having NAD-dependent glycerol-3-phosphate dehydrogenase activity).

[0169] In a further embodiment, the method further includes reducing or inactivating the activity of malic acid oxidase and / or oxaloacetate decarboxylase and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme and / or 5'-orhodophosphate decarboxylase and / or monocarboxylic acid permease and / or dicarboxylic acid transporter in the yeast strain. In one embodiment, the method includes weakening or inactivating the gene encoding 5'-orhodophosphate decarboxylase and / or the gene encoding monocarboxylic acid permease in the yeast strain.

[0170] In one embodiment, the method includes weakening or inactivating genes encoding malates, bifunctional enzymes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation, 5'-phospho-orhodoside decarboxylase, and dicarboxyltransferase (e.g., phospho-orhodoside decarboxylation) in the yeast strain. JEN2 Genes such as JEN2-1 Gene, JEN2-2 One or more of the genes encoding monocarboxylic acid permeases.

[0171] In one embodiment, the gene encoding malicase (e.g., encoding the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having malicase activity) is knocked out, for example by homologous recombination.

[0172] In one embodiment, the gene encoding a bifunctional enzyme of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde (e.g., encoding the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having bifunctional enzyme activity of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde) is knocked out, for example by homologous recombination.

[0173] In one embodiment, the gene encoding 5'-orhodophosphoside decarboxylase (e.g., encoding the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having 5'-orhodophosphoside decarboxylase activity) is knocked out, for example by homologous recombination.

[0174] In one implementation, the gene encoding the dicarboxylic acid transporter... JEN2-1 Genes (e.g., those encoding the amino acid sequence shown in SEQ ID NO: 14 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same amino acid sequence and having dicarboxylic acid transporter activity) are knocked out, for example by homologous recombination.

[0175] In one implementation, the gene encoding the dicarboxylic acid transporter... JEN2-2 Genes (e.g., those encoding the amino acid sequence shown in SEQ ID NO: 15 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same amino acid sequence and having dicarboxylic acid transporter activity) are knocked out, for example by homologous recombination.

[0176] In one embodiment, the gene encoding a monocarboxylic acid permease (e.g., an amino acid sequence encoding the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same and having monocarboxylic acid permease activity) is knocked out, for example by homologous recombination.

[0177] In one embodiment, the present invention provides a method for producing a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing an enzyme encoding NADPH-dependent malate dehydrogenase in the genetically modified malate-producing yeast strain. MDH Genes and genes encoding malate transport proteins, for example SpMAE1 The gene optionally includes the knockout of an endogenous gene encoding orotidine phosphate decarboxylase and / or an endogenous gene encoding a monocarboxylic acid permease and / or an endogenous gene encoding a bifunctional enzyme of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation. Preferably, the gene... MDHThe gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0178] In one embodiment, the present invention provides a method for producing a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing an enzyme encoding NADPH-dependent malate dehydrogenase in the genetically modified malate-producing yeast strain. MDH Genes and genes encoding malate transport proteins, for example SpMAE1 Genes, including the knockout of endogenous genes encoding pyruvate decarboxylase and endogenous genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase, optionally knocking out endogenous genes encoding orotidine phosphate decarboxylase and / or endogenous genes encoding monocarboxylic acid permease and / or endogenous genes encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0179] In one embodiment, the present invention provides a method for producing a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing an enzyme encoding NADPH-dependent malate dehydrogenase in the genetically modified malate-producing yeast strain. MDH Genes and genes encoding malate transport proteins, for example SpMAE1 Genes, and knockout of the gene encoding endogenous malate esterase, optionally, knockout of the gene encoding endogenous 5'-phosphoorhinoside decarboxylase and / or the gene encoding endogenous monocarboxylic acid permease and / or the gene encoding the bifunctional enzyme oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation. Preferably, the gene... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0180] In one embodiment, the present invention provides a method for producing a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing an enzyme encoding NADPH-dependent malate dehydrogenase in the genetically modified malate-producing yeast strain. MDH Genes and genes encoding malate transport proteins, for example SpMAE1Genes, including the knockout of genes endogenously encoding pyruvate decarboxylase, endogenously encoding NAD-dependent glycerol-3-phosphate dehydrogenase, and endogenously encoding malate enzymes; optionally, the knockout of genes endogenously encoding orotidine phosphate decarboxylase and / or endogenously encoding monocarboxylic acid permease and / or endogenously encoding oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzymes. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0181] In one embodiment, the present invention provides a method for generating a genetically modified malic acid-producing yeast strain (e.g., Pichia kudrica, such as CY902), comprising overexpressing the following gene in the genetically modified malic acid-producing yeast strain:

[0182] Encoding NADPH-dependent malate dehydrogenase MDH Gene,

[0183] Genes encoding malate transport proteins, for example SpMAE1 Genes, and

[0184] At least one of the following genes: a gene encoding a biotin transporter, a gene encoding *Escherichia coli* phosphoenolpyruvate carboxylkinase, a gene encoding phosphoenolpyruvate carboxylase (preferably *Escherichia coli* phosphoenolpyruvate carboxylase), a gene encoding *Pichia pastoris* phosphoenolpyruvate carboxylkinase, and a gene encoding pyruvate carboxylase; and

[0185] Optionally, knock out the genes encoding endogenous pyruvate decarboxylase and endogenous NAD-dependent glycerol-3-phosphate dehydrogenase.

[0186] Optionally, knock out the gene encoding endogenous 5'-orhodophosphoside decarboxylase and / or the gene encoding endogenous monocarboxylic acid permease and / or the gene encoding endogenous dicarboxylic acid transporter, for example... JEN2-1 or JEN2-2 Genes and / or endogenous genes encoding bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation and / or endogenous genes encoding malate enzymes.

[0187] In one embodiment, the present invention provides a method for producing a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing an enzyme encoding NADPH-dependent malate dehydrogenase in the genetically modified malate-producing yeast strain. MDH Genes, genes encoding malate transport proteins, for example SpMAE1Genes and genes encoding biotin transport proteins, for example SpVHT1 Optionally, the gene may be knocked out by knocking out the gene encoding endogenous 5'-orhodophosphoside decarboxylase and / or the gene encoding endogenous monocarboxylic acid permease and / or the gene encoding endogenous oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde bifunctional enzyme and / or the gene encoding endogenous malate enzyme. Preferably, the gene may be knocked out by knocking out the gene encoding endogenous 5'-orhodophosphoside decarboxylase and / or the gene encoding endogenous malate enzyme. MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or the gene encoding the biotin transporter contains the sequence shown in SEQ ID NO: 3 or its degenerate sequence.

[0188] In one embodiment, the present invention provides a method for producing a genetically modified malate-producing yeast strain (e.g., *Pichia kudrica*, such as CY902), comprising overexpressing an enzyme encoding NADPH-dependent malate dehydrogenase in the genetically modified malate-producing yeast strain. MDH Genes, genes encoding malate transport proteins, for example SpMAE1 Genes and genes encoding biotin transport proteins, for example SpVHT1 Genes, and knockout of the gene encoding endogenous malate esterase, optionally, knockout of the gene encoding endogenous 5'-phosphoorhinoside decarboxylase and / or the gene encoding endogenous monocarboxylic acid permease and / or the gene encoding the bifunctional enzyme oxaloacetate decarboxylation and 3-hydroxy-3-methylglutaraldehyde condensation. Preferably, the gene... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or the gene encoding the biotin transporter contains the sequence shown in SEQ ID NO: 3 or its degenerate sequence.

