CN114891652A

CN114891652A - Acid-resistant saccharomyces cerevisiae and application thereof

Info

Publication number: CN114891652A
Application number: CN202210296692.3A
Authority: CN
Inventors: 刘龙; 陈坚; 吕雪芹; 堵国成; 李江华; 刘延峰; 孙利
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-08-12
Anticipated expiration: 2042-03-24
Also published as: CN114891652B

Abstract

The invention discloses acid-resistant saccharomyces cerevisiae and application thereof, and belongs to the technical field of metabolic engineering. According to the invention, saccharomyces cerevisiae S288C is taken as an original strain, and the HMG (high molecular weight molecule) in the ROX1 transcription regulatory factor of saccharomyces cerevisiae S288C is knocked out, or G158A in the ROX1 transcription regulatory factor is mutated, so that the acid-resistant saccharomyces cerevisiae is obtained. The invention carries out 5 mutations with different degrees on ROX1, and experimental data show that when ROX1 domain HMG is damaged with different degrees, the acid-resistant effect can be well achieved: growth phenotype (maximum OD) at pH2.32, as compared with wild type strain WT ₆₀₀ ) The improvement is 14-15 times.

Description

Acid-resistant saccharomyces cerevisiae and application thereof

Technical Field

The invention belongs to the technical field of metabolic engineering, and particularly relates to acid-resistant saccharomyces cerevisiae and application thereof.

Background

The saccharomyces cerevisiae becomes one of the main chassis cells for microbial fermentation production due to the advantages of short growth period, strong fermentation capacity, good large-scale production performance and the like. In recent years, researchers have focused on increasing the tolerance of s.cerevisiae to various stresses, such as temperature, pH, osmotic pressure (salts, alcohols), etc., to increase the tolerance of cells in different environments. For example, to obtain a strain of s.cerevisiae that is capable of tolerating high temperatures and producing ethanol, a homotypic strain with high ethanol productivity is crossed with a heterotypic strain exhibiting a thermostable phenotype, resulting in a cross of TJ14, which can synthesize ethanol that is effective at high temperatures. To increase the tolerance of cells to ethanol, which is the most common stress of yeast cells, the mechanisms reported for ethanol tolerance are mainly associated with changes in membrane composition, as well as stabilization or repair of denatured proteins.

In addition, the ability to design cellular stress tolerance based on specific circumstances may have applications in basic research and bio-manufacturing, and acid tolerance of saccharomyces cerevisiae has been of high interest to researchers. For example, strains that are tolerant to aromatic acids (pH3.5) were obtained by laboratory adaptive evolution and further analysis showed that overexpression of the aromatic acid transporter ESBP6 is a key factor in increasing tolerance to low pH. Meanwhile, previous reports indicate that overexpression of the proton pumps PMA1 and PMA2 can increase the acid resistance of yeast, and that Pdr18(ABC transporter) participates in the response of yeast to acetic acid stress. However, proton pumps or other ABC transporters can enhance the acid resistance of the strain, but only increase the tolerance to weak acids (pH 4.0-5.8). Thus, the acid resistance of yeast is far from meeting the demand of the lower pH environment formed by industrial production.

The currently reported microorganism production of organic acids is mainly to add a neutralizing agent (calcium carbonate and the like) to adjust the low pH value in the fermentation process to form organic acid salt, and then complicated procedures such as acidolysis, separation, purification and the like are carried out. On one hand, the cost and the working procedure of product separation and purification are increased, and on the other hand, if the neutralizing agent is added at the early stage, the waste of resources and the environmental pollution are also caused.

Disclosure of Invention

In order to solve the technical problems, the invention provides acid-resistant saccharomyces cerevisiae and application thereof. Therefore, the invention provides a novel acid-resistant mechanism and a construction method, and provides a theoretical basis for construction of acid-resistant underpan cells.

The invention aims to provide acid-resistant saccharomyces cerevisiae, which is obtained by taking saccharomyces cerevisiae S288C as an initial strain and knocking out the HMG (high molecular weight molecule) in the ROX1 transcription regulatory factor of saccharomyces cerevisiae S288C or mutating G158A in the ROX1 transcription regulatory factor; the amino acid sequence of the ROX1 transcription regulatory factor is shown in SEQ ID NO. 11.

In one embodiment of the invention, the nucleotide sequence of the G158A mutated site is shown as SEQ ID NO.1, and the encoded amino acid sequence is shown as SEQ ID NO. 2.

In one embodiment of the invention, one or more of an 8aa-91aa fragment, a 54aa-368aa fragment, a 1aa-53aa fragment, a 1aa-7aa fragment, or a 92aa-368aa fragment in the ROX1 is knocked out.

In one embodiment of the invention, after the 8aa-91aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulatory factor is shown as SEQ ID NO.3, and the coded amino acid sequence is shown as SEQ ID NO. 4.

In one embodiment of the invention, after the 54aa-368aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulatory factor is shown as SEQ ID NO. and the encoded amino acid sequence is shown as SEQ ID NO. 6.

In one embodiment of the invention, after the 1aa-53aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulatory factor is shown as SEQ ID NO.7, and the coded amino acid sequence is shown as SEQ ID NO. 8.

In one embodiment of the invention, after the 1aa-7aa fragment and the 92aa-368aa fragment are knocked out simultaneously, the nucleotide sequence of the ROX1 transcription regulatory factor is shown as SEQ ID NO.9, and the encoded amino acid sequence is shown as SEQ ID NO. 10.

The second purpose of the invention is to provide the product containing the acid-resistant saccharomyces cerevisiae.

The third purpose of the invention is to provide the acid-resistant saccharomyces cerevisiae or the application of the product in the fermentation production of L-malic acid.

In one embodiment of the invention, the conditions of the fermentation are: the fermentation temperature is 28-32 ℃, and the fermentation time is 60-108 h.

