Gene site-directed mutagenesis method and stress resistance breeding application thereof

文档序号：501555 发布日期：2021-05-28 浏览：24次中文

阅读说明：本技术 一种基因定点突变方法及其胁迫抗性育种应用 (Gene site-directed mutagenesis method and stress resistance breeding application thereof ) 是由蔺玉萍刘艳芳郭玉凤张媛媛王钦宏于 2020-04-20 设计创作，主要内容包括：本发明公开了一种基因定点突变方法及其胁迫抗性育种应用。本发明方法首次应用胞苷脱氨酶碱基编辑技术定点突变酿酒酵母基因组上的全局转录调控因子Spt15,扩大了转录因子Spt15的突变范围,实现了转录因子Spt15的精确突变,获得了36株突变体；在高糖高渗、高温和乙醇胁迫条件下成功且高效地筛选到14株、18株和19株耐受性明显提高的菌株,其中14株菌株在这三种胁迫下均是抗性菌株。本发明可解决实际工业发酵中酵母细胞胁迫耐受性不足的缺陷。本发明的方法操作简单方便,选育效率高,为今后耐性机制研究和菌株分子改造与应用奠定了良好基础。(The invention discloses a gene site-directed mutagenesis method and stress resistance breeding application thereof. The method of the invention firstly applies the cytidine deaminase base editing technology to perform site-specific mutagenesis on the global transcription regulatory factor Spt15 on the saccharomyces cerevisiae genome, expands the mutation range of the transcription factor Spt15, realizes the accurate mutagenesis of the transcription factor Spt15, and obtains 36 mutants; 14, 18 and 19 strains with obviously improved tolerance are successfully and efficiently screened under the conditions of high sugar hyperosmotic, high temperature and ethanol stress, wherein the 14 strains are resistant strains under the three stresses. The invention can solve the defect of insufficient stress tolerance of the yeast cells in actual industrial fermentation. The method of the invention has simple and convenient operation and high breeding efficiency, and lays a good foundation for the study of tolerance mechanism and the molecular modification and application of strains in the future.)

1. The mutant protein is obtained by performing any one or more of the following mutations (a1) to (a21) on the Spt15 protein:

(a1) the 140 th amino acid residue from the N end is mutated from A to G;

(a2) mutating the 169 th amino acid residue from the N end from P to A;

(a3) the 238 th amino acid residue from the N end is mutated from R to K;

(a4) the 2 nd amino acid residue from the N end is mutated from A to D;

(a5) the 6 th amino acid residue from the N end is mutated from R to C;

(a6) the 9 th amino acid residue from the N end is mutated from E to K;

(a7) the 26 th amino acid residue from the N end is mutated from W to S;

(a8) the 26 th amino acid residue from the N end is mutated from W to C;

(a9) the 38 th amino acid residue from the N end is mutated from T to I;

(a10) the 56 th amino acid residue from the N end is mutated from D to E;

(a11) the 101 th amino acid residue from the N end is mutated from A to P and the 102 th amino acid residue is mutated from V to I;

(a12) the 102 th amino acid residue from the N end is mutated from V to L;

(a13) the 214 th amino acid residue from the N end is mutated from L to F;

(a14) the 238 th amino acid residue from the N end is mutated from R to T;

(a15) the 20 th amino acid residue from the N end is mutated from P to L;

(a16) the 150 th amino acid residue from the N end is mutated from A to P;

(a17) the 20 th amino acid residue from the N end is mutated from P to R;

(a18) the 38 th amino acid residue from the N end is mutated from T to S;

(a19) mutating from P to L at 65 th amino acid residue from N terminal;

(a20) mutating the 71 th amino acid residue from the N end from V to L;

(a21) the 192 th amino acid residue from the N terminal is mutated from G to S.

2. A polynucleotide encoding the mutant protein of claim 1, or an expression cassette, a recombinant vector or a recombinant microorganism having the polynucleotide.

3. Use of the mutant protein of claim 1, or the polynucleotide of claim 2, or an expression cassette, recombinant vector or recombinant microorganism comprising said polynucleotide in saccharomyces cerevisiae breeding.

4. A method for increasing stress tolerance of Saccharomyces cerevisiae by any one or more of the following methods 1-21;

the method 1 comprises the following steps: mutating the codon of the 140 th amino acid residue A of the Spt15 protein in the saccharomyces cerevisiae genome into the codon of the amino acid residue G, thereby improving the high sugar hyperosmotic stress tolerance, high temperature stress tolerance and/or ethanol stress tolerance of the saccharomyces cerevisiae;

the method 2 comprises the following steps: mutating the codon of 169 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into the codon of coding amino acid residue A, thereby improving the high sugar hyperosmotic stress tolerance, high temperature stress tolerance and/or ethanol stress tolerance of the saccharomyces cerevisiae;

the method 3 comprises the following steps: mutating a codon for encoding 238 th amino acid residue R of Spt15 protein in a saccharomyces cerevisiae genome into a codon for encoding amino acid residue K, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 4 comprises the following steps: mutating a codon encoding the 2 nd amino acid residue A of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding the amino acid residue D, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 5 comprises the following steps: mutating a codon for coding the 6 th amino acid residue R of the Spt15 protein in a saccharomyces cerevisiae genome into a codon for coding an amino acid residue C, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 6 comprises the following steps: mutating a codon encoding the 9 th amino acid residue E of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue K, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 7 comprises the following steps: mutating a codon encoding the 26 th amino acid residue W of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue S, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 8 comprises the following steps: mutating a codon encoding the 26 th amino acid residue W of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue C, thereby improving the high sugar hyperosmotic stress tolerance, the high temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae;

the method 9 comprises the following steps: mutating the codon encoding the 38 th amino acid residue T of the Spt15 protein in a saccharomyces cerevisiae genome into the codon encoding the amino acid residue I, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 10 comprises the following steps: mutating a codon encoding the 56 th amino acid residue D of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding the amino acid residue E, thereby improving the high sugar hyperosmotic stress tolerance, high temperature stress tolerance and/or ethanol stress tolerance of the saccharomyces cerevisiae;

the method 11 comprises the following steps: the method comprises the following steps of (1) mutating a codon for a 101 th amino acid residue A of a Spt15 protein in a saccharomyces cerevisiae genome into a codon for a P encoded amino acid residue, and mutating a codon for a 102 th amino acid residue V of a Spt15 protein in the saccharomyces cerevisiae genome into a codon for an I encoded amino acid residue, so that the high sugar hyperosmotic stress tolerance and/or the high temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae are improved;

the method 12 comprises the following steps: mutating a codon encoding the 102 th amino acid residue V of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue L, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 13 comprises the following steps: mutating a codon encoding 214 th amino acid residue L of Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding amino acid residue F, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 14 comprises the following steps: mutating a codon for encoding 238 th amino acid residue R of Spt15 protein in a saccharomyces cerevisiae genome into a codon for encoding amino acid residue T, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress;

the method 15 comprises the following steps: mutating a codon encoding the 20 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue L, thereby improving the high-temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae;

the method 16 comprises the following steps: mutating the codon encoding the 150 th amino acid residue A of the Spt15 protein in the saccharomyces cerevisiae genome into the codon encoding the amino acid residue P, thereby improving the high-temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae;

the method 17 comprises the following steps: mutating the codon of the 20 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into the codon of the encoded amino acid residue R, thereby improving the high-temperature stress tolerance of the saccharomyces cerevisiae;

the method 18 comprises the following steps: mutating the codon of the 38 th amino acid residue T of the Spt15 protein in a saccharomyces cerevisiae genome into a codon of a coded amino acid residue S, thereby improving the high-temperature stress tolerance of the saccharomyces cerevisiae;

the method 19 comprises the following steps: mutating the codon of the 65 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into the codon of the coding amino acid residue L, thereby improving the ethanol stress tolerance of the saccharomyces cerevisiae;

the method 20 comprises the following steps: mutating the codon of 71 th amino acid residue V of the Spt15 protein in a saccharomyces cerevisiae genome into a codon of a coded amino acid residue L, thereby improving the ethanol stress tolerance of the saccharomyces cerevisiae;

the method 21 comprises the following steps: and (3) mutating the codon for the 192 th amino acid residue G of the Spt15 protein in the saccharomyces cerevisiae genome into the codon for the amino acid residue S, thereby improving the ethanol stress tolerance of the saccharomyces cerevisiae.