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

[0190] In one embodiment, the present invention provides a method for producing a genetically modified *Pichia gondii* strain (e.g., CY902 strain), comprising overexpressing in said strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding malate transport proteins, and optionally, endogenous genes knocked out in the strains. URA3 Genes and / or endogenous MCH4 Genes, wherein the preferred ones are described MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0191] In one embodiment, the present invention provides a method for producing a genetically modified *Pichia gondii* strain (e.g., CY902 strain), comprising overexpressing in said strain an enzyme encoding NADPH-dependent malate dehydrogenase. MDH Genes encoding malate transport proteins, and the knockout of endogenous malates in the strain. PDC1 Genes and endogenous GPD1 Genes, optional knockout of endogenous genes URA3 Genes and / or endogenous MCH4 Genes and / or endogenous JEN2-1 and / or JEN2-2 Genes and / or endogenous PkMAE Gene. Preferably, the... MDH The gene contains the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or the gene encoding the malate transporter contains the sequence shown in SEQ ID NO: 2 or its degenerate sequence.

[0192] In one embodiment, the present invention provides a method for producing a genetically modified *Pichia gondii* strain (e.g., CY902 strain), comprising overexpressing the following in said strain:

[0193] Encoding NADPH-dependent malate dehydrogenase MDH Gene;

[0194] Gene encoding malate transporter;

[0195] The gene encoding phosphoenolpyruvate carboxylase in Escherichia coli;

[0196] The gene encoding the biotin transporter protein; and

[0197] The gene encoding Aspergillus oryzae pyruvate carboxylase, and

[0198] Knock out genes encoding endogenous pyruvate decarboxylase and / or genes encoding endogenous NAD-dependent glycerol-3-phosphate dehydrogenase.

[0199] Optionally, the endogenous in the strain URA3 Genes and / or endogenous MCH4 Genes and / or endogenous JEN2-1 and / or JEN2-2 Genes and / or endogenous PkMAE1 Genes and / or endogenous Pk2365 The gene was knocked out.

[0200] In one aspect, the present invention provides a method for producing L-malic acid, comprising culturing the genetically modified malic acid-producing yeast strain of the present invention or the genetically modified malic acid-producing yeast strain prepared by the method for producing the genetically modified malic acid-producing yeast strain of the present invention under conditions suitable for fermentation to produce L-malic acid, optionally including isolated and purified L-malic acid.

[0201] Conditions known in the art for fermenting malic acid-producing yeast strains for L-malic acid production include, for example, but not limited to, pH, temperature, culture medium composition, fermentation time, etc.

[0202] Culture media known in the art for the fermentation of malic acid-producing yeast strains to produce L-malic acid include, for example, but not limited to, inorganic salt media (about 5-12% w / v glucose, optionally containing about 30 g / L CaCO3).

[0203] Temperatures known in the art for the fermentation of malic acid-producing yeast strains to produce L-malic acid include, for example, about 25-37°C, such as about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, and about 37°C. In one embodiment, the malic acid-producing yeast strain of the present invention is fermented at 30°C to produce L-malic acid.

[0204] The malic acid-producing yeast strain described in this invention can ferment at suitable pH values ​​known in the art, such as pH values ​​less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, and less than about 1.0 (e.g., pH values ​​of about 1.0-7.0, 1.0-6.0, 1.0-5.5, 1.0-5.0, 1.0-4.5, 1.0-). 4.0, 1.0-3.5, 1.0-3.0, 2.0-7.0, 2.0-6.0, 2.0-5.5, 2.0-5.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 3.0-7.0, 3.0-6.0, 3.0-5.5, 3.0-5.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 4.0-7.0, 4.0-6.0, 4.0-5.5, 4.0-5.0, 4.0-4.5). In one embodiment, the malic acid-producing yeast strain of the present invention is fermented at pH < about 3.5 to produce L-malic acid.

[0205] To produce L-malic acid, the malic acid-producing yeast strain of the present invention can ferment for a suitable time, such as about 12-96 hours, including about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, and about 96 hours. In one embodiment, to produce L-malic acid, the malic acid-producing yeast strain of the present invention ferments for about 24-72 hours, for example, about 30 hours.

[0206] The malic acid-producing yeast strain described in this invention can be fermented under shaking conditions (e.g., about 100-300 rpm, such as about 150, about 200, or about 250 rpm) to produce L-malic acid.

[0207] The L-malic acid content in the fermentation broth can be determined by suitable methods known in the art, such as HPLC.

[0208] In one embodiment, the present invention provides a method for producing L-malic acid, comprising operating at acidic pH values ​​(less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, less than about 1.0, for example, pH values ​​of about 1.0-7.0, 1.0-6.0, 1.0-5.5, 1.0-5.0, 1.0-4.5, 1.0-4.0, 1.0-3.5). Genetically modified malic acid-producing yeast strains were cultured in an inorganic salt medium (such as one containing approximately 5% w / v glucose and approximately 30 g / L CaCO3, or approximately 12% w / v glucose) under the following conditions: 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, 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, 1.0-3.0, 2.0-7.0, 2.0-6.0, 2.0-5.5, 2.0-5.0, 2.0-4.5 ...4.5, 2.0-4.5, 2.0-4.5, 2.0-4.5, 2.0-4.5, 2.0-4.5, 2.0-5.5,

[0209] In one embodiment, the method for producing L-malic acid according to the present invention does not require the addition of a neutralizing agent.

[0210] In one aspect, the present invention provides the application of the genetically modified malic acid-producing yeast strain described in this invention, or the genetically modified malic acid-producing yeast strain prepared according to the method for producing the genetically modified malic acid-producing yeast strain described in this invention, in the production of L-malic acid, particularly at acidic pH values ​​(less than about 7.0, less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2.0, less than about 1.5, less than about 1.0, for example, pH values ​​about 1.0-7.0, 1.0-6.0, 1.0-5.5, 1... Applications in the production of L-malic acid under conditions of 0-5.0, 1.0-4.5, 1.0-4.0, 1.0-3.5, 1.0-3.0, 2.0-7.0, 2.0-6.0, 2.0-5.5, 2.0-5.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 3.0-7.0, 3.0-6.0, 3.0-5.5, 3.0-5.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 4.0-7.0, 4.0-6.0, 4.0-5.5, 4.0-5.0, 4.0-4.5) and / or without the addition of a neutralizing agent.

[0211] As used herein, "optional" or "optionally" means that the event or situation subsequently described occurs or does not occur, including both the occurrence and non-occurrence of said event or situation. For example, an optional step means that the step is present or absent.

[0212] As used herein, the term "about" refers to a range of values ​​that includes a specific value and that would be reasonably regarded by those skilled in the art as similar to that specific value. In some embodiments, the term "about" refers to within the standard error of measurements generally accepted in the art. In some embodiments, "about" refers to + / - 10% of a specific value.

[0213] The scope disclosed herein should be considered to specifically disclose all possible subranges and the individual values ​​within those ranges. For example, a description of the range from 1 to 6 should be considered as explicitly disclosing subranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0214] The present invention will be further illustrated below through the following non-limiting embodiments. As will be known to those skilled in the art, many modifications can be made to the present invention without departing from the spirit of the invention, and such modifications also fall within the scope of the present invention.

[0215] Unless otherwise specified, the experimental methods described below are standard methods, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.

[0216] Example 1: Overexpression SpMAE1 Genes and NADPH-dependent MDH Gene

[0217] The gene encoding orotidine 5'-phosphate decarboxylase was isolated by homologous recombination. URA3 Knockout of (CY902 locus number PK2075, encoding the amino acid sequence shown in SEQ ID NO: 13) yielded ΔURA3 Mutants (Xi, Y.; Zhan, T.; Xu, H.; Chen, J.; Bi, C.; Fan, F.; Zhang, X., Characterization of JEN family carboxylate transporters from the acid-tolerant yeast Pichia kudriavzeviiand their applications in succinic acid production. Microb Biotechnol 2021. 0(0), 1–18. doi:10.1111 / 1751-7915.13781). We then constructed the CRISPR / Cas9 plasmid pWSPK-Cas9 (GenBank accession number: MW296878.1) suitable for this bacterium, which contains... URA3 Screening markers can be used to obtain positive transformants through auxotrophic screening.

