CN114891652B

CN114891652B - Acid-resistant saccharomyces cerevisiae and application thereof

Info

Publication number: CN114891652B
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: 2023-09-08
Anticipated expiration: 2042-03-24
Also published as: CN114891652A

Abstract

The invention discloses acid-resistant saccharomyces cerevisiae and application thereof, and belongs to the technical field of metabolic engineering. The invention takes Saccharomyces cerevisiae S288C as an initial strain, knocks out a domain HMG in a ROX1 transcription regulatory factor of the Saccharomyces cerevisiae S288C, or mutates G158A in the ROX1 transcription regulatory factor, thereby obtaining the acid-resistant Saccharomyces cerevisiae. According to the invention, 5 mutations with different degrees are carried out on ROX1, and experimental data show that when the ROX1 structural domain HMG is damaged with different degrees, the good acid resistance effect can be achieved as well: at pH2.32, the growth phenotype (maximum OD 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

Saccharomyces cerevisiae is one of the main chassis cells for microbial fermentation production because of the advantages of short growth cycle, strong fermentation capacity, good mass production performance and the like. In recent years, researchers have focused on increasing the tolerance of Saccharomyces 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 Saccharomyces cerevisiae that is tolerant to high temperatures and produces ethanol, a homotype strain having high ethanol productivity is crossed with a heterotype strain exhibiting a high temperature resistant phenotype to obtain hybrid TJ14, which can synthesize ethanol efficiently at high temperatures. In order to increase the tolerance of cells to ethanol, which is the most common stress of yeast cells, the mechanism of ethanol tolerance has been reported to be mainly related to changes in membrane composition, as well as to stabilization or repair of denatured proteins.

Furthermore, the ability to design cell stress resistance based on specific circumstances may find application in basic research and biological manufacturing, while Saccharomyces cerevisiae has received high attention from researchers. For example, strains capable of tolerating aromatic acids (pH 3.5) were obtained by laboratory adaptive evolution, and further analysis indicated that overexpression of the aromatic acid transporter ESBP6 was a key factor in improving tolerance to low pH. Meanwhile, previous reports indicate that overexpression of proton pumps PMA1 and PMA2 can increase acid resistance of yeast, and that Pdr18 (ABC transporter) participates in the reaction of yeast to acetic acid stress. However, proton pumps or other ABC transporters can enhance the acid resistance of the strain, but only the tolerance to weak acids (ph 4.0-5.8). Thus, the acid resistance of yeast is still far from meeting the needs of the lower pH environment created by industrial production.

The organic acid produced by the microorganism reported at present is mainly prepared by adding a neutralizer (such as calcium carbonate) to adjust the low pH value of the fermentation process to form organic acid salt, and then carrying out complex procedures such as acidolysis, separation, purification and the like. On the 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 in the earlier stage, the waste of resources and the pollution of the environment 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 the construction of acid-resistant chassis cells.

The first object of the invention is to provide an acid-resistant saccharomyces cerevisiae, which is obtained by taking saccharomyces cerevisiae S288C as an initial strain, knocking out a domain HMG in a 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 regulating factor is shown as 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 the 8aa-91aa fragment, 54aa-368aa fragment, 1aa-53aa fragment, 1aa-7aa fragment, or 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 regulator is shown as SEQ ID NO.3, and the encoded amino acid sequence is shown as SEQ ID NO. 4.

In one embodiment of the invention, after 54aa-368aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulator is shown as SEQ ID NO.5, and the encoded amino acid sequence is shown as SEQ ID NO. 6.

In one embodiment of the invention, after 1aa-53aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulator is shown as SEQ ID NO.7, and the encoded 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 regulator is shown as SEQ ID NO.9, and the encoded amino acid sequence is shown as SEQ ID NO. 10.

A second object of the present invention is to provide a product comprising said acid-tolerant Saccharomyces cerevisiae.

The third object 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: the fermentation temperature is 28-32 ℃ and the fermentation time is 60-108h.

