阅读说明：本技术 一种基因在调控菌株对抑制剂耐受性中的应用 (Application of gene in regulating and controlling tolerance of strain to inhibitor ) 是由 元英进 谢泽雄 陈涛 李霞 于 2021-06-28 设计创作，主要内容包括：本发明涉及基因工程技术领域,特别涉及一种基因在调控菌株对抑制剂耐受性中的应用。该基因选自基因YJL120W、YJR130C、YJR131W、YJL078C中的一种或多种；抑制剂选自呋喃类抑制剂、弱酸类抑制剂、酚类抑制剂中的一种或几种。本发明基因是在SCRaMbLE重排的基础上,结合生物信息学分析和RT-qPCR手段进行基因挖掘工作,得到了耐受性相关基因YJL120W、YJL078C、YJR130C和YJR131W,并对它们进行功能验证,最终发现基因YJL120W的过表达,基因YJL078C、YJR130C和YJR131W的删除能提升菌株耐受性。(The invention relates to the technical field of genetic engineering, in particular to application of a gene in regulating and controlling the tolerance of a strain to an inhibitor. The gene is selected from one or more of genes YJL120W, YJR130C, YJR131W and YJL 078C; the inhibitor is one or more selected from furan inhibitor, weak acid inhibitor and phenol inhibitor. The gene is characterized in that gene mining work is carried out by combining bioinformatics analysis and RT-qPCR means on the basis of SCRaMbLE rearrangement, tolerance related genes YJL120W, YJL078C, YJR130C and YJR131W are obtained and are subjected to functional verification, and finally over-expression of the gene YJL120W is found, and the strain tolerance can be improved by deleting the genes YJL078C, YJR130C and YJR 131W.)

1. Use of a gene for regulating the tolerance of a strain to an inhibitor, wherein the gene is selected from one or more of the genes YJL120W, YJR130C, YJR131W and YJL 078C;

the inhibitor is selected from one or more of furan inhibitors, weak acid inhibitors and phenolic inhibitors.

2. Use according to claim 1, wherein the furan-based inhibitor is furfural, the weak acid-based inhibitor is acetic acid and the phenolic inhibitor is phenol.

3. Use according to claim 2, wherein the inhibitor is a mixture of furfural, acetic acid, phenol.

4. Use according to claim 1, wherein the strain is a yeast strain.

5. The use according to claim 4, wherein the strain is a strain of Saccharomyces cerevisiae.

6. The use according to any one of claims 1 to 5, wherein the gene YJL120W positively regulates the resistance of the strain to inhibitors and the genes YJR130C, YJR131W, YJL078C negatively regulates the resistance of the strain to inhibitors.

7. A method of increasing the tolerance of a strain to an inhibitor, comprising one or more of the following:

a) overexpression of gene YJL 120W;

b) knock-out gene YJR 130C;

c) knock-out gene YJR 131W;

d) knock-out gene YJL 078C;

the inhibitor is selected from one or more of furan inhibitors, weak acid inhibitors and phenolic inhibitors.

8. The method of claim 7, wherein the strain is a yeast strain.

9. A bioengineering strain, which is prepared by one or more of the following modification:

a) overexpression of gene YJL 120W;

b) knock-out gene YJR 130C;

c) knock-out gene YJR 131W;

d) knock-out gene YJL 078C;

the inhibitor is selected from one or more of furan inhibitors, weak acid inhibitors and phenolic inhibitors.

10. The bioengineered strain of claim 9, wherein the strain is a yeast strain.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a gene in regulating and controlling the tolerance of a strain to an inhibitor.

Background

The strain modification technology is a technology for providing excellent strains for production and further promoting production development by utilizing principles of genetics, biochemistry and the like to carry out molecular modification and corresponding condition screening on target strains. The strain modification technology mainly comprises methods of gene mutation (such as mutagenesis and the like), gene recombination (such as hybridization, protoplast fusion, genetic engineering and the like), direct gene evolution (such as error-prone PCR, DNA shuffling, exon shuffling and the like) and the like. The modification of the physiological tolerance of the strain has important significance for the development of biorefinery and biological economy.

Yeast is closely related to the life of people, plays a vital role in the fields of brewing, baking, biofuel, pollution treatment, nutriment, biomedicine and the like, and is an ideal industrial strain. Improving the tolerance of the yeast, greatly reducing the industrial fermentation cost and improving the fermentation yield.

At present, the work related to the tolerance of the yeast relates to the work of ethanol tolerance, high temperature resistance, acid resistance, rapamycin tolerance, alkali resistance and the like of the saccharomyces cerevisiae, and the adopted means comprise adaptive evolution, a SCRAMBLE rearrangement technology and the like, and genes such as ACE2, GLN3, YER161C and the like are excavated according to the work. Genes that improve yeast tolerance are to be further explored.