5. A method or use;

the method is a method for obtaining the recombinant microorganism with changed directional characters, and comprises the following steps:

(1) introducing mutation by using a cytidine deaminase base editing system to obtain a plurality of recombinant microorganisms by taking a coding gene of a target protein in a receptor microorganism as an object; each recombinant microorganism has a mutant form; the cytidine deaminase base editing system adopts 1 or more gRNAs, and the design principle of the gRNAs comprises the following (A), (B) and (C): (A) the target sequence is positioned in the coding region of the coding gene; (B) causing a codon encoding at least one existing amino acid residue of the protein of interest to be mutated to a codon encoding another amino acid residue; (C) does not cause a mutation of the codon encoding any existing amino acid residue of the protein of interest to a stop codon; (2) screening multiple recombinant microorganisms obtained in the step (1) to obtain recombinant microorganisms with changed directional characters;

the application is the application of the method in breeding of microorganisms.

6. A recombinant microorganism obtained by the method of claim 5.

7. A method or use;

the method is a method for preparing recombinant saccharomyces cerevisiae bacteria, and is any one or combination of methods 1-16;

the method 1 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 5; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 5 th site from the 5' end is mutated from C to A and the 6 th site is mutated from C to T;

the method 2 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 6; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to T at 16 th site of 5' end;

the method 3 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 7; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 24 th site from the 5' end is mutated from G to A and the 25 th site is mutated from G to A;

the method 4 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 8; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (a) mutation from C to T at the 58 th site and C to T at the 59 th site from the 5' end; (b) mutation from C to G at the 59 th site of the 5' end;

the method 5 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 9; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (c) the 77 th site from the 5' end is mutated from G to C and the 78 th site is mutated from G to A; (d) the 78 th site of the 5' end is mutated from G to C;

the method 6 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 10; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (e) mutation from C to T at 113 th site of 5' end; (f) mutation from C to G at 113 th site of 5' end;

the method 7 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 11; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to G from 168 th site of 5' end;

the method 8 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 12; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 194 th position from the 5' end is mutated from C to T;

the method 9 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 13; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 211 th site is mutated from G to C and the 213 th site is mutated from G to A from the 5' end;

the method 10 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 14; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (g) the 301 th site is mutated from G to C and the 304 th site is mutated from G to A from the 5' end; (h) mutation from G to C from the 304 th site of the 5' end;

the method 11 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 15; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to G at the 419 th site of the 5' end;

the method 12 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 16; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from G to C at 448 th site from the 5' end;

the method 13 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 17; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 505 th site from the 5' end is mutated from C to G;

the method 14 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 18; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from G to A at position 574 and G to A at position 579 from the 5' end;

the method 15 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 19; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to T at 640 th site of 5' end;

the method 16 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 20; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (i) the 713 th site of the 5' end is mutated from G to A; (j) mutating from the 713 th site of the 5' end to C from G;

the application is the application of the method in saccharomyces cerevisiae breeding.

8. The recombinant saccharomyces cerevisiae strain prepared by the method of any one of the claims 7.

9. A method or use;

the method is a method for obtaining mutant protein with changed microorganism directional characters, and comprises the following steps:

(2) screening multiple recombinant microorganisms obtained in the step (1) to obtain recombinant microorganisms with changed directional characters;

(3) sequencing a mutant gene corresponding to the coding gene of the target protein in the recombinant microorganism obtained in the step (2) to obtain a sequence of the mutant protein;

the application is the application of the method in breeding of microorganisms.

10. A mutant protein obtained by the method of claim 8.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a gene site-directed mutagenesis method and stress resistance breeding application thereof.

Background

Saccharomyces cerevisiae has a long fermentation history, and is finally developed into an important cell factory from traditional food fermentation to modern process fermentation for producing medicines, energy substitutes, bulk chemicals and the like. However, in the actual industrial fermentation production, the saccharomyces cerevisiae fermentation is restricted by various stresses, especially the toxic action of stresses such as hypertonicity, high temperature and ethanol. Therefore, improving the stress tolerance of s.cerevisiae is crucial to improving the productivity of the strain. Many breeding strategies for improving the stress tolerance of the saccharomyces cerevisiae include protoplast fusion, mutation breeding, adaptive evolution and the like. In 2006, Alper et al reported that random mutagenesis was performed on the initial transcription factor Spt15 in RNA polymerase II for yeast to perform transcription by a Global transcription machinery engineering (gTME) method, which is based on error-prone PCR, wherein erroneous bases were randomly introduced into the coding region of Spt15 gene, and the whole transcription regulation process was changed by modifying the Global transcription regulation factor, thereby affecting the change of TATA binding ability of promoter region of related gene, so that related gene was overexpressed or suppressed, and phenotypically improving the stress tolerance of saccharomyces cerevisiae to hyperosmotic and high ethanol. The transcription factor Spt15 belongs to a transcription initiation complex, is a TATA binding protein, and binds to RNA polymerase II and other transcription factors to transcribe mRNA genes, thereby affecting gene expression.

Chinese patent publication No. CN105950649B describes a method of mutating SPT15 gene on plasmid by error-prone PCR, and then transforming yeast to screen a strain with high ethanol yield, which only obtains a beneficial mutant. The method utilizes TaqDNA polymerase which has low fidelity and does not have exonuclease activity of 3 '→ 5' end, generates base mismatch in the amplification process, generates random mutation, has limited application range, is difficult to control the mutation, and is harmful to cells due to excessive mutation. Chinese patent publication No. CN107988092A discloses mutant strains and engineering bacteria of Arthrobacter simplex with stress tolerance, and an ethanol-resistant mutant strain of Arthrobacter simplex is bred by a method combining ultraviolet-sodium nitrite composite mutagenesis and pressure domestication. The method has the advantages of long mutation breeding period, unstable characters and low screening efficiency.

The cytidine deaminase base editing (CBE) technology can operate a genome and realize accurate mutation from a base C to a base T. The technology has the advantages of wide application range, simple operation, no DNA double-strand break, no need of exogenous templates, high cell survival rate and the like. The technology has been applied to mutation correction and the like in higher eukaryotes such as mammals and plants. In 2016, Nishida reported a single base editing system for s.cerevisiae, using CBE technology to generate stop codons to evaluate the efficiency of this technology in s.cerevisiae. This study showed that the CBE technique can edit bases in the window-13 to-20 upstream of the PAM, where editing at the-18 position is most efficient. In addition to the base mutations that result from C to T, the CBE technique also found a proportion of C to G and C to a mutations. However, no relevant literature and patent report exists yet for realizing protein mutation by carrying out amino acid mutation on a coding region of a saccharomyces cerevisiae genome gene by utilizing a CBE technology.

Disclosure of Invention

The invention aims to provide a gene site-directed mutagenesis method and stress resistance breeding application thereof.

In a first aspect, the protective mutant protein of the present invention is a protein obtained by mutating Spt15 protein by any one or more of the following (a1) to (a 21): (a1) the 140 th amino acid residue from the N end is mutated from A to G; (a2) mutating the 169 th amino acid residue from the N end from P to A; (a3) the 238 th amino acid residue from the N end is mutated from R to K; (a4) the 2 nd amino acid residue from the N end is mutated from A to D; (a5) the 6 th amino acid residue from the N end is mutated from R to C; (a6) the 9 th amino acid residue from the N end is mutated from E to K; (a7) the 26 th amino acid residue from the N end is mutated from W to S; (a8) the 26 th amino acid residue from the N end is mutated from W to C; (a9) the 38 th amino acid residue from the N end is mutated from T to I; (a10) the 56 th amino acid residue from the N end is mutated from D to E; (a11) the 101 th amino acid residue from the N end is mutated from A to P and the 102 th amino acid residue is mutated from V to I; (a12) the 102 th amino acid residue from the N end is mutated from V to L; (a13) the 214 th amino acid residue from the N end is mutated from L to F; (a14) the 238 th amino acid residue from the N end is mutated from R to T; (a15) the 20 th amino acid residue from the N end is mutated from P to L; (a16) the 150 th amino acid residue from the N end is mutated from A to P; (a17) the 20 th amino acid residue from the N end is mutated from P to R; (a18) the 38 th amino acid residue from the N end is mutated from T to S; (a19) mutating from P to L at 65 th amino acid residue from N terminal; (a20) mutating the 71 th amino acid residue from the N end from V to L; (a21) the 192 th amino acid residue from the N terminal is mutated from G to S.

The Spt15 protein can be specifically a protein shown as SEQ ID No. 4.