In order to solve the problem that the acid-resistant saccharomyces cerevisiae obtained at present is resistant to extremely low pH, the invention firstly provides a transcription regulatory factor ROX1 and a mutant mROX1 thereof, wherein the 158 th position of mROX1 base is mutated from G to A, the corresponding amino acid is mutated from Trp to a stop codon, and the mutation site is positioned in a domain HMG (8aa-91aa) of ROX 1. Through reverse engineering, a corresponding point mutation G158A is carried out in ROX1 of wild saccharomyces cerevisiae, and the important role played by the ROX1 mutant in the acid-resistant process of yeast is verified: growth phenotype (maximum OD) at pH 2.32, as compared with wild type strain WT ₆₀₀ ) The growth rate is increased by 15.41 times and the maximum growth rate is 0.37h ^-1 (ii) a Meanwhile, in order to further analyze the function of the ROX1 in the acid-resistant process and whether other mutations have the acid-resistant effect, other 5 mutations with different degrees are carried out on the ROX1 in the invention, and experimental data show that when the ROX1 domain HMG is damaged with different degrees, the excellent acid-resistant effect can be achieved: growth phenotype (maximum OD) at pH 2.32, as compared with wild type strain WT ₆₀₀ ) The improvement is 14-15 times.

Compared with the prior art, the technical scheme of the invention has the following advantages:

in the invention, the transcriptional regulation ROX1 and the mutant gene thereof are found to be effective in improving the tolerance to low pH in saccharomyces cerevisiae. Experimental data show that compared with wild saccharomyces cerevisiae, different mutants of the transcription regulatory factor ROX1 can improve the acid-resistant phenotype by about 15 times. Meanwhile, an acid-resistant strain WT delta ROX1 is used as a chassis cell to synthesize L-malic acid through metabolic modification, and the yield of the acid-resistant strain WT delta ROX1 malic acid is 2.5 times that of the strain WT without adding a neutralizing agent in the fermentation process. Therefore, the method has higher application value for constructing the acid-resistant saccharomyces cerevisiae.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is acid-proof assay of the transcriptional regulatory factor ROX1 and its mutant mROX1 of the present invention.

FIG. 2 is the acid resistance function analysis of other mutants of the transcription regulatory factor ROX1 of the present invention.

FIG. 3 is the analysis of the acid-tolerant Saccharomyces cerevisiae synthesizing L-malic acid.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1 verification of acid-resistant function of transcriptional regulatory factor ROX1 and mutant mROX1 thereof

(1) The original ROX1 gene is replaced in the original strain WT by using a genome template of wild type Saccharomyces cerevisiae S288C and primers (mROX1-up-F, mROX1-up-R, mROX1-His-F, mROX1-His-R, ROX1-down-F and ROX 1-down-R).

The primer sequence is as follows:

mROX1-up-F：ACGAAGTAGAAGGGCTTACAAC

mROX1-up-R：GGCGTAATCATGGTCATAGCTGTTTCCTGCTTCGTACCAATAATTTTAGAAATGT

mROX1-His-F：CAAACATTTCTAAAATTATTGGTACGAAGCAGGAAACAGCTATGACCATGA

mROX1-His-R：AATATAACGGAAAGAAGAAATGGAAAAAAAAAATAAAACGACGGCCAGTGCCAA

mROX1-down-F：TCGACCTTGGCACTGGCCGTCGTTTTATTTTTTTTTTCCATTTCTTCTTTCCGTT

mROX1-down-R：GAACTGGAAGTCTTTGTAAAAGCT

(2) and (2) replacing the original gene of the original strain S288C with the gene fragment obtained in the step (1) by using a homologous recombination method, and naming the strain as WT-mrOX 1.

(3) To verify the effect of mROX1 on yeast acid resistance, the growth of yeast WT-mROX1 was measured at pH 2.32 and compared to the starting strain WT. As shown in FIG. 2, the growth of WT-mROX1 was significantly improved compared to WT (maximum OD600 increased by 15.41 times and maximum growth rate of 0.37h ^-1 )。

Example 2 analysis of acid-resistant function of other mutants of the transcriptional regulatory factor ROX1

(1) In order to verify the influence of other mutations of ROX1 on acid resistance, 5 mutations with different degrees were selected, and different mutants of ROX1 were constructed in wild type S288C using the following primers, some of which were universal primers as follows:

the primer sequence is as follows:

ROX1-up-F：ACAAATCCCCTGGATATCATTG

ROX1-His-R：AATATAACGGAAAGAAGAAATGGAAAAAAAAAATAAAACGACGGCCAGTGCCAA

ROX1-down-F：TCGACCTTGGCACTGGCCGTCGTTTTATTTTTTTTTTCCATTTCTTCTTTCCGTT

ROX1-down-R：GAACTGGAAGTCTTTGTAAAAGCT

the method comprises the following steps of firstly, adopting universal primers (ROX1-up-F, ROX1-His-R, ROX1-down-F and ROX1-down-R) and the following primers (delta ROX1-up-R and delta ROX1-His-F) to completely knock out ROX1(1aa-368aa) in a WT strain to construct a WT-delta ROX1 strain.

The primer sequence is as follows:

ΔROX1-up-R：CTAGGCGTAATCATGGTCATAGCTGTTTCCTGTGTTGATTGTCTAACTGCGTTCT

ΔROX1-His-F：CACACAAAAGAACGCAGTTAGACAATCAACACAGGAAACAGCTATGACCATGA

secondly, universal primers (ROX1-up-F, ROX1-His-R, ROX1-down-F and ROX1-down-R) and the following primers (delta ROX1-1-up-R and delta ROX1-1-His-F) are adopted to knock out partial sequences (1aa-53aa) of ROX1 in the WT strain to construct the WT-delta ROX1-1 strain.

The primer sequence is as follows:

ΔROX1-1-up-R：CTAGGCGTAATCATGGTCATAGCTGTTTCCTGCTTCGTACCAATAATTTTAGAAATGT

ΔROX1-1-His-F：CATAATTCAAACATTTCTAAAATTATTGGTACGAAGCAGGAAACAGCTATGACCATG

and thirdly, knocking out partial sequences (54aa-368aa) of ROX1 in the WT strain by adopting universal primers (ROX1-up-F, ROX1-His-R, ROX1-down-F and ROX1-down-R) and the following primers (delta mROX1-R and delta mROX1-His-F) to construct the WT-delta mROX1 strain.

The primer sequence is as follows:

ΔmROX1-R：GGCGTAATCATGGTCATAGCTGTTTCCTGCTTCGTACCAATAATTTTAGAAATGT

ΔmROX1-His-F：CAAACATTTCTAAAATTATTGGTACGAAGCAGGAAACAGCTATGACCATGA

and wherein a partial sequence (1aa-7aa, 92aa-368aa) of ROX1 was knocked out in the WT strain to obtain a WT- Δ ROX1(HMG) strain, using a universal primer (ROX1-His-R, ROX1-down-F, ROX1-down-R) and the following primers (ROX1(HMG) -F1, ROX1(HMG) -R1, ROX1(HMG) -F2, ROX1(HMG) -R2, ROX1(HMG) -His-F).