In order to solve the problem that the acid-resistant saccharomyces cerevisiae which is obtained at present tolerates extremely low pH, the invention firstly provides a transcription regulatory factor ROX1 and a mutant mROX1 thereof, wherein the 158 th position of the mROX1 base is mutated from G to A, the corresponding amino acid is mutated from tryptophan Trp to a stop codon, and the mutation site is positioned in the structure of ROX1Domain HMG (8 aa-91 aa). By reverse engineering, the corresponding point mutation G158A is carried out in the ROX1 of the wild-type Saccharomyces cerevisiae, and the important role of the ROX1 mutant in the acid-resistant process of the yeast is verified: at pH2.32, the growth phenotype (maximum OD ₆₀₀ ) The growth rate is increased by 15.41 times and the maximum growth rate is 0.37h ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Meanwhile, in order to further analyze the function of ROX1 in the acid-resistant process and whether other mutations have acid-resistant effect, in the invention, the ROX1 is subjected to other 5 different degrees of mutations, and experimental data show that when the ROX1 structural domain HMG is damaged to different degrees, the good acid-resistant effect can be achieved as well: at pH2.32, the growth phenotype (maximum OD 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 capable of effectively improving the tolerance to low pH in Saccharomyces cerevisiae. Experimental data shows that compared with wild Saccharomyces cerevisiae, different mutants of transcription regulatory factor ROX1 can improve acid resistance phenotype by about 15 times. Meanwhile, the acid-resistant strain WT delta ROX1 is taken as chassis cells, the metabolic transformation is performed to synthesize the L-malic acid, and the yield of the acid-resistant strain WT delta ROX1 malic acid is 2.5 times that of the strain WT under the condition that a neutralizing agent is not added in the fermentation process. Therefore, the method has higher application value for constructing the acid-resistant saccharomyces cerevisiae.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which

FIG. 1 shows an acid resistance verification analysis of the transcription regulatory factor ROX1 and its mutant mROX1 of the present invention.

FIG. 2 shows an acid-resistant function analysis of other mutants of the transcription regulatory factor ROX1 of the invention.

FIG. 3 is an analysis of the synthesis of L-malic acid by Saccharomyces cerevisiae, which is resistant to acid according to the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Example 1 transcription regulatory factor ROX1 and acid resistance verification of mutant mROX1 thereof

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

Primer sequence:

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 (3) replacing the original gene of the original strain S288C by using the gene fragment obtained in the step (1) by using a homologous recombination method, wherein the strain is named as WT-mROX1.

(3) To verify the effect of mROX1 on yeast acid resistance, growth of yeast WT-mROX1 was measured at pH2.32 and compared to the starting strain WT. As shown in FIG. 1, the growth of WT-mROX1 was significantly improved over WT (15.41-fold increase in OD600 maximum, 0.37h maximum growth rate) ^-1 )。

Example 2 analysis of acid-proof function of other mutants of transcription regulatory factor ROX1

(1) To verify the effect of other mutations of ROX1 on acid resistance, 5 different degrees of mutation were selected, and different mutants of ROX1 were constructed in wild saccharomyces cerevisiae S288C using the primers shown below, wherein part of the sequences were universal primers as follows:

primer sequence:

ROX1-up-F：ACAAATCCCCTGGATATCATTG

ROX1-His-R：AATATAACGGAAAGAAGAAATGGAAAAAAAAAATAAAACGACGGCCAG TGCCAA

ROX1-down-F：TCGACCTTGGCACTGGCCGTCGTTTTATTTTTTTTTTCCATTTCTTCTTTC CGTT

ROX1-down-R：GAACTGGAAGTCTTTGTAAAAGCT

(1) wherein the WT-DeltaROX 1 strain was constructed by completely knocking out ROX1 (1 aa-368 aa) in the WT strain using general primers (ROX 1-up-F, ROX1-His-R, ROX1-down-F and ROX 1-down-R) and the following primers (DeltaROX 1-up-R, deltaROX 1-His-F).

Primer sequence:

ΔROX1-up-R：CTAGGCGTAATCATGGTCATAGCTGTTTCCTGTGTTGATTGTCTAACTGCG TTCT

ΔROX1-His-F：CACACAAAAGAACGCAGTTAGACAATCAACACAGGAAACAGCTATGACCATGA

(2) wherein the WT-DeltaROX 1-1 strain was constructed by knocking out a partial sequence of ROX1 (1 aa-53 aa) in the WT strain using a general primer (ROX 1-up-F, ROX1-His-R, ROX1-down-F, ROX 1-down-R) and the following primer (DeltaROX 1-1-up-R, deltaROX 1-1-His-F).

Primer sequence:

ΔROX1-1-up-R：CTAGGCGTAATCATGGTCATAGCTGTTTCCTGCTTCGTACCAATAATTTTAGAAATGT

ΔROX1-1-His-F：CATAATTCAAACATTTCTAAAATTATTGGTACGAAGCAGGAAACAGCTATGACCATG

(3) wherein the WT-DeltaROX 1 strain was constructed by knocking out a partial sequence of ROX1 (54 aa-368 aa) in the WT strain using a general primer (ROX 1-up-F, ROX1-His-R, ROX1-down-F, ROX 1-down-R) and the following primer (DeltamROX 1-R, deltamROX 1-His-F).