The Spt15 protein may specifically be a protein derived from SEQ ID No.4 by substituting and/or deleting and/or adding the amino acid sequence of SEQ ID No.4 by one or several amino acid residues at positions other than the positions described in (a1) to (a21) and having the same function.

In a second aspect, the present invention also protects a polynucleotide encoding a mutant protein as hereinbefore described.

The polynucleotide for coding the mutant protein of (a1) is a DNA molecule obtained by mutating 419 th position from 5' end of SEQ ID No.3 from C to G; the polynucleotide for coding the mutant protein (a2) is a DNA molecule obtained by mutating 505 th site of SEQ ID No.3 from 5' end from C to G; the polynucleotide for coding the mutant protein (a3) is a DNA molecule obtained by mutating the 713 th site from the 5' end of SEQ ID No.3 from G to A; the polynucleotide for coding the mutant protein (a4) is specifically a DNA molecule obtained by mutating the 5 th position from C to A and the 6 th position from C to T from the 5' end of SEQ ID No. 3; the polynucleotide encoding the mutant protein of (a5) is specifically a DNA molecule obtained by mutating the 16 th position from the 5' end of SEQ ID No.3 from C to T; the polynucleotide for coding the mutant protein (a6) is a DNA molecule obtained by mutating the 24 th position from G to A and the 25 th position from G to A of the 5' end of SEQ ID No. 3; the polynucleotide for coding the mutant protein (a7) is a DNA molecule obtained by mutating the 77 th site from the 5' end of SEQ ID No.3 from G to C and the 78 th site from G to A; the polynucleotide for coding the mutant protein (a8) is a DNA molecule obtained by mutating the 78 th site from the 5' end of SEQ ID No.3 from G to C; the polynucleotide encoding the mutant protein of (a9) is specifically a DNA molecule obtained by mutating the 113 th site from the 5' end of SEQ ID No.3 from C to T; the polynucleotide for coding the mutant protein (a10) is a DNA molecule obtained by mutating the 168 th site from the 5' end of SEQ ID No.3 from C to G; the polynucleotide for coding the mutant protein (a11) is a DNA molecule obtained by mutating the 301 th position from G to C and the 304 th position from G to A of the 5' end of SEQ ID No. 3; the polynucleotide for coding the mutant protein (a12) is a DNA molecule obtained by mutating the 304 th position from the 5' end of SEQ ID No.3 from G to C; the polynucleotide encoding the mutant protein of (a13) is specifically a DNA molecule obtained by mutating the 640 th position from the 5' end of SEQ ID No.3 from C to T; the polynucleotide encoding the mutant protein of (a14) is specifically a DNA molecule obtained by mutating the 713 th site from the 5' end of SEQ ID No.3 from G to C; the polynucleotide for coding the mutant protein (a15) is specifically a DNA molecule obtained by mutating the 58 th site of the 5' end of SEQ ID No.3 from C to T and the 59 th site from C to T; the polynucleotide encoding the mutant protein of (a16) is specifically a DNA molecule obtained by mutating the 448 th position from the 5' end of SEQ ID No.3 from G to C; the polynucleotide for coding the mutant protein of (a17) is a DNA molecule obtained by mutating the 59 th position from the 5' end of SEQ ID No.3 from C to G; the polynucleotide encoding the mutant protein of (a18) is specifically a DNA molecule obtained by mutating the 113 th site from the 5' end of SEQ ID No.3 from C to G; the polynucleotide encoding the mutant protein of (a19) is specifically a DNA molecule obtained by mutating the 194 th site of the 5' end of SEQ ID No.3 from C to T; the polynucleotide for coding the mutant protein (a20) is a DNA molecule obtained by mutating the 211 th position from G to C and the 213 th position from G to A of the 5' end of SEQ ID No. 3; the polynucleotide for coding the mutant protein (a21) is specifically a DNA molecule obtained by mutating the 574 th position from the 5' end of SEQ ID No.3 from G to A and the 579 th position from G to A.

In a third aspect, the invention also protects an expression cassette, a recombinant vector or a recombinant microorganism having a polynucleotide as described hereinbefore. The recombinant microorganism may specifically be a recombinant saccharomyces cerevisiae. The recombinant s.cerevisiae having a polynucleotide encoding the mutant protein described in (a1) - (a14) above has higher tolerance to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress than the s.cerevisiae having a polynucleotide encoding the wild-type Spt15 protein. The high temperature stress tolerance and/or ethanol stress tolerance of the recombinant s.cerevisiae having a polynucleotide encoding the mutant protein described in (a15) - (a16) above that of the s.cerevisiae having a polynucleotide encoding the wild-type Spt15 protein. The high temperature stress tolerance of recombinant s.cerevisiae having a polynucleotide encoding the mutant protein described in (a17) - (a18) above was higher than that of s.cerevisiae having a polynucleotide encoding the wild-type Spt15 protein. The ethanol stress tolerance of the recombinant s.cerevisiae having a polynucleotide encoding the mutant protein described in (a19) - (a21) above was higher than that of the s.cerevisiae having a polynucleotide encoding the wild-type Spt15 protein. The recombinant saccharomyces cerevisiae can be obtained BY modifying a saccharomyces cerevisiae BY4741 strain.

In a fourth aspect, the present invention protects the use of a mutant protein or polynucleotide or expression cassette, recombinant vector or recombinant microorganism as described hereinbefore in the breeding of Saccharomyces cerevisiae. The aim of the breeding is to breed strains of Saccharomyces cerevisiae with increased stress tolerance. The stress tolerance may in particular be a high sugar hyperosmotic stress tolerance and/or a high temperature stress tolerance and/or an ethanol stress tolerance.

In a fifth aspect, the present invention provides a method of increasing stress tolerance of Saccharomyces cerevisiae by any one or combination of methods 1-21; the method 1 comprises the following steps: mutating the codon of the 140 th amino acid residue A of the Spt15 protein in the saccharomyces cerevisiae genome into the codon of the amino acid residue G, thereby improving the high sugar hyperosmotic stress tolerance, high temperature stress tolerance and/or ethanol stress tolerance of the saccharomyces cerevisiae; the method 2 comprises the following steps: mutating the codon of 169 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into the codon of coding amino acid residue A, thereby improving the high sugar hyperosmotic stress tolerance, high temperature stress tolerance and/or ethanol stress tolerance of the saccharomyces cerevisiae; the method 3 comprises the following steps: mutating a codon for encoding 238 th amino acid residue R of Spt15 protein in a saccharomyces cerevisiae genome into a codon for encoding amino acid residue K, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 4 comprises the following steps: mutating a codon encoding the 2 nd amino acid residue A of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding the amino acid residue D, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 5 comprises the following steps: mutating a codon for coding the 6 th amino acid residue R of the Spt15 protein in a saccharomyces cerevisiae genome into a codon for coding an amino acid residue C, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 6 comprises the following steps: mutating a codon encoding the 9 th amino acid residue E of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue K, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 7 comprises the following steps: mutating a codon encoding the 26 th amino acid residue W of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue S, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 8 comprises the following steps: mutating a codon encoding the 26 th amino acid residue W of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue C, thereby improving the high sugar hyperosmotic stress tolerance, the high temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae; the method 9 comprises the following steps: mutating the codon encoding the 38 th amino acid residue T of the Spt15 protein in a saccharomyces cerevisiae genome into the codon encoding the amino acid residue I, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 10 comprises the following steps: mutating a codon encoding the 56 th amino acid residue D of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding the amino acid residue E, thereby improving the high sugar hyperosmotic stress tolerance, high temperature stress tolerance and/or ethanol stress tolerance of the saccharomyces cerevisiae; the method 11 comprises the following steps: the method comprises the following steps of (1) mutating a codon for a 101 th amino acid residue A of a Spt15 protein in a saccharomyces cerevisiae genome into a codon for a P encoded amino acid residue, and mutating a codon for a 102 th amino acid residue V of a Spt15 protein in the saccharomyces cerevisiae genome into a codon for an I encoded amino acid residue, so that the high sugar hyperosmotic stress tolerance and/or the high temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae are improved; the method 12 comprises the following steps: mutating a codon encoding the 102 th amino acid residue V of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue L, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 13 comprises the following steps: mutating a codon encoding 214 th amino acid residue L of Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding amino acid residue F, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 14 comprises the following steps: mutating a codon for encoding 238 th amino acid residue R of Spt15 protein in a saccharomyces cerevisiae genome into a codon for encoding amino acid residue T, thereby improving the tolerance of the saccharomyces cerevisiae to high sugar hyperosmotic stress and/or high temperature stress and/or ethanol stress; the method 15 comprises the following steps: mutating a codon encoding the 20 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into a codon encoding an amino acid residue L, thereby improving the high-temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae; the method 16 comprises the following steps: mutating the codon encoding the 150 th amino acid residue A of the Spt15 protein in the saccharomyces cerevisiae genome into the codon encoding the amino acid residue P, thereby improving the high-temperature stress tolerance and/or the ethanol stress tolerance of the saccharomyces cerevisiae; the method 17 comprises the following steps: mutating the codon of the 20 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into the codon of the encoded amino acid residue R, thereby improving the high-temperature stress tolerance of the saccharomyces cerevisiae; the method 18 comprises the following steps: mutating the codon of the 38 th amino acid residue T of the Spt15 protein in a saccharomyces cerevisiae genome into a codon of a coded amino acid residue S, thereby improving the high-temperature stress tolerance of the saccharomyces cerevisiae; the method 19 comprises the following steps: mutating the codon of the 65 th amino acid residue P of the Spt15 protein in a saccharomyces cerevisiae genome into the codon of the coding amino acid residue L, thereby improving the ethanol stress tolerance of the saccharomyces cerevisiae; the method 20 comprises the following steps: mutating the codon of 71 th amino acid residue V of the Spt15 protein in a saccharomyces cerevisiae genome into a codon of a coded amino acid residue L, thereby improving the ethanol stress tolerance of the saccharomyces cerevisiae; the method 21 comprises the following steps: and (3) mutating the codon for the 192 th amino acid residue G of the Spt15 protein in the saccharomyces cerevisiae genome into the codon for the amino acid residue S, thereby improving the ethanol stress tolerance of the saccharomyces cerevisiae.

