C4-dicarboxylate transporter mutant and its application

Abstract

The present invention provides C4-dicarboxylate transporter mutants and their applications. Specifically, amino acid residues at at least one of the wild-type C4-dicarboxylate transporter sites, T26, R78, L85, T100, W312, A314, P318, K260, K331, and K339, are mutated. These mutants can be used to increase the specificity of malate and succinate transport and / or increase malate and succinate production, thus possessing significant application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a C4-dicarboxylic acid transporter mutant and an application thereof. Background Art

[0002] Malic acid (2-hydroxy-1,4-butanedioic acid) and succinic acid (succinic acid, 1,2-ethanedicarboxylic acid) are two important C4-dicarboxylic acids. Due to their wide range of applications, they have become important platform compounds. Compared to chemical extraction methods that use fossil fuels such as petroleum as raw materials, microbial fermentation is renewable, efficient, and clean. Therefore, the production of C4-dicarboxylic acids through microbial fermentation is becoming a growing research hotspot.

[0003] The transport of C4-dicarboxylic acids plays a crucial role in the accumulation of target products. Intracellular C4-dicarboxylic acids are primarily transported to the extracellular space by transporters, thereby reducing the toxic effects of high intracellular acid concentrations on cells and accumulating the target product in the fermentation broth. Therefore, transporters have become a key target for metabolic engineering of C4-dicarboxylic acid fermentation strains. C4-dicarboxylic acid transporters are widely present in bacteria, yeast, and filamentous fungi, such as DcuC from Escherichia coli and SpMae1 from Saccharomyces cerevisiae, as well as their homologs. In Aspergillus carbonii, heterologous expression of SpMae1 increased L-malate production by 8-fold and succinate production by 3-fold. Overexpression of AoMae (the homolog of SpMae1 in Aspergillus oryzae) in Myceliophthora thermophila increased L-malate production by 20-fold. Therefore, improving the transport efficiency of C4-dicarboxylic acid plays a crucial role in organic acid synthesis. On the other hand, malate and succinate, two C4-dicarboxylic acids, differ structurally by only a single hydroxyl group, resulting in poor C4-dicarboxylic acid transport specificity. For example, DcuC, SpMae1, and AoMae all indiscriminately transport succinate and L-malate within the cell, a characteristic that reduces the purity of the target product. The accumulation of byproducts increases the difficulty and cost of subsequent separation and purification, limiting the industrialization of microbial fermentation for the production of malate and succinate. Therefore, significantly improving the efficiency and specificity of C4-dicarboxylic acid transport is an urgent issue to be addressed in the large-scale production of malate and succinate. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a C4-dicarboxylic acid transporter mutant and its application. Starting from the C4-dicarboxylic acid transporter AoMae derived from Aspergillus oryzae, key amino acid sites are screened through protein sequence analysis, structural simulation and molecular docking analysis, and single-point mutations are performed on the screened amino acid sites. Combined with the organic acid production analysis of the strain, the screened proteins have specific transport capabilities for malic acid or succinic acid or significantly improved substrate transport efficiency.

[0005] In this regard, the present invention provides a C4-dicarboxylic acid transporter mutant, which can transport C4-dicarboxylic acids such as malate, succinate or fumarate in cells to the outside of cells. Its amino acid sequence is mutated at at least one of the amino acid residues in the T26, R78, L85, T100, W312, A314, P318, K260, K331 and K339 positions relative to the wild-type sequence; or the amino acid sequence of the C4-dicarboxylic acid transporter mutant has the mutation site in the mutated amino acid sequence and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence, preferably an amino acid sequence with more than 90%, more than 95% or more than 98% homology. The wild-type C4-dicarboxylic acid transporter is derived from Schizosaccharomyces pombe ( Schizosaccharomyces pombe ), Neurospora crassa ( Neurospora crassa ), Aspergillus sojae ( Aspergillus sojae ), Myceliophthora thermophila ( Myceliophthora thermophila ), Trichoderma reesei ( Trichoderma reesei ), Aspergillus niger ( Aspergillus niger ), Aspergillus carbonarius ( Aspergillus carbonarius ), Aspergillus oryzae ( Aspergillus oryzae ) or Aspergillus flavus; More specifically, the wild-type C4-dicarboxylate transporter is derived from Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) of SpMae1, derived from Neurospora crassa ( Neurospora crassa ) of NcMae, derived from Aspergillus sojae ( Aspergillus sojae ) of AsMae, derived from Myceliophthora thermophila ( Myceliophthora thermophila ) of MtMae, derived from Trichoderma reesei ( Trichoderma reesei ) of TrMae, derived from Aspergillus niger ( Aspergillus niger ) of AnMae, derived from Carbonaceous Aspergillus ( Aspergillus carbonarius ) of AcMae, Aspergillus oryzae ( Aspergillus oryzae ) of AoMae or Aspergillus flavus ( Aspergillus flavus ); Further preferably, the wild-type C4-dicarboxylate transporter is derived from Aspergillus oryzae ( Aspergillus oryzae ) of AoMae.

[0006] In one embodiment, the amino acid sequence of the C4-dicarboxylate transporter mutant includes the following site: the threonine T corresponding to position 26 of SEQ ID NO: 1 is mutated to glutamic acid E. Specifically, the C4-dicarboxylate transporter mutant has the following substitution corresponding to SEQ ID NO: 1: T26E.

[0007] In one embodiment, the amino acid sequence of the C4-dicarboxylate transporter mutant includes the following site: the arginine R at position 78 corresponding to SEQ ID NO: 1 is mutated to aspartic acid D, phenylalanine F, cysteine C, glutamine Q, methionine M, glutamate E, histidine H, asparagine N, lysine K, tryptophan W, tyrosine Y, or threonine T. Specifically, the C4-dicarboxylate transporter mutant undergoes substitution at any of the following sites corresponding to SEQ ID NO: 1: R78D, R78F, R78C, R78Q, R78M, R78E, R78H, R78N, R78K, R78W, R78Y, or R78T.

[0008] In one embodiment, the amino acid sequence of the C4-dicarboxylic acid transporter mutant includes the following site: the leucine L corresponding to position 85 of SEQ ID NO: 1 is mutated to methionine M. Specifically, the C4-dicarboxylic acid transporter mutant has the following substitution corresponding to SEQ ID NO: 1: L85M.