The primer sequence is as follows:

ROX1(HMG)-F1：ACAAATCCCCTGGATATCATTG

ROX1(HMG)-R1：AACAGAATAAATGCGTTCTTGGGTCTTGGAATCTTTGTTGATTGTCTAACTGCGTTC

ROX1(HMG)-F2：TCACACAAAAGAACGCAGTTAGACAATCAACAAAGATTCCAAGACCCAAGAAC

ROX1(HMG)-R2：CTAGGCGTAATCATGGTCATAGCTGTTTCCTGTTCCTTCAAAAGTAGTTGCTTCT

ROX1(HMG)-His-F：AGTCTAAGAAGAAGCAACTACTTTTGAAGGAACAGGAAACAGCTATGACCATGA

and fifthly, knocking out partial sequence (8aa-91aa) of ROX1 in the WT strain by using a universal primer (ROX1-up-F) and the following primers (delta HMG-R, delta HMG-His-F, delta HMG-His-R, delta HMG-down-F and delta HMG-down-R) to construct the WT-delta HMG strain.

The primer sequence is as follows:

ΔHMG-R：GGCGTAATCATGGTCATAGCTGTTTCCTGCTTCGTACCAATAATTTTAGAAATGTT

ΔHMG-His-F：ATTCAAACATTTCTAAAATTATTGGTACGAAGCAGGAAACAGCTATGACCATGA

ΔHMG-His-R：GTTGCTCGATTTCCTTCAAAAGTAGTTGCTTCTTTAAAACGACGGCCAGTGCCAA

ΔHMG-down-F：AAGTCGACCTTGGCACTGGCCGTCGTTTTAAAGAAGCAACTACTTTTGAAGGAA

ΔHMG-down-R：TCATTTCGGAGAAACTAGGCT

(2) the 5 ROX1 mutants obtained in step (1) were tested for growth at pH 2.32 and compared with the original strain WT. As a result, as shown in FIG. 3, the ROX1 mutant strain series, except for the strain WT- Δ ROX1(HMG) having an intact HMG domain, had a remarkable effect on acid resistance, and the growth phenotype (maximum OD) was comparable to that of the wild-type strain WT ₆₀₀ ) The improvement is 14-15 times, which shows that the mutation of the domain HMG has obvious improvement on the acid-resistant growth of the yeast.

The results show that the transcription regulation factor and the mutant thereof play an important role in the acid-resistant process of the yeast, the influence of other sites on the acid-resistant growth of cells is further analyzed, and the mutation of the HMG (human growth hormone) domain is found to be the key of the acid resistance of the yeast.

Test example

The obtained strain is subjected to an application experiment for synthesizing L-malic acid

(1) In order to verify the productivity of the acid-tolerant strain obtained, taking L-malate as an example, a malate dehydrogenase MDH3 Δ SKL (malate dehydrogenase3, MDH3) gene, a pyruvate carboxylase PYC2(pyruvate carboxylase, PYC2) gene, which is 3 amino acids SKL from which the end of malate dehydrogenase MDH3 was removed, was ligated to pY26TEF-GPD plasmid using the following primers; the resulting recombinant vector pY26TEF-GPD-mdh 3. delta. SKL-pyc 2. The prepared recombinant vector pY26TEF-GPD-mdh3 delta SKL-pyc2 is respectively introduced into acid-resistant saccharomyces cerevisiae WT-delta ROX1 and wild type saccharomyces cerevisiae WT to prepare the genetically engineered bacterium CT-M1, and the genetically engineered bacterium CT-M1 is transformed in the wild type yeast in the same way and is named as WT-M1.

The primer sequence is as follows:

Scepyc2+mdh3-F1:

TCTGGCGAAGAATTGTGGTGGTGGTGGTGGTGTCAAGAGTCTAGGATGAAACTCTTG

Scepyc2+mdh3-R1:

ATAGCAATCTAATCTAAGTTTTCTAGAACTAGATGGTCAAAGTCGCAATTCTTGG

Scemdh3+pyc2-F2:

GGGCTGCAGGAATTCGATATCAAATGAGCAGTAGCAAGAAATTGG

Scemdh3+pyc2-R2:

ACATGACTCGAGGTCGACGGTATCGATAAGCTTACTTTTTTTGGGATGGGGGTAGG

Scepyc2+mdh3-F3:

CCAAGAATTGCGACTTTGACCATCTAGTTCTAGAAAACTTAGATTAGATTGCT

Scepyc2+mdh3-R3:

CTAAGACCGGCCAATTTCTTGCTACTGCTCATTTGATATCGAATTCCTGCAGCC

Scepyc2+mdh3-F4:

GAAACCCTACCCCCATCCCAAAAAAAGTAAGCTTATCGATACCGTCGACCT

Scepyc2+mdh3-R4:

AATATTGAAAAAGGCAAGAGTTTCATCCTAGACTCTTGACACCACCACCACCACCACAA

(2) the following primers are adopted to over-express malic acid transporter SpMAE1 from Schizosaccharomyces pombe, and the malic acid transporter SpMAE1 is introduced into saccharomyces cerevisiae CT-M1 by utilizing a homologous recombination technology to prepare the genetically engineered bacterium CT-M2. Meanwhile, the control strain is a wild yeast WT-M1 which is subjected to the same transformation and is named as WT-M2.

