A yeast site-directed mutant gene THI5 M , recombinant vector, recombinant bacteria and application thereof

Abstract

The application provides a yeast site-directed mutant gene THI5 M , a recombinant vector, a recombinant bacterium and application thereof, and belongs to the technical field of bioengineering. The nucleotide sequence of the yeast site-directed mutant gene THI5 M of the application is shown in SEQ ID NO. 1, the amino acid sequence encoded by the sequence is shown in SEQ ID NO. 2, and the amino acid sequence is obtained by mutating alanine at the 70th position of the amino acid encoded by the THI5 gene into proline and mutating cysteine at the 196th position into tyrosine. In the application, the site-directed mutation technology is used to mutate alanine at the 70th position of the THI5 gene in the BY4741 strain into proline and mutate cysteine at the 196th position into tyrosine. The acid resistance performance of the site-directed mutant strain DS-2 is obviously improved, and the strain can be used as a potential chassis microorganism for fermenting short-chain organic acids, and is especially suitable for the late-stage modification and production of L-malic acid, lactic acid and pyruvic acid.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a yeast site-directed mutation gene THI5. M Recombinant vectors, recombinant bacteria and their applications. Background Technology

[0002] Saccharomyces cerevisiae, as an important model organism, was the first eukaryote to have its entire genome sequenced. This achievement accelerated the process of gene identification and homology comparison in Saccharomyces cerevisiae and forms the basis for studying gene function in other species. Currently, research on the gene function of Saccharomyces cerevisiae has a complete set of mature gene manipulation techniques.

[0003] In recent years, with the rapid development of synthetic biology, the production efficiency of Saccharomyces cerevisiae as a platform cell factory has been greatly improved. It is now widely used in the large-scale production of various food, chemical products and pharmaceuticals. Since Saccharomyces cerevisiae does not produce a large amount of organic acids and has low nutritional requirements, it is more acid-resistant than commonly used prokaryotes. Its development as a chassis organism for organic acid products has attracted much attention.

[0004] Acid stress is the most common abiotic stress encountered by yeast cells during the fermentation of acidic products such as organic acids. The most direct impact of weak organic acids on *Saccharomyces cerevisiae* is the inhibition of yeast growth and metabolic functions during fermentation. To alleviate the inhibitory effects of acid stress on cell growth and product synthesis, large amounts of neutralizing agents are often added during production, which not only increases costs and complicates downstream purification processes but also easily causes environmental pollution. Improving the acid tolerance of strains and elucidating the molecular mechanisms of *Saccharomyces cerevisiae*'s tolerance to weak acid stress are key steps in the rational design of fermentation conditions and the engineering of more robust industrial yeast strains. Acid stress is the most common abiotic stress encountered by yeast cells during the fermentation of acidic products such as organic acids, severely affecting cell growth and metabolism. To alleviate the inhibitory effects of acid stress on cell growth and product synthesis, large amounts of neutralizing agents are often added during production, which not only increases costs and complicates downstream purification processes but also easily causes environmental pollution. Studying acid tolerance mechanisms to obtain acid-tolerant *Saccharomyces cerevisiae* can effectively solve this problem.

[0005] Extensive functional genomics research has explored the mechanisms by which Saccharomyces cerevisiae responds to environmental stress. To adapt to acidic environments, Saccharomyces cerevisiae exhibits corresponding stress responses. Under acid stress, it regulates gene expression and metabolism through signaling molecules and signal transduction pathways. Comparison of genomes among different strains can effectively identify variant sequences leading to phenotypic differences, thus facilitating the genetic modification of industrial strains to obtain desired traits. However, further research is needed to identify key factors for cellular acid tolerance and construct stable, high-yield, energy-efficient, and easy-to-use acid-tolerant Saccharomyces cerevisiae. Nevertheless, no studies have been reported on the impact of THI5 gene mutations on acid-tolerant yeast fermentation. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a site-directed mutant gene THI5 in yeast. M Recombinant vectors, recombinant strains and their applications: Saccharomyces cerevisiae containing this mutant gene can ferment in an acidic environment, and compared with the standard Saccharomyces cerevisiae strain, it can also ferment to produce L-malic acid, lactic acid and pyruvic acid.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides a site-directed mutant gene THI5 in yeast. M The yeast site-directed mutant gene THI5 M The nucleotide sequence is shown in SEQ ID NO.1.