The Spt15 protein can be specifically a protein shown as SEQ ID No. 4.

The Saccharomyces cerevisiae may be Saccharomyces cerevisiae BY4741 strain.

In a sixth aspect, the present invention provides a method for obtaining a recombinant microorganism with altered directional traits, comprising the steps of:

in the method, the mutation of the codon causes mutation of an amino acid residue, thereby causing change of the directional character.

When multiple grnas are designed, their target sequences may be located in different regions of the coding region of the coding gene, respectively.

In the method, the microorganism can be specifically yeast, more specifically saccharomyces cerevisiae, such as saccharomyces cerevisiae BY4741 strain. In the method, the target protein can be Spt15 protein, shown as SEQ ID No.4, and the coding gene is shown as SEQ ID No. 3. In the method, the targeted trait alteration may specifically be an increase in stress tolerance. The stress tolerance may in particular be a high sugar hyperosmotic stress tolerance and/or a high temperature stress tolerance and/or an ethanol stress tolerance.

In the method, when the target protein is Spt15 protein, the gRNA may specifically be any combination of the following 16 grnas: gRNA1 with a target sequence of SEQ ID No.5, gRNA2 with a target sequence of SEQ ID No.6, gRNA3 with a target sequence of SEQ ID No.7, gRNA4 with a target sequence of SEQ ID No.8, gRNA5 with a target sequence of SEQ ID No.9, gRNA6 with a target sequence of SEQ ID No.10, gRNA7 with a target sequence of SEQ ID No.11, gRNA8 with a target sequence of SEQ ID No.12, gRNA9 with a target sequence of SEQ ID No.13, gRNA10 with a target sequence of SEQ ID No.14, gRNA11 with a target sequence of SEQ ID No.15, gRNA12 with a target sequence of SEQ ID No.16, gRNA13 with a target sequence of SEQ ID No.17, gRNA14 with a target sequence of SEQ ID No.18, gRNA15 with a target sequence of SEQ ID No.19 and gRNA16 with a target sequence of SEQ ID No. 20.

The invention also protects the application of the method in microbial breeding. The breeding of the microorganism may specifically be aimed at screening for a microorganism with increased stress tolerance. The microorganism may specifically be a yeast, more specifically a saccharomyces cerevisiae, such as saccharomyces cerevisiae BY4741 strain. The stress tolerance may in particular be a high sugar hyperosmotic stress tolerance and/or a high temperature stress tolerance and/or an ethanol stress tolerance.

The invention also protects the recombinant microorganism prepared by the method. In the embodiment of the invention, 36 recombinant saccharomyces cerevisiae strains are obtained by the method, and 21 recombinant saccharomyces cerevisiae strains with enhanced high sugar hyperosmotic stress tolerance, high temperature stress tolerance and/or ethanol stress tolerance are screened from the recombinant saccharomyces cerevisiae strains.

In a seventh aspect, the invention provides a method for preparing recombinant saccharomyces cerevisiae, which is any one or combination of methods 1 to 16; the method 1 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 5; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 5 th site from the 5' end is mutated from C to A and the 6 th site is mutated from C to T; the method 2 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 6; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to T at 16 th site of 5' end; the method 3 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 7; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 24 th site from the 5' end is mutated from G to A and the 25 th site is mutated from G to A; the method 4 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 8; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (a) mutation from C to T at the 58 th site and C to T at the 59 th site from the 5' end; (b) mutation from C to G at the 59 th site of the 5' end; the method 5 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 9; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (c) the 77 th site from the 5' end is mutated from G to C and the 78 th site is mutated from G to A; (d) the 78 th site of the 5' end is mutated from G to C; the method 6 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 10; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (e) mutation from C to T at 113 th site of 5' end; (f) mutation from C to G at 113 th site of 5' end; the method 7 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 11; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to G from 168 th site of 5' end; the method 8 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 12; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 194 th position from the 5' end is mutated from C to T; the method 9 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 13; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 211 th site is mutated from G to C and the 213 th site is mutated from G to A from the 5' end; the method 10 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 14; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (g) the 301 th site is mutated from G to C and the 304 th site is mutated from G to A from the 5' end; (h) mutation from G to C from the 304 th site of the 5' end; the method 11 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 15; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to G at the 419 th site of the 5' end; the method 12 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 16; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from G to C at 448 th site from the 5' end; the method 13 comprises the following steps: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 17; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: the 505 th site from the 5' end is mutated from C to G; the method 14 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 18; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from G to A at position 574 and G to A at position 579 from the 5' end; the method 15 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 19; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has the following mutations: mutation from C to T at 640 th site of 5' end; the method 16 comprises the steps of: introducing mutation by using a cytidine deaminase base editing system by taking a Spt15 gene in saccharomyces cerevisiae recipient bacteria as an object to obtain saccharomyces cerevisiae recombinant bacteria; the cytidine deaminase base editing system adopts specific gRNA, and the target sequence of the gRNA is SEQ ID No. 20; compared with the saccharomyces cerevisiae recipient strain, the Spt15 gene of the saccharomyces cerevisiae recombinant strain has any mutation as follows: (i) the 713 th site of the 5' end is mutated from G to A; (j) the 713 th site of the 5' end is mutated from G to C.

The gene Spt15 is a DNA molecule shown in SEQ ID No.3 or a DNA molecule which has the same source as SEQ ID No.3, 90 percent of homology and the same function.

The method is a method for improving the stress tolerance of the saccharomyces cerevisiae strain. Compared with saccharomyces cerevisiae receptor bacteria, the saccharomyces cerevisiae recombinant bacteria obtained by the method have improved high-sugar hyperosmotic stress tolerance and/or high-temperature stress tolerance and/or ethanol stress tolerance.

The invention also protects the application of the method in saccharomyces cerevisiae breeding. The breeding can specifically aim at screening saccharomyces cerevisiae with improved stress tolerance. The saccharomyces cerevisiae can be a saccharomyces cerevisiae BY4741 strain. The stress tolerance may in particular be a high sugar hyperosmotic stress tolerance and/or a high temperature stress tolerance and/or an ethanol stress tolerance.

The invention also protects the saccharomyces cerevisiae recombinant strain prepared by the method.

In an eighth aspect, the present invention provides a method for obtaining a mutant protein with altered microbial directional traits, comprising the steps of: (1) introducing mutation by using a cytidine deaminase base editing system to obtain a plurality of recombinant microorganisms by taking a coding gene of a target protein in a receptor microorganism as an object; each recombinant microorganism has a mutant form; the cytidine deaminase base editing system adopts 1 or more gRNAs, and the design principle of the gRNAs comprises the following (A), (B) and (C): (A) the target sequence is positioned in the coding region of the coding gene; (B) causing a codon encoding at least one existing amino acid residue of the protein of interest to be mutated to a codon encoding another amino acid residue; (C) does not cause a mutation of the codon encoding any existing amino acid residue of the protein of interest to a stop codon; (2) screening multiple recombinant microorganisms obtained in the step (1) to obtain recombinant microorganisms with changed directional characters; (3) sequencing a mutant gene corresponding to the coding gene of the target protein in the recombinant microorganism obtained in the step (2) to obtain a sequence of the mutant protein;

in the method, the mutation of the codon causes mutation of an amino acid residue, thereby causing change of the directional character.