[0009] In one embodiment, the amino acid sequence of the C4-dicarboxylate transporter mutant includes the following site: the threonine T at position 100 corresponding to SEQ ID NO: 1 is mutated to alanine A, cysteine C, glycine G, methionine M, glutamic acid E, histidine H, isoleucine I, lysine K, arginine R, tryptophan W, tyrosine Y, or valine V. Specifically, the C4-dicarboxylate transporter mutant undergoes substitution at any of the following sites corresponding to SEQ ID NO: 1: T100A, T100C, T100G, T100M, T100E, T100H, T100I, T100K, T100R, T100W, T100Y, or T100V.

[0010] In one embodiment, the amino acid sequence of the C4-dicarboxylate transporter mutant includes the following position: the tryptophan W corresponding to position 312 of SEQ ID NO: 1 is mutated to alanine A. Specifically, the C4-dicarboxylate transporter mutant has the following substitution corresponding to SEQ ID NO: 1: W312A.

[0011] In one embodiment, the amino acid sequence of the C4-dicarboxylic acid transporter mutant includes the following site: alanine A corresponding to position 314 of SEQ ID NO: 1 is mutated to histidine H. Specifically, the C4-dicarboxylic acid transporter mutant has the following substitution corresponding to SEQ ID NO: 1: A314H.

[0012] In one embodiment, the amino acid sequence of the C4-dicarboxylate transporter mutant includes the following site: proline P corresponding to position 318 of SEQ ID NO: 1 is mutated to glycine G. Specifically, the C4-dicarboxylate transporter mutant has the following substitution corresponding to SEQ ID NO: 1: P318G.

[0013] In one embodiment, the amino acid sequence of the C4-dicarboxylic acid transporter mutant comprises the following sites: lysine K at positions 260, 331, and 339 corresponding to SEQ ID NO: 1 are simultaneously mutated to arginine R. Specifically, the C4-dicarboxylic acid transporter mutant corresponds to SEQ ID NO: 1 and simultaneously undergoes substitutions at the following three sites: K260R, K331R, and K339R.

[0014] As an exemplary embodiment of the present invention, the amino acid sequence of the C4-dicarboxylate transporter mutant is specifically any one of the following (1-30):

[0015] (1) Threonine at position 26 of SEQ ID NO: 1 was mutated to glutamic acid, and the other amino acid residues remained unchanged;

[0016] (2) Arginine at position 78 of SEQ ID NO: 1 was mutated to aspartic acid, and the other amino acid residues remained unchanged;

[0017] (3) Arginine at position 78 of SEQ ID NO: 1 was mutated to phenylalanine, and the other amino acid residues remained unchanged;

[0018] (4) Arginine at position 78 of SEQ ID NO: 1 was mutated to cysteine, and the other amino acid residues remained unchanged;

[0019] (5) Arginine at position 78 of SEQ ID NO: 1 was mutated to glutamine, and the other amino acid residues remained unchanged;

[0020] (6) Arginine at position 78 of SEQ ID NO: 1 was mutated to methionine, and the other amino acid residues remained unchanged;

[0021] (7) Arginine at position 78 of SEQ ID NO: 1 was mutated to glutamic acid, and the other amino acid residues remained unchanged;

[0022] (8) Arginine at position 78 of SEQ ID NO: 1 was mutated to histidine, and the other amino acid residues remained unchanged;

[0023] (9) Arginine at position 78 of SEQ ID NO: 1 was mutated to asparagine, and the other amino acid residues remained unchanged;

[0024] (10) Arginine at position 78 of SEQ ID NO: 1 was mutated to lysine, and the other amino acid residues remained unchanged;

[0025] (11) Arginine at position 78 of SEQ ID NO: 1 was mutated to tryptophan, and the other amino acid residues remained unchanged;

[0026] (12) Arginine at position 78 of SEQ ID NO: 1 was mutated to tyrosine, and the other amino acid residues remained unchanged;

[0027] (13) Arginine at position 78 of SEQ ID NO: 1 was mutated to threonine, and the other amino acid residues remained unchanged;

[0028] (14) Leucine at position 85 of SEQ ID NO: 1 was mutated to methionine, and the other amino acid residues remained unchanged;

[0029] (15) Threonine at position 100 of SEQ ID NO: 1 was mutated to alanine, and the other amino acid residues remained unchanged;

[0030] (16) Threonine at position 100 of SEQ ID NO: 1 was mutated to cysteine, and the other amino acid residues remained unchanged;

[0031] (17) Threonine at position 100 of SEQ ID NO: 1 was mutated to glycine, and the other amino acid residues remained unchanged;

[0032] (18) Threonine at position 100 of SEQ ID NO: 1 was mutated to methionine, and the other amino acid residues remained unchanged;

[0033] (19) Threonine at position 100 of SEQ ID NO: 1 was mutated to glutamic acid, and the other amino acid residues remained unchanged;

[0034] (20) Threonine at position 100 of SEQ ID NO: 1 was mutated to histidine, and the other amino acid residues remained unchanged;

[0035] (21) Threonine at position 100 of SEQ ID NO: 1 was mutated to isoleucine, and the other amino acid residues remained unchanged;

[0036] (22) Threonine at position 100 of SEQ ID NO: 1 was mutated to lysine, and the other amino acid residues remained unchanged;

[0037] (23) Threonine at position 100 of SEQ ID NO: 1 was mutated to arginine, and the other amino acid residues remained unchanged;

[0038] (24) Threonine at position 100 of SEQ ID NO: 1 was mutated to tryptophan, and the other amino acid residues remained unchanged;

[0039] (25) Threonine at position 100 of SEQ ID NO: 1 was mutated to tyrosine, and the other amino acid residues remained unchanged;

[0040] (26) Threonine at position 100 of SEQ ID NO: 1 was mutated to valine, and the other amino acid residues remained unchanged;

[0041] (27) The tryptophan at position 312 of SEQ ID NO: 1 was mutated to alanine, and the other amino acid residues remained unchanged;

[0042] (28) Alanine at position 314 of SEQ ID NO: 1 was mutated to histidine, and the other amino acid residues remained unchanged;

[0043] (29) Proline 318 of SEQ ID NO: 1 was mutated to glycine, and the other amino acid residues remained unchanged;

[0044] (30) Lysine at positions 260, 331, and 339 of SEQ ID NO: 1 were simultaneously mutated to arginine, while the other amino acid residues remained unchanged;

[0045] The present invention also provides a polynucleotide encoding the above-mentioned C4-dicarboxylate transporter mutant.