The primer sequence is as follows:

SpMae1-F1：CAGAAAAACAGATGTGCCCAAATC

SpMae1-R1：TCCTAGGCGTAATCATGGTCATAGCTGTTTCCTGGATCCTAAACTGCGTCATAGTAAG

SpMae1-F2：AGTATCAAAGAAACTTACTATGACGCAGTTTAGGATCCAGGAAACAGCTATGACCATG

SpMae1-R2：AAGAGTAAAAAAGGAGTAGAAACATTTTGGAGCTCTAAAACGACGGCCAGTGCCAA

SpMae1-F3：TGCAAGTCGACCTTGGCACTGGCCGTCGTTTTAGAGCTCCAAAATGTTTCTACTCC

SpMae1-R3：AATAAACAAGGGGCTTTACGATGGAGTAGTAGACCTGCAAATTAAAGCCTTCGAGC

SpMae1-F4：GAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGTCTACTACTCCATCGTAAAG

SpMae1-R4：TGAGGAATTTACAATAAGGTGGTTCC

(3) the engineered strain CT-M2 obtained above was shaken in 250mLFermentation verification is carried out in a bottle, the fermentation temperature is 30 ℃, the fermentation time is 96h, and the adopted fermentation medium is as follows: 10g/L corn steep liquor, 50g/L soybean peptone, 100g/L glucose, 2.5g/L K ₂ HPO ₄ ，1.5g/L KH ₂ PO ₄ ，0.75g/LMgSO ₄ ·7H ₂ And O. Without addition of neutralising agent CaCO ₃ The L-malic acid production was determined and compared with the strain WT-M2. The results showed that CT-M2(14.03g/L) produced 2.5 times greater than WT-M2(5.61 g/L).

The sequence is as follows:

SEQ ID NO.1

atgaatcctaaatcctctacacctaagattccaagacccaagaacgcatttattctgttcagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtgtggaaataccccataattcaaacatttctaaaattattggtacgaagtagaagggcttacaaccggaagataaggcacactgggaaaatctagcggagaaggagaaactagaacatgaaaggaagtatcctgaatacaaatacaagccggtaagaaagtctaagaagaagcaactacttttgaaggaaatcgagcaacagcagcagcaacaacagaaagaacagcagcagcagaaacagtcacaaccgcaattacaacagccctttaacaacaatatagttcttatgaaaagagcacattctctttcaccatcttcctcggtgtcaagctcgaacagctatcagttccaattgaacaatgatcttaagaggttgcctattccttctgttaatacttctaactatatggtctccagatctttaagtggactacctttgacgcatgataagacggcaagagacctaccacagctgtcatctcaactaaattctattccatattactcagctccacacgacccttcaacgagacatcattacctcaacgtcgctcaagctcaaccaagggctaactcgacccctcaattgccctttatttcatccattatcaacaacagcagtcaaacaccggtaactacaactaccacatccacaacaactgcgacatcttctcctgggaaattctcctcttctccgaactcctctgtactggagaacaacagattaaacagtatcaacaattcaaatcaatatttacctccccctctattaccttctctgcaagattttcaactggatcagtaccagcagctaaagcagatgggaccaacttatattgtcaaaccactgtctcacaccaggaacaatctattgtccacaactacccctacgcatcatcacattcctcatataccaaaccaaaacattcctctacatcaaattataaactcaagcaacactgaggtcaccgctaaaactagcctagtttctccgaaatga

SEQ ID NO.2

MNPKSSTPKIPRPKNAFILFRQHYHRILIDEWTAQGVEIPHNSNISKIIGTK*KGLQPEDKAHWENLAEKEKLEHERKYPEYKYKPVRKSKKKQLLLKEIEQQQQQQQKEQQQQKQSQPQLQQPFNNNIVLMKRAHSLSPSSSVSSSNSYQFQLNNDLKRLPIPSVNTSNYMVSRSLSGLPLTHDKTARDLPQLSSQLNSIPYYSAPHDPSTRHHYLNVAQAQPRANSTPQLPFISSIINNSSQTPVTTTTTSTTTATSSPGKFSSSPNSSVLENNRLNSINNSNQYLPPPLLPSLQDFQLDQYQQLKQMGPTYIVKPLSHTRNNLLSTTTPTHHHIPHIPNQNIPLHQIINSSNTEVTAKTSLVSPK

SEQ ID NO.3

atgaatcctaaatcctctacaaagaagcaactacttttgaaggaaatcgagcaacagcagcagcaacaacagaaagaacagcagcagcagaaacagtcacaaccgcaattacaacagccctttaacaacaatatagttcttatgaaaagagcacattctctttcaccatcttcctcggtgtcaagctcgaacagctatcagttccaattgaacaatgatcttaagaggttgcctattccttctgttaatacttctaactatatggtctccagatctttaagtggactacctttgacgcatgataagacggcaagagacctaccacagctgtcatctcaactaaattctattccatattactcagctccacacgacccttcaacgagacatcattacctcaacgtcgctcaagctcaaccaagggctaactcgacccctcaattgccctttatttcatccattatcaacaacagcagtcaaacaccggtaactacaactaccacatccacaacaactgcgacatcttctcctgggaaattctcctcttctccgaactcctctgtactggagaacaacagattaaacagtatcaacaattcaaatcaatatttacctccccctctattaccttctctgcaagattttcaactggatcagtaccagcagctaaagcagatgggaccaacttatattgtcaaaccactgtctcacaccaggaacaatctattgtccacaactacccctacgcatcatcacattcctcatataccaaaccaaaacattcctctacatcaaattataaactcaagcaacactgaggtcaccgctaaaactagcctagtttctccgaaatga

SEQ ID NO.4

MNPKSSTKKQLLLKEIEQQQQQQQKEQQQQKQSQPQLQQPFNNNIVLMKRAHSLSPSSSVSSSNSYQFQLNNDLKRLPIPSVNTSNYMVSRSLSGLPLTHDKTARDLPQLSSQLNSIPYYSAPHDPSTRHHYLNVAQAQPRANSTPQLPFISSIINNSSQTPVTTTTTSTTTATSSPGKFSSSPNSSVLENNRLNSINNSNQYLPPPLLPSLQDFQLDQYQQLKQMGPTYIVKPLSHTRNNLLSTTTPTHHHIPHIPNQNIPLHQIINSSNTEVTAKTSLVSPK

SEQ ID NO.5

atgaatcctaaatcctctacacctaagattccaagacccaagaacgcatttattctgttcagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtgtggaaataccccataattcaaacatttctaaaattattggtacgaagSEQ ID NO.6