When multiple grnas are designed, their target sequences may be located in different regions of the coding region of the coding gene, respectively.

The invention also protects the application of the method in microbial breeding. The breeding of the microorganism specifically aims to screen mutant proteins capable of improving the stress tolerance of the microorganism. The microorganism may specifically be a yeast, more specifically a saccharomyces cerevisiae, such as saccharomyces cerevisiae BY4741 strain. The stress tolerance may in particular be a high sugar hyperosmotic stress tolerance and/or a high temperature stress tolerance and/or an ethanol stress tolerance.

The invention also protects the mutant protein obtained by the method. In the embodiment of the invention, the Spt15 protein is used as the target by the method of the eighth aspect, and 21 mutant proteins, specifically the mutant proteins shown in the first aspect, are obtained in total.

The cytidine deaminase base editing system specifically comprises a cytidine deaminase base editor and a gRNA expression vector. The cytidine deaminase base editor can be specifically a plasmid pRS315e _ pGal-nCas9(D10A) -PmCDA 1.

The method of the invention firstly applies the cytidine deaminase base editing technology to perform site-specific mutagenesis on the global transcription regulatory factor Spt15 on the saccharomyces cerevisiae genome, expands the mutation range of the transcription factor Spt15, realizes the accurate mutagenesis of the transcription factor Spt15, and obtains 36 mutants; 14, 18 and 19 strains with obviously improved tolerance are successfully and efficiently screened under the conditions of high sugar hyperosmotic, high temperature and ethanol stress, wherein the 14 strains are resistant strains under the three stresses. Meanwhile, the method of the invention can also be used for constructing other protein mutations and mutant strains. The invention can solve the defect of insufficient stress tolerance of the yeast cells in actual industrial fermentation. The method of the invention has simple and convenient operation and high breeding efficiency, and lays a good foundation for the study of tolerance mechanism and the molecular modification and application of strains in the future.

Drawings

Fig. 1 shows gRNA design.

FIG. 2 is a plasmid map of pRS423-gRNA (pCAS).

FIG. 3 shows statistics of URA3 and ADE1 gene editing situations. (A) Mutation of the target sites of the genes URA3 and ADE1, wherein the lower case letters represent the mutated bases, and the amino acid sequences of the genes are wild type N20 and PAM sequences; (B) cell viability for genes ADE1 and URA 3; (C) mutation rates edited by passage of genes URA3 and ADE 1.

FIG. 4 is a principal component analysis of fermentation data under conditions of high sugar hyperosmotic stress; methods using biological replicates (2 replicates); all strains were evaluated for fermentation in 4 batches, including the first (circle), the second (triangle), the third (square), the fourth (diamond); tolerant strains are indicated by the upward arrow and susceptible strains by the downward arrow.

FIG. 5 is a principal component analysis of fermentation data under high temperature stress conditions in an embodiment of the present invention; methods using biological replicates (2 replicates); all strains were evaluated for fermentation in 4 batches, including the first (circle), the second (triangle), the third (square), the fourth (diamond); tolerant strains are indicated by the upward arrow and susceptible strains by the downward arrow.

FIG. 6 is a principal component analysis of fermentation data under ethanol stress conditions in an embodiment of the invention; (ii) a Methods using biological replicates (2 replicates); all strains were evaluated for fermentation in 4 batches, including the first (circle), the second (triangle), the third (square), the fourth (diamond); tolerant strains are indicated by the upward arrow and susceptible strains by the downward arrow.

FIG. 7 is an alignment analysis of protein structures of stress tolerant mutants. The wild-type protein structure is on the left and the mutant protein structure is on the right. Within the circle are mutated amino acids.

Figure 8 is the number of significantly differentially expressed genes.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Plasmid pCAS: addgene, plasmid number: # 60847.

(ii) pRS423 plasmid: described in Christianson TW, Sikorski RS, Dante M, Shoro JH, high P.multifunctional year high-copy-number shunt vectors, Gene,1992,110(1): 119-; the public is available from the institute of biotechnology for Tianjin industry, the Chinese academy of sciences.

Recombinase clonxpress II: nanjing nuokexin Biotechnology, Inc., cat #: and C112-02.

Zymo Research Frozen-EZ Yeast TransformationⅡKit^TMYeast transformation kit: boaoruijing (beijing) science and technology development ltd, cat no: t2001.

Cytidine deaminase plasmid pRS315e _ pGal-nCas9(D10A) -PmCDA 1: addgene, plasmid number: # 79617.

The pRS315 plasmid is described in the literature: sikorski, r.s., and Hieter, P. (1989). a system of short vectors and a year host strain designed for efficacy management of DNA in Saccharomyces cerevisiae 122, 19-27; the public is available from the institute of biotechnology for Tianjin industry, the Chinese academy of sciences.

Saccharomyces cerevisiae BY4741a strain (MATa; his 3. DELTA.1; leu 2. DELTA.0; met 15. DELTA.0; URA 3. DELTA.0; pdc1: URA 3): are described in the literature: zhang G, Lin Y, Qi X, Li L, Wang Q, Ma Y. talens-associated multiplex editing for an acceptable geometry evaluation to improve layer Biology. ACS Synthetic Biology 2015,4(10) 1101-1111; the public is available from the institute of biotechnology for Tianjin industry, the Chinese academy of sciences.

Saccharomyces cerevisiae BY4741 strain (MATa; his 3. DELTA.1; leu 2. DELTA.0; met 15. DELTA.0; ura 3. DELTA.0): EUROSCARF.

-Leu-His Minus Media: beijing pan keno technologies, Inc., cat #: YGM 003A-6.

SC Complete Medium: beijing pan keno technologies, Inc., cat #: YGM 003A-1.

Example 1 method of site-directed mutagenesis of the coding region of a Gene by base editing of cytidine deaminase

This example carries out the gene editing for the endogenous genes URA3, ADE1 and SPT15 of Saccharomyces cerevisiae

The URA3 gene is shown in SEQ ID No. 1.

The ADE1 gene is shown in SEQ ID No. 2.

The gene SPT15 is shown in SEQ ID No.3, and the encoded protein is shown in SEQ ID No. 4.

First, selection of target site

The corresponding gRNA sequences were found by searching for candidate base editing target sites of endogenous genes URA3, ADE1 and SPT15 using the ATUM gRNADesign Tool website (https:// www.atum.bio/eCommerce/cas9/input), and the design concept is shown in FIG. 1. All gRNA sequences of URA3, ADE1, and SPT15 gene coding regions were searched on the ATUM website, manually looking for windows located 13 to 20 before PAM. When base editing introducing a stop codon is carried out, a triplet codon CAA \ CAG \ CGA \ CCA is searched in a window from 13 to 20 sites in front of PAM, and when C is edited and mutated into T by CBE base, the stop codon TAA \ TAG \ TGA \ CCA is generated. When the purpose of amino acid mutation is not to generate a stop codon but to change the function of the protein, a triplet codon (except CAA \ CAG \ CGA \ CCA) containing a base C is searched in a window from 13 to 20 sites in front of PAM, and when C is edited and mutated into T by a CBE base, the codon mutation causes the mutation of the coded amino acid. Through the search, two target sites of URA3 gene, two target sites of ADE1 gene and 24 target sites of SPT15 gene were co-selected for subsequent experiments.

Second, construction of gRNA plasmid

1. Amplification of gRNA expression cassettes

Carrying out PCR amplification by taking a plasmid pCAS containing a gRNA expression cassette as a template and XmaI-F and BamHI-R as primers; and recovering the PCR amplification product to obtain the gRNA expression cassette.

XmaI-F (forward primer): 5'-cccccgggtctttgaaaagataatgtat-3', respectively;

BamHI-R (reverse primer): 5'-cgggatcctatccactagacagaagttt-3' are provided.

2. After the pRS423 plasmid was digested with XmaI and BamHI, the digested product was recovered by gel extraction using an agarose gel recovery kit.