Disclosure of Invention

In view of the above, the present invention provides an application of a gene in regulating and controlling the tolerance of a strain to an inhibitor. Through functional verification, overexpression of the gene YJL120W is found, and knockout of the genes YJL078C, YJR130C and YJR131W can improve strain tolerance.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of a gene in regulating and controlling the tolerance of a strain to an inhibitor, wherein the gene is selected from one or more of genes YJL120W, YJR130C, YJR131W and YJL 078C;

the inhibitor is one or more selected from furan inhibitor, weak acid inhibitor and phenol inhibitor.

Preferably, the furan inhibitor is furfural, the weak acid inhibitor is acetic acid, and the phenolic inhibitor is phenol.

In the specific embodiment provided by the invention, the inhibitor is a mixture of furfural, acetic acid and phenol.

Preferably, the strain is a yeast strain.

In the specific embodiment provided by the invention, the strain is a saccharomyces cerevisiae strain.

In the present invention, the gene YJL120W positively regulates the resistance of the strain to the inhibitor, and the genes YJR130C, YJR131W, YJL078C negatively regulates the resistance of the strain to the inhibitor.

The invention also provides a method for improving the tolerance of a strain to an inhibitor, which comprises one or more of the following modifications:

a) overexpression of gene YJL 120W;

b) knock-out gene YJR 130C;

c) knock-out gene YJR 131W;

d) knock-out gene YJL 078C;

the inhibitor is one or more selected from furan inhibitor, weak acid inhibitor and phenol inhibitor.

Preferably, the furan inhibitor is furfural, the weak acid inhibitor is acetic acid, and the phenolic inhibitor is phenol.

In the specific embodiment provided by the invention, the inhibitor is a mixture of furfural, acetic acid and phenol.

Preferably, the strain is a yeast strain.

In the specific embodiment provided by the invention, the strain is a saccharomyces cerevisiae strain.

The invention also provides a bioengineering strain, which is prepared by one or more of the following improvements:

a) overexpression of gene YJL 120W;

b) knock-out gene YJR 130C;

c) knock-out gene YJR 131W;

d) knock-out gene YJL 078C;

the inhibitor is one or more selected from furan inhibitor, weak acid inhibitor and phenol inhibitor.

Preferably, the furan inhibitor is furfural, the weak acid inhibitor is acetic acid, and the phenolic inhibitor is phenol.

In the specific embodiment provided by the invention, the inhibitor is a mixture of furfural, acetic acid and phenol.

Preferably, the strain is a yeast strain.

In the specific embodiment provided by the invention, the strain is a saccharomyces cerevisiae strain.

The invention provides an application of a gene in regulating and controlling the tolerance of a strain to an inhibitor, wherein the gene is selected from one or more of genes YJL120W, YJR130C, YJR131W and YJL 078C; the inhibitor is one or more selected from furan inhibitor, weak acid inhibitor and phenol inhibitor. The invention has the following technical effects:

the gene introduced by the invention is characterized in that on the basis of SCRaMbLE rearrangement, bioinformatics analysis and RT-qPCR means are combined to carry out gene mining work, tolerance-related genes YJL120W, YJL078C, YJR130C and YJR131W are obtained and are subjected to functional verification, and finally over-expression of the gene YJL120W is found, and deletion of the genes YJL078C, YJR130C and YJR131W can improve the tolerance of the strain, so that over-expression of the gene YJL120W has a remarkable effect on improvement of the tolerance, and mechanism analysis is carried out on the gene YJL120W, and finally over-expression of the gene YJL120W can regulate and control the increase of the gene expression amount in the oxidative stage of a pentose phosphate pathway, improve the content of intracellular NADPH, promote degradation of furfural and finally improve the tolerance of the strain.

Drawings

FIG. 1 shows the determination of post-rearrangement screening conditions;

FIG. 2 shows the overall scheme of the tolerant strain library construction;

FIG. 3 shows the characterization of the rearranged selected strain;

FIG. 4 shows synV and synX variant event statistics;

FIG. 5 shows strain construction and validation and gene mining of deletion segment 71, wherein FIG. 5a shows deletion design and validation of segment 71; FIG. 5b shows growth characterization of strains deleted for segment 71; FIG. 5c shows yCT388 b, wherein the expression level of the gene upstream and downstream of segment 71 is changed; FIG. 5d shows the validation of the tolerance of an over-expressed strain of gene YJL 120W;

FIG. 6 shows the characterization of gene YJR130C and YJR131W knockouts;

FIG. 7 shows the characterization of the gene YJL078C knock-out;

FIG. 8 shows the characterization of overexpression of gene YJL 120W; wherein FIGS. 8A, 8B show culture conditions in SC-His; FIGS. 8C and 8D show the culture conditions for SC-His + 60% FAP;

FIG. 9 shows the culture conditions for overexpression of gene YJL120W in SC-His + 50% water wash;

FIG. 10 shows a characterization of knockout gene YJR130C to promote tolerance of Saccharomyces cerevisiae to FAP; wherein FIGS. 10A, 10B show SC medium conditions; FIGS. 10C, 10D show SC medium conditions for 40% FAP;

FIG. 11 shows a characterization that knock-out gene YJR131W promotes the tolerance of Saccharomyces cerevisiae to FAP; wherein FIGS. 11A, 11B show SC medium conditions; FIGS. 11C, 11D show SC medium conditions for 40% FAP;

FIG. 12 shows a characterization that knock-out gene YJL078C promotes tolerance of Saccharomyces cerevisiae to FAP; wherein FIGS. 12A, 12B show SC-His media conditions; FIGS. 12C, 12D show SC-His media conditions for 40% FAP;

FIG. 13 shows the effect of YJL120W on pentose phosphate pathway gene expression.