[0008] Preferably, the yeast site-directed mutation gene THI5 M The amino acid sequence is shown in SEQ ID NO.2.

[0009] This invention provides a recombinant vector containing the yeast site-directed mutant gene THI5. M .

[0010] This invention provides a recombinant strain containing the yeast site-directed mutant gene THI5. M Or the recombinant vector.

[0011] Preferably, the starting strain of the recombinant bacteria includes Saccharomyces cerevisiae BY4741.

[0012] This invention also provides the yeast site-directed mutant gene THI5. M The application of the recombinant vector or the recombinant bacteria in acidic fermentation.

[0013] Preferably, the pH value of the acidic environment is 2.3-3.5.

[0014] Preferably, the acidic environment includes one or more of L-malic acid, citric acid, lactic acid, succinic acid, pyruvic acid, and acetic acid.

[0015] This invention provides the yeast site-directed mutant gene THI5. M The application of the recombinant vector or the recombinant bacteria in the production of organic acids.

[0016] Preferably, the organic acid includes one or more of L-malic acid, lactic acid, and pyruvic acid.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a site-directed mutant gene THI5 in yeast. M Recombinant vectors, recombinant strains, and their applications: Ultraviolet irradiation of *Saccharomyces cerevisiae* BY4741 was used to induce mutagenesis. Flow cytometry sorting yielded strain DS-1, which exhibited significantly improved acid tolerance. Genome sequencing revealed a mutation in its THI5 gene, where alanine at position 70 (A, Ala) was replaced by proline (P, Pro). Cysteine ​​at position 196 (C, Cys) was replaced by tyrosine (Y, Tyr). Using site-directed mutagenesis, alanine at position 70 (A, Ala) in strain BY4741 was mutated to proline (P, Pro), and cysteine ​​at position 196 (C, Cys) was mutated to tyrosine (Y, Tyr). The site-directed mutagenesis strain DS-2 showed significantly improved acid tolerance and can serve as a potential chassis microorganism for fermenting short-chain organic acids, particularly suitable for the later-stage modification and production of L-malic acid, lactic acid, and pyruvic acid. Attached Figure Description

[0018] Figure 1 This is a gel electrophoresis image of THI5 gene amplification.

[0019] Figure 2 The spectrum of the PESC-TEFI-HIS vector.

[0020] Figure 3 The spectrum of the PESC-THI5 vector.

[0021] Figure 4 This is a sequencing verification diagram of a positive clone.

[0022] Figure 5 OD values ​​of BY4741 and DS-2 strains in YPD medium and YPD medium at different pH values. 600 Condition. Detailed Implementation

[0023] This invention provides a site-directed mutant gene THI5 in yeast. M The yeast site-directed mutant gene THI5 MThe nucleotide sequence is shown in SEQ ID NO.1. The mutant gene THI5 described in this invention... M In the THI5 gene, GCC is mutated to CCC at positions 208-210, and TGT is mutated to TAT at positions 586-588. The amino acid sequence encoded by the nucleotide sequence of the present invention is shown in SEQ ID NO. 2, wherein the amino acid sequence in the THI5 gene is mutated at position 70 (alanine, Ala) to proline (P, Pro) and position 196 (cysteine, C, Cys) to tyrosine (Y, Tyr).

[0024] The present invention also provides a recombinant vector containing the yeast site-directed mutant gene THI5. M The recombinant vector used in this invention is PESC-TEFI-HIS, synthesized by Genscript Biotech.

[0025] The present invention also provides a recombinant strain containing the yeast site-directed mutant gene THI5. M Or the recombinant vector. The starting strains of the recombinant bacteria of the present invention include, but are not limited to, Saccharomyces cerevisiae BY4741.

[0026] This invention also provides the yeast site-directed mutant gene THI5. M The application of the recombinant vector or the recombinant bacteria in acidic fermentation. The pH of the acidic environment described in this invention is 2.3-3.5. The acidic environment described in this invention includes one or more of L-malic acid, citric acid, lactic acid, succinic acid, pyruvic acid, and acetic acid. The yeast site-directed mutagenesis gene THI5 described in this invention... M It can improve the acid resistance of brewing yeast.