3. Construction of pRS423-gRNA (pCAS) plasmid

After the gRNA expression cassette prepared in step 1 and the vector pRS423 enzyme digestion product recovered in step 2 were ligated and circularized with recombinase ClonExpress II, they were immediately placed in an ice-water bath to transform E.coli competent DH-5. alpha. and positive clones were selected to obtain pRS423-gRNA (pCAS) plasmid (see FIG. 2 for plasmid map).

4. Construction of gRNA expression plasmid

Forward and reverse primers for constructing gRNA expression plasmids are designed, each primer is 40bp in length and comprises a20 bp homology arm and N20 (gRNA). In each pair of primers in table 1, upper case letters represent grnas specifically targeting the target gene, and lower case letters represent 20bp homology arms; S-NGG-CBE-is a primer for constructing a gRNA expression plasmid with an SPT15 target site.

TABLE 1

And (3) taking pRS423-gRNA (pCAS) plasmids as templates, respectively adopting the primers to perform reverse PCR, recovering products, cyclizing the products by using recombinase ClonExpress II, immediately placing the products in an ice water bath, transforming escherichia coli competence DH-5 alpha, and screening positive clones to obtain gRNA expression plasmids (the sequencing verification is correct). 2 gRNA expression plasmids for editing URA3, 2 gRNA expression plasmids for editing ADE1, and 24 gRNA expression plasmids for base SPT15 were obtained in total.

Third, Gene editing

1. Using the Zymo Research Frozen-EZ Yeast Transformation II Kit^TMYeast transformation kit 1 microgram cytidine deaminase plasmid pRS315e _ pGal-nCas9(D10A) -PmCDA1 and the 1 microgram gRNA expression plasmid prepared in step two were transformed into Saccharomyces cerevisiae (the plasmid for editing URA3 was transformed into Saccharomyces cerevisiae BY4741a strain, and the plasmids for editing ADE1 and SPT15 were transformed into Saccharomyces cerevisiae BY4741 strain), and clones containing the two plasmids were obtained.

2. Inoculating the clone obtained in the step 1 to an SD-His-Leu culture medium (-Leu-His Minus Media 8g/L, 20g/L agar powder and NaOH for regulating the pH value to about 5.0) containing 20g/L glucose for activation, and culturing at 30 ℃ and 250r/min for 24h to grow to saturation; by OD₆₀₀Inoculating the strain to SD-His-Leu culture medium containing 20g/L raffinose at an initial inoculation amount of 0.3, activating again, and culturing at 30 deg.C and 250r/min for 24 hr to saturate; take 0.9OD₆₀₀Cells were washed 2 times with sterile water, inoculated into SD-His-Leu medium containing 10g/L raffinose and 20g/L galactose for induction editing, and transferred one generation every 24 h. And 5, carrying out subsequent screening and statistics on mutant cells.

(1) Screening of URA3 mutant cells

Take 0.3OD₆₀₀Cells were serially diluted 10 times and coated with SD-His-Leu +1.5 g/L5-FOA screening plates. If the gene URA3 is edited, the disrupted URA3 strain can form a single colony on a screening plate, the single colony is picked for PCR sequencing verification, sequencing primers are URA3-testF and URA3-testR, and the mutation rate of the cells is counted.

URA3-testF (forward primer): 5'-cattacgaatgcacacggtg-3', respectively;

URA3-testR (reverse primer): 5'-caaatatgcttcccagcctg-3' are provided.

(2) Screening of ADE1 mutant cells

Take 0.3OD₆₀₀Cells are continuously diluted by 10 times and coated with an SD-His-Leu +5mg/LAde screening plate, ADE1 gene-disrupted strains turn red on the plate, single colonies are picked for PCR sequencing verification, sequencing primers are ADE1-testF and ADE1-testR, the number of single clones on the plate is counted, and the mutation rate of the cells is calculated.

ADE1-testF (forward primer): 5'-tcgtatctctgcatatgacg-3', respectively;

ADE1-testR (reverse primer): 5'-ggagtacagttttacagcca-3' are provided.

(3) Screening of mutant Spt15 Strain

The edited cells were serially diluted and coated with SD-His-Leu plates containing 20g/L glucose, and single clones on the plates were randomly picked for colony PCR, and then the PCR products were subjected to Sanger sequencing using SPT15-F-wz and SPT15-R-wz as sequencing primers. And (3) aligning with the original SPT15 sequence, and detecting the mutation condition.

SPT15-F-wz (forward primer): 5'-tttagactgctctgcggaaga-3', respectively;

SPT15-R-wz (reverse primer): 5'-gagacgatccaccagatatgc-3' are provided.

3. Toxicity detection for editing systems

Editing cells were serially diluted 10-fold and plated on the following two plates: 1) SD-His-Leu plate containing 20g/L glucose; 2) SD-His-Leu plates containing 10g/L raffinose and 20g/L galactose, and the number of clones on the plates was counted to calculate the survival rate of the cells.

The statistics of URA3 and ADE1 gene editing are shown in figure 3. The results showed that the genes URA3 and ADE1 underwent point mutation of C > T at the target site, producing early stop codons TAG and TAA, the URA3 editing efficiency and cell survival rate were 8.8% and 95.3% highest, respectively, and the ADE1 editing efficiency and cell survival rate were 53.1% and 82.8% highest, respectively. The results show that the gene editing system can be used for carrying out gene editing on the saccharomyces cerevisiae.

The result of the transcription factor Spt15 gene editing showed that C > T, C > G and C > a mutations occurred at the target site, resulting in 36 Spt15 mutants in total. Specific information of the 36 Spt15 mutants is shown in table 2 (in table 2, the gene mutation site is described by using SEQ ID No.3 as a pre-mutation sequence, and the protein mutation site is described by using SEQ ID No.4 as a pre-mutation sequence).

TABLE 2

Example 2 evaluation of fermentation Performance of the Spt15 mutant Strain

The strains to be detected are as follows: the 36 Spt15 mutant strain and the control strain prepared in example 1.

Control strains were obtained BY transforming 1. mu.g of pRS423 plasmid and 1. mu.g of pRS315 plasmid into Saccharomyces cerevisiae strain BY 4741.

Inoculating the strain to be tested into 50mL shake flask containing 20mL seed culture medium (-Leu-His Minus Media 8g/L, glucose 20g/L), culturing at 30 deg.C and 250r/min for 24 hr, and collecting initial OD₆₀₀Transferring the seed solution of about 0.4 into a 100mL shake flask containing 50mL fermentation medium, and fermenting at 220r/min for 60 hr under different stress conditions (high sugar and high temperature, ethanol)In the preparation process, 1mL of the culture broth is sampled at the time points of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h and 60h, and the growth and metabolism conditions (ethanol content and residual glucose amount in the fermentation broth) of the strain are detected by using a microplate reader and a High Performance Liquid Chromatograph (HPLC).

Fermentation medium used under conditions of high sugar hyperosmotic stress: SC Complete Medium 8g/L, glucose 250 g/L. Culture temperature under conditions of high sugar hyperosmotic stress: at 30 ℃.

Fermentation medium used under high temperature stress conditions: SC Complete Medium 8g/L, glucose 20 g/L. Culture temperature under high temperature osmotic stress conditions: at 40 ℃.

Fermentation medium used under ethanol stress conditions: SC Complete Medium 8g/L, glucose 20g/L, 8% (v/v) ethanol. Culture temperature under ethanol stress conditions: at 30 ℃.

The HPLC detection conditions were as follows: the HPLC is American Agilent 1200, the chromatographic column is OOD-0223-KO (100 × 7.8mM) of Philomen, USA, the sample introduction amount is 5 μ L, the mobile phase is 5mM sulfuric acid solution, the flow rate is 0.6mL/min, the detector is a differential detector, the detection time is 10min, and the chromatographic column temperature is 55 ℃.

To more intuitively and clearly demonstrate the stress fermentability of the control and mutant strains, the effect of the strains on glucose consumption and ethanol production in the high-sugar hyperosmotic, high-temperature and high-ethanol fermentation processes (0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h and 60h) was evaluated using Principal Component Analysis (PCA). The results are shown in FIGS. 4 to 6. The first and second major components account for 75.72% (PC1) and 17.63% (PC2) of the total variation, respectively, and basically, the strains with decreased and increased tolerance were distributed on the left and right sides of the control strain, respectively.

The results in fig. 4 show that 36 mutant strains had 14 resistant strains (respectively, strains numbered 1, 3, 5, 8, 9, 10, 13, 17, 18, 22, 27, 33, 35, 36 in table 2) and 9 sensitive strains (respectively, strains numbered 2, 4, 12, 16, 19, 21, 24, 28, 34 in table 2) compared to the control strain under high sugar stress, wherein the resistant strains accounted for 38.9%.