[0218] With CY902 ΔURA3 As the starting strain, the SpMAE1 transporter protein from *Schizosaccharomyces cerevisiae* (Uniprot database search number: P50537) and the malate dehydrogenase SbMDH from sorghum (Uniprot database search number: P17606) were overexpressed successively. SpMAE1 Gene (SEQ ID NO: 2) and SbMDH The gene (SEQ ID NO: 1) was synthesized by Nanjing Genscript Biotech Co., Ltd., and optimized based on the codon preference of CY902. SpMAE1 Gene integration into the CY902 genome MCH4 The gene locus, and the promoter and terminator used, are the CY902's own glyceraldehyde-3-phosphate dehydrogenase 3 gene. TDH3 promoter and galactose permease gene GAL2 The terminator. SbMDH The locus of gene integration in the CY902 genome is numbered as follows: Pk2365 The site used, along with the promoter and terminator, are the CY902 gene itself, specifically the fructose-1,6-bisphosphate aldolase gene. FBA1 promoter and inositol-3-phosphate synthase gene INO1 The terminator. The specific construction method is as follows:

[0219] 1. Constructing donor DNA fragments for homologous recombination.

[0220] Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_MCH4_F and 1_UP_MCH4_R1 (see Table 1). MCH4Upstream homologous arm fragment 1 (fragment 1); amplification system and procedure were performed according to the TAKARA PrimeSTAR® HS DNA polymerase product instructions; primer 2_P was used. TDH3 _F and 2_P TDH3 _R (see Table 1) amplification of CY902 itself TDH3 Gene promoter sequence (fragment 2); containing SpMAE1 Using the plasmid containing the synthesized sequence as a template, the SpMAE1 coding sequence (fragment 3, SEQ ID NO: 2) of *Schizosaccharomyces cerevisiae* was amplified using primers 3_SpMAE1_F and 3_SpMAE1_R (see Table 1); using CY902 genomic DNA as a template, primer 4_T... GAL2 _F and 4_T GAL2 _R (see Table 1) amplification of CY902 itself GAL2 Gene terminator sequence (fragment 4); amplified using primers 5_DW_MCH4_F1 and 5_DW_MCH4_R (see Table 1). MCH4 Downstream homologous arm fragment 1 (fragment 5). Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_MCH4_F and 1_UP_MCH4_R2 (see Table 1). MCH4 Upstream homologous arm fragment 2 (fragment 6) of the gene; amplified using primers 5_DW_MCH4_F2 and 5_DW_MCH4_R (see Table 1). MCH4 Downstream homologous arm fragment 2 (fragment 7).

[0221] Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_2365_F and 1_UP_2365_R1 (see Table 1). Pk2365 Upstream homologous arm fragment 1 (fragment 8); using primer 2_P FBA1 _F and 2_P FBA1 _R (see Table 1) amplification of CY902 itself FBA1 Promoter sequence (segment 9); containing SbMDH Using the plasmid containing the synthesized sequence as a template, the SbMDH coding sequence of sorghum (fragment 10, SEQ ID NO: 1) was amplified using primers 3_SbMDH_F and 3_SbMDH_R (see Table 1); using CY902 genomic DNA as a template, primer 4_T... INO1 _F and 4_T INO1 _R (see Table 2) amplification of CY902 itself INO1 Gene terminator sequence (fragment 11); CY902 itself was amplified using primers 5_DW_2365_F1 and 5_DW_2365_R (see Table 1). Pk2365Downstream homologous arm fragment 1 (fragment 12). Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_2365_F and 1_UP_2365_R2 (see Table 1). Pk2365 Upstream homologous arm fragment 2 (fragment 13); CY902 itself was amplified using primers 5_DW_2365_F2 and 5_DW_2365_R (see Table 1). Pk2365 Downstream homologous arm fragment 2 (fragment 14).

[0222] 2. Construct a system for editing CY902 itself. MCH4 and Pk2365 plasmid at the site

[0223] Using the pWSPK-Cas9 plasmid (GenBank accession number: MW296878.1) as a template, a 9052 bp plasmid backbone (referred to as pWSPK_backbone (SEQ ID NO: 23)) without an sgRNA sequence was amplified using primers pWSPK-F and pWSPK-R (see Table 1). The 5' end sequence of the sgRNA (GenBank accession number: MW296878.1) (referred to as MCH4_sgRNA_1) was amplified using primers sgRNA-1F and MCH4_sgRNA-1R (see Table 1). This fragment contains […]. MCH4 The gene contains a unique 20nt prototype spacer sequence. Using the pWSPK-Cas9 plasmid as a template, the 3' end sequence of a 500bp sgRNA (abbreviated as sgRNA_2, SEQ ID NO: 103) was amplified using primers sgRNA-2F and sgRNA-2R (see Table 1). The plasmid backbone pWSPK_backbone, sgRNA fragments MCH4_sgRNA_1 and sgRNA_2 were ligated using a seamless cloning kit (Beyotime Biotechnology Co., Ltd., Shanghai, product number: D7010S). The seamless cloning ligation product was transformed into Trans1-T1 competent cells (TransGen Biotech Co., Ltd., Beijing, product number: CD501-02), and the resulting positive plasmid was named pWSPK_MCH4. Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (abbreviated as 2365_sgRNA_1) was amplified using primers sgRNA-1F and 2365_sgRNA-1R (see Table 1). The 3' end of this fragment contains Pk2365 The gene has a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2, and 2365_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_2365.

[0224] Table 1. Overexpression SpMAE1 and SbMDH Primers required for gene and CRISPR / Cas9 plasmid construction

[0225]

[0226] 3. Construct overexpression SpMAE1 and SbMDH Gene strains

[0227] The pWSPK_MCH4 plasmid and fragments 6 and 7 were electroporated into CY902 according to the yeast electroporation method (doi:10.1111 / 1751-7915.13781). ΔURA3 Positive transformants were obtained from the strain and named MA101-1 (genotype: CY902). ΔURA3 , ΔPkMCH4 The pWSPK_MCH4 plasmid and fragments 1-5 were electroconverted into CY902. ΔURA3 Positive transformants were obtained from the strain and named MA101-2 (genotype: CY902). ΔURA3 , PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).

[0228] The pWSPK_2365 plasmid and fragments 13 and 14 were transformed into strain MA101-2 via yeast electroporation. Positive transformants were obtained through screening and named strain MA102-1 (genotype: MA101-2). ΔPk2365 The pWSPK_2365 plasmid and fragments 8-12 were transformed into strain MA101-2 via yeast electroporation. Positive transformants were obtained and named strain MA102-2 (genotype: MA101-2). Pk2365::P PkFBA1 -ORF SbMDH -T PkINO1 ).

[0229] Example 2: Evaluation of L-malic acid production capacity of MA101 and MA102 series

[0230] 1. Fermentation with neutralizing agent

[0231] strains MA101-1, MA101-2, MA102-1, and MA102-2 were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 30 g / L CaCO3) and fermented in shake flasks at 30 °C and 250 rpm for 30 h. The L-malic acid yield was measured by HPLC and is shown in Table 2 below.

[0232] Table 2. L-malic acid shake-flask fermentation yield of MA101 and MA102 series strains

[0233]

[0234] 2. Fermentation without neutralizer

[0235] strains MA101-1, MA101-2, MA102-1, and MA102-2 were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30 °C and 250 rpm for 30 h. The L-malic acid yield was measured by HPLC and is shown in Table 3 below.