[0027] This invention also provides the yeast site-directed mutant gene THI5. M The application of the recombinant vector or the recombinant bacteria in the production of organic acids. The organic acids described in this invention include one or more of L-malic acid, acetone, and lactic acid.

[0028] In this invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0029] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0030] Example 1: Screening for Mutant Genes brewing yeast ( Saccharomyces cerevisiae The culture medium (BY4741 Beijing Coolplay Technology Co., Ltd.) was cultured in YPD liquid medium (Qingdao Haibo Biotechnology) to obtain the logarithmic phase culture medium; the logarithmic phase culture medium was centrifuged, the supernatant was discarded, and the bacterial cells were obtained. The bacterial cells were then subjected to 10 liters of physiological saline. -2 10 -3 10 -4 10 -5 10 -6 Serial dilutions were performed to obtain dilutions of different factors. 100 μL of each dilution was plated onto YPD solid medium (Qingdao Haibo Biotechnology) plates and incubated upside down at 30°C. The dilution gradient was determined based on colony counts ranging from 100 to 300 for subsequent experiments. The determined dilution gradient was 10-1. -4 .

[0031] Preheat the UV lamp for 30 minutes, then take 3 mL of the solution and dilute it at a gradient of 10. -4 The bacterial suspension was placed in 60mm petri dishes and subjected to UV treatment for 0s, 30s, 60s, 90s, 120s, and 150s, with three parallel experiments for each treatment time. 100μL of the mutagenized bacterial suspension was then evenly spread onto YPD solid medium plates and incubated upside down at 30℃ for 24h. Colony counting was performed, and the UV treatment time with a lethality rate of 90%-95% was used as the subsequent UV mutagenesis treatment time; the determined UV mutagenesis time was 120s.

[0032] Using the UV mutagenesis time determined above, UV irradiation was applied to *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae Mutagenesis was performed on the original strain (BY4741) to obtain a mutant strain. An acid tolerance test was then conducted on the obtained mutant strain: the original strain and the mutant strain were cultured separately in media with different pH values, and the results were determined based on OD... 600nm The acid resistance of strain DS-1 was significantly improved. Genome sequencing of strain DS-1 revealed a mutation in its THI5 gene, in which alanine at position 70 (A, Ala) was replaced by proline (P, Pro), and cysteine ​​at position 196 (C, Cys) was replaced by tyrosine (Y, Tyr).

[0033] Example 2: THI5 sequence amplification 1. DNA extraction: Saccharomyces cerevisiae was streaked onto YPD solid medium (Qingdao Haibo Biotechnology) for culture. Saccharomyces cerevisiae (BY4741 Beijing Cooler Technology Co., Ltd.), single clones were selected, inoculated into YPD liquid medium, and cultured for 24 hours. Then, yeast genomic DNA was extracted using the yeast genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0034] 2. Primer Design: Based on the whole genome data of strain S288C from the National Center for Biotechnology Information (NCBI) database, primers THI5-F and THI5-R were designed to amplify the THI5 gene (simultaneously introducing the restriction enzyme sites NotI and SacI). The THI5 gene primers are as follows: THI5-F: nucleotide sequence as shown in SEQ ID NO.3 of the sequence listing; THI5-R: nucleotide sequence as shown in SEQ ID NO.4 of the sequence listing.

[0035] 3. PCR Amplification: Using the extracted genomic DNA from strain S288C as a template, the THI5 gene was amplified using primers THI5-F and THI5-R. The PCR amplification reaction system was as follows: total volume 50 μL, DNA template 2 μL, THI5-F and THI5-R primers 1 μL each, dNTPs 4 μL, PCR buffer (with MgSO4) 2+ 5 μL of Taq polymerase, 0.25 μL of Taq polymerase, and 6.75 μL of ddH2O were added. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, 35 cycles (94℃ denaturation for 30 s, 65℃ annealing for 35 s and 72℃ extension for 2 min), followed by a final extension at 72℃ for 10 min, and storage at 4℃. The amplified sequence was verified by agarose gel electrophoresis. Two samples were tested in parallel. The resulting gel bands were located between 1000-1500 bp, closer to 1000 bp, which is basically consistent with the 1037 bp of the THI5 gene (e.g., ...). Figure 1 (As shown).