Detailed Description

The invention discloses an application of a gene in regulating and controlling the tolerance of a strain to an inhibitor, and a person skilled in the art can use the content to reference the content and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the present invention, the gene sequences of YJL120W, YJL078C, YJR130C and YJR131W have been disclosed, and the specific sequences are as follows:

YJL120W：

ATGACATTCGCAACCCAAGTTGGCGAAGTCAGAAGCAAGGATACTGGGAGCTATA ATTGGTTTGACCATTTTTTCTTGTGTGTTTACCTCGCTCTTGGAATTAGCAAATGGCC TTCTTGCATGAAATTGTATCGAGTTTGCTTTATTTTTCTTTTTACGGGCGGATTCTTT CTATTCTGGCTTTCCTATAACAGAGATCATGAAAGAAGTTCCAGCTTACGGATCAA GAAAGTACCTATACATATACAAAAATCTGATTACTTTCCCAGCTCGACTTGGATAG CTGTTCTTGTTTTCTCTTGGCGACACATTTTTTGTTTCTGA

YJL078C：

ATGCTGGAGTTTCCAATATCAGTTCTGCTAGGATGCCTAGTAGCCGTCAAGGCACA AACCACGTTTCCAAACTTCGAGAGCGATGTGCTGAACGAGCATAACAAGTTCAGAG CGCTACATGTTGACACAGCGCCGCTCACCTGGTCCGACACTCTGGCCACCTATGCG CAGAACTACGCCGACCAATATGATTGTTCGGGTGTCTTAACGCATTCCGATGGCCC ATATGGTGAGAACCTTGCCCTTGGTTACACAGACACGGGAGCGGTGGACGCCTGGT ACGGGGAGATAAGCAAGTATAATTATTCAAATCCCGGATTTTCTGAATCCACGGGT CACTTCACACAGGTGGTTTGGAAGTCAACCGCCGAGATTGGATGTGGTTATAAATA TTGTGGTACGACATGGAACAATTATATTGTGTGCTCCTACAACCCTCCTGGAAACT ACCTGGGTGAGTTTGCAGAGGAAGTGGAACCACTTATAAGCACTGTTTCCTCGTCC TCATCCTCGTCCTCTTCTACCTCAACTACATCAGACACAGTCTCCACCATCTCATCC AGTATTATGCCCGCTGTAGCGCAAGGGTATACAACAACGGTATCGTCTGCGGCTAG CAGCAGTTCTTTAAAATCGACGACCATAAACCCTGCCAAGACCGCTACCCTCACTG CGTCCTCTTCTACCGTAATTACTAGTAGCACAGAATCAGTTGGATCCTCCACTGTCT CATCAGCCTCAAGCTCTTCTGTCACTACTTCCTATGCTACCTCCTCGAGTACCGTCG TCTCTAGTGATGCTACTTCATCCACTACCACCACCTCATCGGTTGCTACATCGTCCA GTACCACTTCTTCCGACCCTACCTCGAGCACTGCTGCTGCTTCTTCTTCTGATCCTGC CTCAAGTTCCGCTGCCGCTTCCTCCAGCGCGAGTACCGAGAACGCCGCTTCTTCTAG CAGCGCCATCTCGAGCTCTTCATCAATGGTTTCTGCTCCTTTGAGTAGTACTCTTAC TACTTCCACCGCAAGCTCCAGAAGTGTAACTTCCAATTCAGTTAATTCTGTTAAGTT TGCAAACACAACTGTGTTTTCTGCTCAAACAACCTCTTCTGTAAGCGCCTCATTATC ATCATCTGTAGCTGCTGACGATATTCAGGGTAGCACTTCCAAGGAGGCCACAAGCT CAGTTTCCGAACATACTAGTATAGTAACTAGTGCAACTAATGCTGCCCAATATGCA ACGAGACTTGGGTCATCTTCCAGAAGTTCTTCCGGGGCCGTCTCTTCCTCAGCTGTG TCGCAATCTGTTCTGAATTCCGTTATAGCCGTCAACACCGACGTATCTGTAACCTCA GTTAGTAGCACAGCCCATACCACAAAGGACACCGCCACCACTTCAGTAACCGCCTC AGAAAGTATCACTTCGGAAACTGCTCAGGCTTCAAGTTCAACAGAGAAGAATATTA GTAACAGTGCCGCCACATCGAGTAGCATTTACTCCAACAGTGCTTCTGTGTCAGGA CACGGTGTAACATACGCTGCCGAATACGCCATTACATCCGAGCAATCCTCTGCGCT TGCCACATCTGTGCCTGCTACAAATTGCTCTAGTATCGTGAAGACCACAACTTTAG AAAATTCGAGTACCACAACCATCACAGCCATTACTAAGAGTACTACAACCTTGGCC ACTACTGCTAACAACTCCACAAGGGCAGCTACCGCAGTAACCATAGATCCCACATT GGACCCTACCGACAACTCAGCTAGTCCAACCGACAATGCTAAACACACCTCTACAT ATGGATCTTCTTCCACAGGCGCATCTTTAGATAGCTTACGCACAACCACCAGTATTA GTGTCTCAAGCAACACCACACAGTTAGTCTCTACCTGCACTTCCGAGAGCGATTAT TCCGATAGTCCTAGCTTCGCCATCTCCACTGCCACCACCACTGAAAGCAATCTGATC ACAAACACCATCACAGCTTCTTGTAGTACGGATAGTAATTTCCCTACCTCCGCTGCT TCTTCTACAGATGAGACGGCCTTCACTAGAACAATCTCGACATCTTGTAGCACTTTG AACGGCGCCTCAACCCAAACCAGTGAGCTAACCACATCGCCTATGAAAACCAACA CGGTGGTTCCAGCTTCTTCTTTCCCTTCAACTACAACCACTTGTCTAGAAAATGATG ACACTGCCTTTTCTAGTATCTACACTGAAGTCAACGCCGCAACTATCATTAACCCCG GAGAAACATCTTCTCTCGCTAGCGATTTCGCCACATCTGAAAAGCCAAACGAGCCC ACTTCTGTCAAATCCACCTCAAACGAAGGCACCTCTTCCACAACAACAACCTACCA ACAGACTGTTGCTACACTGTATGCCAAGCCCTCCAGCACAAGCCTAGGTGCAAGAA CAACTACTGGTAGCAACGGTCGTTCAACTACCAGCCAACAAGACGGGTCTGCCATG CATCAGCCAACTTCCTCGATCTACACTCAACTAAAAGAAGGCACATCAACCACCGC AAAACTTTCTGCATACGAAGGTGCTGCAACACCTCTTTCCATTTTCCAGTGCAATAG TCTAGCTGGAACGATTGCCGCTTTTGTCGTAGCTGTTCTGTTCGCCTTCTAG