[0236] Table 3. L-malic acid shake-flask fermentation yield of MA101 and MA102 series strains

[0237]

[0238] Given that strain MA102-2 yielded the highest L-malic acid production, it was used for the construction of subsequent L-malic acid strains.

[0239] Example 3: Knockout in strain MA102-2 PDC1 , GPD1 Gene

[0240] 1. Build a tool for knocking out PDC1 , GPD1 DNA fragments

[0241] Using CY902 genomic DNA as a template, amplification was performed using primers PDC1_1F and PDC1_1R, PDC1_2F and PDC1_2R (see Table 4). PDC1 Upstream and downstream homologous arm fragments of the gene (abbreviated as PDC1_1, PDC1_2); amplified using primers GPD1_1F and GPD1_1R, GPD1_2F and GPD1_2R (see Table 4). GPD1 Upstream and downstream homologous arm fragments of the gene (abbreviated as GPD1_1, GPD1_2).

[0242] 2. Build for editing PDC1 , GPD1 CRISPR / Cas9 plasmids for genes

[0243] The 5' end sequence of sgRNA (abbreviated as PDC1_sgRNA_1, SEQ ID NO: 102) was amplified using primers sgRNA-1F and PDC1_sgRNA-1R (see Table 4). The 3' end of this fragment contains... PDC1The gene contains a unique 20nt prototype spacer sequence. The plasmid backbone pWSPK_backbone, sgRNA fragments PDC1_sgRNA_1, and sgRNA_2 were ligated using a seamless cloning kit. The seamless cloning ligation product was transformed into Trans1-T1 competent cells, and the resulting positive plasmid was named pWSPK_PDC1. The 5' end sequence of the gRNA (referred to as GPD1_sgRNA_1) was amplified using primers sgRNA-1F and GPD1_sgRNA-1R (see Table 4). This fragment contains a unique 20nt prototype spacer sequence at its 3' end. GPD1 The gene's unique 20nt prototype spacer sequence allows for seamless cloning and fusion of pWSPK_backbone, sgRNA_2, and GPD1_sgRNA_1 into a single gene for editing. GPD1 The pWSPK_GPD1 plasmid of the gene.

[0244] Table 4. PDC1 , GPD1 Primers required for gene knockout and CRISPR / Cas9 plasmid construction

[0245]

[0246] 3. Construction PDC1 , GPD1 Gene knockout strains

[0247] Fragments PDC1_1, PDC1_2, and plasmid pWSPK_PDC1 were simultaneously transformed into MA102-2. Positive transformants were identified using primers PDC1_1F / PDC1_2R and named strain MA103 (genotype: MA102-2). ΔPDC1 The pWSPK_GPD1 plasmid and fragments GPD1_1 and GPD1_2 were electroporated into strain MA103. Positive transformants were obtained by screening on SD-URA medium and named strain MA104 (genotype: MA103). ΔGPD1 ).

[0248] Example 4: Overexpression of carboxylase gene in strain MA102-2

[0249] exist JEN2-2 Three carboxylases were overexpressed at the sites, including (I) Escherichia coli ( Escherichia coli (I) Phosphoenolpyruvate carboxylkinase EcPCK (Uniprot database search number: P22259); (II) E. coli phosphoenolpyruvate carboxylase EcPPC (Uniprot database search number: P00864). CY902's own PkPCK1 (locus number PK0402, SEQ ID NO: 6) was used as a control. The promoter and terminator used were respectively the CY902's own enolase gene. ENO1 promoters and cell wall glycoprotein (GPI) genes SED1 The terminator. In JEN2-1 The sites were overexpressed with AoPYC (Uniprot database search number: Q2UGL1) (SEQ ID NO: 4) derived from Aspergillus oryzae, and PkPYC (SEQ ID NO: 5) of CY902 itself was used as a control. The promoters and terminators used were respectively... TDH3 promoters and GAL2 Termination of contract. EcPCK , EcPPC , AoPYC The gene was synthesized by Nanjing Genscript Biotech Co., Ltd., and optimized based on the codon bias of CY902. The specific construction method is as follows:

[0250] 1. Constructing donor DNA fragments for homologous recombination.

[0251] Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R1 (see Table 5). JEN2-2 Upstream homologous arm fragment 1 (fragment 15); using primer 2_P ENO1 _F and 2_P ENO1 _R (see Table 5) amplification ENO1 Gene promoter sequence (fragment 16); containing EcPCK Using the plasmid containing the synthesized sequence as a template, the EcPCK coding sequence of *E. coli* (fragment 17-1, SEQ ID NO: 7) was amplified using primers 3_EcPCK_F and 3_EcPCK_R (see Table 5); EcPPC Using the plasmid containing the synthesized sequence as a template, the EcPPC coding sequence (fragment 17-2, SEQ ID NO: 8) of *E. coli* was amplified using primers 3_EcPPC_F and 3_EcPPC_R (see Table 5); using CY902 genomic DNA as a template, the PkPCK coding sequence (fragment 17-3) was amplified using primers 3_PkPCK_F and 3_PkPCK_R (see Table 5); and primer 4_T... SED1 _F and 4_T SED1 _R (see Table 5) amplification SED1 Gene terminator sequence (fragment 18); amplified using primers 5_DW_JEN2-2_F1 and 5_DW_JEN2-2_R (see Table 5). JEN2-2 Downstream homologous arm fragment 1 (fragment 19); amplified using primers 1_UP_JEN2-2_F and 1_UP_JEN2-2_R2 (see Table 5). JEN2-2Upstream homologous arm fragment 2 (fragment 20) of the gene; amplified using primers 5_DW_JEN2-2_F2 and 5_DW_JEN2-2_R (see Table 5). JEN2-2 Downstream homologous arm fragment 2 (fragment 21).

[0252] Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R1 (see Table 5). JEN2-1 Upstream homologous arm fragment 1 (fragment 22); amplified the CY902 self-PkPYC1 coding sequence (fragment 23-1) using primers 3_PkPYC1_F and 3_PkPYC1_R (see Table 5); with... AoPYC Using the plasmid containing the synthesized sequence as a template, the AoPYC coding sequence (fragment 23-2, SEQ ID NO: 4) from Aspergillus oryzae was amplified using primers 3_AoPYC_F and 3_AoPYC_R (see Table 5); using CY902 genomic DNA as a template, primers 5_DW_JEN2-1_F1 and 5_DW_JEN2-1_R (see Table 5) were used to amplify... JEN2-1 Downstream homologous arm fragment 1 (fragment 24). Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_JEN2-1_F and 1_UP_JEN2-1_R2 (see Table 5). JEN2-1 Upstream homologous arm fragment 2 (fragment 25); amplified using primers 5_DW_JEN2-1_F2 and 5_DW_JEN2-1_R (see Table 5). JEN2-1 Downstream homologous arm fragment 2 (fragment 26).

[0253] Table 5. Primers required for overexpression of carboxylase gene and construction of CRISPR / Cas9 plasmid

[0254]

[0255] 2. Build for editing JEN2-1 and JEN2-2 CRISPR / Cas9 plasmids for genes

[0256] Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (abbreviated as JEN2-1_sgRNA_1) was amplified using primers sgRNA-1F and JEN2-1_sgRNA-1R (see Table 5). The 3' end of this fragment contains JEN2-1 The gene has a unique 20nt prototype spacer sequence. The pWSPK_backbone, sgRNA_2, and JEN2-1_sgRNA_1 from Example 1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_JEN2-1.

[0257] Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (abbreviated as JEN2-2_sgRNA_1) was amplified using primers sgRNA-1F and JEN2-2_sgRNA-1R (see Table 5). The 3' end of this fragment contains JEN2-2 The gene has a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2 and JEN2-2_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, named pWSPK_JEN2-2.