[0046] The present invention also provides a recombinant vector, an expression cassette, a transgenic cell line and / or a recombinant bacterium containing the polynucleotide of the C4-dicarboxylic acid transporter mutant.

[0047] The present invention also provides the use of the aforementioned C4-dicarboxylic acid transporter mutant, a recombinant vector containing the aforementioned polynucleotide, an expression cassette, a transgenic cell line, and / or a recombinant bacterium in the production of malic acid and / or succinic acid. Preferably, the transport vector is used for producing malic acid and / or succinic acid, for example, to transport intracellular malic acid and / or succinic acid into a fermentation broth.

[0048] Preferably, the application is to increase the yield of malic acid or succinic acid; in another preferred embodiment, the application is to increase the purity of malic acid or succinic acid.

[0049] More specifically, the application of mutants wherein the threonine T at position 100 is mutated to alanine A, or the lysine K at positions 260, 331, and 339 are simultaneously mutated to arginine R in improving malic acid production; the application of mutants wherein the threonine T at position 100 is mutated to alanine A in improving succinic acid production; the application of mutants wherein the threonine at position 100 is mutated to cysteine C, glycine G, glutamic acid E, isoleucine I, lysine K, arginine R, tryptophan W, or the arginine R at position 78 is mutated to aspartic acid D, or the leucine L at position 85 ... methionine M; threonine T at position 26 mutates to glutamate E or arginine R at position 78 mutates to lysine K, threonine T, tryptophan W, phenylalanine F, histidine H, glutamate E, aspartic acid N, cysteine C, glutamine Q, methionine M, tyrosine Y or threonine at position 100 mutates to methionine M, histidine H, tyrosine Y, valine V or tryptophan W at position 312 mutates to alanine A, alanine at position 314 mutates to histidine H or proline P at position 318 mutates to glycine G.

[0050] The present invention also provides a method for producing malic acid and / or succinic acid, which is achieved by fermenting a transformed strain containing the recombinant expression vector, and optionally further comprising the step of collecting or purifying the malic acid and / or succinic acid. Preferably, the strain is Myceliophthora thermophila. Specifically, the malic acid and / or succinic acid is collected or purified from the fermentation broth.

[0051] The present invention, through molecular engineering of the C4-dicarboxylic acid transporter, obtains C4-dicarboxylic acid transporter mutants with improved or enhanced functions and properties. These mutants can be categorized into four types: the first type: C4-dicarboxylic acid transporter mutants with significantly enhanced malic acid transport capacity, thereby increasing malic acid production; the second type: C4-dicarboxylic acid transporter mutants with significantly enhanced malic acid specificity, thereby increasing malic acid purity during fermentation; the third type: C4-dicarboxylic acid transporter mutants with significantly enhanced succinic acid transport capacity, thereby increasing succinic acid production; and the fourth type: C4-dicarboxylic acid transporter mutants with significantly enhanced succinic acid specificity, thereby increasing succinic acid purity in fermentation broth. Therefore, the present invention has significant application value in the production of organic acids such as malic acid and succinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : AoMae protein structure simulation and point mutation site screening.

[0053] Figure 2 :Malic acid production is increased after partial point mutation of C4-dicarboxylate transporter AoMae.

[0054] Figure 3 :The purity of malic acid increased significantly after mutation of some sites of C4-dicarboxylate transporter AoMae.

[0055] Figure 4 :The purity of succinate was significantly increased after mutation of some sites of C4-dicarboxylate transporter AoMae.

[0056] Figure 5 : Conservation analysis of key amino acid sites T26, R78, L85, T100, W312, A314, P318, K260, K331 and K339.

[0057] Terms and Definitions

[0058] In the context of the present invention, the "C4-dicarboxylate transporter" in the present invention refers to a protein that can transport C4-dicarboxylic acids such as malate, succinate, and fumarate out of the cell. As long as it has the function of transporting C4-dicarboxylic acids such as malate, succinate, and fumarate out of the cell, there is no particular limitation on its amino acid sequence and source. By way of example only and not limitation, it can be, for example, a protein derived from Aspergillus oryzae ( A.oryzae ) of AoMae (XM_001820829.3), derived from Schizosaccharomyces pombe ( S.pombe ) of SpMae1 (NP_594777.1), derived from Neurospora crassa ( N.crassa ) of NcMae (XP_958365.2), derived from Trichoderma reesei ( T. reesei ) of TrMae (XP_006963989.1), derived from Aspergillus niger ( A.niger ) of AnMae (XM_001398094), derived from Aspergillus sojae ( A.sojae ) of AsMae (CP035528.1), derived from Aspergillus carbonarius ( A. carbonarius ) of AcMae (KY178298), derived from Myceliophthora thermophila ( M.thermophila ) of MtMae (XP_003663832.1) and from Aspergillus flavus ( A.flavus )'s AfMae (XP_747269.1) and so on.

[0059] In the present invention, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0060] The term "vector" in the present invention refers to a recombinant expression vector or genome into which a coding polynucleotide sequence can be inserted. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements. Those skilled in the art can use well-known methods to construct an expression vector containing a "C4-dicarboxylic acid transporter mutant" encoding DNA sequence and appropriate transcription / translation control signals, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0061] The "host cell" of the present invention includes a host cell containing the above-mentioned expression vector or a host cell in which the coding sequence of the mutant protein of the present invention is integrated into the genome. The host cell includes a host cell derived from a microbial host having a high similarity in the sequence and functionality of the C4-dicarboxylic acid transporter. By way of example only and not limitation, it may be, for example, Saccharomyces cerevisiae ( S. cerevisiae ), Schizosaccharomyces pombe ( S. pombe ), Escherichia coli ( Escherichia coli ), Bacillus subtilis ( Bacillus subtilis ), Aspergillus oryzae ( A. oryzae ), Rhizopus oryzae ( Rhizopus oryzae )、Myceliophthora thermophila( M. thermophila )wait.