YJR130C：

ATGATATCTAGAACCATTGGTGAATCTATTCCGCCCAACACAAAACATGCTGTTTC CGTGTGTTTGCCCACATGGGAAGCTACAGTAGGCTATGAAGAGGGTGAAAGCAGT ATAATTAACTCGCTTACTACCGGATATCCAAGATTTTTCATACACAAGTCAATAAA GAAATTATGTGAAATTTTATCCGCTAAGTACTCCATGGAAGATGAAGCTTGTCTTTG CTTCCCCTCTTACAAGGTTGCCAATAGGTGCAGAGAGTTTATCAAAGTGAAAACGG GTCTTTCTACCAAAGTACGCATCTTACAACTATGTACACCTAAACCTATGAACCAA GAGGAAAAGCTCTGGAGAAGGGAATGTAAAATCACGGTGGTGTTTGTAGACCAGG AAATATTCCCCGTGATGAAGCAGTATTGGCAACATTCTGGTGAGATTGTATCTAGC AGAATGGCGGAGTATATTTTACATGAATTACAAGTCAAGGATAATCTTAAGAAGAT GGAAACAGTGGATAACGGTAAAAAATTTATGACCGAAGATGAAAACAGAGTGAAC GAAGAATATATAGAAACGAGATTTGGGAGAAATTTAAATTTTCTTGCTGCTGATAA AGCCAAATATCTAATACGAAAGAGGATTGCCACAAAAGTTGTTGAAAAAATTGAC TCTGAAGGTCTATCGGACCTATTCTCTTTTGAACACTATAACGAGAGCAACGGTCC ATTCAATGTTGGCAGTGGAGAGGCTCTGGATGATGATCAGTTGAATTCGGACATAC CTGCCGAAACAATTACTTCTATGGGAGAAAGTGGCTCAAACAGTACTTTTGAAAAC ACAGCTACCGATGACTTGAAGTTTCATGTCAACCCTAATACGGATGTTTACTTATTT CCAAGTGGAATGGCGTCCATTTTCACTGCCCACAGGCTGCTTTTAAACTTCGATGCC AAGCGCTTATCAAGGTCTTCTAGTAGACAAGATAAGTTGATTGGATATGGACCTCC GTTCAAGAAGACCGTCATGTTTGGATTCCCATATACAGATACTCTGAGCATTCTTCG GAAGTTCAATCACACACATTTCTTGGGACAGGGAGATTCAACGTCAATGAACGCTC TGAAAAATATTTTACATTCTGGTGAGCAAATACTGGCGGTATTTATAGAAGCTCCA TCGAACCCACTGTTGAAAATGGGAGATTTACAAGAACTGAAAAGGCTATCAGACTT GTATTCATTTTACATAGTGGTCGATGAAACTGTGGGTGGCTTTGTCAATATAGATGT ATTACCGTATGCCGATATTGTTTGTAGTTCGCTAACGAAAATCTTCAGCGGCGACTC GAACGTTATTGCAGGATCCTTAGTACTAAATCCTAGAGGAAAGATTTATGAGTTTG CTAGAAAATTTATGAAGACTGAAGACGGATATGAAGATTGCCTATGGTGCGAAGA TGCGTTATGCCTGGAAAGAAATTCGAGAGACTTCGTGGAACGTACAATCAAGGTGA ATACAAACACTGATATATTGTTAAAAAGGGTGTTATTGCCTCAGGTGGGAAAATTG TTCAAGAAAATATATTACCCCAGTTTAACATCAGAGGATACCAAGCGGAATTATGA CAGCGTTATGTCTACAAAAGATGGTGGGTATGGAGGTTTATTTTCACTAACTTTTTT CAATATTGAAGAGGCAAAGAAGTTTTTCAATAATCTGGAATTGTGCAAAGGACCTT CTCTCGGTACGAATTTCACTTTGGCGTGTCCATATGCTATTATTGCCCACTACCAGG AATTAGACGAGGTAGCTCAATATGGTGTCGAAACAAATCTTGTAAGAGTGAGCGTC GGTTTGGAAAATAGCGACGTTTTATGCAATGTTTTTCAGCGTGCCATTGAGAAAGC TTTAGGGGAATAA