The results in fig. 5 show that 36 mutant strains had 18 resistant strains (respectively, strains numbered 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 17, 18, 22, 25, 27, 33, 35, 36 in table 2) and 15 sensitive strains (respectively, strains numbered 2, 4, 12, 15, 16, 19, 20, 21, 23, 24, 26, 28, 30, 32, 34 in table 2) under high temperature stress compared to the control strain, wherein the resistant strains accounted for 50.0%.

The results in fig. 6 show that 36 mutant strains had 19 resistant strains (respectively, strains numbered 1, 3, 5, 7, 8, 9, 10, 13, 14, 15, 17, 18, 22, 25, 27, 31, 33, 35, 36 in table 2) and 17 sensitive strains (respectively, strains numbered 2, 4, 6, 11, 12, 16, 19, 20, 21, 23, 24, 26, 28, 29, 30, 32, 34 in table 2) compared to the control strain under ethanol stress, wherein the resistant strains accounted for 52.8%.

Under all three stress conditions, 14 mutants showed increased tolerance, and are numbered 1, 3, 5, 8, 9, 10, 13, 17, 18, 22, 27, 33, 35, and 36 in table 2.

The statistical results of fold-changes in ethanol accumulation, cell growth, and glucose consumption in exponential growth phase of the stress tolerant and sensitive mutants compared to the control strain are shown in tables 3 and 4, respectively. It should be noted that tables 3 and 4 are based on the analysis results of the individual fermentation time points, and FIGS. 4, 5 and 6 are based on the analysis results of all the fermentation time points, and the stress tolerance of the strain is more reflected than the analysis results of the individual fermentation time points.

TABLE 3

TABLE 4

Example 3 protein structure alignment analysis of significant stress tolerant Spt15 mutants

In order to study the effect of mutant amino acids on protein structure in Spt15 mutation with significantly improved stress tolerance, Spt15 mutant protein structure alignment analysis was performed on mutants 22(a140G), 27(P169A) and 35(R238K) screened in the above examples.

First, using the online analysis tool, MutFunc (http:// MutFunc. com), it was found that the a140G mutation affected the interaction between Spt15 and Brf1, while P169A and R238K affected the conserved regions of the protein. And then utilizeMaestro10.6 analyzed the interaction of Spt15 mutants with DNA and with other transcription factors. The wild-type Spt15 protein structure of Saccharomyces cerevisiae (PDB: 1RM1) was used as a template to mimic the protein structure of the mutant strain. Firstly, preparing protein, optimizing an original protein structure, dehydrating, hydrogenating, filling side chains and minimizing energy; secondly, mutation; then energy was minimized and to save time, only the effect of this mutation on residues in the nearby 5 angstrom range was analyzed using the fasta parameter; finally, RMSD analysis was performed to calculate the structural changes of each residue in the protein before and after mutation.

The results are shown in fig. 7, where the a140G mutation is located on the beta sheet of Spt15, the P169A mutation and the R238K mutation are located on the random coil of Spt 15. A140G changed the spatial structure of the nearby amino acid R137, which is one of the conserved amino acids of the interaction of Spt15 and Brf 1. Brf1 is one of the three subunits of RNA polymerase III transcription initiation factor TFIIIB, binds to TATA Binding Protein (TBP) and TFIIIC, recruits RNA polymerase III as a promoter. Thus, a140G may influence transcription initiation by affecting the interaction of Spt15 with Brf 1. Both mutations, P169A and R238K, alter the spatial structure of R171. R171 is adjacent to L172 and is a conserved amino acid which contributes to the stability of the protein structure. Therefore, the two mutations, P169A and R238K, probably affect the stability of the Spt15 protein, thereby affecting the gene transcription in which Spt15 participates and changing the stress tolerance of the Saccharomyces cerevisiae strain.

Example 4 transcriptome analysis

In order to study the global regulation of the Spt15 mutation with significantly improved stress tolerance on the transcriptome, 3 stress tolerant mutants, mutants 22(a140G), 27(P169A), 35(R238K), and control strain BY4741 were cultured under normal conditions (30 ℃,20 g/L glucose), hyperosmolar and high temperature conditions, respectively, to the logarithmic growth phase for transcriptome sequencing.

First, 3 stress-tolerant mutants, mutants 22(a140G), 27(P169A) and 35(R238K), were subcultured continuously in SD medium to discard plasmids before the official examination. Carrying out continuous passage for 15 generations, culturing for 12h each generation, coating an SD plate, culturing for about 48h at 30 ℃, generating obviously visible monoclones, and selecting monoclonal PCR sequencing to verify the mutation condition of the Spt15 expression cassette, wherein sequencing primers are LYF-SCC4-F and LYF-PEA 2-R.

LYF-SCC4-F (forward primer): 5'-actccaccatctcaaacgtg-3', respectively;

LYF-PEA2-R (reverse primer): 5'-caagttcaccttcctctgtag-3' are provided.

Transcriptome sample preparation: single colonies were picked and inoculated into 3mL SD medium for about 24h to initiate OD₆₀₀Approximatively 0.4 was transferred to 50mL SD medium for about 24h, then transferred to stress and non-stress conditions for culture, with two mutants per strain in parallel. The sampling time is respectively as follows: culturing at 30 deg.C for 9h, at 40 deg.C for 15h, and culturing under 250g/L glucose stress for 21 h.

Transcriptome data analysis: the extraction of total RNA was performed in Hangzhou Union Biometrics (China, Zhejiang). The RefSeq download from NCBI (sequence Assembly version R64, RefSeq Assembly accession: GCF-000146045.2) includes 16 chromosomes and 1 mitochondrial chromosome, with the S.cerevisiae S288c genome as the reference genome. Each library produced on average 17.8. + -. 0.4 million clean reads with an average mapping rate of 63.4. + -. 4.0% which is approximately 140 times the reference genome coverage (total size 12.17 Mb). Clean reads were aligned to the reference transcriptome using Bowtie (version 2.2.3). The number of reads per kilobase length from a gene per million Reads (RPKM) was calculated from the length of the gene and mapped to the gene, and transcript quantification was estimated from the mapping using RSEM. The expression values of genes are generally expressed as FPKM, with the sequencing depth and the gene length being corrected in sequence (ref: TRAPNELL C, WILLIAMS B A, PERTEA G, et al. transcription analysis and quantification by RNA-Seq retrieved independent primers and isogram switching cell differentiation [ J ]. Nat Biotechnology, 2010,28(5): 511-5.). Differences in gene expression between the Spt15 mutant and wild strains were compared under the same culture or stress conditions. When DESeq2 was used to perform a differentially expressed gene analysis, mutant strains were considered to express Significant Differentially Expressed Genes (SDEGs) when the gene expression level was 2-fold greater or equal to that of the control strain (P.ltoreq.0.05). Compared to the wild-type Spt15, a140G, P169A, and R238K resulted in a significant expression up-regulated gene factor of between 202 and 452 and a significant expression down-regulated gene factor of between 97 and 404 (fig. 8).

Functional annotation of SDEGs was performed using FunSpec (http:// fundpec. med. utoronto. ca. /), to analyze which pathways are affected by significant gene transcription due to Spt15 mutation under stress conditions.

The function of the differentially expressed genes between the Spt15 mutant and wild strains compared to normal conditions was analyzed as follows: for mutant 22(a140G), under hyperosmotic stress, up-and down-regulated genes affected multiple pathways, up-regulated gene regulation of protein (re) folding, methionine biosynthesis, cellular amino acid biosynthesis, heavy head protein folding and sulfate assimilation processes, down-regulated gene regulation of cell division, cell cycle, cell wall tissue, DNA replication, protein kinase activity, meiosis and mitosis. Under high temperature stress, the up-regulated gene regulates the assembly of the RNA polymerase II transcription pre-initiation complex, and the down-regulated gene regulates the biological process, stress reaction and purine metabolic process. The biological function analysis specific information of the differentially expressed genes in mutant A140G is shown in Table 5.

TABLE 5

And (4) surface note: k: number of genes in the input cluster in a given category; f: total number of genes in a given class.