[0062] In the present invention, the terms "mutant" and "variant" and "mutant protein" can be used interchangeably, and "modification" or "mutation" can be used interchangeably. These expressions refer to amino acids relative to unmodified or modified proteins, such as the C4-dicarboxylic acid transporter mutant of SEQ ID NO: 1 as the starting sequence, or derived from such proteins, comprising changes at one or more positions, i.e., substitutions, insertions and / or deletions, and still retaining their activity. Mutant proteins can be obtained by various techniques known in the art. In particular, exemplary techniques for modifying DNA sequences encoding wild-type proteins include, but are not limited to, directed mutagenesis, random mutagenesis, and construction of synthetic oligonucleotides.

[0063] The term "substitution" with respect to an amino acid position or residue means that the amino acid at the specified position has been replaced by another amino acid. Substitutions can be conservative or non-conservative.

[0064] The mutations are described according to their mutation at specific residues, the positions of which are determined by alignment with the amino acid sequence of the starting protein SEQ ID NO: 1 or the reference sequence SEQ ID NO: 1. In the context of the present invention, any variants carrying these same mutations at functionally equivalent residues are also contemplated.

[0065] As used herein, the term "corresponding to" has the meaning commonly understood by persons of ordinary skill in the art. Specifically, "corresponding to" refers to a position in one sequence that corresponds to a specified position in the other sequence after alignment for homology or sequence identity. In the present invention, "corresponding to SEQ ID NO: 1" refers to the position of a mutation site determined by comparison with SEQ ID NO: 1.

[0066] Those skilled in the art will understand that "corresponding to SEQ ID NO: 1" merely indicates that SEQ ID NO: 1 is used as a reference for determining the position of the mutation site, and does not mean that the mutant protein of the present invention can be obtained only by modifying the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, those skilled in the art can use the amino acid sequence of any C4-dicarboxylic acid transporter known in the art as a starting sequence to obtain the C4-dicarboxylic acid transporter mutant of the present invention. As long as the obtained C4-dicarboxylic acid transporter mutant is mutated at the site involved in the present invention when compared with SEQ ID NO: 1 and maintains the transport capacity of the C4-dicarboxylic acid transporter, that is, within the scope of the present invention, all sites other than the mutation site do not need to be completely identical to SEQ ID NO: 1.

[0067] As used herein, "starting protein" or "starting sequence" refers to the C4-dicarboxylate transporter or its amino acid sequence prior to modification or mutation according to the present invention. Amino acid mutations or substitutions are indicated herein using the format "XnY," where X represents the wild-type amino acid at position n in SEQ ID NO:1, n represents the position of the amino acid in SEQ ID NO:1, and Y represents the mutated amino acid at position n in SEQ ID NO:1. For example, "T26E" indicates that the threonine T at position 26 in SEQ ID NO:1 has been replaced with the glutamic acid E. DETAILED DESCRIPTION

[0068] The following will further describe the technical solution of the present invention in detail with reference to specific examples. It should be understood that the following examples are only for illustrative purposes and explanation of the present invention, and should not be construed as limiting the scope of protection of the present invention.

[0069] Unless otherwise noted, all raw materials and reagents used in the following examples were commercially available or prepared by known methods. Malic acid and succinic acid were purchased from Sigma-Aldrich. The experimental procedures used in the following examples, unless otherwise noted, were generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or according to the conditions recommended by the manufacturer.

[0070] Example 1: Identification of key amino acid sites in the C4-dicarboxylate transporter AoMae

[0071] TMHMM Server v. 2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ) analysis showed that the C4-dicarboxylate transporter AoMae (Genbank ID: XM_001820829.3) is a membrane protein composed of 380 amino acids with nine transmembrane domains. I-TASSER (https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ) protein structure prediction showed that the protein with the highest structural similarity to AoMae in the protein structure database is Haemophilus influenzae ( Haemophilus influenza ) protein HiTehA (PDB: 3M7E, z-score = 12.78, cutoff = 6.9). Using 3M7E as a reference, the AoMae protein was structurally simulated to obtain its 3D structural model ( Figure 1A). Molecular docking was performed with L-malic acid and succinic acid as substrates, respectively, and the simulated AoMae protein structure was obtained. Possible key amino acid sites were screened: T26, R78, L85, T100, W312, A314, P318, K260, K331, and K339.

[0072] Example 2: Construction of C4-dicarboxylate transporter AoMae overexpression vector

[0073] Using the genome of the starting strain Myceliophthora thermophila ATCC42464 (purchased from the American type culture collection) as a template, PCR amplified the 1.4 kb upstream promoter (named Ptef promoter) encoding the translation elongation factor reading frame (MYCTH_2298136) using the following primers:

[0074] pAN52-Ptef-F:TTGAAGTAATCTTCTGCAGATCTTTAATTAACTCGAGCATGTACCTTGACGTCCTCCGA,

[0075] pAN52-Ptef-R:TAAGTGGATCCGAATTCGATATCGTTTAAACACTAGTTCTGAAGAACGAAACTGGCGAC.

[0076] After the PCR reaction was completed, the Gibson kit was used to connect the Bgl II and Bcu I. After double enzyme digestion, linearize the vector pAN52-neo and transform it into Escherichia coli DH5α. Select single clones for PCR verification. Finally, enzyme digestion and sequencing verification are performed to obtain promoter-linked vector pAN52-neo. tef The vector was named pAN52-Ptef.

[0077] The C4-dicarboxylate transporter gene was amplified from the cDNA of Aspergillus oryzae DSM1863 (DSMZ, purchased from German Microorganisms and Cell Culture GmbH). Aomae (XM_001820829.3, SEQ ID NO: 1), and ligated into Bcu I and EcoR After enzyme digestion, the linearized vector pAN52-Ptef was transformed into Escherichia coli DH5α, and single clones were selected for PCR verification. Finally, enzyme digestion and sequencing were performed to obtain the promoter. tef Under regulation Aomae The gene expression vector was named pAN52-Ptef-AoMae. AomaeIt does not contain mutation sites and is the initial expression vector. The primers are as follows:

[0078] AoMae-F: AGTCGCCAGTTTTCGTTCTTCAGAACTAGTGTTTTAAACATGCTGACACCTCCCAAGTTTG,

[0079] AoMae-R: TAAAGATGAGGATGTATCTGATTAGGATATCGAATTCGGATCCACTTAACGTTACTGA.