YJR131W：

ATGAAGAACTCTGTCGGTATTTCAATTGCAACCATTGTTGCTATCATAGCAGCTATA TACTATGTGCCATGGTACGAACACTTTGAGAGAAAGTCACCGGGGGCCGGAGAAA TGAGAGATCGGATTGAAAGCATGTTCTTGGAATCGTGGAGAGACTATTCCAAGCAT GGCTGGGGATACGATGTGTATGGACCTATTGAGCACACTTCCCATAATATGCCTCG TGGCAACCAGCCGTTAGGCTGGATTATCGTAGATTCAGTGGATACCTTGATGTTAA TGTATAACTCCTCCACACTATACAAAAGTGAGTTCGAGGCAGAAATTCAGAGATCG GAGCATTGGATAAACGATGTTTTGGATTTTGATATTGATGCCGAAGTGAATGTTTTT GAAACTACTATTAGAATGCTAGGTGGTTTATTATCCGCATATCATCTATCTGATGTT TTAGAAGTAGGTAATAAGACTGTCTACTTGAACAAAGCAATAGATTTGGGGGATAG GCTTGCTTTGGCGTTCTTATCCACTCAGACCGGAATTCCATACTCAAGTATAAACCT TCATAGTGGCCAAGCGGTTAAGAACCATGCAGATGGGGGGGCATCTTCTACCGCAG AATTCACTACGCTACAAATGGAATTCAAATATCTGGCGTATTTGACAGGAAATCGT ACTTATTGGGAGCTGGTGGAGCGTGTTTACGAGCCATTATACAAAAATAACGATCT TCTAAATACCTACGATGGATTGGTTCCAATTTATACATTTCCAGATACTGGGAAGTT TGGTGCTTCGACTATCCGGTTCGGATCAAGAGGTGATTCTTTTTATGAGTATTTACT AAAACAATATTTATTGACGCACGAAACACTTTATTATGATCTGTACAGAAAATCCA TGGAAGGTATGAAAAAGCATTTATTAGCACAATCCAAACCCTCTTCTCTGTGGTAC ATTGGGGAAAGAGAACAAGGTCTACATGGACAACTTTCTCCTAAGATGGACCACCT CGTGTGCTTTATGGGGGGATTGTTAGCATCAGGCTCTACTGAGGGCCTTTCTATTCA TGAAGCCCGAAGACGTCCGTTTTTCTCTCTTTCCCTTGAAAGAAAAAGTGACTGGG ATTTGGCTAAAGGGATAACTGACACATGTTATCAAATGTACAAGCAGTCTTCCTCG GGGCTTGCGCCTGAAATCGTTGTCTTCAATGATGGAAACATAAAACAGGATGGTTG GTGGCGGTCGTCTGTGGGTGATTTTTTTGTTAAACCACTCGATAGGCACAACCTACA AAGACCAGAAACGGTGGAATCGATTATGTTCATGTATCATTTATCTCATGATCACA AATATCGTGAATGGGGGGCGGAAATCGCAACTAGCTTCTTTGAAAATACCTGTGTT GATTGTAATGACCCAAAATTAAGGCGGTTCACCAGTTTAAGTGATTGTATCACGTT ACCTACAAAGAAATCTAACAATATGGAAAGTTTCTGGTTGGCAGAGACTTTAAAGT ATTTATATATATTGTTTTTAGACGAGTTTGATTTGACCAAAGTTGTTTTCAACACAG AAGCTCATCCTTTTCCAGTATTAGACGAAGAAATATTAAAATCGCAGTCTCTGACC ACAGGTTGGTCGTTGTAG

the raw materials, reagents and instruments used in the present invention are commercially available.