MNPKSSTPKIPRPKNAFILFRQHYHRILIDEWTAQGVEIPHNSNISKIIGTK

SEQ ID NO.7

aagggcttacaaccggaagataaggcacactgggaaaatctagcggagaaggagaaactagaacatgaaaggaagtatcctgaatacaaatacaagccggtaagaaagtctaagaagaagcaactacttttgaaggaaatcgagcaacagcagcagcaacaacagaaagaacagcagcagcagaaacagtcacaaccgcaattacaacagccctttaacaacaatatagttcttatgaaaagagcacattctctttcaccatcttcctcggtgtcaagctcgaacagctatcagttccaattgaacaatgatcttaagaggttgcctattccttctgttaatacttctaactatatggtctccagatctttaagtggactacctttgacgcatgataagacggcaagagacctaccacagctgtcatctcaactaaattctattccatattactcagctccacacgacccttcaacgagacatcattacctcaacgtcgctcaagctcaaccaagggctaactcgacccctcaattgccctttatttcatccattatcaacaacagcagtcaaacaccggtaactacaactaccacatccacaacaactgcgacatcttctcctgggaaattctcctcttctccgaactcctctgtactggagaacaacagattaaacagtatcaacaattcaaatcaatatttacctccccctctattaccttctctgcaagattttcaactggatcagtaccagcagctaaagcagatgggaccaacttatattgtcaaaccactgtctcacaccaggaacaatctattgtccacaactacccctacgcatcatcacattcctcatataccaaaccaaaacattcctctacatcaaattataaactcaagcaacactgaggtcaccgctaaaactagcctagtttctccgaaatga

SEQ ID NO.8

KGLQPEDKAHWENLAEKEKLEHERKYPEYKYKPVRKSKKKQLLLKEIEQQQQQQQKEQQQQKQSQPQLQQPFNNNIVLMKRAHSLSPSSSVSSSNSYQFQLNNDLKRLPIPSVNTSNYMVSRSLSGLPLTHDKTARDLPQLSSQLNSIPYYSAPHDPSTRHHYLNVAQAQPRANSTPQLPFISSIINNSSQTPVTTTTTSTTTATSSPGKFSSSPNSSVLENNRLNSINNSNQYLPPPLLPSLQDFQLDQYQQLKQMGPTYIVKPLSHTRNNLLSTTTPTHHHIPHIPNQNIPLHQIINSSNTEVTAKTSLVSPK

SEQ ID NO.9

cctaagattccaagacccaagaacgcatttattctgttcagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtgtggaaataccccataattcaaacatttctaaaattattggtacgaagtggaagggcttacaaccggaagataaggcacactgggaaaatctagcggagaaggagaaactagaacataaaaggaagtatcctgaatacaaatacaagccggtaagaaagtctaag

SEQ ID NO.10

PKIPRPKNAFILFRQHYHRILIDEWTAQGVEIPHNSNISKIIGTKWKGLQPEDKAHWENLAEKEKLEHERKYPEYKYKPVRKSK

SEQ ID NO.11

MNPKSSTPKIPRPKNAFILFRQHYHRILIDEWTAQGVEIPHNSNISKIIGTKWKGLQPEDKAHWENLAEKEKLEHERKYPEYKYKPVRKSKKKQLLLKEIEQQQQQQQKEQQQQKQSQPQLQQPFNNNIVLMKRAHSLSPSSSVSSSNSYQFQLNNDLKRLPIPSVNTSNYMVSRSLSGLPLTHDKTARDLPQLSSQLNSIPYYSAPHDPSTRHHYLNVAQAQPRANSTPQLPFISSIINNSSQTPVTTTTTSTTTATSSPGKFSSSPNSSVLENNRLNSINNSNQYLPPPLLPSLQDFQLDQYQQLKQMGPTYIVKPLSHTRNNLLSTTTPTHHHIPHIPNQNIPLHQIINSSNTEVTAKTSLVSPK

it should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

SEQUENCE LISTING

<110> university in south of the Yangtze river

<120> acid-resistant saccharomyces cerevisiae and application thereof

<130> 11

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 1107

<212> DNA

<213> (Artificial Synthesis)

<400> 1

atgaatccta aatcctctac acctaagatt ccaagaccca agaacgcatt tattctgttc 60

agacagcact accacaggat cttaatagac gaatggaccg ctcaaggtgt ggaaataccc 120

cataattcaa acatttctaa aattattggt acgaagtaga agggcttaca accggaagat 180

aaggcacact gggaaaatct agcggagaag gagaaactag aacatgaaag gaagtatcct 240

gaatacaaat acaagccggt aagaaagtct aagaagaagc aactactttt gaaggaaatc 300

gagcaacagc agcagcaaca acagaaagaa cagcagcagc agaaacagtc acaaccgcaa 360

ttacaacagc cctttaacaa caatatagtt cttatgaaaa gagcacattc tctttcacca 420

tcttcctcgg tgtcaagctc gaacagctat cagttccaat tgaacaatga tcttaagagg 480

ttgcctattc cttctgttaa tacttctaac tatatggtct ccagatcttt aagtggacta 540

cctttgacgc atgataagac ggcaagagac ctaccacagc tgtcatctca actaaattct 600

attccatatt actcagctcc acacgaccct tcaacgagac atcattacct caacgtcgct 660

caagctcaac caagggctaa ctcgacccct caattgccct ttatttcatc cattatcaac 720

aacagcagtc aaacaccggt aactacaact accacatcca caacaactgc gacatcttct 780

cctgggaaat tctcctcttc tccgaactcc tctgtactgg agaacaacag attaaacagt 840

atcaacaatt caaatcaata tttacctccc cctctattac cttctctgca agattttcaa 900

ctggatcagt accagcagct aaagcagatg ggaccaactt atattgtcaa accactgtct 960

cacaccagga acaatctatt gtccacaact acccctacgc atcatcacat tcctcatata 1020

ccaaaccaaa acattcctct acatcaaatt ataaactcaa gcaacactga ggtcaccgct 1080

aaaactagcc tagtttctcc gaaatga 1107

<210> 2

<211> 367

<212> PRT

<213> (Artificial Synthesis)

