A malate transporter mutant and its application

Abstract

The invention provides a malate transporter mutant, a genetically engineered bacterium expressing the mutant and applications thereof.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a malate transporter mutant and application thereof. Background Art

[0002] Organic acids, natural metabolites of organisms, can be converted into a variety of valuable products, such as food, medicine, cosmetics, detergents, polymers and textiles. Among them, L-malic acid, as an intermediate of microbial metabolism and a new generation of acidulants and flavor enhancers, is widely used in the food and beverage industry. In recent years, with the continuous development and progress of microbial transformation technology, people are increasingly using microbial fermentation to produce L-malic acid.

[0003] In the process of producing L-malic acid by microbial fermentation, because its products include carboxyl (-COOH) groups, it is very easy to form a low pH, which seriously inhibits the metabolic activity and production efficiency of cells. At present, the main strategy to solve such key problems is to add neutralizers (calcium carbonate CaCO3, magnesium carbonate MgCO3, sodium hydroxide NaOH or ammonia NH3·H2O, etc.), and the products formed exist in the form of malate. Subsequently, the malate is purified under the premise of acid hydrolysis with HCl or H2SO4 to obtain higher purity L-malic acid. The acid hydrolysis process not only complicates the downstream purification process, but also easily causes environmental pollution. Therefore, the potential of microbial L-malic acid production has not yet been fully realized, and the low pH formed by the intermediate product is the main bottleneck of industrial-scale production. In addition, the low efficiency of L-malic acid synthesis is also a problem that needs to be solved in the production of L-malic acid by microbial fermentation.

[0004] In addition, in order to further realize the cell factory with high yield of L-malic acid, transport is another key factor. In known reports, the proteins with malic acid transport function mainly include two types: SpMAE1 derived from Schizosaccharomyces pombe and tetracarboxylic acid transporter C4T318 derived from Aspergillus oryzae. Since C4T318 is mostly used in molds and has the transport function for most tetracarboxylic acids, some other by-products of tetracarboxylic acid may be transported during the production of malic acid. Therefore, SpMAE1 is mostly selected in the yeast fermentation process. However, the preliminary study of the present invention found that after overexpressing SpMAE1, the concentration of L-malic acid in yeast cells was still only about 11.2g / L.

[0005] Chinese Patent No. ZL202110494752.8 constructed recombinant brewer's yeast engineered bacteria CTPM1, CTPM2 and CTPM3, but there is still the problem of low L-malic acid fermentation yield (the L-malic acid yields were 3.9 g / L, 8.6 g / L and 16.5 g / L, respectively, under 250 ml shake flask culture).

[0006] Therefore, there is still a need in the art to construct a recombinant acid-tolerant Saccharomyces cerevisiae engineered bacterium that produces higher yields of L-malic acid during fermentation. Summary of the invention

[0007] In order to solve the above problems, in a first aspect, the present invention aims to provide a malate transporter mutant (SpMAE1-F253A), wherein the mutant has a mutation site F253A.

[0008] In a specific embodiment, the amino acid sequence of the mutant of the present invention is shown as SEQ ID No.1, and more specifically, the amino acid sequence of the wild-type malate transporter (SpMAE1) (from Schizosaccharomyces pombe) before mutation relative to the mutant is shown as SEQ ID No.2.

[0009] In a second aspect, the present invention aims to provide a genetically engineered bacterium, wherein the genetically engineered bacterium expresses the malate transporter mutant as described in the first aspect.

[0010] In a preferred embodiment, the malate transporter mutant is expressed by a constitutive promoter in the genetically engineered bacteria of the present invention.

[0011] In a specific embodiment, one or more of L-lactate dehydrogenase, pyruvate kinase, malate dehydrogenase and pyruvate carboxylase are overexpressed in the genetically engineered bacteria of the present invention. In a preferred embodiment, one or more of L-lactate dehydrogenase, pyruvate kinase, malate dehydrogenase and pyruvate carboxylase are expressed by a constitutive promoter in the genetically engineered bacteria of the present invention.

[0012] In a specific embodiment, two or more pyruvate carboxylases are overexpressed in the genetically engineered bacteria of the present invention; in a preferred embodiment, pyruvate carboxylase 1 having an amino acid sequence as shown in SEQ ID No.3 and pyruvate carboxylase 2 having an amino acid sequence as shown in SEQ ID No.4 are overexpressed in the genetically engineered bacteria.