[0036] Example 3: Ligation, Transformation and Identification of Cloning Vectors After the PCR amplification in Example 2, 3 μL of dNTP and 0.5 μL of Taq enzyme were added directly to the high-fidelity enzyme PCR tube (50 μL). The reaction was continued at 72°C for 15 min, and the amplified gene fragment was then purified and recovered using a gel extraction kit (TaKaRa). The recovered product was ligated into the pMD19-T cloning vector (TaKaRa), and the ligation vector was transformed into E. coli DH5α competent cells using the calcium chloride method. Positive transformations were selected using blue-white screening. The monoclonal recombinant plasmid pMD19-T-THI5 was extracted using a plasmid extraction kit (Tiangen Biotech Co., Ltd.) and sequenced. The plasmid sequencing results showed that the obtained THI5 sequence was correct and had 100% homology with the gene sequence in the gene bank (NCBI Reference Sequence: Z48220.1).

[0037] Example 4 Construction of the recombinant expression vector PESC-THI5 Construction of expression vector PESC-TEFI-HIS: Using the PESC-HIS vector, the GAL1 promoter was replaced with the TEFI-GAP promoter, and the restriction enzyme sites were EcoRI and BamHI. The TEFI-GAP promoter sequence is shown in SEQ ID NO.5. The constructed PESC-TEFI-HIS vector was synthesized by Genscript Biotech (e.g., Figure 2 (As shown).

[0038] The monoclonal recombinant plasmid pMD19-T-THI5 constructed in Example 3 and the PESC-TEFI-HIS vector constructed above were double-digested using Beyotime Biotechnology's rapid endonucleases NotI and SacI. The digestion products were purified and recovered using a gel extraction kit (TaKaRa). The digestion products were ligated using 1 μL of Beyotime Biotechnology's T4 ligase and transformed into *E. coli* DH5α. The cells were inoculated onto Amp-resistant plates for screening. After 12 h, single clones were picked for colony PCR identification. Positive clones were picked and cultured in LB broth containing Amp resistance at 37°C and 160 rpm for 12 h. The cells were collected by centrifugation, and the plasmid was extracted. Sequencing verification was performed using primers THI5-F (nucleotide sequence as shown in SEQ ID NO. 3) and THI5-R (nucleotide sequence as shown in SEQ ID NO. 4) (Sangon Biotech Co., Ltd.). The results showed that the THI5 sequence was correct, and the constructed recombinant expression vector was named PESC-THI5 (e.g., ...). Figure 3 (As shown).

[0039] Example 5 Construction of site-directed mutant strains 1. Mutant Primer Design: Using PESC-THI5 as the starting vector, and based on the principle of reverse overlap extension PCR, the mutation site was introduced in a one-step process. Two pairs of mutant primers, THI5-F1 / THI5-R1 and THI5-F2 / THI5-R2, were designed, corresponding to amino acids 70 and 196, respectively. The specific primers are as follows: THI5 M -F1: The nucleotide sequence is shown as SEQ ID NO.6 in the sequence listing, THI5 M -R1: Nucleotide sequence as shown in SEQ ID NO.7 of the sequence listing; THI5 M -F2: The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing, THI5 M -R2: The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing.

[0040] 2. PCR Amplification: Using the PESC-THI5 vector as a template, PCR amplification was performed sequentially using two pairs of mutant primers, THI5-F1 / THI5-R1 and THI5-F2 / THI5-R2. The PCR amplification reaction system was: a total volume of 50 μL, 2 μL template, and THI5... M -F1 and THI5 M -R1 2μL, 25μL 2×PhantaFlash Master Mix, and 19μL ddH2O were added respectively. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 30 cycles (94℃ denaturation for 30 s, 55℃ annealing for 30 s and 70℃ extension for 45 s), followed by a final extension at 72℃ for 5 min (denoted as PCR product 1); the total volume was 50 μL, with 2μL template and THI5 added. M -F2 and THI5 M -R2 2μL, 2×PhantaFlash Master Mix 25μL, ddH2O 19μL respectively. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 30 cycles (94℃ denaturation for 30 s, 55℃ annealing for 30 s and 70℃ extension for 45 s), and after the cycle, extension at 72℃ for 5 min (denoted as PCR product 2).