<400> 2

Met Asn Pro Lys Ser Ser Thr Pro Lys Ile Pro Arg Pro Lys Asn Ala

1 5 10 15

Phe Ile Leu Phe Arg Gln His Tyr His Arg Ile Leu Ile Asp Glu Trp

20 25 30

Thr Ala Gln Gly Val Glu Ile Pro His Asn Ser Asn Ile Ser Lys Ile

35 40 45

Ile Gly Thr Lys Lys Gly Leu Gln Pro Glu Asp Lys Ala His Trp Glu

50 55 60

Asn Leu Ala Glu Lys Glu Lys Leu Glu His Glu Arg Lys Tyr Pro Glu

65 70 75 80

Tyr Lys Tyr Lys Pro Val Arg Lys Ser Lys Lys Lys Gln Leu Leu Leu

85 90 95

Lys Glu Ile Glu Gln Gln Gln Gln Gln Gln Gln Lys Glu Gln Gln Gln

100 105 110

Gln Lys Gln Ser Gln Pro Gln Leu Gln Gln Pro Phe Asn Asn Asn Ile

115 120 125

Val Leu Met Lys Arg Ala His Ser Leu Ser Pro Ser Ser Ser Val Ser

130 135 140

Ser Ser Asn Ser Tyr Gln Phe Gln Leu Asn Asn Asp Leu Lys Arg Leu

145 150 155 160

Pro Ile Pro Ser Val Asn Thr Ser Asn Tyr Met Val Ser Arg Ser Leu

165 170 175

Ser Gly Leu Pro Leu Thr His Asp Lys Thr Ala Arg Asp Leu Pro Gln

180 185 190

Leu Ser Ser Gln Leu Asn Ser Ile Pro Tyr Tyr Ser Ala Pro His Asp

195 200 205

Pro Ser Thr Arg His His Tyr Leu Asn Val Ala Gln Ala Gln Pro Arg

210 215 220

Ala Asn Ser Thr Pro Gln Leu Pro Phe Ile Ser Ser Ile Ile Asn Asn

225 230 235 240

Ser Ser Gln Thr Pro Val Thr Thr Thr Thr Thr Ser Thr Thr Thr Ala

245 250 255

Thr Ser Ser Pro Gly Lys Phe Ser Ser Ser Pro Asn Ser Ser Val Leu

260 265 270

Glu Asn Asn Arg Leu Asn Ser Ile Asn Asn Ser Asn Gln Tyr Leu Pro

275 280 285

Pro Pro Leu Leu Pro Ser Leu Gln Asp Phe Gln Leu Asp Gln Tyr Gln

290 295 300

Gln Leu Lys Gln Met Gly Pro Thr Tyr Ile Val Lys Pro Leu Ser His

305 310 315 320

Thr Arg Asn Asn Leu Leu Ser Thr Thr Thr Pro Thr His His His Ile

325 330 335

Pro His Ile Pro Asn Gln Asn Ile Pro Leu His Gln Ile Ile Asn Ser

340 345 350

Ser Asn Thr Glu Val Thr Ala Lys Thr Ser Leu Val Ser Pro Lys

355 360 365

<210> 3

<211> 855

<212> DNA

<213> (Artificial Synthesis)

<400> 3

atgaatccta aatcctctac aaagaagcaa ctacttttga aggaaatcga gcaacagcag 60

cagcaacaac agaaagaaca gcagcagcag aaacagtcac aaccgcaatt acaacagccc 120

tttaacaaca atatagttct tatgaaaaga gcacattctc tttcaccatc ttcctcggtg 180

tcaagctcga acagctatca gttccaattg aacaatgatc ttaagaggtt gcctattcct 240

tctgttaata cttctaacta tatggtctcc agatctttaa gtggactacc tttgacgcat 300

gataagacgg caagagacct accacagctg tcatctcaac taaattctat tccatattac 360

tcagctccac acgacccttc aacgagacat cattacctca acgtcgctca agctcaacca 420

agggctaact cgacccctca attgcccttt atttcatcca ttatcaacaa cagcagtcaa 480

acaccggtaa ctacaactac cacatccaca acaactgcga catcttctcc tgggaaattc 540

tcctcttctc cgaactcctc tgtactggag aacaacagat taaacagtat caacaattca 600

aatcaatatt tacctccccc tctattacct tctctgcaag attttcaact ggatcagtac 660

cagcagctaa agcagatggg accaacttat attgtcaaac cactgtctca caccaggaac 720

aatctattgt ccacaactac ccctacgcat catcacattc ctcatatacc aaaccaaaac 780

attcctctac atcaaattat aaactcaagc aacactgagg tcaccgctaa aactagccta 840

gtttctccga aatga 855

<210> 4

<211> 284

<212> PRT

<213> (Artificial Synthesis)

<400> 4

Met Asn Pro Lys Ser Ser Thr Lys Lys Gln Leu Leu Leu Lys Glu Ile

1 5 10 15

Glu Gln Gln Gln Gln Gln Gln Gln Lys Glu Gln Gln Gln Gln Lys Gln

20 25 30

Ser Gln Pro Gln Leu Gln Gln Pro Phe Asn Asn Asn Ile Val Leu Met

35 40 45

Lys Arg Ala His Ser Leu Ser Pro Ser Ser Ser Val Ser Ser Ser Asn

50 55 60

Ser Tyr Gln Phe Gln Leu Asn Asn Asp Leu Lys Arg Leu Pro Ile Pro

65 70 75 80

Ser Val Asn Thr Ser Asn Tyr Met Val Ser Arg Ser Leu Ser Gly Leu

85 90 95

Pro Leu Thr His Asp Lys Thr Ala Arg Asp Leu Pro Gln Leu Ser Ser

100 105 110

Gln Leu Asn Ser Ile Pro Tyr Tyr Ser Ala Pro His Asp Pro Ser Thr

115 120 125

Arg His His Tyr Leu Asn Val Ala Gln Ala Gln Pro Arg Ala Asn Ser

130 135 140

Thr Pro Gln Leu Pro Phe Ile Ser Ser Ile Ile Asn Asn Ser Ser Gln

145 150 155 160

Thr Pro Val Thr Thr Thr Thr Thr Ser Thr Thr Thr Ala Thr Ser Ser

165 170 175

Pro Gly Lys Phe Ser Ser Ser Pro Asn Ser Ser Val Leu Glu Asn Asn

180 185 190

Arg Leu Asn Ser Ile Asn Asn Ser Asn Gln Tyr Leu Pro Pro Pro Leu

195 200 205

Leu Pro Ser Leu Gln Asp Phe Gln Leu Asp Gln Tyr Gln Gln Leu Lys

210 215 220

Gln Met Gly Pro Thr Tyr Ile Val Lys Pro Leu Ser His Thr Arg Asn

225 230 235 240

Asn Leu Leu Ser Thr Thr Thr Pro Thr His His His Ile Pro His Ile

245 250 255

Pro Asn Gln Asn Ile Pro Leu His Gln Ile Ile Asn Ser Ser Asn Thr

260 265 270

Glu Val Thr Ala Lys Thr Ser Leu Val Ser Pro Lys

275 280

<210> 5

<211> 156

<212> DNA

<213> (Artificial Synthesis)

