CN115386503A - High-yield ethyl crotonate saccharomyces cerevisiae strain and construction method and application thereof - Google Patents

High-yield ethyl crotonate saccharomyces cerevisiae strain and construction method and application thereof Download PDF

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CN115386503A
CN115386503A CN202210506230.XA CN202210506230A CN115386503A CN 115386503 A CN115386503 A CN 115386503A CN 202210506230 A CN202210506230 A CN 202210506230A CN 115386503 A CN115386503 A CN 115386503A
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陈叶福
康新玥
张果
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Tianjin University of Science and Technology
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Abstract

The invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and particularly relates to a method for constructing a saccharomyces cerevisiae strain capable of producing ethyl crotonate at high yield and application thereof. The 3-hydroxybutyryl-CoA dehydrogenase Hbd, the 3-hydroxybutyryl-CoA dehydratase Crt and the alcohol acyltransferase AAT are over-expressed in the original strain to obtain 1 strain Ck-HCVL with the ability of producing the ethyl crotonate, and compared with the original strain which does not produce the ethyl crotonate, the yield of the modified strain reaches 58.6 +/-6.19 mg/L. The strain Ck-HC-DVL-E is obtained after the Erg10 and two copies of AAT genes are over-expressed, the yield of the crotonic acid ethyl ester reaches 122.99 +/-6.55 mg/L, the yield is improved by 109.9 percent compared with the Ck-HCVL strain, an unexpected technical effect is achieved, and a solution is provided for producing the crotonic acid ethyl ester by utilizing microorganisms.