[0080] Example 3: Construction of C4-dicarboxylate transporter AoMae mutant

[0081] Primers are used to introduce mutation sites, and then the mutant gene is obtained by Overlap PCR. Specifically, primers are designed at the codon position corresponding to the amino acid to be mutated. The codon of the mutated amino acid in the primer sequence is designed to be the corresponding mutation codon. Both the F-end and R-end primers need to be mutated. Mutation primers are designed at each mutation site in turn, and each DNA fragment is amplified by PCR. Then, Overlap PCR is performed on two fragments to obtain the full-length mutant gene. The vector pAN52-Ptef is connected by Gibson, and the ligation product is transformed into Escherichia coli DH5α. Single clones are selected for PCR verification, and finally enzyme digestion and sequencing verification or correct single clones are performed. The specific mutation site primers are as follows:

[0082] (1) The T26E mutation was introduced using primers T26E-F: CCGGGAGAGGCTTCGCCATTTCGAGTGGGCCTGGTACACATTA and T26E-R: TAATGTGTACCAGGCCCACTCGAAATGGCGAAGCCTCTCCCGG.

[0083] (2) The R78D mutation was introduced using primers R78D-F: TCTGCTCTACCATGGCTATAGACTTCATCCTGCACGGCAACC and R78D-R: GGTTGCCGTGCAGGATGAAGTCTATAGCCATGGTAGAGCAGA.

[0084] (3) The R78F mutation was introduced using primers R78F-F: TCTGCTCTACCATGGCTATATTCTTCATCCTGCACGGCAACC and R78F-R: GGTTGCCGTGCAGGATGAAGAATATAGCCATGGTAGAGCAGA.

[0085] (4) The R78C mutation was introduced using primers R78C-F: TCTGCTCTACCATGGCTATATGCTTCATCCTGCACGGCAACC and R78C-R: GGTTGCCGTGCAGGATGAAGCATATAGCCATGGTAGAGCAGA.

[0086] (5) The R78Q mutation was introduced using primers R78Q-F: TCTGCTCTACCATGGCTATACAATTCATCCTGCACGGCAACC and R78Q-R: GGTTGCCGTGCAGGATGAATTGTATAGCCATGGTAGAGCAGA.

[0087] (6) The R78M mutation was introduced using primers R78M-F: TCTGCTCTACCATGGCTATAATGTTCATCCTGCACGGCAACC and R78M-R: GGTTGCCGTGCAGGATGAACATTATAGCCATGGTAGAGCAGA.

[0088] (7) The R78E mutation was introduced using primers R78E-F: TCTGCTCTACCATGGCTATAGAGTTCATCCTGCACGGCAACC and R78E-R: GGTTGCCGTGCAGGATGAACTCTATAGCCATGGTAGAGCAGA.

[0089] (8) The R78H mutation was introduced using primers R78H-F: TCTGCTCTACCATGGCTATACACTTCATCCTGCACGGCAACC and R78H-R: GGTTGCCGTGCAGGATGAAGTGTATAGCCATGGTAGAGCAGA.

[0090] (9) The R78N mutation was introduced using primers R78N-F: TCTGCTCTACCATGGCTATAAACTTCATCCTGCACGGCAACC and R78N-R: GGTTGCCGTGCAGGATGAAGTTTATAGCCATGGTAGAGCAGA.

[0091] (10) The R78K mutation was introduced using primers R78K-F: TCTGCTCTACCATGGCTATAAAGTTCATCCTGCACGGCAACC and R78K-R: GGTTGCCGTGCAGGATGAACTTTATAGCCATGGTAGAGCAGA.

[0092] (11) The R78W mutation was introduced using primers R78W-F: TCTGCTCTACCATGGCTATATGGTTCATCCTGCACGGCAACC and R78W-R: GGTTGCCGTGCAGGATGAACCATATAGCCATGGTAGAGCAGA.

[0093] (12) The R78Y mutation was introduced using primers R78Y-F: TCTGCTCTACCATGGCTATATACTTCATCCTGCACGGCAACC and R78Y-R: GGTTGCCGTGCAGGATGAAGTATATAGCCATGGTAGAGCAGA.

[0094] (13) The R78T mutation was introduced using primers R78T-F: TCTGCTCTACCATGGCTATAACCTTCATCCTGCACGGCAACC and R78T-R: GGTTGCCGTGCAGGATGAAGGTTATAGCCATGGTAGAGCAGA.

[0095] (14) The L85M mutation was introduced using primers L85M-F: GGTTCATCCTGCACGGCAACATGCTGGAGTCCCTCCGTCATG and L85M-R: CATGACGGAGGGACTCCAGCATGTTGCCGTGCAGGATGAACC.

[0096] (15) The T100A mutation was introduced using primers T100A-F: GACCGCGAGGGTCTCTTCTTCCCGACCTTCTGGCTCTCCGTCGCAACC and T100A-R: GGTTGCGACGGAGAGCCAGAAGGTCGGGAAGAAGAGACCCTCGCGGTC.

[0097] (16) The T100C mutation was introduced using primers T100C-F: GAGGGTCTCTTCTTCCCGTGCTTCTGGCTCTCCGTCGCAACC and T100C-R: GGTTGCGACGGAGAGCCAGAAGCACGGGAAGAAGAGACCCTC.

[0098] (17) The T100G mutation was introduced using primers T100G-F: GAGGGTCTCTTCTTCCCGGGATTCTGGCTCTCCGTCGCAACC and T100G-R: GGTTGCGACGGAGAGCCAGAATCCCGGGAAGAAGAGACCCTC.

[0099] (18) The T100M mutation was introduced using primers T100M-F: GAGGGTCTCTTCTTCCCGATGTTCTGGCTCTCCGTCGCAACC and T100M-R: GGTTGCGACGGAGAGCCAGAACATCGGGAAGAAGAGACCCTC.

[0100] (19) The T100E mutation was introduced using primers T100E-F: GAGGGTCTCTTCTTCCCGGAGTTCTGGCTCTCCGTCGCAACC and T100E-R: GGTTGCGACGGAGAGCCAGAACTCCGGGAAGAAGAGACCCTC.

[0101] (20) The T100H mutation was introduced using primers T100H-F: GAGGGTCTCTTCTTCCCGCACTTCTGGCTCTCCGTCGCAACC and T100H-R: GGTTGCGACGGAGAGCCAGAAGTGCGGGAAGAAGAGACCCTC.