<400> 5

atgaatccta aatcctctac acctaagatt ccaagaccca agaacgcatt tattctgttc 60

agacagcact accacaggat cttaatagac gaatggaccg ctcaaggtgt ggaaataccc 120

cataattcaa acatttctaa aattattggt acgaag 156

<210> 6

<211> 52

<212> PRT

<213> (Artificial Synthesis)

<400> 6

Met Asn Pro Lys Ser Ser Thr Pro Lys Ile Pro Arg Pro Lys Asn Ala

1 5 10 15

Phe Ile Leu Phe Arg Gln His Tyr His Arg Ile Leu Ile Asp Glu Trp

20 25 30

Thr Ala Gln Gly Val Glu Ile Pro His Asn Ser Asn Ile Ser Lys Ile

35 40 45

Ile Gly Thr Lys

50

<210> 7

<211> 948

<212> DNA

<213> (Artificial Synthesis)

<400> 7

aagggcttac aaccggaaga taaggcacac tgggaaaatc tagcggagaa ggagaaacta 60

gaacatgaaa ggaagtatcc tgaatacaaa tacaagccgg taagaaagtc taagaagaag 120

caactacttt tgaaggaaat cgagcaacag cagcagcaac aacagaaaga acagcagcag 180

cagaaacagt cacaaccgca attacaacag ccctttaaca acaatatagt tcttatgaaa 240

agagcacatt ctctttcacc atcttcctcg gtgtcaagct cgaacagcta tcagttccaa 300

ttgaacaatg atcttaagag gttgcctatt ccttctgtta atacttctaa ctatatggtc 360

tccagatctt taagtggact acctttgacg catgataaga cggcaagaga cctaccacag 420

ctgtcatctc aactaaattc tattccatat tactcagctc cacacgaccc ttcaacgaga 480

catcattacc tcaacgtcgc tcaagctcaa ccaagggcta actcgacccc tcaattgccc 540

tttatttcat ccattatcaa caacagcagt caaacaccgg taactacaac taccacatcc 600

acaacaactg cgacatcttc tcctgggaaa ttctcctctt ctccgaactc ctctgtactg 660

gagaacaaca gattaaacag tatcaacaat tcaaatcaat atttacctcc ccctctatta 720

ccttctctgc aagattttca actggatcag taccagcagc taaagcagat gggaccaact 780

tatattgtca aaccactgtc tcacaccagg aacaatctat tgtccacaac tacccctacg 840

catcatcaca ttcctcatat accaaaccaa aacattcctc tacatcaaat tataaactca 900

agcaacactg aggtcaccgc taaaactagc ctagtttctc cgaaatga 948

<210> 8

<211> 315

<212> PRT

<213> (Artificial Synthesis)

<400> 8

Lys Gly Leu Gln Pro Glu Asp Lys Ala His Trp Glu Asn Leu Ala Glu

1 5 10 15

Lys Glu Lys Leu Glu His Glu Arg Lys Tyr Pro Glu Tyr Lys Tyr Lys

20 25 30

Pro Val Arg Lys Ser Lys Lys Lys Gln Leu Leu Leu Lys Glu Ile Glu

35 40 45

Gln Gln Gln Gln Gln Gln Gln Lys Glu Gln Gln Gln Gln Lys Gln Ser

50 55 60

Gln Pro Gln Leu Gln Gln Pro Phe Asn Asn Asn Ile Val Leu Met Lys

65 70 75 80

Arg Ala His Ser Leu Ser Pro Ser Ser Ser Val Ser Ser Ser Asn Ser

85 90 95

Tyr Gln Phe Gln Leu Asn Asn Asp Leu Lys Arg Leu Pro Ile Pro Ser

100 105 110

Val Asn Thr Ser Asn Tyr Met Val Ser Arg Ser Leu Ser Gly Leu Pro

115 120 125

Leu Thr His Asp Lys Thr Ala Arg Asp Leu Pro Gln Leu Ser Ser Gln

130 135 140

Leu Asn Ser Ile Pro Tyr Tyr Ser Ala Pro His Asp Pro Ser Thr Arg

145 150 155 160

His His Tyr Leu Asn Val Ala Gln Ala Gln Pro Arg Ala Asn Ser Thr

165 170 175

Pro Gln Leu Pro Phe Ile Ser Ser Ile Ile Asn Asn Ser Ser Gln Thr

180 185 190

Pro Val Thr Thr Thr Thr Thr Ser Thr Thr Thr Ala Thr Ser Ser Pro

195 200 205

Gly Lys Phe Ser Ser Ser Pro Asn Ser Ser Val Leu Glu Asn Asn Arg

210 215 220

Leu Asn Ser Ile Asn Asn Ser Asn Gln Tyr Leu Pro Pro Pro Leu Leu

225 230 235 240

Pro Ser Leu Gln Asp Phe Gln Leu Asp Gln Tyr Gln Gln Leu Lys Gln

245 250 255

Met Gly Pro Thr Tyr Ile Val Lys Pro Leu Ser His Thr Arg Asn Asn

260 265 270

Leu Leu Ser Thr Thr Thr Pro Thr His His His Ile Pro His Ile Pro

275 280 285

Asn Gln Asn Ile Pro Leu His Gln Ile Ile Asn Ser Ser Asn Thr Glu

290 295 300

Val Thr Ala Lys Thr Ser Leu Val Ser Pro Lys

305 310 315

<210> 9

<211> 252

<212> DNA

<213> (Artificial Synthesis)

<400> 9

cctaagattc caagacccaa gaacgcattt attctgttca gacagcacta ccacaggatc 60

ttaatagacg aatggaccgc tcaaggtgtg gaaatacccc ataattcaaa catttctaaa 120

attattggta cgaagtggaa gggcttacaa ccggaagata aggcacactg ggaaaatcta 180

gcggagaagg agaaactaga acataaaagg aagtatcctg aatacaaata caagccggta 240

agaaagtcta ag 252

<210> 10

<211> 84

<212> PRT

<213> (Artificial Synthesis)