Wherein, the original strains yXZX1875 and ySC041 and strains yCT89-2, yCT89-3, yCT104-3, yCT138-1, yCT153, yCT405, yCT646, yCT649, yCT651, BY4741 and BY4741+ pRS413 are provided BY Yuanying laboratories of Tianjin university, the composite inhibitor is furfural, acetic acid and phenol mixture (FAP), is a representative substance of inhibiting components in cellulose hydrolysate, plays a serious inhibiting role on yeast growth, and the cellulose washing solution is prepared BY pretreating corn straws BY sodium hydroxide.

Concentration of FAP mother liquor: 5.3g/L of acetic acid, 1.3g/L of furfural and 0.5g/L of phenol.

TABLE 1 FAP composition at different concentrations

Concentration of	Acetic acid (g/L)	Furfural (g/L)	Phenol (g/L)
				30％	1.59	0.39	0.15
40％	2.12	0.612	0.2
				50％	2.65	0.65	0.25
60％	3.18	0.78	0.3

In the following examples, overexpression and knock-out of genes are all performed by conventional techniques in the art.

The invention is further illustrated by the following examples:

example 1 determination of Complex inhibitor tolerance detection and screening conditions for starting strains

The starting strain yXZX1875 has circular synthetic type V chromosome and linear synthetic type X chromosome (ring _ synV & synX); the starting strain ySC041 has circular synthetic type chromosome V and circular synthetic type chromosome X (ring _ synV & ring _ synX).

yXZX1875 and ySC041 were diluted in solid plates containing different concentrations of FAP to observe their growth at different concentrations. The final screening pressure for genomic rearrangements was obtained at 40% FAP concentration, as shown in figure 1.

Example 2 genomic rearrangement

Plasmids pRS413 and pCRE4 were transformed into the starting strains yXZX1875, ySC041, followed by chromosome-induced rearrangement.

SCRAMBLE-induced rearrangements

1.3 single colonies of the strain which are verified to have been introduced into pCRE4 and the plasmid carrying the target gene are picked and inoculated into 5mL of SC-His medium, and are placed on a shaker at 30 ℃ for overnight culture at 220 r/min.

Cells were collected by centrifugation at 2.5000 rpm and washed 3 times with sterile water to ensure no glucose residue.

3. Transferring the bacterial liquid to 5mL of 2% SGal-His galactose medium containing 1 μ M estradiol to control OD₆₀₀For 1, the mixture is placed on a shaking table at 220r/min for induction for 8 hours at the temperature of 30 ℃.

Cells were collected by centrifugation at 4.5000 rpm and washed 3 times with sterile water to ensure no galactose and estradiol remained.

5. Spreading on inhibitor-containing plate according to a certain dilution ratio, and culturing in 30 deg.C incubator for 3-5 days.

At the moment, a batch of strain libraries are obtained preliminarily, strains with good growth vigor are stored, and the improvement of the tolerance is carried out by two strategies. The first strategy is to perform adaptive evolution on the strains for a period of time and then perform the next round of rearrangement, and the second strategy is to directly perform the next round of rearrangement, so that the strains with further improved tolerance are obtained.

Example 3 continuous passage degeneration to obtain a phenotypically Stable Strain library

To ensure strain-enhanced phenotype stabilization, serial passage regression procedures were performed after genomic rearrangement, as the strain contained the pCRE4 plasmid, so the regression experiments were performed in SC-His.

1. The rearranged and selected strains were inoculated into a 96-well plate containing 200. mu.L of SC-His medium and cultured overnight at 30 ℃ with a microplate constant temperature shaker.

2. A new 96-well plate was prepared, 180. mu.L of SC-His medium was added, and 20. mu.L of the bacterial suspension was transferred from the 96-well plate in the first step, followed by culture.

3. The operation was repeated 2 six times.

4. Transferring the bacterial liquid to 200 μ L SC-His culture medium added with a certain amount of inhibitor, and controlling OD₆₀₀0.5, and culturing for 3-4 days at 30 ℃.

5. Measurement of the OD of the Strain₆₀₀The strain capable of rapidly growing is coated in SC-His culture mediumFor subsequent experiments, and the prepared glycerol bacteria are stored in a refrigerator at-80 ℃.

Culturing in a culture medium containing an inhibitor with concentration higher than that of the original strain to be screened after 7 days, monitoring the growth condition of the strain, screening the strain with faster growth, diluting the strain on a YPD plate containing 60% FAP, and finally finding that the growth vigor of the rearranged and screened strain on the plate is obviously superior to that of the original strain, which indicates that the tolerant phenotype of the rearranged and screened strain is stable.

The whole operation flow is shown in FIG. 2, and finally 54 strains with stably enhanced tolerance are obtained, a phenotype stable tolerant strain library is formed, and the expansion of the strain library can be continued according to the method. The 5 phenotypically superior strains selected from the library were characterized as shown in FIG. 3, and these 5 strains were subsequently subjected to systematic analysis.