Primer sequence:

ΔmROX1-R：GGCGTAATCATGGTCATAGCTGTTTCCTGCTTCGTACCAATAATTTTAGAAATGT

ΔmROX1-His-F：CAAACATTTCTAAAATTATTGGTACGAAGCAGGAAACAGCTATGACCATGA

(4) wherein the WT-. DELTA.ROX 1 (HMG) strain was constructed by knocking out a partial sequence of ROX1 (1 aa-7aa,92aa-368 aa) in the WT strain using a general primer (ROX 1-His-R, ROX1-down-F, ROX 1-down-R) and the following primers (ROX 1 (HMG) -F1, ROX1 (HMG) -R1, ROX1 (HMG) -F2, ROX1 (HMG) -His-F).

Primer sequence:

ROX1(HMG)-F1：ACAAATCCCCTGGATATCATTG

ROX1(HMG)-R1：AACAGAATAAATGCGTTCTTGGGTCTTGGAATCTTTGTTGATTGTCTAAC TGCGTTC

ROX1(HMG)-F2：TCACACAAAAGAACGCAGTTAGACAATCAACAAAGATTCCAAGACCCA AGAAC

ROX1(HMG)-R2：CTAGGCGTAATCATGGTCATAGCTGTTTCCTGTTCCTTCAAAAGTAGTTG CTTCT

ROX1(HMG)-His-F：AGTCTAAGAAGAAGCAACTACTTTTGAAGGAACAGGAAACAGCTATGA CCATGA

(5) wherein the WT-. DELTA.HMG strain was constructed by knocking out a part of the ROX1 sequence (8 aa-91 aa) in the WT strain using the general primer (ROX 1-up-F) and the following primers (. DELTA.HMG-R,. DELTA.HMG-His-F,. DELTA.HMG-His-R,. DELTA.HMG-down-F,. DELTA.HMG-down-R).

Primer sequence:

ΔHMG-R：GGCGTAATCATGGTCATAGCTGTTTCCTGCTTCGTACCAATAATTTTAGAA ATGTT

ΔHMG-His-F：ATTCAAACATTTCTAAAATTATTGGTACGAAGCAGGAAACAGCTATGACC ATGA

ΔHMG-His-R：GTTGCTCGATTTCCTTCAAAAGTAGTTGCTTCTTTAAAACGACGGCCAGT GCCAA

ΔHMG-down-F：AAGTCGACCTTGGCACTGGCCGTCGTTTTAAAGAAGCAACTACTTTTGA AGGAA

ΔHMG-down-R：TCATTTCGGAGAAACTAGGCT

(2) The 5 ROX1 mutants obtained in the above step (1) were subjected to growth measurement at pH2.32 and compared with the starting strain WT. As shown in FIG. 2, the ROX1 mutant strains showed remarkable effects on acid resistance, and the mutant strains showed a growth phenotype (maximum OD) as compared with the wild-type strain WT, except for the strain WT-. DELTA.ROX1 (HMG) having the complete HMG domain ₆₀₀ ) The improvement of 14-15 times indicates that the mutation of the domain HMG has a remarkable improvement on the acid-resistant growth of yeast.

The results show that in the invention, the transcription regulating 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 domain HMG is found to be the key of acid resistance of the yeast.

Test case

The strain obtained is subjected to L-malic acid synthesis application experiment

(1) To verify the productivity of the resulting acid-tolerant strain, the malate dehydrogenase mdh3Δskl (malate dehydrogenase, MDH 3) gene, the pyruvate carboxylase PYC2 (pyruvate carboxylase, PYC 2) gene, which is the 3 amino acids SKL from which the malate dehydrogenase MDH3 was removed at the end, was ligated to the pY26TEF-GPD plasmid using the following primers, as an example; the resulting recombinant vector pY26TEF-GPD-mdh3ΔSKL-pyc2. The recombinant vector pY26TEF-GPD-mdh3ΔSKL-pyc2 prepared is respectively introduced into acid-resistant saccharomyces cerevisiae WT- ΔROX1 and wild-type saccharomyces cerevisiae WT to prepare genetically engineered bacterium CT-M1, and meanwhile, the genetically engineered bacterium CT-M1 is subjected to the same transformation in the wild-type yeast and named as WT-M1.