[0258] 3. Constructing strains overexpressing carboxylase genes

[0259] The pWSPK_JEN2-2 plasmid and fragments 20 and 21 were electroporated into strain MA102-2, and positive transformants were obtained by screening and named MA105-1 (genotype: MA102-2). ΔPkJEN2-2 The pWSPK_JEN2-2 plasmid and fragments 15-19 were electroporated into strain MA102-2, and positive transformants were obtained by screening and named MA105-2 (genotype: MA102-2). JEN2-2::P PkENO1 -ORF EcPCK -T PkSED1 MA105-3 (genotype: MA102-2, JEN2-2::P PkENO1 -ORF EcPPC - T PkSED1 MA105-4 (genotype: MA102-2, JEN2-2::P PkENO1 -ORF PkPCK -T PkSED1 The pWSPK_JEN2-1 plasmid and fragments 25 and 26 were electroporated into strain MA102-2, and positive transformants were obtained by screening and named MA105-5 (genotype: MA102-2). ΔPkJEN2-1 The pWSPK_JEN2-1 plasmid and fragments 22, 2, 23, 4, and 24 were electroporated into strain MA102-2, and positive transformants were obtained by screening and named MA105-6 (genotype: MA102-2). PkJEN2-1::P PkTDH3 - ORF PkPYC1 -T PkGAL2MA105-7 (genotype: MA102-2, PkJEN2-1::P PkTDH3 -ORF AoPYC -T PkGAL2 );

[0260] Example 5: Overexpression SpVHT1 Gene

[0261] strain MA102-2 PkMAE1 The site overexpressed the biotin transporter SpVHT1 derived from Schizosaccharomyces cerevisiae (Uniprot database search number: O13880). SpVHT1 The gene sequence (SEQ ID NO: 3) was optimized by Nanjing Genscript Biotech Co., Ltd. based on the codon bias of CY902. The promoter and terminator used are as follows: TDH3 promoters and GAL2 Termination of contract.

[0262] 1. Constructing donor DNA fragments for homologous recombination.

[0263] Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_MAE1_F and 1_UP_MAE1_R1 (see Table 6). PkMAE1 upstream homologous arm fragment 1 (fragment 27); containing SpVHT1 Using the plasmid containing the synthesized sequence as a template, the SpVHT1 coding sequence (fragment 28) of *Schizosaccharomyces cerevisiae* was amplified using primers 3_SpVHT1_F and 3_SpVHT1_R (see Table 6); using CY902 genomic DNA as a template, primers 5_DW_MAE1_F1 and 5_DW_MAE1_R (see Table 6) were used to amplify... PkMAE1 Downstream homologous arm fragment 1 (fragment 29). Using CY902 genomic DNA as a template, amplification was performed using primers 1_UP_MAE1_F and 1_UP_MAE1_R2 (see Table 6). PkMAE1 Upstream homologous arm fragment 2 (fragment 30); amplified using primers 5_DW_MAE1_F2 and 5_DW_MAE1_R (see Table 6). PkMAE1 Downstream homologous arm fragment 2 (fragment 31).

[0264] 2. Build for editing PkMAE1 CRISPR / Cas9 plasmids for genes

[0265] Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (abbreviated as MAE1_sgRNA_1) was amplified using primers sgRNA-1F and MAE1_sgRNA-1R (see Table 6). The 3' end of this fragment contains PkMAE1 The gene has a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2, and MAE1_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_MAE1.

[0266] Table 6. SpVHT1 Primers required for gene overexpression fragment construction and CRISPR / Cas9 plasmid construction

[0267]

[0268] 3. Construct overexpression SpVHT1 Gene strains

[0269] The pWSPK_MAE1 plasmid and fragments 30 and 31 were transformed into strain MA102-2 using yeast electroporation. Positive transformants were obtained and named strain MA106-1 (genotype: MA102-2). ΔPkMAE1 The pWSPK_MAE1 plasmid and fragments 27, 2, 28, 4, and 29 were transformed into strain MA102-2 using yeast electroporation. Positive transformants were obtained and named strain MA106-2 (genotype: MA102-2). PkMAE1::P PkTDH3 -ORF SpVHT1 -T PkGAL2 ).

[0270] Example 6: Replacement in MA101 and MA102 series strains PkURA3 Gene

[0271] Monocarboxylic acid permease in MA101 and MA102 series strains (NCBI Reference Sequence: XP_029319985.1) PkMCH5 The 5'-phospho-orhinoside decarboxylase gene was reintroduced at the gene locus. PkURA3 (SEQ ID NO: 104). The promoter and terminator used are... PkURA3 Its own promoter (SEQ ID NO: 105) and terminator (SEQ ID NO: 106). The specific construction method is as follows:

[0272] 1. Constructing donor DNA fragments for homologous recombination.

[0273] Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_MCH5_F and 1_UP_MCH5_R1 (see Table 7). PkMCH5 Upstream homologous arm fragment 1 (fragment 32); amplified using primers 2_PkURA3_F and 2_PkURA3_R (see Table 7). URA3 Promoter, coding frame, and terminator sequences (fragment 33); CY902 itself was amplified using primers 3_DW_MCH5_F1 and 3_DW_MCH5_R (see Table 7). PkMCH5 Downstream homologous arm fragment 1 (fragment 34). Using CY902 genomic DNA as a template, CY902 itself was amplified using primers 1_UP_MCH5_F and 1_UP_MCH5_R2 (see Table 7). PkMCH5 Upstream homologous arm fragment 2 (fragment 35); CY902 itself was amplified using primers 3_DW_MCH5_F2 and 3_DW_MCH5_R (see Table 7). PkMCH5 Downstream homologous arm fragment 2 (fragment 36).

[0274] 2. Build for editing PkMCH5 plasmids of gene loci

[0275] Using pWSPK-Cas9 plasmid as a template, the 5' end sequence of sgRNA (referred to as MCH5_sgRNA_1) was amplified using primers sgRNA-1F and MCH5_sgRNA-1R (see Table 7). The 3' end of this fragment contains PkMCH5 The gene promoter has a unique 20nt prototype spacer sequence. pWSPK_backbone, sgRNA_2, and MCH5_sgRNA_1 were ligated using a seamless cloning kit to obtain a positive clone plasmid, which was named pWSPK_MCH5.

[0276] Table 7. Replenishment PkURA3 Primers required for gene and CRISPR / Cas9 plasmid construction

[0277]

[0278] 3. Construct replenishment PkURA3 Gene strains

[0279] The pWSPK_MCH5 plasmid and fragments 35 and 36 were transformed into strains MA101-1, MA101-2, MA102-1, and MA102-2 using yeast electroporation. Positive transformants were obtained and named strains MA107-1 (genotype: MA101-1). Δ PkMCH5 strain MA107-2 (genotype: MA101-2, ΔPkMCH5 strain MA108-1 (genotype: MA102-1, Δ PkMCH5 ) and strain MA108-2 (genotype: MA102-2, ΔPkMCH5 The pWSPK_MCH5 plasmid and fragments 32-34 were transformed into strains MA101-1, MA101-2, MA102-1, and MA102-2 using yeast electroporation. Positive transformants were obtained and named strains MA107-3 (genotype: MA101-1). PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA107-4 (genotype: MA101-2, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA108-3 (genotype: MA102-1, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 ) and strain MA108-4 (genotype: MA102-2, PkMCH5::P PkURA3 - ORF PkURA3 -T PkURA3 ).