<400> 10

Pro Lys Ile Pro Arg Pro Lys Asn Ala Phe Ile Leu Phe Arg Gln His

1 5 10 15

Tyr His Arg Ile Leu Ile Asp Glu Trp Thr Ala Gln Gly Val Glu Ile

20 25 30

Pro His Asn Ser Asn Ile Ser Lys Ile Ile Gly Thr Lys Trp Lys Gly

35 40 45

Leu Gln Pro Glu Asp Lys Ala His Trp Glu Asn Leu Ala Glu Lys Glu

50 55 60

Lys Leu Glu His Glu Arg Lys Tyr Pro Glu Tyr Lys Tyr Lys Pro Val

65 70 75 80

Arg Lys Ser Lys

<210> 11

<211> 368

<212> PRT

<213> (Artificial Synthesis)

<400> 11

Met Asn Pro Lys Ser Ser Thr Pro Lys Ile Pro Arg Pro Lys Asn Ala

1 5 10 15

Phe Ile Leu Phe Arg Gln His Tyr His Arg Ile Leu Ile Asp Glu Trp

20 25 30

Thr Ala Gln Gly Val Glu Ile Pro His Asn Ser Asn Ile Ser Lys Ile

35 40 45

Ile Gly Thr Lys Trp Lys Gly Leu Gln Pro Glu Asp Lys Ala His Trp

50 55 60

Glu Asn Leu Ala Glu Lys Glu Lys Leu Glu His Glu Arg Lys Tyr Pro

65 70 75 80

Glu Tyr Lys Tyr Lys Pro Val Arg Lys Ser Lys Lys Lys Gln Leu Leu

85 90 95

Leu Lys Glu Ile Glu Gln Gln Gln Gln Gln Gln Gln Lys Glu Gln Gln

100 105 110

Gln Gln Lys Gln Ser Gln Pro Gln Leu Gln Gln Pro Phe Asn Asn Asn

115 120 125

Ile Val Leu Met Lys Arg Ala His Ser Leu Ser Pro Ser Ser Ser Val

130 135 140

Ser Ser Ser Asn Ser Tyr Gln Phe Gln Leu Asn Asn Asp Leu Lys Arg

145 150 155 160

Leu Pro Ile Pro Ser Val Asn Thr Ser Asn Tyr Met Val Ser Arg Ser

165 170 175

Leu Ser Gly Leu Pro Leu Thr His Asp Lys Thr Ala Arg Asp Leu Pro

180 185 190

Gln Leu Ser Ser Gln Leu Asn Ser Ile Pro Tyr Tyr Ser Ala Pro His

195 200 205

Asp Pro Ser Thr Arg His His Tyr Leu Asn Val Ala Gln Ala Gln Pro

210 215 220

Arg Ala Asn Ser Thr Pro Gln Leu Pro Phe Ile Ser Ser Ile Ile Asn

225 230 235 240

Asn Ser Ser Gln Thr Pro Val Thr Thr Thr Thr Thr Ser Thr Thr Thr

245 250 255

Ala Thr Ser Ser Pro Gly Lys Phe Ser Ser Ser Pro Asn Ser Ser Val

260 265 270

Leu Glu Asn Asn Arg Leu Asn Ser Ile Asn Asn Ser Asn Gln Tyr Leu

275 280 285

Pro Pro Pro Leu Leu Pro Ser Leu Gln Asp Phe Gln Leu Asp Gln Tyr

290 295 300

Gln Gln Leu Lys Gln Met Gly Pro Thr Tyr Ile Val Lys Pro Leu Ser

305 310 315 320

His Thr Arg Asn Asn Leu Leu Ser Thr Thr Thr Pro Thr His His His

325 330 335

Ile Pro His Ile Pro Asn Gln Asn Ile Pro Leu His Gln Ile Ile Asn

340 345 350

Ser Ser Asn Thr Glu Val Thr Ala Lys Thr Ser Leu Val Ser Pro Lys

355 360 365

Claims

1. Acid-tolerant saccharomyces cerevisiae is characterized in that saccharomyces cerevisiae S288C is used as an original strain, and the HMG (high molecular weight molecule) in the ROX1 transcription regulatory factor of saccharomyces cerevisiae S288C is knocked out, or G158A in the ROX1 transcription regulatory factor is mutated to obtain the acid-tolerant saccharomyces cerevisiae; the amino acid sequence of the ROX1 transcription regulatory factor is shown in SEQ ID NO. 11.

2. The acid-tolerant Saccharomyces cerevisiae according to claim 1, wherein the nucleotide sequence of the G158A mutated site is shown as SEQ ID No.1, and the encoded amino acid sequence is shown as SEQ ID No. 2.

3. The acid-tolerant Saccharomyces cerevisiae of claim 1, wherein one or more of an 8aa-91aa fragment, a 54aa-368aa fragment, a 1aa-53aa fragment, a 1aa-7aa fragment, or a 92aa-368aa fragment of the ROX1 is knocked out.

4. The acid-tolerant Saccharomyces cerevisiae according to claim 3, wherein after the 8aa-91aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulatory factor is shown as SEQ ID No.3, and the encoded amino acid sequence is shown as SEQ ID No. 4.

5. The acid-tolerant Saccharomyces cerevisiae of claim 3, wherein the nucleotide sequence of the ROX1 transcription regulatory factor is shown in SEQ ID NO. and the encoded amino acid sequence is shown in SEQ ID NO.6 after the 54aa-368aa fragment is knocked out.

6. The acid-tolerant Saccharomyces cerevisiae according to claim 3, wherein after the 1aa-53aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulatory factor is shown as SEQ ID No.7, and the encoded amino acid sequence is shown as SEQ ID No. 8.

7. The acid-tolerant Saccharomyces cerevisiae of claim 3, wherein the nucleotide sequence of the ROX1 transcription regulatory factor is shown in SEQ ID NO.9 and the encoded amino acid sequence is shown in SEQ ID NO.10 after the 1aa-7aa fragment and the 92aa-368aa fragment are knocked out simultaneously.

8. Product comprising the acid-tolerant Saccharomyces cerevisiae according to any of claims 1 to 7.

9. Use of an acid tolerant s.cerevisiae as claimed in any one of claims 1 to 7, or a product as claimed in claim 8, for the fermentative production of L-malic acid.

10. Use according to claim 9, characterized in that the conditions of the fermentation are: the fermentation temperature is 28-32 ℃, and the fermentation time is 60-108 h.