Primer sequence:

Scepyc2+mdh3-F1:

TCTGGCGAAGAATTGTGGTGGTGGTGGTGGTGTCAAGAGTCTAGGA TGAAACTCTTG

Scepyc2+mdh3-R1:

ATAGCAATCTAATCTAAGTTTTCTAGAACTAGATGGTCAAAGTCGCA ATTCTTGG

Scemdh3+pyc2-F2:

GGGCTGCAGGAATTCGATATCAAATGAGCAGTAGCAAGAAATTGG

Scemdh3+pyc2-R2:

ACATGACTCGAGGTCGACGGTATCGATAAGCTTACTTTTTTTGGGAT GGGGGTAGG

Scepyc2+mdh3-F3:

CCAAGAATTGCGACTTTGACCATCTAGTTCTAGAAAACTTAGATTAG ATTGCT

Scepyc2+mdh3-R3:

CTAAGACCGGCCAATTTCTTGCTACTGCTCATTTGATATCGAATTCCT GCAGCC

Scepyc2+mdh3-F4:

GAAACCCTACCCCCATCCCAAAAAAAGTAAGCTTATCGATACCGTC GACCT

Scepyc2+mdh3-R4:

AATATTGAAAAAGGCAAGAGTTTCATCCTAGACTCTTGACACCACC ACCACCACCACAA

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

Primer sequence:

SpMae1-F1：CAGAAAAACAGATGTGCCCAAATC

SpMae1-R1：TCCTAGGCGTAATCATGGTCATAGCTGTTTCCTGGATCCTAAACTGCGTC ATAGTAAG

SpMae1-F2：AGTATCAAAGAAACTTACTATGACGCAGTTTAGGATCCAGGAAACAGCT ATGACCATG

SpMae1-R2：AAGAGTAAAAAAGGAGTAGAAACATTTTGGAGCTCTAAAACGACGGCC AGTGCCAA

SpMae1-F3：TGCAAGTCGACCTTGGCACTGGCCGTCGTTTTAGAGCTCCAAAATGTTT CTACTCC

SpMae1-R3：AATAAACAAGGGGCTTTACGATGGAGTAGTAGACCTGCAAATTAAAGCC TTCGAGC

SpMae1-F4：GAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGTCTACTACTC CATCGTAAAG

SpMae1-R4：TGAGGAATTTACAATAAGGTGGTTCC

(3) And (3) carrying out fermentation verification on the engineering strain CT-M2 in a 250mL shaking flask, wherein the fermentation temperature is 30 ℃, the fermentation time is 96 hours, and the adopted fermentation culture 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 O. No addition of neutralizing agent CaCO ₃ On the premise of measuring the L-malic acid yield, and comparing and analyzing with the strain WT-M2. As a result, FIG. 3 shows that the yield of CT-M2 (14.03 g/L) was 2.5 times that of WT-M2 (5.61 g/L).

Sequence:

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

atgaatcctaaatcctctacacctaagattccaagacccaagaacgcatttattctgttcagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtgtggaaataccccataattcaaacatttctaaaattattggtacgaag

SEQ ID NO.6

MNPKSSTPKIPRPKNAFILFRQHYHRILIDEWTAQGVEIPHNSNISKIIGTK

SEQ ID NO.7

aagggcttacaaccggaagataaggcacactgggaaaatctagcggagaaggagaaactagaacatgaaaggaagtatcctgaatacaaatacaagccggtaagaaagtctaagaagaagcaactacttttgaaggaaatcgagcaacagcagcagcaacaacagaaagaacagcagcagcagaaacagtcacaaccgcaattacaacagccctttaacaacaatatagttcttatgaaaagagcacattctctttcaccatcttcctcggtgtcaagctcgaacagctatcagttccaattgaacaatgatcttaagaggttgcctattccttctgttaatacttctaactatatggtctccagatctttaagtggactacctttgacgcatgataagacggcaagagacctaccacagctgtcatctcaactaaattctattccatattactcagctccacacgacccttcaacgagacatcattacctcaacgtcgctcaagctcaaccaagggctaactcgacccctcaattgccctttatttcatccattatcaacaacagcagtcaaacaccggtaactacaactaccacatccacaacaactgcgacatcttctcctgggaaattctcctcttctccgaactcctctgtactggagaacaacagattaaacagtatcaacaattcaaatcaatatttacctccccctctattaccttctctgcaagattttcaactggatcagtaccagcagctaaagcagatgggaccaacttatattgtcaaaccactgtctcacaccaggaacaatctattgtccacaactacccctacgcatcatcacattcctcatataccaaaccaaaacattcctctacatcaaattataaactcaagcaacactgaggtcaccgctaaaactagcctagtttctccgaaatga