[0013] In a specific embodiment, two or more malate dehydrogenases are overexpressed in the genetically engineered bacteria of the present invention; in a preferred embodiment, malate dehydrogenase 1 having an amino acid sequence as shown in SEQ ID No.5 and malate dehydrogenase 3 having an amino acid sequence as shown in SEQ ID No.6 are overexpressed in the genetically engineered bacteria.

[0014] In a specific embodiment, the amino acid sequence of the L-lactate dehydrogenase in the genetically engineered bacteria of the present invention is shown as SEQ ID No.7 and / or the amino acid sequence of the pyruvate kinase is shown as SEQ ID No.8.

[0015] In a specific embodiment, the expression of pyruvate decarboxylase and / or alcohol dehydrogenase is knocked down in the genetically engineered bacteria of the present invention; in a preferred embodiment, the amino acid sequence of the pyruvate decarboxylase is shown in SEQ ID No.9 and / or the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID No.10.

[0016] In a specific embodiment, the genetically engineered bacteria of the present invention is an engineered yeast of Saccharomyces cerevisiae. In a preferred embodiment, the Saccharomyces cerevisiae is an acid-resistant Saccharomyces cerevisiae.

[0017] In a third aspect, the present invention aims to provide use of the malate transporter mutant in the first aspect and the genetically engineered bacteria in the second aspect in fermentation production of L-malic acid.

[0018] Compared with the prior art, the above technical solution of the present invention has the following advantages:

[0019] (1) Compared with the original wild-type malate transporter, the malate transporter mutant of the present invention can significantly improve the transport efficiency of the malate transporter, promote the output of L-malic acid, and thus increase the yield of L-malic acid in the fermentation broth supernatant.

[0020] (2) The genetically engineered bacteria of the present invention have been modified in terms of increasing the supply of key precursor pyruvate, weakening the metabolic flux of the competitive ethanol synthesis pathway, and introducing a heterologous synthesis pathway. The L-malic acid production of the modified engineered bacteria has been significantly improved. Without adding any neutralizing agent, the L-malic acid production of the brewer's yeast engineered bacteria of the present invention can reach a maximum of 202.1 g / L, the conversion rate is 1.2 mol / mol, and the production intensity is 1.46 g / L / h. In addition, the inhibition of low pH of the product on cell production and metabolism is overcome during the production process, thereby realizing "green production" without neutralizing agents to a large extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is the result of PCR verification of the engineered bacterial strains CTPM3 and CTPM4.

[0022] FIG. 2 is the result of PCR verification of the genetically engineered bacterial strain CTPM5 of the present invention; wherein Figure 2A is the result of PCR verification of the nucleotide sequence encoding pyruvate kinase. Figure 2B It is the result of PCR verification of the nucleotide sequence encoding L-lactate dehydrogenase.

[0023] FIG3 is the result of PCR verification of the genetically engineered bacterial strain CTPM6 of the present invention; wherein Figure 3A is the result of PCR verification of the nucleotide sequence encoding pyruvate decarboxylase, Figure 3B It is the result of PCR verification of the nucleotide sequence encoding alcohol dehydrogenase.

[0024] Figure 4 This is the result of PCR verification of the genetically engineered bacterial strain CTPM7 of the present invention.

[0025] Figure 5 It is a curve chart showing the changes of L-malic acid and pH in the fermentation broth over time when the genetically engineered bacterial strain CTPM7 of the present invention is cultured in a 3L fermenter. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0027] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0028] The technical solution of the present invention is further illustrated below through specific implementation methods.

[0029] Example 1: Effect of malate transporter mutants on fermentation production of L-malic acid

[0030] First, the acid-tolerant Saccharomyces cerevisiae strain CTPth-2 (deposited in the China Center for Type Culture Collection, with the accession number CCTCC M 2021258) was obtained through directional screening. This strain was used as the starting strain and the Loxp homologous recombination method was used. The genomic integration site was 416d to construct a recombinant Saccharomyces cerevisiae engineered bacterium CTPM2 (see Chinese Patent No. ZL202110494752.8) that overexpresses pyruvate carboxylase 2 (PYC2, amino acid sequence SEQ ID No. 4, from Saccharomyces cerevisiae strain, Saccharomyces cerevisiae) and malate dehydrogenase 3 (MDH3, amino acid sequence SEQ ID No. 6, from Saccharomyces cerevisiae strain, Saccharomyces cerevisiae S288C).

