阅读说明：本技术 一种酿酒酵母中2-苯乙醇的制备方法 (Preparation method of 2-phenethyl alcohol in saccharomyces cerevisiae ) 是由 王肇悦 姜明月 杜正达 郭雪娜 何秀萍 于 2019-04-16 设计创作，主要内容包括：本发明公开了一种酿酒酵母中2-苯乙醇的制备方法。本发明保护一种提高酿酒酵母生产2-苯乙醇的产量的方法,包括如下步骤：提高酿酒酵母中转录因子Cat8p的表达量和/或活性,从而提高酿酒酵母生产2-苯乙醇的产量。本发明还保护将转录因子Cat8p的编码基因导入酿酒酵母后得到的重组菌株。本发明提供的产2-苯乙醇的酿酒酵母菌株与宿主菌相比,艾氏途径关键调控因子及关键酶基因表达水平增强,细胞的2-苯乙醇合成能力显著提高,且该酵母菌株发酵条件简单,发酵周期短,有利于该菌株工业化生产2-苯乙醇。(The invention discloses a preparation method of 2-phenethyl alcohol in saccharomyces cerevisiae. The invention discloses a method for improving the yield of 2-phenethyl alcohol produced by saccharomyces cerevisiae, which comprises the following steps: the expression quantity and/or activity of transcription factor Cat8p in the saccharomyces cerevisiae is improved, so that the yield of 2-phenethyl alcohol produced by the saccharomyces cerevisiae is improved. The invention also protects a recombinant strain obtained by introducing the coding gene of the transcription factor Cat8p into the saccharomyces cerevisiae. Compared with the host bacteria, the saccharomyces cerevisiae strain for producing the 2-phenethyl alcohol provided by the invention has the advantages that the expression level of key regulatory factors and key enzyme genes in the aldrin pathway is enhanced, the synthesis capacity of the 2-phenethyl alcohol of the cell is obviously improved, the fermentation condition of the saccharomyces cerevisiae strain is simple, the fermentation period is short, and the industrial production of the 2-phenethyl alcohol by the strain is facilitated.)

1. A method for improving the yield of 2-phenethyl alcohol produced by saccharomyces cerevisiae comprises the following steps: the expression quantity and/or activity of transcription factor Cat8p in the saccharomyces cerevisiae is improved, so that the yield of 2-phenethyl alcohol produced by the saccharomyces cerevisiae is improved.

2. The method of claim 1, wherein: the amino acid sequence of the transcription factor Cat8p is (a1) or (a2) or (a3):

(a1) an amino acid sequence shown as a sequence 5 in a sequence table;

(a2) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in a sequence 5 in a sequence table;

(a3) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the sequence 5 of the sequence table and has the same function.

3. The method of claim 1 or 2, wherein: the expression level and/or activity of the transcription factor Cat8p in the saccharomyces cerevisiae are/is improved by introducing a coding gene of the transcription factor Cat8p into the saccharomyces cerevisiae.

4. The method of claim 3, wherein: in the saccharomyces cerevisiae, a constitutive promoter is used for promoting the expression of the coding gene.

5. The transcription factor Cat8p or the coding gene thereof is applied to improving the yield of 2-phenethyl alcohol produced by saccharomyces cerevisiae.

6. The application of the expression cassette containing the coding gene of the transcription factor Cat8p in improving the yield of 2-phenethyl alcohol produced by saccharomyces cerevisiae; in the expression cassette, expression of the coding gene is promoted by a constitutive promoter.

7. A recombinant bacterium is obtained by introducing a coding gene of a transcription factor Cat8p into saccharomyces cerevisiae.

8. A recombinant bacterium is obtained by introducing an expression cassette containing a coding gene of a transcription factor Cat8p into saccharomyces cerevisiae; in the expression cassette, expression of the coding gene is promoted by a constitutive promoter.

9. Use of the recombinant bacterium of claim 7 or 8 for the production of 2-phenylethyl alcohol.

10. A process for producing 2-phenylethyl alcohol, comprising the steps of: culturing the recombinant bacterium according to claim 7 or 8 to obtain 2-phenylethyl alcohol from the culture product.

Technical Field

The invention relates to the field of genetic engineering, in particular to a preparation method of 2-phenethyl alcohol in saccharomyces cerevisiae.