SEQ ID NO.8

KGLQPEDKAHWENLAEKEKLEHERKYPEYKYKPVRKSKKKQLLLKEIEQQQQQQQKEQQQQKQSQPQLQQPFNNNIVLMKRAHSLSPSSSVSSSNSYQFQLNNDLKRLPIPSVNTSNYMVSRSLSGLPLTHDKTARDLPQLSSQLNSIPYYSAPHDPSTRHHYLNVAQAQPRANSTPQLPFISSIINNSSQTPVTTTTTSTTTATSSPGKFSSSPNSSVLENNRLNSINNSNQYLPPPLLPSLQDFQLDQYQQLKQMGPTYIVKPLSHTRNNLLSTTTPTHHHIPHIPNQNIPLHQIINSSNTEVTAKTSLVSPK

SEQ ID NO.9

cctaagattccaagacccaagaacgcatttattctgttcagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtgtggaaataccccataattcaaacatttctaaaattattggtacgaagtggaagggcttacaaccggaagataaggcacactgggaaaatctagcggagaaggagaaactagaacataaaaggaagtatcctgaatacaaatacaagccggtaagaaagtctaag

SEQ ID NO.10

PKIPRPKNAFILFRQHYHRILIDEWTAQGVEIPHNSNISKIIGTKWKGLQ PEDKAHWENLAEKEKLEHERKYPEYKYKPVRKSK

SEQ ID NO.11

MNPKSSTPKIPRPKNAFILFRQHYHRILIDEWTAQGVEIPHNSNISKIIGTKWKGLQPEDKAHWENLAEKEKLEHERKYPEYKYKPVRKSKKKQLLLKEIEQQQQQQQKEQQQQKQSQPQLQQPFNNNIVLMKRAHSLSPSSSVSSSNSYQFQLNNDLKRLPIPSVNTSNYMVSRSLSGLPLTHDKTARDLPQLSSQLNSIPYYSAPHDPSTRHHYLNVAQAQPRANSTPQLPFISSIINNSSQTPVTTTTTSTTTATSSPGKFSSSPNSSVLENNRLNSINNSNQYLPPPLLPSLQDFQLDQYQQLKQMGPTYIVKPLSHTRNNLLSTTTPTHHHIPHIPNQNIPLHQIINSSNTEVTAKTSLVSPK

it is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

SEQUENCE LISTING

<110> university of Jiangnan

<120> an acid-resistant Saccharomyces cerevisiae and application thereof

<130> 11

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 1107

<212> DNA

<213> (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> (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> (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> (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> (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> (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> (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> (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> (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> (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> (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. The acid-resistant saccharomyces cerevisiae is characterized in that saccharomyces cerevisiae S288C is taken as an initial strain, and 8aa-91aa fragments, 54aa-368aa fragments and 1aa-53aa fragments in a ROX1 transcription regulatory factor of the saccharomyces cerevisiae S288C are knocked out to obtain the acid-resistant saccharomyces cerevisiae; the amino acid sequence of the ROX1 transcription regulating factor is shown as SEQ ID NO. 11.

2. The acid-resistant saccharomyces cerevisiae is characterized in that saccharomyces cerevisiae S288C is taken as an initial strain, and G158A in a ROX1 transcription regulatory factor is mutated to obtain the acid-resistant saccharomyces cerevisiae; 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-resistant saccharomyces cerevisiae according to claim 1, wherein after the 8aa-91aa fragment is knocked out, the nucleotide sequence of the ROX1 transcription regulator is shown as SEQ ID NO.3, and the encoded amino acid sequence is shown as SEQ ID NO. 4.

4. The acid-resistant saccharomyces cerevisiae according to claim 1, wherein the nucleotide sequence of the ROX1 transcription regulating factor is shown in SEQ ID No.5 and the encoded amino acid sequence is shown in SEQ ID No.6 after the 54aa-368aa fragment is knocked out.

5. The acid-resistant saccharomyces cerevisiae according to claim 1, wherein after knocking out 1aa-53aa fragment, the nucleotide sequence of the ROX1 transcription regulator is shown as SEQ ID No.7, and the encoded amino acid sequence is shown as SEQ ID No. 8.

6. A product comprising the acid-tolerant saccharomyces cerevisiae according to any of claims 1-5.

7. Use of the acid-tolerant saccharomyces cerevisiae according to any of claims 1-5, or the product of claim 6, for the fermentative production of L-malic acid.

8. The use according to claim 7, characterized in that the conditions of the fermentation: the fermentation temperature is 28-32 ℃, and the fermentation time is 60-108h.