[0031] (1) Construction of acid-tolerant Saccharomyces cerevisiae engineered strains CTPM3 and CTPM4

[0032] (a) Chemically synthesized nucleotide sequences encoding the wild-type malate transporter SpMAE1 (sequence: SEQ ID No. 11), the nucleotide sequence encoding the malate transporter mutant SpMAE1-F253A (sequence: SEQ ID No. 12), and the nucleotide sequence encoding the amino acid Leu tag (sequence: SEQ ID No. 13).

[0033] (b) preparing a competent state of the engineered yeast Saccharomyces cerevisiae CTPM2, and respectively integrating the nucleotide sequence encoding the malate transporter SpMAE1 and the amino acid Leu tag and the nucleotide sequence encoding the malate transporter mutant SpMAE1-F253A and the amino acid Leu tag obtained in step (a) into the CTPM2 competent cells (the strain integrating the transporter SpMAE1 and the amino acid Leu tag was named CTPM3, and the engineered strain integrating the transporter mutant SpMAE1-F253A and the amino acid Leu tag was named CTPM4) by using the Loxp homologous recombination method, and the constructed strain cells were spread on the SD LEU screening solid plate, and after a single colony grew on the plate, PCR verification was performed using primers Y-Spmae1-F (sequence is SEQ ID No. 14) and Y-Spmae1-R (sequence is SEQ ID No. 15), and the verification results were as follows: Figure 1 shown.

[0034] (c) The single colony verified by PCR in step (b) was inoculated into YPD liquid culture medium and cultured for 16 hours, then streaked onto a YPD solid plate containing 5-FOA and cultured at 30°C for 3 days. The grown single colonies were then transferred to YPD solid plates and SD Ura screening solid plates for comparison and verification. The single colony that grew normally on the YPD plate but could not grow on the SD Ura screening plate was the correct genetically engineered bacterium.

[0035] (2) Comparison of L-malic acid fermentation culture

[0036] The engineered bacterial strains CTPM3 and CTPM4 obtained above were respectively cultured in 250 mL shake flasks, and the L-malic acid content in the fermentation supernatant was detected by high performance liquid chromatography (HPLC). The required culture medium, culture conditions and detection method are as follows:

[0037] Seed liquid culture

[0038] Bacterial volume: Pick a single colony on the YEPD solid plate, inoculate the engineered strain into a 50mL shaking tube with a liquid volume of 7.5mL, and culture in YPD medium.

[0039] Culture medium formula: YPD medium, glucose 20g / L, trypsin 20g / L, yeast powder 10g / L.

[0040] Culture conditions: 30°C, 220 rpm, culture for 30-36 hours until the logarithmic growth phase.

[0041] 250mL shake flask fermentation

[0042] Bacterial volume: Take 1.5mL of seed liquid and put it in a 250mL baffled shake flask, filling the bottle with 50mL of liquid.

[0043] Culture medium formula: sucrose 100 g / L, soy peptone 15 g / L, sodium glutamate 2 g / L, (NH4)2SO4 15 g / L, KH2PO4, ZnSO4·7H2O 0.72 g / L, MgSO4·7H2O 6 g / L, methionine 0.15 g / L.

[0044] Culture conditions: 250 mL baffled shake flask, 50 mL of liquid, 30°C, 220 rpm.

[0045] Method for detecting L-malic acid by high performance liquid chromatography (HPLC)

[0046] Detection method: At the end of fermentation, the L-malic acid titer in the supernatant was determined by high performance liquid chromatography using an Agilent 1260 series binary liquid chromatography system equipped with an HPX-87H column (Bio-Rad, Hercules, CA, USA), and 5 mM H2SO4 was used as the mobile phase. Approximately 1 mL of fermentation broth was collected at a flow rate of 0.6 mL / min at 40°C, centrifuged at 12,000 rpm for 10 min, and the supernatant was collected to determine the extracellular concentration of L-malic acid using HPLC.

[0047] The results showed that the L-malic acid production in the supernatant of the fermentation broth of strains CTPM3 and CTPM4 after 250 mL shake flask fermentation was 16.5 g / L and 42.1 g / L, respectively.