Example 4 genomic variation information analysis

synV and synX are divided into 175 and 248 segments by loxPsym sites distributed on the chromosome, and chromosomal rearrangements by SCRAMBLE are caused by deletions, duplications, inversions, translocations occurring between different segments. The difference between the sequences on both sides of the loxPsym site and the reference sequence is counted by bioinformatics, and finally, genome variation data of 5 strains of yCT89-2, yCT89-3, yCT104-3, yCT153 and yCT138-1 which occur in synV and synX are obtained.

Table 2 figure 4 raw data

The structural variations of each strain occurring on synV and synX were determined from the above table in combination with bioinformatics software and PCR validation.

Example 5 mining of FAP-tolerant genes

For structural variations such as segment inversion, deletion, replication and the like, the overexpression of the upstream gene YJL120W is discovered from the deletion of 183738-184470bp on synX, so as to improve the strain tolerance, as shown in FIG. 5; the deletion of the genes YJR130C and YJR131W, which were discovered from the deletion of 631427-637507bp of synX, improved strain tolerance, as shown in FIG. 6; the deletion of the gene YJL078C near the inversion interface excavated from the inversion of 276636-585463bp of synX improves strain tolerance, as shown in FIG. 7. The specific operation is as follows:

deletion of 183738-184470bp (segment 71) from synX occurred in both the rearrangement strains selected based on yXZX1875 and the rearrangement strains selected based on ySC041, and it is presumed that deletion of this region promotes tolerance to the complex inhibitor. To verify this hypothesis, the 731bp fragment homologous to segment 71 in the genome was deleted in wild type strain BY4741 BY means of yeast homologous recombination using URA3 as a screening tag and the correct knockout strain was designated yCT 388.

The expression levels of yCT388 and three genes YJL121C, YJL120W and YJL117W upstream and downstream of the region in BY4741 were detected BY RT-qPCR technology, and whether the genes were affected or not was observed. Respectively extracting RNA from the strains BY4741 and yCT388, carrying out reverse transcription on the RNA to obtain cDNA, and detecting the expression levels of the three genes BY taking the cDNA as a template. The results show that the expression level of YJL121C in yCT388 is reduced to 0.33 of BY4741, while the expression level of YJL120W is increased to 10.36 times of BY4741, and YJL117W is hardly affected.

The prior literature indicates that the deletion of YJL121C causes the furfural tolerance of a saccharomyces cerevisiae strain to be reduced, so that the reduction of the expression level of the gene does not improve the furfural tolerance of the strain to a composite inhibitor, and the overexpression of YJL120W probably plays a role. In order to verify the function of YJL120W, the gene needs to be overexpressed inside the strain to observe whether the tolerance of the strain to the composite inhibitor is improved, and the result shows that the growth condition of yCT405 is obviously improved compared with that of a control strain, which indicates that the tolerance of the saccharomyces cerevisiae to the composite inhibitor can be improved by overexpression of YJL 120W. Genes YJR130C, YJR131W and YJL078C related to tolerance were also mined in the above-described manner.

Example 6 characterization of Gene YJL120W overexpression to improve tolerance of Saccharomyces cerevisiae to FAP

Strain yCT405 was a strain in which gene YJL120W was overexpressed, and gene YJL120W was assembled to pRS413 and BY4741 was introduced. As shown in FIG. 8, in the SC-His medium without inhibitor, the strains yCT405 and BY4741+ pRS413 were fermented, and as can be seen from the growth curves, under normal conditions, the growth rates of the two strains are almost consistent, the logarithmic phase can be reached within about 4h, the plateau phase can be reached within about 16h, and the final growth density of the strains is between 5 and 6. The consumption rates of the strains on glucose are basically consistent and are respectively 1.30g/L/h and 1.34g/L/h, the ethanol production rates are respectively 0.67 g/L/h and 0.63g/L/h, and the ethanol yields are respectively 91.63% and 83.44%;

under the culture condition of 60% FAP, the strains yCT405 and BY4741+ pRS413 are strongly inhibited, and the inhibition effect of a control strain is particularly obvious. yCT405, the delay period is prolonged again, the logarithmic phase is not reached until about 12h, the plateau phase is reached about 36h, and the growth density of the strain is reduced to between 2 and 3. The delay period of the starting strain is as long as about 136 hours, after the logarithmic phase, the growth is extremely slow, the strain enters the plateau phase after about 172 hours, and the growth density of the strain is reduced to between 2 and 3. The glucose consumption rate yCT405 was approximately 0.52g/L/h, the starting strain was approximately 0.12 g/L/h, the ethanol production rates were 0.25g/L/h and 0.05g/L/h, respectively, and the ethanol yields were 95% and 91%, respectively.