[0280] Example 7: Replacement in MA103-MA106 series strains PkURA3 Gene

[0281] Referring to Example 6, in the MA103-MA106 series strains PkMCH5 Gene locus replacement PkURA3 strain MA109 (genotype: MA103) was obtained. PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA110 (genotype: MA104, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA111-1 (genotype: MA105-1, PkMCH5::P PkURA3 - ORF PkURA3 -T PkURA3 strain MA111-2 (genotype: MA105-2, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA111-3 (genotype: MA105-3, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA111-4 (genotype: MA105-4, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA111-5 (genotype: MA105-5, PkMCH5::P PkURA3 - ORF PkURA3 -T PkURA3 strain MA111-6 (genotype: MA105-6, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA111-7 (genotype: MA105-7, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA112-1 (genotype: MA106-1, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA112-2 (genotype: MA106-2, PkMCH5::P PkURA3 - ORF PkURA3 -T PkURA3 ).

[0282] Example 8: Evaluation of L-malic acid production capacity of MA101-MA112 series strains

[0283] 1. Fermentation with neutralizing agent

[0284] The MA101-MA112 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 30 g / L CaCO3) and fermented in shake flasks at 30℃ and 250 rpm for 30 h. The yield of L-malic acid was measured by HPLC.

[0285] Table 8. Malic acid shake-flask fermentation yield of MA101-MA112 series strains

[0286]

[0287] 2. Fermentation without neutralizer

[0288] The MA101-MA112 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30℃ and 250 rpm for 30 h. The yield of L-malic acid was measured by HPLC.

[0289] Table 9. Malic acid shake-flask fermentation yield of MA101-MA112 series strains

[0290]

[0291] Example 9: Optimization of L-malic acid producing bacteria

[0292] Referring to Example 4, three carboxylase genes were overexpressed in MA104 to obtain strain MA113-1 (genotype: MA104, ΔPkJEN2-2 strain MA113-2 (genotype: MA104, JEN2-2::P PkENO1 -ORF EcPCK -T PkSED1 strain MA113-3 (genotype: MA104, JEN2-2::P PkENO1 -ORF EcPPC -T PkSED1 strain MA113-4 (genotype: MA104, JEN2- 2::P PkENO1 -ORF PkPCK -T PkSED1 In strain MA113-3, two sources of overexpression were observed. PYC strain MA114-1 (genotype: MA113-3) was obtained. ΔPkJEN2-1 ); strain MA114-2 (genotype: MA113-3, PkJEN2- 1::P PkTDH3 -ORF PkPYC1 -T PkGAL2 strain MA114-3 (genotype: MA113-3, PkJEN2-1::P PkTDH3 -ORF AoPYC - T PkGAL2 ).

[0293] Referring to Example 5, biotin transporter SpVHT1 was overexpressed in MA114-3 to obtain strain MA115-1 (genotype: MA114-3, ΔPkMAE1 strain MA115-2 (genotype: MA114-3, PkMAE1::P PkTDH3 -ORF SpVHT1 - T PkGAL2 ).

[0294] Referring to Example 6, the 5'-phospho-orhodoside decarboxylase gene was reintroduced into strain MA115-2. PkURA3 strain MA116 (genotype: MA115-2) was obtained. PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 ).

[0295] Example 10: Evaluation of L-malic acid production capacity of strain MA116

[0296] The MA116 strain was fermented in a 5L tank (yeast inorganic salt medium, 20% w / v glucose, 70 g / L CaCO3), with no pH control. The malic acid yield reached 204 g / L after 120 hours.

[0297] Example 11: In Pichia pastoris CICC32244 ΔURA3 strain overexpression SpMAE1 and SbMDH Gene

[0298] Referring to Example 1, the orotidine 5'-phosphate decarboxylase encoding gene of CICC32244 (purchased from China Industrial Microbial Culture Collection Center) was synthesized via homologous recombination. URA3 Knockout was performed, resulting in CICC32244. ΔURA3 Mutant.

[0299] The pWSPK_MCH4 plasmid and fragments 6 and 7 were electroporated into CICC32244 using the yeast electroporation method. ΔURA3 Positive transformants were obtained from the strain and named MA117-1 (genotype: CICC32244). ΔURA3 , ΔPkMCH4 The pWSPK_MCH4 plasmid and fragments 1-5 were electroporated into CICC32244. ΔURA3 Positive transformants were obtained from the strain and named MA117-2 (genotype: CICC32244). ΔURA3 , PkMCH4::P PkTDH3 -ORF SpMAE1 -T PkGAL2 ).

[0300] The pWSPK_2365 plasmid and fragments 13 and 14 were transformed into strain MA117-2 via yeast electroporation. Positive transformants were obtained through screening and named strain MA118-1 (genotype: MA117-2). ΔPk2365 The pWSPK_2365 plasmid and fragments 8-12 were transformed into strain MA117-2 via yeast electroporation. Positive transformants were obtained and named strain MA118-2 (genotype: MA117-2). Pk2365::P PkFBA1 -ORF SbMDH -T PkINO1 ).

[0301] Example 12: Replacement in MA117 and MA118 series strains PkURA3 Gene

[0302] Referring to Example 6, in the MA117 and MA118 series strains PkMCH5 Gene locus replacement PkURA3 strain MA119-1 (genotype: MA117-1) was obtained. PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 strain MA119-2 (genotype: MA117-2, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3strain MA120-1 (genotype: MA118-1, PkMCH5::P PkURA3 - ORF PkURA3 -T PkURA3 strain MA120-2 (genotype: MA118-2, PkMCH5::P PkURA3 -ORF PkURA3 -T PkURA3 ).

[0303] Example 13: Evaluation of malic acid production capacity of MA117-MA120 series strains

[0304] 1. Fermentation with neutralizing agent

[0305] The MA117-MA120 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 30 g / L CaCO3) and fermented in shake flasks at 30℃ and 250 rpm for 30 h. The yield of L-malic acid was measured by HPLC.

[0306] Table 10. Malic acid shake-flask fermentation yield of MA117-MA120 series strains

[0307]

[0308] 2. Fermentation without neutralizer

[0309] The MA117-MA120 series strains were inoculated into 30 mL of yeast inorganic salt medium (5% w / v glucose, 0 g / L CaCO3) and fermented in shake flasks at 30℃ and 250 rpm for 30 h. The yield of L-malic acid was measured by HPLC.

[0310] Table 11. Malic acid shake-flask fermentation yield of MA117-MA120 series strains

[0311]

Claims

1. A genetically modified malate-producing yeast strain having or enhanced malate transporter activity and having or enhanced NADPH-dependent malate dehydrogenase (EC50) 1.1.1.82) activity, and also possesses pyruvate decarboxylase with reduced or inactivated activity, wherein the malic acid producing yeast strain is *Pichia kudrica* ( Pichia kudriavzevii ).

2. The malic acid-producing yeast strain according to claim 1, wherein the malic acid-producing yeast strain further has or has enhanced activity of at least one of the following: (i) pyruvate carboxylase (EC 6.4.1.1), (ii) phosphoenolpyruvate carboxylkinase (EC 6.4.1.1). 4.1.1.49, (iii) Phosphoenolpyruvate carboxylase (EC) 4.1.1.31), and (iv) biotin transporter.

3. The malic acid-producing yeast strain according to claim 1, wherein the malic acid transporter protein is selected from SpMAE1 protein, C4T318 protein, and AsDct protein, and / or The NADPH-dependent malate dehydrogenase is derived from plants, or from Euglena or Thermobacterium.

4. The malic acid-producing yeast strain according to claim 2, wherein the phosphoenolpyruvate carboxylase is *Escherichia coli* phosphoenolpyruvate carboxylase, and / or The pyruvate carboxylase is derived from Aspergillus oryzae or Kudria zwibichii yeast.

5. The malic acid-producing yeast strain according to claim 3, wherein the NADPH-dependent malic acid dehydrogenase is derived from a C4 plant.

6. The malic acid-producing yeast strain according to claim 3, wherein the NADPH-dependent malic acid dehydrogenase is derived from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, or Amaranthaceae families.