In mutant 27(P169A), the gene is up-regulated to regulate biological processes under hyperosmotic and high temperature stress as compared to normal conditions. Down-regulated genes regulate cytoplasmic division and transmembrane transport under hyperosmotic stress, and down-regulated genes regulate glycogen biosynthesis, citrate metabolism processes and trehalose biosynthesis under high temperature stress, which is inconsistent with accumulation of trehalose under high temperature stress as reported in the literature (AUESUKAREE C, DAMNERNSAWAD A, KRUATRACHE M, et al genome-wide identification of genes involved in metabolism to yield environmental stress in Saccharomyces cerevisiae [ J ]. Journal of applied genes, 2009,50(3):301-10.), probably the strain produces tolerance mainly by transcriptional regulation of other biological processes. The biological function analysis specific information of the differentially expressed genes in mutant 27(P169A) is shown in Table 6.

TABLE 6

And (4) surface note: k: number of genes in the input cluster in a given category; f: total number of genes in a given class.

In mutant 35(R238K), differentially expressed genes regulate multiple pathways under normal conditions, compared to the fact that under hyperosmotic stress, up-regulation of gene regulation transmembrane transport and iron ion homeostasis, and down-regulation of gene regulation of cell cycle, cell division, cell wall tissue and protein phosphorylation. Under high temperature stress, the up-regulated gene does not affect any biological pathway, and the down-regulated gene regulates biological processes, iron cell transport, iron ion homeostasis, hexose metabolic processes, stress reactions, and purine base metabolic processes. The biological function analysis specific information of the differentially expressed genes in the mutant R238K is shown in Table 7.

TABLE 7

And (4) surface note: k: number of genes in the input cluster in a given category; f: total number of genes in a given class.

In conclusion, by comparing the regulation pathways involved by differentially expressed genes in the normal condition and the stress condition, the biological processes of protein folding, protein refolding, repeated synthesis of protein folding, sulfate assimilation process, amino acid biosynthesis, assembly of complex before RNA polymerase II transcription initiation, ribosome biosynthesis, methionine biosynthesis, sulfate assimilation and the like are found, and the regulation of the pathways by the up-regulated genes has a promoting effect on the resistance of cells to the stress environment, so that the mutant generates different phenotypes by up-regulating or down-regulating the important pathways.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> gene site-directed mutagenesis method and stress resistance breeding application thereof

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 597

<212> DNA

<213> Saccharomyces cerevisiae

<400> 1

atggagggca cagttaagcc gctaaaggca ttatccgcca agtacaattt tttactcttc 60

gaagacagaa aatttgctga cattggtaat acagtcaaat tgcagtactc tgcgggtgta 120

tacagaatag cagaatgggc agacattacg aatgcacacg gtgtggtggg cccaggtatt 180

gttagcggtt tgaagcaggc ggcggaagaa gtaacaaagg aacctagagg ccttttgatg 240

ttagcagaat tgtcatgcaa gggctcccta gctactggag aatatactaa gggtactgtt 300

gacattgcga agagcgacaa agattttgtt atcggcttta ttgctcaaag agacatgggt 360

ggaagagatg aaggttacga ttggttgatt atgacacccg gtgtgggttt agatgacaag 420

ggagacgcat tgggtcaaca gtatagaacc gtggatgatg tggtctctac aggatctgac 480

attattattg ttggaagagg actatttgca aagggaaggg atgctaaggt agagggtgaa 540

cgttacagaa aagcaggctg ggaagcatat ttgagaagat gcggccagca aaactaa 597

<210> 2

<211> 921

<212> DNA

<213> Saccharomyces cerevisiae

<400> 2

atgtcaatta cgaagactga actggacggt atattgccat tggtggccag aggtaaagtt 60

agagacatat atgaggtaga cgctggtacg ttgctgtttg ttgctacgga tcgtatctct 120

gcatatgacg ttattatgga aaacagcatt cctgaaaagg ggatcctatt gaccaaactg 180

tcagagttct ggttcaagtt cctgtccaac gatgttcgta atcatttggt cgacatcgcc 240

ccaggtaaga ctattttcga ttatctacct gcaaaattga gcgaaccaaa gtacaaaacg 300

caactagaag accgctctct attggttcac aaacataaac taattccatt ggaagtaatt 360

gtcagaggct acatcaccgg atctgcttgg aaagagtacg taaaaacagg tactgtgcat 420

ggtttgaaac aacctcaagg acttaaagaa tctcaagagt tcccagaacc aatcttcacc 480

ccatcgacca aggctgaaca aggtgaacat gacgaaaaca tctctcctgc ccaggccgct 540

gagctggtgg gtgaagattt gtcacgtaga gtggcagaac tggctgtaaa actgtactcc 600

aagtgcaaag attatgctaa ggagaagggc atcatcatcg cagacactaa attcgaattc 660

ggtattgacg aaaagaccaa tgaaattatt ctagtggacg aggtgctaac gccagactcc 720

tctagattct ggaacggtgc ctcttataag gtaggagaat cccaagattc ttacgataag 780

caatttttaa gagactggct tactgctaat aagttgaacg gtgttaacgg cgtcaaaatg 840

ccccaagaca ttgtcgacag gacaagggcc aaatatatag aggcttatga aacattgaca 900

gggtctaaat ggtctcacta a 921

<210> 3

<211> 723

<212> DNA

<213> Saccharomyces cerevisiae

<400> 3

atggccgatg aggaacgttt aaaggagttt aaagaggcaa acaagatagt gtttgatcca 60

aataccagac aagtatggga aaaccagaat cgagatggta caaaaccagc aactactttc 120

cagagtgaag aggacataaa aagagctgcc ccagaatctg aaaaagacac ctccgccaca 180

tcaggtattg ttccaacact acaaaacatt gtggcaactg tgactttggg gtgcaggtta 240

gatctgaaaa cagttgcgct acatgcccgt aatgcagaat ataaccccaa gcgttttgct 300

gctgtcatca tgcgtattag agagccaaaa actacagctt taatttttgc ctcagggaaa 360

atggttgtta ccggtgcaaa aagtgaggat gactcaaagc tggccagtag aaaatatgca 420

agaattatcc aaaaaatcgg gtttgctgct aaattcacag acttcaaaat acaaaatatt 480

gtcggttcgt gtgacgttaa attccctata cgtctagaag ggttagcatt cagtcatggt 540

actttctcct cctatgagcc agaattgttt cctggtttga tctatagaat ggtgaagccg 600

aaaattgtgt tgttaatttt tgtttcagga aagattgttc ttactggtgc aaagcaaagg 660

gaagaaattt accaagcttt tgaagctata taccctgtgc taagtgaatt tagaaaaatg 720

tga 723

<210> 4

<211> 240

<212> PRT

<213> Saccharomyces cerevisiae

<400> 4

Met Ala Asp Glu Glu Arg Leu Lys Glu Phe Lys Glu Ala Asn Lys Ile

1 5 10 15

Val Phe Asp Pro Asn Thr Arg Gln Val Trp Glu Asn Gln Asn Arg Asp

20 25 30

Gly Thr Lys Pro Ala Thr Thr Phe Gln Ser Glu Glu Asp Ile Lys Arg

35 40 45

Ala Ala Pro Glu Ser Glu Lys Asp Thr Ser Ala Thr Ser Gly Ile Val

50 55 60

Pro Thr Leu Gln Asn Ile Val Ala Thr Val Thr Leu Gly Cys Arg Leu

65 70 75 80

Asp Leu Lys Thr Val Ala Leu His Ala Arg Asn Ala Glu Tyr Asn Pro

85 90 95

Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Lys Thr Thr

100 105 110

Ala Leu Ile Phe Ala Ser Gly Lys Met Val Val Thr Gly Ala Lys Ser

115 120 125

Glu Asp Asp Ser Lys Leu Ala Ser Arg Lys Tyr Ala Arg Ile Ile Gln

130 135 140

Lys Ile Gly Phe Ala Ala Lys Phe Thr Asp Phe Lys Ile Gln Asn Ile

145 150 155 160

Val Gly Ser Cys Asp Val Lys Phe Pro Ile Arg Leu Glu Gly Leu Ala

165 170 175

Phe Ser His Gly Thr Phe Ser Ser Tyr Glu Pro Glu Leu Phe Pro Gly

180 185 190

Leu Ile Tyr Arg Met Val Lys Pro Lys Ile Val Leu Leu Ile Phe Val

195 200 205

Ser Gly Lys Ile Val Leu Thr Gly Ala Lys Gln Arg Glu Glu Ile Tyr

210 215 220

Gln Ala Phe Glu Ala Ile Tyr Pro Val Leu Ser Glu Phe Arg Lys Met

225 230 235 240

<210> 5

<211> 20

<212> DNA