[0048] Example 2: Method for constructing the genetically engineered bacteria of the present invention

[0049] (1) Construction of acid-tolerant Saccharomyces cerevisiae engineered strain CTPM5

[0050] (a) Chemically synthesize a nucleotide sequence encoding pyruvate kinase PYK (SEQ ID No. 16, from Saccharomyces cerevisiae) and a nucleotide sequence encoding an amino acid His tag (SEQ ID No. 17), a nucleotide sequence encoding L-lactate dehydrogenase (SEQ ID No. 18, from Saccharomyces cerevisiae) and a nucleotide sequence encoding an amino acid Leu tag (SEQ ID No. 13). The primer sequences are as follows:

[0051] (b) preparing a competent cell of Saccharomyces cerevisiae engineered bacteria CTPM4, and integrating the nucleotide sequence encoding pyruvate kinase and an amino acid His tag and the nucleotide sequence encoding L-lactate dehydrogenase CYB and an amino acid tag Leu obtained in (a) into the competent cells of CTPM4 using the Loxp homologous recombination method, with the integration site being 1622b. The constructed strain cells were spread on a SDHIS+LEU screening solid plate, and after a single colony grew on the plate, PCR verification was performed using primers Y-PYK-F (sequence: SEQ ID No. 19) and Y-PYK-R (sequence: SEQ ID No. 20), Y-CYB-F1 (sequence: SEQ ID No. 21) and Y-CYB-R1 (sequence: SEQ ID No. 22). The verification results are as follows: Figure 2A and B as shown.

[0052] (c) The single colony verified by PCR in step (b) was inoculated into YPD liquid culture medium and cultured for 16 hours, then streaked onto YPD solid plates containing 5-FOA and cultured at 30°C for 3 days. The grown single colonies were then transferred to YPD solid plates and SD Ura screening solid plates for comparison and verification. The single colony that grew normally on the YPD plate but could not grow on the SD Ura screening plate was the correct genetically engineered bacterium and was named CTPM5.

[0053] The engineered bacterial strain CTPM5 obtained above was fermented in a 250 mL shake flask, and the L-malic acid content in the fermentation supernatant was detected by high performance liquid chromatography (HPLC) to be 51 g / L. The required culture medium, culture conditions and detection method are as described in Example 1.

[0054] (2) Construction of acid-tolerant Saccharomyces cerevisiae engineered strain CTPM6

[0055] (a) Chemically synthesize a nucleotide sequence encoding pyruvate decarboxylase PDC6 (SEQ ID No. 23, from Saccharomyces cerevisiae) and a nucleotide encoding an amino acid Leu tag (SEQ ID No. 13), a nucleotide sequence encoding alcohol dehydrogenase ADH1 (SEQ ID No. 25, from Saccharomyces cerevisiae) and a nucleotide encoding an amino acid His tag (SEQ ID No. 17).

[0056] (b) The competent state of the engineered yeast Saccharomyces cerevisiae CTPM5 was prepared, and the Loxp insertion method was used to homologously replace the original pyruvate decarboxylase PDC6 and alcohol dehydrogenase ADH1 in the strain genome (the Loxp sequences were all SEQ ID No. 24) to knock out the pyruvate decarboxylase and alcohol dehydrogenase in CTPM5. The constructed strain cells were spread on the SD HIS screening solid plate. After a single colony grew on the plate, PCR verification was performed using primers Y-PDC6-F (sequence SEQ ID No. 26), Y-PDC6-R (sequence SEQ ID No. 27) and Y-adh1-F (sequence SEQ ID No. 28), Y-adh1-R (sequence SEQ ID No. 29), respectively. The verification results were as follows: Figure 3A and B as shown.

[0057] (c) The single colony verified by PCR in step (b) was inoculated into YPD liquid culture medium and cultured for 16 hours, then streaked onto YPD solid plates containing 5-FOA and cultured at 30°C for 3 days. The grown single colonies were then transferred to YPD solid plates and SD Ura screening solid plates for comparison and verification. The single colony that grew normally on the YPD plate but could not grow on the SD Ura screening plate was the correct genetically engineered bacterium and was named CTPM6.