Background

2-phenethyl alcohol (2-phenylethanol) is aromatic alcohol with rose flavor, exists in various plant essential oils such as rose, jasmine and the like, is a natural flavor substance of various fermented foods such as wine, yellow wine, beer, bread and the like, and is a key factor for determining the quality of the fermented foods. The fragrance of 2-phenethyl alcohol is popular with people, is the mainstream style of international essences and fragrances, is widely applied in the fields of food, daily chemical products and the like, and is second to vanillin in use amount, and is the second largest fragrance component. In addition, 2-phenethyl alcohol is used as a bactericide and an angiotensin converting enzyme inhibitor, and has important application in the pharmaceutical industry.

At present, the 2-phenethyl alcohol produced industrially mainly comprises a chemical synthesis method and a natural synthesis method, and the by-product of the chemical synthesis method is difficult to remove, thereby seriously influencing the quality and the application range of the product. The natural synthesis method comprises plant extraction and microbial fermentation, and the plant extraction method has high production cost and low yield although the product purity is high, and cannot meet the increasing market demand.

The microbial fermentation method has low cost, short period and high efficiency, and is a main way for improving the production of 2-phenethyl alcohol. Saccharomyces cerevisiae, as a safe model organism, is one of microorganisms with high 2-phenylethyl alcohol synthesis capacity, and has good adaptability to various environmental stresses, so that the Saccharomyces cerevisiae is an ideal strain for producing 2-phenylethyl alcohol by a microbial fermentation method. The existing research mainly focuses on natural screening of strains, traditional microbial breeding or fermentation process improvement, and has the problems of limited 2-phenethyl alcohol synthesis capacity and difficulty in large-scale industrial production.

Disclosure of Invention

The invention aims to provide a preparation method of 2-phenethyl alcohol in saccharomyces cerevisiae.

The invention provides a method for improving the yield of 2-phenethyl alcohol produced by saccharomyces cerevisiae, which comprises the following steps: the expression quantity and/or activity of transcription factor Cat8p in the saccharomyces cerevisiae is improved, so that the yield of 2-phenethyl alcohol produced by the saccharomyces cerevisiae is improved.

The amino acid sequence of the transcription factor Cat8p is (a1) or (a2) or (a3):

(a1) an amino acid sequence shown as a sequence 5 in a sequence table;

(a2) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in a sequence 5 in a sequence table;

(a3) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the sequence 5 of the sequence table and has the same function.

The expression level and/or activity of the transcription factor Cat8p in the saccharomyces cerevisiae are/is improved by introducing a coding gene of the transcription factor Cat8p into the saccharomyces cerevisiae.

The coding gene (CAT8 gene) of the transcription factor Cat8p is as follows (b1) or (b2) or (b3):

(b1) DNA molecules shown from 657 th to 4958 th positions of a 5' end of a sequence 2 in a sequence table;

(b2) a DNA molecule which has 75 percent or more homology with the nucleotide sequence defined by (b1) and codes the transcription factor CAT 8;

(b3) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under strict conditions and encodes the transcription factor CAT 8.

The stringent conditions can be hybridization and membrane washing with a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ in DNA or RNA hybridization experiments.

In the saccharomyces cerevisiae, a constitutive promoter is used for promoting the expression of the coding gene. The constitutive promoter may be particularly a TEF1 promoter.

The "introduction of the coding gene of the transcription factor Cat8p into the saccharomyces cerevisiae" can be realized by specifically introducing an expression vector for expressing a CAT8 gene into the saccharomyces cerevisiae. In the expression vector, the expression of CAT8 gene can be particularly promoted by a constitutive promoter.

The constitutive promoter may specifically be the ADH1 promoter. The expression vector can be specifically a circular plasmid shown in a sequence 2 of a sequence table.

The constitutive promoter may be particularly a TEF1 promoter. The expression vector can be specifically a circular plasmid shown in a sequence 3 of a sequence table.

The invention also protects the application of the transcription factor Cat8p or the coding gene thereof in improving the yield of 2-phenethyl alcohol produced by saccharomyces cerevisiae.

The invention also protects the application of the expression cassette containing the coding gene of the transcription factor Cat8p in improving the yield of 2-phenethyl alcohol produced by saccharomyces cerevisiae; in the expression cassette, expression of the coding gene is promoted by a constitutive promoter.