[0102] (21) The T100I mutation was introduced using primers T100I-F: GAGGGTCTCTTCTTCCCGATCTTCTGGCTCTCCGTCGCAACC and T100I-R: GGTTGCGACGGAGAGCCAGAAGATCGGGAAGAAGAGACCCTC.

[0103] (22) The T100K mutation was introduced using primers T100K-F: GAGGGTCTCTTCTTCCCGAAGTTCTGGCTCTCCGTCGCAACC and T100K-R: GGTTGCGACGGAGAGCCAGAACTTCGGGAAGAAGAGACCCTC.

[0104] (23) The T100R mutation was introduced using primers T100R-F: GAGGGTCTCTTCTTCCCGCGCTTCTGGCTCTCCGTCGCAACC and T100R-R: GGTTGCGACGGAGAGCCAGAAGCGCGGGAAGAAGAGACCCTC.

[0105] (24) The T100W mutation was introduced using primers T100W-F: GAGGGTCTCTTCTTCCCGTGGTTCTGGCTCTCCGTCGCAACC and T100W-R: GGTTGCGACGGAGAGCCAGAACCACGGGAAGAAGAGACCCTC.

[0106] (25) The T100Y mutation was introduced using primers T100Y-F: GAGGGTCTCTTCTTCCCGTACTTCTGGCTCTCCGTCGCAACC and T100Y-R: GGTTGCGACGGAGAGCCAGAAGTACGGGAAGAAGAGACCCTC.

[0107] (26) The T100Y mutation was introduced using primers T100V-F: GAGGGTCTCTTCTTCCCGGTCTTCTGGCTCTCCGTCGCAACC and T100V-R: GGTTGCGACGGAGAGCCAGAAGACCGGGAAGAAGAGACCCTC.

[0108] (27) The W312A mutation was introduced using primers W312A-F: GAGGCCTTCCACCTCAACGCCTGGGCCATGGTCTTCCCCAAC and W312A-R: GTTGGGGAAGACCATGGCCCAGGCGTTGAGGTGGAAGGCCTC.

[0109] (28) The A314H mutation was introduced using primers A314H-F: CCACCTCAACTGGTGGCACATGGTCTTCCCCAACACCGGC and A314H-R: GCCGGTGTTGGGGAAGACCATGTGCCACCAGTTGAGGTGG.

[0110] (29) The P318G mutation was introduced using primers P318G-F: CTGGTGGGCCATGGTCTTCGGCAACACCGGCTTCACCCTGGC and P318G-R: GCCAGGGTGAAGCCGGTGTTGCCGAAGACCATGGCCCACCAG.

[0111] (30) The K260R mutation was induced using primers K260R-F: GGCCTCCCCGAAGACTTCCGACTGCTCCACGACGCCCACGC and K260R-R: GCGTGGGCGTCGTGGAGCAGTCGGAAGTCTTCGGGGAGGCC.

[0112] (30) The K331R mutation was detected using primers K331R-F: GCCACCATCACCCTGGGCCGCGCTCTCAACAGTAACGGTGT and K331R-R: ACACCGTTACTGTTGAGAGCGCGGCCCAGGGTGATGGTGGC.

[0113] (30) The K339R mutation was detected using primers K339R-F: CTCTCAACAGTAACGGTGTGCGCGGCGTCGGTTCCGCCATGTC and K339R-R: GACATGGCGGAACCGACGCCGCGCACACCGTTACTGTTGAGAG.

[0114] Example 4: Construction of a C4-dicarboxylic acid transporter fermentation strain of Myceliophthora thermophila

[0115] (1) Myceliophthora thermophila ATCC42464 was cultured on XM plates [50× Vogel's salt solution 20 mL, xylose 20 g, agar 15 g, constant volume to 1 L, and sterilized by high temperature and high pressure. 50× Vogel's salt solution (1 L): Na3citrate·2H2O 130 g, KNO3 126 g, (NH4)H2PO4 144 g, KH2PO4 80 g, MgSO4·7H2O 10 g, CaCl2·2H2O 5 g, trace element salt solution 5 mL, biotin (0.1 mg / mL) 2.5 mL, constant volume to 1 L].

[0116] (2) Protoplast transformation of Myceliophthora thermophila

[0117] a. Mycelium Preparation

[0118] Mature spores of Myceliophthora thermophila were collected with 0.05% Tween-80 sterilized water, filtered through lens paper to remove the mycelium, and then spread on GM plate medium (20 mL of 50× Vogel's salt solution, 20 g of glucose, 15 g of agar, fixed volume to 1 L, sterilized by high temperature and high pressure) covered with cellophane paper and cultured at 45°C for 20 h.

[0119] b. Protoplast Preparation

[0120] Place the cellophane with mycelium in 30 mL of lysis solution (formula: 0.15 g lysis enzyme dissolved in 30 mL of solution A, filter sterilized; solution A: 1.0361 g KH2PO4, 21.864 g sorbitol, dissolved in 90 mL of deionized water, adjust the pH to 5.6 with KOH, quantify to 100 mL, and sterilize with high temperature and high pressure), and lyse at 28°C for 2 h, gently shaking every 20 min.

[0121] Then, after filtering through lens paper, centrifuge at 2000 rpm and 4°C for 10 min, discard the supernatant, add 5 mL of solution B (0.735 g CaCl2·2H2O, 18.22 g sorbitol, 1 mL Tris HCl 1M pH 7.5, dissolved in 90 mL deionized water, adjust the pH to 7.6 with hydrochloric acid, quantify to 100 mL, and sterilize at high temperature and high pressure), centrifuge at 2000 rpm and 4°C for 10 min; discard the supernatant, and add 200 μL of solution B.

[0122] c. Protoplast transformation

[0123] Pre-cooled 50mL centrifuge tubes were added with 200 μL protoplasts, 10 μL Bgl Add 50 μL of pre-chilled PEG (12.5 g PEG6000, 0.368 g CaCl2·2H2O, to a volume of 100 mL) to the linearized plasmid. Place on ice for 20 minutes, then add 2 mL of pre-chilled PEG. Incubate at room temperature for 5 minutes. Add 4 mL of Solution B and gently swirl to mix. Add the transformation medium containing the appropriate antibiotic, shake well, and pour over the prepared lower medium. Once solidified, incubate in a constant temperature incubator at 45°C. After 3 days, single mycelia were picked under a stereomicroscope and grown on the corresponding resistance plate. Verification was then performed by PCR.