7. The malic acid-producing yeast strain according to claim 3, wherein the NADPH-dependent malic acid dehydrogenase is derived from sorghum ( Sorghum bicolor ),corn( Zea mays ),sugar cane( Saccharum officinarum ),pea( Pisum sativum ), chickpeas ( Cicer arietinum ),spinach( Spinacia oleracea ), small worm ( Euglena gracilis ) or thermoautotrophic methanotherapeutic bacteria ( Methanothermobacter thermautotrophicus ).

8. The genetically modified malic acid-producing yeast strain of claim 1, further comprising the following reduced or inactivated characteristics: (i) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), and / or (ii) 5'-phospho-orhinoside decarboxylase (EC 4.1.1.23), and / or (iii) Monocarboxylic acid permeases, and / or (iv) dicarboxylic acid transporters, and / or (v) malic acid oxidase (EC 1.1.1.38), and / or (vi) Oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

9. The genetically modified malic acid-producing yeast strain according to any one of claims 1-8, having at least one of the following: (i) The overexpressed nucleic acid sequence encoding NADPH-dependent malate dehydrogenase, (ii) The overexpressed nucleic acid sequence encoding the malate transporter. (iii) The overexpressed nucleic acid sequence encoding pyruvate carboxylase, (iv) The overexpressed nucleic acid sequence encoding phosphoenolpyruvate carboxylase, (v) The overexpressed nucleic acid sequence encoding the biotin transporter protein. (vi) The overexpressed nucleic acid sequence encoding phosphoenolpyruvate carboxykinase. (vii) The gene encoding endogenous pyruvate decarboxylase was knocked out. (viii) The gene encoding the endogenous NAD-dependent glycerol-3-phosphate dehydrogenase was knocked out. (ix) The gene encoding endogenous 5'-orhinophosphate decarboxylase was knocked out. (x) The gene encoding the endogenous monocarboxylic acid permease was knocked out. (xi) The gene encoding the endogenous dicarboxylic acid transporter was knocked out. (xii) The gene encoding endogenous malic acid oxidase was knocked out. (xiii) The gene encoding the endogenous bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation was knocked out.

10. The genetically modified malic acid-producing yeast strain of claim 9, wherein it has at least one of the following: (i) The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase is the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity. (ii) The nucleic acid sequence encoding the malic acid transporter is the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having malic acid transporter activity. (iii) The amino acid sequence of the pyruvate carboxylase is as shown in SEQ ID NO: 5 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and having pyruvate carboxylase activity; or the nucleic acid sequence encoding the pyruvate carboxylase is the sequence shown in SEQ ID NO: 4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity and encoding an amino acid sequence having pyruvate carboxylase activity. (iv) The nucleic acid sequence encoding phosphoenolpyruvate carboxylase is the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxylase activity. (v) The nucleic acid sequence encoding the biotin transporter is the sequence shown in SEQ ID NO: 3 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having biotin transporter activity. (vi) The amino acid sequence of the phosphoenolpyruvate carboxykinase is as shown in SEQ ID NO: 6 or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encodes an amino acid sequence having phosphoenolpyruvate carboxykinase activity; or the nucleic acid sequence encoding phosphoenolpyruvate carboxykinase is the sequence shown in SEQ ID NO: 7 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encodes an amino acid sequence having phosphoenolpyruvate carboxykinase activity.

11. The genetically modified malic acid-producing yeast strain according to any one of claims 1-8, wherein, In the genetically modified malic acid-producing yeast strain, (a) The nucleic acid sequences encoding NADPH-dependent malate dehydrogenase and the nucleic acid sequences encoding malate transport proteins are overexpressed; or (b) The nucleic acid sequences encoding NADPH-dependent malate dehydrogenase and malate transporter are overexpressed, and the nucleic acid sequences endogenously encoding pyruvate decarboxylase and / or endogenously encoding NAD-dependent glycerol-3-phosphate dehydrogenase are knocked out; or (c) The nucleic acid sequences encoding NADPH-dependent malate dehydrogenase and malate transporter are overexpressed, and the endogenous nucleic acid sequence encoding malate enzyme is knocked out; or (d) The following nucleic acid sequences are overexpressed: The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase; The nucleic acid sequence encoding the malic acid transporter protein; and At least one of the following nucleic acid sequences: a nucleic acid sequence encoding a biotin transporter, a nucleic acid sequence encoding Escherichia coli phosphoenolpyruvate carboxylkinase, a nucleic acid sequence encoding phosphoenolpyruvate carboxylase, a nucleic acid sequence encoding Pichia pastoris phosphoenolpyruvate carboxylkinase, and a nucleic acid sequence encoding pyruvate carboxylase. (e) The following nucleic acid sequences are overexpressed: The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase; Nucleic acid sequence encoding malic acid transporter protein; The nucleic acid sequence encoding Escherichia coli phosphoenolpyruvate carboxylase; The nucleic acid sequence encoding the biotin transporter protein; and The nucleic acid sequence encoding Aspergillus oryzae pyruvate carboxylase. as well as The genes encoding endogenous pyruvate decarboxylase and / or endogenous NAD-dependent glycerol-3-phosphate dehydrogenase were knocked out.

12. The genetically modified malic acid-producing yeast strain of claim 11, wherein, In the genetically modified malic acid-producing yeast strain, The nucleic acid sequence encoding phosphoenolpyruvate carboxylase is the same as the nucleic acid sequence encoding Escherichia coli phosphoenolpyruvate carboxylase, or The pyruvate carboxylase is derived from Aspergillus oryzae or Pichia kudrica, or The nucleic acid sequence encoding pyruvate carboxylase encodes an amino acid sequence as shown in SEQ ID NO: 5 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having the pyruvate carboxylase activity; or a sequence as shown in SEQ ID NO: 4 or its degenerate sequence; or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity; or The nucleic acid sequence encoding Aspergillus oryzae pyruvate carboxylase is the sequence shown in SEQ ID NO: 4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity.

13. The genetically modified malic acid-producing yeast strain of any one of claims 1-8, wherein the malic acid-producing yeast strain is *Kudelazvichis kudriazines*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20885.

14. A method for producing a genetically modified malate-producing yeast strain, comprising conferring or enhancing malate transporter activity and NADPH-dependent malate dehydrogenase (EC5) activity in the strain. 1.1.1.82) activity, also includes weakening or inactivating pyruvate decarboxylase in the strain, wherein the malic acid producing yeast strain is *Pichia kudrica*.

15. The method of claim 14, further comprising conferring or enhancing at least one of the following activities: (i) pyruvate carboxylase (EC6.4.1.1) activity, (ii) phosphoenolpyruvate carboxylkinase (EC6.4.1.1) activity. 4.1.1.49) activity, (iii) phosphoenolpyruvate carboxylase activity (EC) 4.1.1.31), and (iv) biotin transporter activity.

16. The method of claim 14, wherein the malate transporter is selected from SpMAE1 protein, C4T318 protein, and AsDct protein, and / or The NADPH-dependent malate dehydrogenase is derived from plants, or from Euglena or Thermobacterium.

17. The method of claim 15, wherein the pyruvate carboxylase is derived from *Aspergillus oryzae* or *Pichia kudrica*, and / or The phosphoenolpyruvate carboxylase activity is the same as that of Escherichia coli phosphoenolpyruvate carboxylase.

18. The method of claim 16, wherein the NADPH-dependent malate dehydrogenase is derived from a C4 plant.

19. The method of claim 16, wherein the NADPH-dependent malate dehydrogenase is derived from plants of the Poaceae, Cyperaceae, Asteraceae, Euphorbiaceae, Chenopodiaceae, Portulacaceae, or Amaranthaceae families.