Strains yCT405 and BY4741+ pRS413 were fermented in a 50% aqueous medium. The results show that yCT405 enters the logarithmic phase at about 4h and the plateau phase at about 20h under the inhibition condition, and the final growth density of the cells is between 4 and 5. The initial strain has a long delay period, the initial strain enters a logarithmic phase only within about 40 hours, the initial strain enters a plateau phase within about 76 hours, and the final growth density of the cells is between 3 and 4. yCT405 was about 1.12g/L/h, the starting strain was about 0.24g/L/h, the ethanol production rates were 0.58g/L/h and 0.1g/L/h, respectively, and the ethanol yields were 99.12% and 86.14%, respectively (FIG. 9).

From the above results, yCT405 was excellent in performance, and the fermentation parameters were only slightly lower than those of the no-inhibition condition, indicating that overexpression of YJL120W has a great improvement in strain tolerance.

Example 7 characterization of knockout of Gene YJR130C to increase tolerance of Saccharomyces cerevisiae to FAP

Strain yCT646 was BY4741 with the knockout gene YJR 130C. As shown in FIG. 10, in the absence of inhibition, yCT646 and BY4741 were almost identical in growth, they both entered the log phase at around 4h and the plateau phase at around 18h, the final growth density of the strain was between 5 and 6, the glucose consumption rates were 1.02g/L/h and 1.26g/L/h, the ethanol production rates were 0.43g/L/h and 0.54g/L/h, and the ethanol yields were 85.71% and 84.06%, respectively;

the growth of both strains was inhibited at 40% FAP compared to normal conditions, wherein yCT646 showed a faster growth rate, the lag phase was maintained for about 12h, the plateau phase was reached at about 32h, and the final cell growth density was about 3. BY4741 grows more slowly under this condition, the time for entering logarithmic phase is about 28h, about 48h enters plateau phase, and the final cell density is about 4. yCT646 has a glucose consumption rate of 0.54g/L/h, an ethanol production rate of about 0.26g/L/h, an ethanol yield of 93.93%, a glucose consumption of BY4741 of about 0.4 g/L/h, an ethanol production rate of about 0.19g/L/h, and an ethanol yield of 96.01%.

Example 8 characterization of knockout of the YJR131W Gene to increase tolerance of Saccharomyces cerevisiae to FAP

Strain yCT649 is BY4741 with the YJR131W knocked out. As shown in FIG. 11, in the absence of inhibition, yCT649 entered the log phase at around 4h and the plateau phase at around 20h, which are nearly identical to BY4741, with a glucose consumption rate of about 1.01g/L/h, an ethanol production rate of about 0.42g/L/h, and an ethanol yield of about 83.23%. This indicates that the knockout of YJR131W also did not have too great an effect on the strain;

under the condition of 40% FAP, the growth of yCT649 is obviously inhibited, but the characterization of each parameter is better than BY 4741. yCT649 entered the log phase at around 12h and plateau phase at around 32h, with a final cell density of around 3. The glucose consumption rate was about 0.56g/L/h, the ethanol production rate was about 0.22g/L/h, and the ethanol yield was about 80.83%.

Example 9 characterization of knockout of Gene YJL078C to increase tolerance of Saccharomyces cerevisiae to FAP

Strain yCT651 was a BY4741 strain with the gene YJL078C knocked out. As shown in FIG. 12, yCT651 also compares the growth and fermentation performance with BY4741, in SC medium, the characterization parameters of the two strains are similar, yCT651 enters the log phase at about 6h, enters the platform phase at about 22h, the glucose consumption rate is about 1.26g/L/h, the ethanol production rate is about 0.48g/L/h, and the ethanol yield is about 89.12%, which indicates that the knockout of YJL078C has no great influence on the growth of the strain itself;

yCT651 also has obvious inhibition effect in the environment of 40% FAP, but various parameters of growth and fermentation performance are better than BY 4741. yCT651 entered log phase around 12h and plateau phase around 32h, with final cell density between 3 and 4. The glucose consumption rate was about 0.59g/L/h, the ethanol production rate was about 0.27g/L/h, and the ethanol yield was about 97.36%.

Example 10 analysis of tolerance mechanism of gene YJL120W

NADPH can accelerate the conversion of furfural into furfuryl alcohol with low toxicity, so that the inhibition effect on cells is reduced, and NADPH in yeast is mainly produced through the oxidation stage of the pentose phosphate pathway, so that the correlation between the gene YJL120W and the pentose phosphate pathway can be researched. Thus, the expression levels of the 19 genes in the pentose phosphate pathway of yCT405 and BY4741 were examined, and as shown in FIG. 13, RT-qPCR results showed that the expression levels of the genes YNL241C, YCR073W-A, YGR248W, YNR034W, YGR256W and YHR183W in the oxidation stage were all increased to some extent, whereas the expression levels of only YBR117C in the genes in the non-oxidation stage were increased and the expression levels of the remaining genes were decreased to some extent. Therefore, the overexpression of YJL120W in the strain is supposed to probably regulate and control the increase of the gene expression amount in the oxidative stage of the pentose phosphate pathway, the increase of the NADPH content, the degradation of furfural is promoted, and the growth and fermentation of the strain are recovered.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.