The constitutive promoter may specifically be the ADH1 promoter. The expression cassette may be specifically represented by positions 251 to 5169 from the 5' end of sequence listing 2.

The promoter may specifically be the TEF1 promoter. The expression cassette may be specifically shown as positions 5117 to 10117 from the 5' end of 3 of the sequence listing.

The invention also protects a recombinant bacterium, which is obtained by introducing the coding gene of the transcription factor Cat8p into saccharomyces cerevisiae.

The "introduction of the coding gene of the transcription factor Cat8p into the saccharomyces cerevisiae" can be realized by specifically introducing an expression vector for expressing a CAT8 gene into the saccharomyces cerevisiae.

The invention also protects a recombinant bacterium, which is obtained by introducing an expression cassette containing the coding gene of the transcription factor Cat8p into saccharomyces cerevisiae; in the expression cassette, expression of the coding gene is promoted by a constitutive promoter.

The constitutive promoter may specifically be the ADH1 promoter. The expression cassette may be specifically represented by positions 251 to 5169 from the 5' end of sequence listing 2.

The constitutive promoter may be particularly a TEF1 promoter. The expression cassette may be specifically shown as positions 5117 to 10117 from the 5' end of 3 of the sequence listing.

The "introduction of the expression cassette containing the coding gene of the transcription factor Cat8p into Saccharomyces cerevisiae" can be specifically realized by introducing an expression vector containing the expression cassette containing the coding gene of the transcription factor Cat8p into Saccharomyces cerevisiae.

The expression vector can be specifically a circular plasmid shown in a sequence 2 of a sequence table.

The expression vector can be specifically a circular plasmid shown in a sequence 3 of a sequence table.

The coding gene of any ADH1 promoter is shown as 251-646 th site from 5' end of sequence 2 in a sequence table.

The coding gene of any one of the TEF1 promoters is shown as 5117 th to 5564 th from 5' end of a sequence 3 of a sequence table.

Any one of the above Saccharomyces cerevisiae may be specifically Saccharomyces cerevisiae YS 58.

The invention also protects the application of any recombinant bacterium in the production of 2-phenethyl alcohol.

The invention also provides a method for producing 2-phenethyl alcohol, which comprises the following steps: culturing any one of the recombinant bacteria to obtain the 2-phenethyl alcohol from the culture product. The culture product can be a culture system of the recombinant bacteria or a supernatant obtained by centrifuging the culture system.

Compared with the host bacteria, the saccharomyces cerevisiae strain for producing the 2-phenethyl alcohol provided by the invention has the advantages that the expression level of key regulatory factors and key enzyme genes in the aldrin pathway is enhanced, the synthesis capacity of the 2-phenethyl alcohol of the cell is obviously improved, the fermentation condition of the saccharomyces cerevisiae strain is simple, the fermentation period is short, and the industrial production of the 2-phenethyl alcohol by the strain is facilitated.

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 YEp 352: reference documents: hill JE, Meyers AM, Koerner TJ, et al (1993). Ayeast/E.coli cut vectors with multiple unique restriction sites. Yeast,9: 163-; the public is available from the institute for microorganisms of the Chinese academy of sciences.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) YS 58: reference documents: tennissen AW R H, Holub E, van den Hucht J, van der Berg J A, Steensma H Y (1993) Sequence of the open reading frame of the FLO1 from Saccharomyces cerevisiae, Yeast,9:423 + 427; the public is available from the institute for microorganisms of the Chinese academy of sciences.

Fermentation medium: 4g/100ml glucose, 0.5g/100ml MgSO₄,0.5g/100ml KH₂PO₄0.5g/100ml phenylalanine; the solvent is water.

Uracil-deficient SC selection medium containing 0.5% phenylalanine: contains 5g/L phenylalanine, 6.7g/L yeast nitrogen source base, 10g/L glucose, 40mg/L histidine, 40mg/L tryptophan and 40mg/L leucine; the solvent is water.

SC complete medium containing 0.5% phenylalanine: contains 5g/L phenylalanine, 6.7g/L yeast nitrogen source base, 10g/L glucose, 40mg/L histidine, 40mg/L tryptophan, 40mg/L leucine and 20mg/L uracil; the solvent is water.

The CAT8 gene is shown in 657 th to 4958 th positions from 5' end of a sequence 2 in a sequence table, and the coded protein (transcription factor Cat8p) is shown in the sequence 5 in the sequence table.