[0041] 3. Processing of amplified products and transformation of strains: PCR products 1 and 2 were treated with DpnI to eliminate the original vector template. The DpnI reaction system was: 50 μL total volume, 1 μL DpnI, 5 μL Quick Cut Buffer, and 44 μL of PCR product 1 / 2. The DpnI reaction program was: 37℃ for 2 h, 75℃ for 15 min, and 4℃ infinity. The purified product PESC-THI5 was then transformed. M DH5α was transformed and inoculated onto antibiotic plates containing Amp for selection. Positive clones were selected for sequencing to verify whether mutations had occurred. Sequencing results are as follows: Figure 4 As shown, by Figure 4 It can be seen that in the THI5 gene amplification product obtained by the mutation primer, alanine at position 70 (A, Ala, GCC) is mutated to proline (P, Pro, CCC), and cysteine ​​at position 196 (C, Cys, TGT) is mutated to tyrosine (Y, Tyr, TAT).

[0042] The correct PESC-THI5 was verified using the rapid preparation kit for competent Saccharomyces cerevisiae from Zhuangmeng Biotechnology Co., Ltd. M The recombinant vector was transformed into Saccharomyces cerevisiae BY4741, and transformants were screened using SD-HIS yeast auxotrophic medium (Beijing Cooler Master Technology Co., Ltd.) to obtain the site-directed mutant strain DS-2.

[0043] Example 6: Experiment to verify the acid resistance of the strain Saccharomyces cerevisiae BY4741 and the site-directed mutant strain DS-2 obtained in Example 5 were activated on YPD plates, then single colonies were picked respectively and inoculated into 10 mL YPD shaking tubes (with a liquid volume of 3 mL). Cultured until the bacterial OD 600 reached the range of 1-1.5, and then inoculated respectively at an inoculum amount of 1 v / v% into 250 mL shake flasks containing YPD medium (pH=6.5) and YPD media with different pH values (pH 5.3, pH 4.1, pH 3.5, pH 3, pH 2.8, pH 2.5 and pH 2.3) (with a liquid volume of 50 mL), and cultured at 30°C and 200 r / min. The growth of different bacterial cells was observed, and samples were taken to detect the OD value of the bacterial cells when the bacterial cells became significantly turbid (as shown in Table 1). Wherein, the above YPD media with different pH values were adjusted with L-malic acid, It can be seen from Table 1 that Saccharomyces cerevisiae BY4741 and the site-directed mutant strain DS-2 grew well within 20 h in YPD medium and YPD media with pH 5.5, pH 4.5 and pH 3.5, with no significant difference; when 2.3 < pH < 3.5, the growth of the site-directed mutant strain DS-2 was significantly better than that of the BY4741 strain.

[0044] Table 1 Growth of BY4741 and DS-2 strains in media with different pH values

[0045] Example 7 Verification experiment of acid resistance of strains to different organic acids Saccharomyces cerevisiae BY4741 and the site-directed mutant strain DS-2 obtained in Example 5 were activated on YPD plates, then single colonies were picked respectively and inoculated into 10 mL shaking tubes (with a liquid volume of 3 mL) filled with YPD medium. The OD value was detected until it reached the range of 1-1.5, and then inoculated respectively at an inoculum amount of 1 v / v% into 250 mL shake flasks of YPD medium and pH 3 YPD medium (with a liquid volume of 50 mL), cultured at 30°C and 200 r / min for 36 h, then sampled to detect OD 600 , and the detection results are shown in Figure 5 . Wherein, the pH 3 YPD medium was adjusted with L-malic acid, citric acid, lactic acid, succinic acid, pyruvic acid and acetic acid respectively.