20. The method of claim 16, wherein the NADPH-dependent malate dehydrogenase is derived from sorghum, corn, sugarcane, peas, chickpeas, spinach, Euglena, or thermoautotrophic methanotherapeutic bacteria.

21. The method of claim 18, further comprising weakening or inactivating the following in the strain: (i) NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), and / or (ii) 5'-phospho-orhinoside decarboxylase (EC 4.1.1.23), and / or (iii) Monocarboxylic acid permeases, and / or (iv) dicarboxylic acid transporters, and / or (v) malic acid oxidase (EC 1.1.1.38), and / or (vi) Oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate aldol condensation bifunctional enzyme (EC 4.1.3.17 or 4.1.1.112).

22. The method of any one of claims 14-21, comprising: In the malic acid-producing yeast strain. (i) Overexpression of a nucleic acid sequence encoding NADPH-dependent malate dehydrogenase, and / or (ii) Overexpression of a nucleic acid sequence encoding a malate transporter, and / or (iii) Overexpression of a nucleic acid sequence encoding pyruvate carboxylase, and / or (iv) Overexpression of a nucleic acid sequence encoding phosphoenolpyruvate carboxylase, and / or (v) Overexpression of a nucleic acid sequence encoding a biotin transporter protein, and / or (vi) Overexpression of a nucleic acid sequence encoding phosphoenolpyruvate carboxykinase, and / or (vii) Knock out the gene encoding endogenous pyruvate decarboxylase, and / or (viii) Knock out the gene encoding endogenous NAD-dependent glycerol-3-phosphate dehydrogenase, and / or (ix) Knockout of the gene encoding endogenous 5'-phospho-orhinoside decarboxylase, and / or (x) Knockout of the gene encoding an endogenous monocarboxylic acid permease, and / or (xi) Knock out the gene encoding the endogenous dicarboxylic acid transporter, and / or (xii) Knock out the gene encoding endogenous malic acid oxidase, and / or (xiii) Knock out the endogenous gene encoding the bifunctional enzymes of oxaloacetate decarboxylation and 3-hydroxy-3-methylglutarate acetate.

23. The method of claim 22, comprising: In the malic acid-producing yeast strain. (i) The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase is the sequence shown in SEQ ID NO: 1 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having NADPH-dependent malate dehydrogenase activity, and / or (ii) The nucleic acid sequence encoding the malate transporter is the sequence shown in SEQ ID NO: 2 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having malate transporter activity, and / or (iii) The nucleic acid sequence encoding pyruvate carboxylase encodes an amino acid sequence as shown in SEQ ID NO: 5 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and having the pyruvate carboxylase activity; or the nucleic acid sequence encoding pyruvate carboxylase is the sequence shown in SEQ ID NO: 4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity, and / or (iv) The nucleic acid sequence encoding phosphoenolpyruvate carboxylase is the sequence shown in SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxylase activity, and / or (v) The nucleic acid sequence encoding the biotin transporter is the sequence shown in SEQ ID NO: 3 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having biotin transporter activity, and / or (vi) The nucleic acid sequence encoding phosphoenolpyruvate carboxykinase is the sequence shown in SEQ ID NO: 7 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxykinase activity, or the phosphoenolpyruvate carboxykinase is the sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having phosphoenolpyruvate carboxykinase activity.

24. The method of any one of claims 14-21, comprising: In the malic acid-producing yeast strain. (a) Overexpression of the nucleic acid sequence encoding NADPH-dependent malate dehydrogenase and the nucleic acid sequence encoding malate transporter; or (b) Overexpression of nucleic acid sequences encoding NADPH-dependent malate dehydrogenase and malate transporter proteins, and knockout of endogenous genes encoding pyruvate decarboxylase and endogenous genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase; or (c) Overexpression of nucleic acid sequences encoding NADPH-dependent malate dehydrogenase and malate transporter, and knockout of endogenous genes encoding malate enzymes; (d) Overexpression of the following nucleic acid sequence: The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase; Nucleic acid sequence encoding malic acid transporter protein; At least one of the following nucleic acid sequences: a nucleic acid sequence encoding a biotin transporter, a nucleic acid sequence encoding *Escherichia coli* phosphoenolpyruvate carboxylkinase, a nucleic acid sequence encoding phosphoenolpyruvate carboxylase, a nucleic acid sequence encoding *Pichia pastoris* phosphoenolpyruvate carboxylkinase, and a nucleic acid sequence encoding pyruvate carboxylase; or (e) Overexpression of the following nucleic acid sequence: The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase; Nucleic acid sequence encoding malic acid transporter protein; At least one of the following nucleic acid sequences: a nucleic acid sequence encoding *Escherichia coli* phosphoenolpyruvate carboxylkinase, a nucleic acid sequence encoding phosphoenolpyruvate carboxylase, a nucleic acid sequence encoding *Pichia pastoris* phosphoenolpyruvate carboxylkinase; and a nucleic acid sequence encoding pyruvate carboxylase, and Knock out the genes encoding endogenous pyruvate decarboxylase and endogenous NAD-dependent glycerol-3-phosphate dehydrogenase; or (f) Overexpression of the following nucleic acid sequence: The nucleic acid sequence encoding NADPH-dependent malate dehydrogenase; Nucleic acid sequence encoding malic acid transporter protein; The nucleic acid sequence encoding Escherichia coli phosphoenolpyruvate carboxylase; The nucleic acid sequence encoding the biotin transporter protein; and The nucleic acid sequence encoding Aspergillus oryzae pyruvate carboxylase. as well as Knock out the gene encoding endogenous pyruvate decarboxylase and / or the gene encoding endogenous NAD-dependent glycerol-3-phosphate dehydrogenase.

25. The method of claim 24, wherein the nucleic acid sequence encoding phosphoenolpyruvate carboxylase is a nucleic acid sequence encoding *Escherichia coli* phosphoenolpyruvate carboxylase, and / or The pyruvate carboxylase is derived from Aspergillus oryzae or Kudria zwibichii yeast.

26. The method of claim 24, wherein the nucleic acid sequence encoding pyruvate carboxylase encodes an amino acid sequence as shown in SEQ ID NO: 5 or having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and having pyruvate carboxylase activity, or is a sequence as shown in SEQ ID NO: 4 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the same sequence and encoding an amino acid sequence having pyruvate carboxylase activity.

27. The method of claim 24, wherein the nucleic acid sequence encoding Aspergillus oryzae pyruvate carboxylase is the sequence shown in SEQ ID NO: 4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with it and encoding an amino acid sequence having pyruvate carboxylase activity.

28. The method of any one of claims 14-21, wherein the malic acid producing yeast strain is *Kudelazvichis kudriazines*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20885.

29. A method for producing L-malic acid, comprising culturing a genetically modified malic acid-producing yeast strain according to any one of claims 1-13 or a genetically modified malic acid-producing yeast strain prepared according to any one of claims 14-28.

30. The method of claim 29, wherein the culture is carried out in a pH range of 2.0-5.0 and / or with little or no addition of a neutralizing agent.

31. The method of claim 29, wherein the culture is carried out in a pH range of 2.0-3.5 and / or with little or no addition of a neutralizing agent.

32. The method of any one of claims 29-31, comprising separating and purifying the generated L-malic acid.

33. The use of the genetically modified yeast strain of any one of claims 1-13 or the genetically modified yeast strain prepared by the method of any one of claims 16-32 in the production of L-malic acid.

34. The application of claim 33, wherein L-malic acid is produced under conditions of pH 2.0-5.0 and / or with little or no addition of a neutralizing agent.

35. The application of claim 33, wherein L-malic acid is produced under conditions of pH 2.0-3.5 and / or with little or no addition of a neutralizing agent.

Images