[0058] The engineered bacterial strain CTPM6 obtained above was fermented in a 250 mL shake flask, and the L-malic acid content in the fermentation supernatant was detected by high performance liquid chromatography (HPLC) to be 62 g / L. The required culture medium, culture conditions and detection method are as described in Example 1.

[0059] (3) Construction of acid-tolerant Saccharomyces cerevisiae engineered strain CTPM7

[0060] (a) Chemically synthesize nucleotide sequences encoding malate dehydrogenase 1 (MDH1) (SEQ ID No. 30, from Aspergillus oryzae), pyruvate carboxylase 1 (PYC1) (SEQ ID No. 31, from Aspergillus oryzae) and nucleotides encoding an amino acid His tag (SEQ ID No. 17).

[0061] (b) preparing a competent cell of Saccharomyces cerevisiae engineered bacteria CTPM6, and integrating the coding nucleotide sequences of malate dehydrogenase, pyruvate dehydrogenase 1 and amino acid His tag obtained in (a) into the genome 208a site by using the Loxp homologous recombination method, and integrating them into the competent cells of CTPM6, and spreading the constructed strain cells on a SD HIS screening solid plate, and after a single colony grows on the plate, using primers Y-Aor-mdh-F1 (sequence is SEQ ID No.32), Y-Aor-mdh-R1 (sequence is SEQ ID No.33) and Y-Aor-pyc-F2 (sequence is SEQ ID No.34), Y-Aor-pyc-R2 (sequence is SEQ ID No.35) for PCR verification, and the verification results are as follows: Figure 4 .

[0062] (c) The single colony verified by PCR in step (b) was inoculated into YPD liquid culture medium and cultured for 16 hours, then streaked onto YPD solid plates containing 5-FOA and cultured at 30°C for 3 days. The grown single colonies were then transferred to YPD solid plates and SD Ura screening solid plates for comparison and verification. The single colony that grew normally on the YPD plate but could not grow on the SD Ura screening plate was the correct genetically engineered bacterium and was named CTPM7.

[0063] The engineered bacterial strain CTPM7 obtained above was fermented in a 250 mL shake flask, and the L-malic acid content in the fermentation supernatant was detected by high performance liquid chromatography (HPLC), which was 76 g / L. The culture medium, culture conditions and detection method required for the 250 mL shake flask fermentation culture are as described in Example 1.

[0064] The engineered bacterial strain CTPM7 obtained above was also cultured in a 3-L fermenter. The culture medium and culture conditions required for the 3-L fermenter culture are as follows, and samples were taken at regular intervals to detect the pH value and L-malic acid content of the fermentation broth supernatant (see Figure 5 ).

[0065] 3-L fermenter culture

[0066] Seed volume: Take 120 mL of seed liquid and put it in a 3-L fermenter with a liquid volume of 1.2 L.

[0067] Culture medium formula: sucrose 60 g / L, soy peptone 35 g / L, sodium glutamate 2 g / L, (NH4)2SO4 15 g / L, KH2PO4, ZnSO4·7H2O 0.72 g / L, MgSO4·7H2O 6 g / L, methionine 0.15 g / L.

[0068] Culture conditions: 3-L fermenter, 1.2 L liquid volume, 30 °C culture, 2 v / v / m aeration, carbon source flow supplement, concentration controlled at about 20 g / L.

[0069] The results showed that during the fermentation process without adding neutralizer, the pH of the fermentation liquid dropped below 3.0, the production of engineered bacteria was not inhibited, and the content of L-malic acid continued to increase steadily, with the final content being 202.1 g / L.

[0070] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in the present application.

Claims

1. A malate transporter mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID No. 1, and the mutant has a mutation site F253A.