[0046] It can be seen from Figure 5 that the acid resistance of the site-directed mutant strain DS-2 is far better than that of Saccharomyces cerevisiae BY4741, and the strain exhibits different tolerance and preference for different organic acids, and grows well in the environment of L-malic acid and lactic acid.

[0047] Example 8 Comparative experiment of organic acid production by strains The site-directed mutant strain DS-2 obtained in Example 5 and Saccharomyces cerevisiae BY4741 were respectively cultured in 250 mL shake flasks for 48 h. The contents of pyruvate, L-lactic acid, citric acid and malic acid in the fermentation broth were rapidly detected using a Beijing Box Biotechnology pyruvate, L-lactic acid, citric acid and malic acid detection kit. The required culture medium and culture conditions are as follows: Preparation of seed culture: Saccharomyces cerevisiae BY4741 and the site-directed mutant strain DS-2 were separately prepared into glycerol tube strains for preservation. The preserved strains were inoculated into YPD solid medium for activation. Single colonies of the activated strains were selected and inoculated into 3 mL YPD liquid medium shake tubes (15 mL volume) and cultured at 30℃ and 220 rpm until OD500. 600 Seed solution was obtained within the range of 1.5-2.

[0048] Shake-flask fermentation: 1.5 mL of seed culture was inoculated into a 250 mL shake flask (50 mL) containing fermentation medium at a volume ratio of 3%. Fermentation was carried out at 30℃ and 220 rpm. Residual sugar was sampled periodically during fermentation. 80% glucose stock solution was added to control the glucose content at 20 g / L-30 g / L based on sugar consumption. YPD medium was used as the fermentation medium. After 48 h of fermentation, the fermentation broth was centrifuged at 8000 rpm for 2 min, and the supernatant was collected. The contents of pyruvate, L-lactic acid, and L-malic acid in the fermentation broth were rapidly detected using a kit. The results are shown in Table 2. Table 2 shows that L-malic acid, lactic acid, pyruvate, and citric acid were detected in the fermentation broths of both BY4741 and DS-2 strains. The original chassis cells of strain BY4741 showed a weak ability to produce short-chain organic acids, while the site-directed mutant strain DS-2 not only exhibited better acid tolerance but also significantly improved acid production. The concentrations of pyruvate, L-malic acid, and lactic acid in the fermentation broth of strain DS-2 were significantly higher than those of the original BY4741 strain. Therefore, strain DS-2 can serve as a potential chassis microorganism for fermenting short-chain organic acids, particularly suitable for the later-stage modification and production of pyruvate, L-malic acid, and lactic acid.

[0049] Table 2. Organic acid content in fermentation broths of BY4741 and DS-2 strains

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A yeast site-directed mutant gene THI5 M Its characteristics are, The yeast site-directed mutation gene THI5 M The nucleotide sequence is shown in SEQ ID NO.

1.

2. The yeast site-directed mutagenesis gene THI5 as described in claim 1 M Its characteristics are, The yeast site-directed mutation gene THI5 M The amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector contains the yeast site-directed mutant gene THI5 as described in claim 1. M .

4. A recombinant bacterium, characterized in that, The recombinant strain contains the yeast site-directed mutant gene THI5 as described in claim 1. M Or the recombinant vector as described in claim 3.

5. The recombinant bacteria as described in claim 4, characterized in that, The starting strain of the recombinant bacteria includes Saccharomyces cerevisiae BY4741.

6. The yeast site-directed mutant gene THI5 as described in claim 1 or 2 M The application of the recombinant vector of claim 3 or the recombinant bacteria of claim 4 or 5 in fermentation in an acidic environment.

7. The application as described in claim 6, characterized in that, The pH value of the acidic environment is 2.3-3.

5.

8. The application as described in claim 6, characterized in that, The acidic environment includes one or more of L-malic acid, citric acid, lactic acid, succinic acid, pyruvic acid, and acetic acid.

9. The yeast site-directed mutant gene THI5 as described in claim 1 or 2 M The application of the recombinant vector of claim 3 or the recombinant bacteria of claim 4 or 5 in the production of organic acids.

10. The application as described in claim 9, characterized in that, The organic acid includes one or more of L-malic acid, lactic acid, and pyruvic acid.

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