[0124] Example 5: Determination of the ability of Myceliophthora thermophila to synthesize organic acids through C4-dicarboxylic acid fermentation

[0125] (1) Acid production culture

[0126] The strain to be tested was inoculated into a medium containing glucose as a carbon source (formula: glucose 75 g, peptone 8.0 g, 0.15 g KH2PO4, 0.15 g K2HPO4, 0.10 g CaCl2·2H2O, 0.10 g MgSO 4·7H2O, 80.0 g calcium carbonate, 1 mL of 0.5 g / L biotin, and 1 mL of trace element solution (dissolved in water and adjusted to 100 mL) were placed in a deep-well plate. Transformed sporozoites were collected with sterile water and filtered through two layers of sterile lens paper. The number of spores was calculated, and the inoculum size was 2.5 × 10 5 The culture medium volume was 3.5 mL / well, 45°C, 700 rpm, and samples were taken for determination of malic acid and succinic acid contents after 3.5 days of culture.

[0127] (2) Sample processing

[0128] After fermentation, 0.5 mL of 1 M H2SO4 was added to each well and the cells were acidified at 80°C for 30 min, with thorough shaking every 10 min. The supernatant was then filtered through a 0.22 μm filter and the malic and succinic acid contents were determined.

[0129] (3) Determination of malic acid and succinic acid content

[0130] The treated samples were subjected to high performance liquid chromatography to determine the contents of malic acid and succinic acid. The detector was a differential refractive index detector, the chromatographic column was an HPX-87H column (Bio-Rad), and the Empower3 software was used. The HPLC parameters were as follows: column temperature 35°C, detector temperature 40°C, mobile phase 5 mM H2SO4, flow rate 0.5 mL / min, and malic acid and succinic acid standards were used for quantification, respectively.

[0131] The results showed that when the threonine (T) at position 100 of the C4-dicarboxylate transporter was mutated to alanine (A), the malic acid production increased by 32%, and when the lysine (K) at positions 260, 331, and 339 were simultaneously mutated to arginine (R), the L-malic acid production increased by 40% (Table 1, Figure 3 ).

[0132] Table 1 Increased L-malic acid production after mutation of some sites in C4-dicarboxylate transporter

[0133] mutation site L-Malic acid (g / L) oMa 17.98 T100A 23.61 K260R & K331R & K339R 25.20

[0134] When the threonine (T) at position 100 of the C4-dicarboxylate transporter was mutated to alanine (A), the succinate production increased to 10.32 g / L (Table 2).

[0135] Table 2 Increased succinate production after mutation of some sites in C4-dicarboxylate transporter

[0136] mutation site Succinic acid (g / L) oMa 10.06 T100A 10.32

[0137] When any of the following sites of the C4-dicarboxylate transporter were mutated, the specificity of L-malate transport increased (Table 3, Figure 4 ): Threonine at position 100 mutates to cysteine (C), glycine (G), glutamic acid (E), isoleucine (I), lysine (K), arginine (R), tryptophan (W), or arginine at position 78 (R) mutates to aspartic acid (D), or leucine at position 85 (L) mutates to methionine (M).

[0138] Table 3: C4-Dicarboxylate transporter specifically transports L-malate after partial site mutation

[0139] mutation site L-Malic acid (g / L) Succinic acid (g / L) L-Malic acid purity (%) oMa 17.98 10.06 64.12 T100C 10.76 4.32 71.37 T100G 12.63 4.01 75.89 T100E 2.81 0.39 87.89 T100I 2.73 1.01 72.95 T100K 1.53 0.49 75.74 T100R 0.88 0.12 87.61 T100W 0.86 0.02 98.18 R78D 1.34 0.21 86.65 L85M 13.96 5.12 73.17

[0140] When any of the following sites of the C4-dicarboxylate transporter were mutated, the succinate transport specificity increased (Table 4, Figure 5 ): Threonine (T) at position 26 mutates to glutamic acid (E), or arginine (R) at position 78 mutates to lysine (K), threonine (T), tryptophan (W), phenylalanine (F), histidine (H), glutamic acid (E), aspartic acid (N), cysteine (C), glutamine (Q), methionine (M), tyrosine (Y), or threonine at position 100 mutates to methionine (M), histidine (H), tyrosine (Y), valine (V), or tryptophan (W) at position 312 mutates to alanine (A), alanine at position 314 mutates to histidine (H), or proline (P) at position 318 mutates to glycine (G).

[0141] Table 4C4-Dicarboxylate transporter specifically transports succinate after partial site mutation

[0142] mutation site L-Malic acid (g / L) Succinic acid (g / L) Succinic acid purity (%) oMa 17.98 10.06 35.88 T100M 6.32 5.36 45.09 T100H 2.34 3.53 45.90 P318G 1.90 2.546 57.24 T100Y 0.00 2.18 60.11 T100V 4.64 3.81 100.00 R78K 0.00 0.414 100.00 R78T 0.00 0.106 100.00 R78W 0.00 0.106 100.00 R78F 0.00 0.103 100.00 R78H 0.00 0.102 100.00 R78E 0.00 0.128 100.00 R78N 0.00 0.124 100.00 R78C 0.00 0.122 100.00 R78Q 0.00 0.135 100.00 R78M 0.00 0.145 100.00 R78Y 0.00 0.125 100.00 A314H 0.00 0.111 100.00 T26E 0.00 0.231 100.00 W312A 0 0.378 100.00

[0143] Example 6. Conservation Analysis of Key Amino Acid Sites T26, R78, L85, T100, W312, A314, P318, K260, K331, and K339