2. A method for preparing a genetically engineered bacterium, characterized in that: The steps include: The acid-tolerant Saccharomyces cerevisiae strain CTPth-2 was used as a starting strain, wherein the acid-tolerant Saccharomyces cerevisiae strain CTPth-2 was deposited in China Center for Type Culture Collection with the deposit number CCTCC M 2021258, and a recombinant Saccharomyces cerevisiae engineering strain CTPM2 overexpressing pyruvate carboxylase 2 shown in amino acid sequence SEQ ID No. 4 and malate dehydrogenase 3 shown in amino acid sequence SEQ ID No. 6 was constructed; Based on the nucleotide sequence of SEQ ID No. 12 that can encode the malate transporter mutant SpMAE1-F253A integrated into CTPM2, a recombinant Saccharomyces cerevisiae CTPM4 that can integrate and transport the malate transporter mutant SpMAE1-F253A of the amino acid sequence of claim 1 as shown in SEQ ID No. 1 is obtained; Based on the integration of CTPM4, the nucleotide sequence encoding pyruvate kinase as shown in SEQ ID No.16 and the nucleotide sequence encoding L-lactate dehydrogenase as shown in SEQ ID No.18 were obtained to obtain a recombinant Saccharomyces cerevisiae CTPM5 capable of integrating and transporting pyruvate kinase and L-lactate dehydrogenase; Based on the knockout of CTPM5, the sequence encoding pyruvate decarboxylase is the nucleotide sequence shown in SEQ ID No. 23 and the sequence encoding alcohol dehydrogenase 1 is the nucleotide sequence shown in SEQ ID No. 25, thereby obtaining recombinant Saccharomyces cerevisiae CTPM6; Based on CTPM6, the nucleotide sequence encoding malate dehydrogenase 1 as shown in SEQ ID No.30 and the nucleotide sequence encoding pyruvate carboxylase 1 as shown in SEQ ID No.31 were integrated to obtain recombinant Saccharomyces cerevisiae CTPM7 capable of integrating and transporting malate dehydrogenase 1 and pyruvate carboxylase 1.

3. A genetically engineered bacterium prepared by the preparation method according to claim 2, characterized in that: The genetically engineered bacteria is at least one of recombinant Saccharomyces cerevisiae CTPM4, recombinant Saccharomyces cerevisiae CTPM5, recombinant Saccharomyces cerevisiae CTPM6, and recombinant Saccharomyces cerevisiae CTPM7, and the recombinant Saccharomyces cerevisiae CTPM4 can express the malate transporter mutant according to claim 1.

4. The genetically engineered bacteria according to claim 2, characterized in that: Recombinant Saccharomyces cerevisiae CTPM5 overexpressed L-lactate dehydrogenase and pyruvate kinase, and recombinant Saccharomyces cerevisiae CTPM7 overexpressed malate dehydrogenase 1 and pyruvate carboxylase 1.

5. The genetically engineered bacterium according to claim 3, characterized in that: The malate transporter mutant is expressed in the genetically engineered bacteria using a constitutive promoter.

6. The genetically engineered bacterium according to claim 4, characterized in that: The recombinant Saccharomyces cerevisiae CTPM5 expresses the L-lactate dehydrogenase and the pyruvate kinase using a constitutive promoter, and the recombinant Saccharomyces cerevisiae CTPM7 overexpresses the malate dehydrogenase 1 and the pyruvate carboxylase 1.

7. The genetically engineered bacteria according to claim 2, characterized in that: The recombinant Saccharomyces cerevisiae CTPM7 overexpresses pyruvate carboxylase 1 whose amino acid sequence is shown in SEQ ID No.3, and the recombinant Saccharomyces cerevisiae engineered bacteria CTPM2 overexpresses pyruvate carboxylase 2 whose amino acid sequence is shown in SEQ ID No.

4.

8. The genetically engineered bacteria according to claim 2, characterized in that: The recombinant Saccharomyces cerevisiae CTPM7 overexpresses malate dehydrogenase 1 whose amino acid sequence is shown in SEQ ID No.5, and the recombinant Saccharomyces cerevisiae engineered bacteria CTPM2 overexpresses malate dehydrogenase 3 whose amino acid sequence is shown in SEQ ID No.

6.

9. The genetically engineered bacterium according to claim 2, characterized in that: The amino acid sequence of the L-lactate dehydrogenase is shown in SEQ ID No.7 and / or the amino acid sequence of the pyruvate kinase is shown in SEQ ID No.

8.

10. The genetically engineered bacterium according to claim 2, characterized in that: The amino acid sequence of the pyruvate decarboxylase is shown in SEQ ID No.9 and / or the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID No.

10.

11. The genetically engineered bacterium according to any one of claims 3 to 8, characterized in that: The brewer's yeast is acid-resistant brewer's yeast.

12. Use of the malate transporter mutant according to claim 1, or the preparation method according to claim 2, or the genetically engineered bacteria according to any one of claims 3 to 11 in fermentation production of L-malic acid.

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