[0144] Homologous proteins of the C4-dicarboxylate transporter AoMae exist in different species, such as those from Schizosaccharomyces pombe ( S.pombe ) of SpMae1 (NP_594777.1), derived from Neurospora crassa ( N.crassa ) of NcMae (XP_958365.2), derived from Aspergillus sojae ( A.sojae ) of AsMae (CP035528.1), derived from Myceliophthora thermophila ( M.thermophila ) of MtMae (XP_003663832.1), derived from Trichoderma reesei ( T. reesei ) of TrMae (XP_006963989.1), derived from Aspergillus niger ( A.niger ) of AnMae (XM_001398094), derived from Aspergillus carbonarius ( A. carbonarius ) of AcMae (KY178298), and from Aspergillus flavus ( A.flavus ) of AfMae (XP_747269.1). According to BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) amino acid sequence analysis, AoMae and AsMae (CP035528.1) have 98.42% amino acid sequence similarity (coverage 100%), AoMae and MtMae (XP_003663832.1) have 55.8% amino acid sequence similarity (coverage 71%), AoMae and NcMae (XP_958365.2) have 54.89% amino acid sequence similarity (coverage 95%), and AoMae and SpMae1 have 10. The amino acid sequence similarity between AoMae and TrMae (XP_006963989.1) was 35.85% (coverage 95%), the amino acid sequence similarity between AoMae and TrMae (XP_006963989.1) was 56.87% (coverage 95%), the amino acid sequence similarity between AoMae and AnMae (XM_001398094) was 71.08% (coverage 97%), the amino acid sequence similarity between AoMae and AcMae (KY178298) was 68.38% (coverage 97%), and the amino acid sequence similarity between AoMae and AfMae (XP_747269.1) was 74.67% (coverage 98%). Sequence analysis results showed that the amino acid sequences of the C4-dicarboxylic acid transporter AoMae and its homologous proteins from different species differed greatly and had low sequence similarity. For example, the sequence similarity between AoMae and MtMae, AcMae, NcMae, AfMae, AnMae, TrMae, and SpMae was less than 80%, especially the sequence similarity between AoMae and SpMae was less than 40%. However, amino acid positions T26, R78, T100, W312, A314, and P318 were conserved in the C4-dicarboxylic acid transporters AoMae, SpMae1, NcMae, AsMae, MtMae, TrMae, AnMae, AcMae, and AfMae ( Figure 5), combined with the results of the above five examples, it is shown that the amino acid sites T26, R78, T100, W312, A314, and P318 have the same important value in the specificity of transporting malate and succinate and / or improving the production of malate and succinate in the C4-dicarboxylate transporters SpMae1, NcMae, AsMae, MtMae, TrMae, AnMae, AcMae, and AfMae as in the C4-dicarboxylate transporter AoMae.

[0145] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A C4-dicarboxylate transporter mutant, characterized in that Any one of the following mutations occurs on the basis of the wild-type C4-dicarboxylate transporter; Threonine T at position 26 is mutated to glutamic acid E; Arginine R at position 78 is mutated to aspartic acid D, or phenylalanine F, or cysteine C, or glutamine Q, or methionine M, or glutamic acid E, or histidine H, or asparagine N, or lysine K, or tryptophan W, or tyrosine Y, or threonine T; Leucine L at position 85 is mutated to methionine M; Threonine T at position 100 is mutated to alanine A, or cysteine C, or glycine G, or methionine M, or glutamic acid E, or histidine H, or isoleucine I, or lysine K, or arginine R, or tryptophan W, or tyrosine Y, or valine V; Tryptophan W at position 312 is mutated to alanine A; Alanine A at position 314 is mutated to histidine H; Proline P at position 318 is mutated to glycine G; The wild-type C4-dicarboxylic acid transporter is derived from Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) of SpMae1, derived from Neurospora crassa ( Neurospora crassa ) of NcMae, derived from Aspergillus sojae ( Aspergillus sojae ) of AsMae, derived from Myceliophthora thermophila ( Myceliophthora thermophila ) of MtMae or Aspergillus oryzae ( Aspergillus oryzae ) of AoMae; Alternatively, the following mutations occur on the basis of the wild-type C4-dicarboxylate transporter: lysine K at positions 260, 331, and 339 are simultaneously mutated to arginine R; and the wild-type C4-dicarboxylate transporter is derived from Aspergillus oryzae ( Aspergillus oryzae ) of AoMae.

2. The C4-dicarboxylic acid transporter mutant according to claim 1, wherein The wild-type C4-dicarboxylate transporter is derived from Aspergillus oryzae ( Aspergillus oryzae ) of AoMae.

3. A polynucleotide encoding the C4-dicarboxylic acid transporter mutant according to claim 1 or 2.

4. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the polynucleotide of the C4-dicarboxylic acid transporter mutant according to claim 3.

5. Use of the C4-dicarboxylic acid transporter mutant according to claim 1, or its encoding polynucleotide, or a recombinant vector, expression cassette, transgenic cell line and / or recombinant bacteria containing the encoding nucleotide in the preparation of malic acid and / or succinic acid.

6. The use according to claim 5, characterized in that The invention is used for increasing the yield of malic acid or succinic acid and / or increasing the purity of malic acid or succinic acid.

7. The use according to claim 6, characterized in that Application of mutants in which threonine T at position 100 is mutated to alanine A, or lysine K at positions 260, 331, and 339 are simultaneously mutated to arginine R, in improving malic acid production; Use of mutants in which the threonine at position 100 is mutated to cysteine C, glycine G, glutamic acid E, isoleucine I, lysine K, arginine R, or tryptophan W, or the arginine R at position 78 is mutated to aspartic acid D, or the leucine L at position 85 is mutated to methionine M, in improving the purity of malic acid; Application of mutants in improving the purity of succinic acid, wherein the threonine T at position 26 is mutated to glutamic acid E, or the arginine R at position 78 is mutated to lysine K, threonine T, tryptophan W, phenylalanine F, histidine H, glutamic acid E, aspartic acid N, cysteine C, glutamine Q, methionine M, tyrosine Y, or the threonine at position 100 is mutated to methionine M, histidine H, tyrosine Y, valine V, or the tryptophan W at position 312 is mutated to alanine A, the alanine at position 314 is mutated to histidine H, or the proline P at position 318 is mutated to glycine G.

8. A method for producing malic acid and / or succinic acid, which is achieved by fermenting a transformed strain containing a recombinant expression vector containing the polynucleotide according to claim 3.

9. The method according to claim 8, wherein The process also includes the steps of collecting or purifying malic acid and / or succinic acid.

10. The method according to claim 8 or 9, characterized in that The strain is Myceliophthora thermophila.

11. The method according to claim 9, wherein Malic acid and / or succinic acid are collected or purified from the fermentation broth.

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