Description

High-yield ethyl crotonate saccharomyces cerevisiae strain and construction method and application thereof
The technical field is as follows:
the invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and particularly relates to a high-yield ethyl crotonate saccharomyces cerevisiae strain as well as a construction method and application thereof.
Background art:
the ethyl crotonate has strong sour and burnt fragrance and fruit fragrance, and has rum and ether fragrance. The natural product exists in apple, pawpaw, strawberry, mango, rum, wine, cocoa and the like, can be applied to the formula of edible essence, and is mainly used for preparing fruit wine essence. In white spirit, wine and other alcoholic beverages, esters are the most important of various minor components, and can improve the aroma richness and aroma quality of the wine. Among these esters, acetate is one of the most abundant, short-medium chain fatty acid ethyl esters are key flavor components in alcoholic beverages, and are the main carriers of alcoholic beverage aroma.
The ethyl crotonate is industrially used as a precursor of an aromatic group in cosmetics, shampoos, detergents and detergents, can also be used as an organic synthesis intermediate and a solvent, and has wide application in the field of drug synthesis.
In saccharomyces cerevisiae, there are three main pathways for synthesizing short-medium chain acyl coenzyme a, including an intracellular fatty acid de novo synthesis pathway, an exogenous fatty acid absorption activation pathway, and a fatty acid degradation (β oxidation) pathway, and the regulation of these production pathways is very complicated, so that the short-medium chain fatty acid ethyl ester production capacity is low. The saccharomyces cerevisiae hardly produces ethyl crotonate, and if a crotonyl coenzyme A synthesis way is constructed in the saccharomyces cerevisiae and a high-efficiency alcohol acyltransferase capable of synthesizing corresponding ethyl ester from acyl coenzyme A and ethanol is introduced, a saccharomyces cerevisiae strain producing ethyl crotonate can be constructed.
Disclosure of Invention
The first purpose of the invention is to solve the problem that the saccharomyces cerevisiae does not synthesize ethyl crotonate in wine production, and provide a construction method of a saccharomyces cerevisiae strain capable of producing ethyl crotonate. Constructing a crotonyl-coenzyme A generation way in the saccharomyces cerevisiae to ensure that the saccharomyces cerevisiae generates crotonyl-coenzyme A; and introducing a high-efficiency alcohol acyltransferase into the saccharomyces cerevisiae on the basis, thereby constructing a complete path of the ethyl crotonate and realizing the production of the ethyl crotonate. Furthermore, the overexpression of endogenous acetyl coenzyme A acyltransferase gene increases the preconditions, and double-copy expression is carried out on the alcohol acyltransferase gene in the production process of the ethyl crotonate, so that the yield of the ethyl crotonate is greatly improved.
In order to solve the problems, the invention provides a Saccharomyces cerevisiae gene engineering strain for producing ethyl crotonate, which is constructed by taking Saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC32315 as an initial strain and by heterogeneously over-expressing a 3-hydroxybutyryl-CoA dehydrogenase gene Hbd and a 3-hydroxybutyryl-CoA dehydratase gene Crt, a heterogeneously over-expressing an alcohol acyltransferase gene AAT and a endogenous acetyl-CoA acyltransferase gene Erg 10.
Preferably, the gene VLAAT is expressed in single or double copies.
Preferably, the 3-hydroxybutyryl-CoA dehydrogenase (3-hydroxybutyryl-CoA dehydrogenase) gene Hbd is derived from Clostridium kluyveri DSM 555.
More preferably, the 3-hydroxybutyryl-coa dehydrogenase Gene Hbd has a Gene ID of: 5394457, the codon optimized nucleotide sequence of Saccharomyces cerevisiae is shown as SEQ ID NO:1 is shown.
Preferably, the 3-hydroxybutyryl-CoA dehydratase gene (3-hydroxybutyryl CoA dehydratase) Crt is derived from Clostridium kluyveri DSM 555.
More preferably, the 3-hydroxybutyryl-coa dehydratase Gene Crt, whose Gene ID is: 5391750, the codon optimized nucleotide sequence of Saccharomyces cerevisiae is shown as SEQ ID NO:2, respectively.
Preferably, the alcohol acetyltransferase gene (alcohol acetyltransferase) AAT is derived from grape (published by NCBI under the name vitas labrusca × vitas vinifera).
More preferably, the alcohol acetyltransferase gene AAT, genBank thereof is: KX963771.1, and the nucleotide sequence after saccharomyces cerevisiae codon optimization is shown as SEQ ID NO:3, respectively.
Preferably, the acetyl-CoA acylase (acetyl-CoA acylase) Erg10 gene is derived from Saccharomyces cerevisiae.
More preferably, the acetyl-coa acyltransferase Gene Erg10 has the Gene ID: 856079, the nucleotide sequence is as shown in SEQ ID NO:4, respectively.
Preferably, the starting yeast strain is Saccharomyces cerevisiae (Saccharomyces cerevisiae) cic 32315;
the heterologous overexpressed 3-hydroxybutyryl-coa dehydrogenase gene Hbd is aimed at the synthesis of 3-hydroxybutyryl-coa from acetoacetyl-coa.
The heterologous overexpressed 3-hydroxybutyryl-coa dehydratase gene Crt is intended to synthesize crotonyl-coa from 3-hydroxybutyryl-coa.
The heterologous over-expression alcohol acyltransferase gene AAT aims at introducing alcohol acyltransferase so as to construct a complete pathway of crotonic acid ethyl ester and realize the production of crotonic acid ethyl ester.
The overexpression of the acetyl-CoA acyltransferase gene Erg10, for the synthesis of acetoacetyl-CoA from acetyl-CoA.
The alcohol acyltransferase gene AAT in the opposite way is double-copy expression, and because the key last step of synthesizing the crotonic acid ethyl ester by using the alcohol acyltransferase in the crotonyl-coenzyme A synthesis way can limit the generation of the crotonic acid ethyl ester, the alcohol acyltransferase gene is double-copied, so that the yield of the crotonic acid ethyl ester of the saccharomyces cerevisiae is obviously improved.
The overexpression of the endogenous gene Erg10 gene is that the synthesis of precursor acetyl coenzyme A catalyzed by Erg10 in the synthesis pathway of crotonyl coenzyme A may limit the generation of crotonic acid ethyl ester, so that the overexpression of the Erg10 gene is performed, and the yield of crotonic acid ethyl ester of saccharomyces cerevisiae is remarkably improved.
The second purpose of the invention is to provide a method for constructing the saccharomyces cerevisiae gene engineering strain for producing the crotonyl-CoA by using a Crispr gene editing technology, firstly, 3-hydroxybutyryl-CoA dehydrogenase (Hbd) and 3-hydroxybutyryl-CoA dehydratase (Crt) perform heterologous expression at a YCR011C, YBR C site in saccharomyces cerevisiae to construct a yeast strain Ck-HC with a crotonyl-CoA generating way; secondly, carrying out heterologous integration expression on the site of the grape alcohol acyltransferase VLAAT in the YHR142W strain to obtain a yeast strain Ck-HCVL for producing the crotonic acid ethyl ester; thirdly, over-expressing acetyl-CoA acyltransferase gene Erg10 at LPP1 site of Ck-HCVL in order to synthesize acetoacetyl-CoA from acetyl-CoA to obtain strain Ck-HCVL-E; finally, the expression of double-copy VLAAT is carried out at the 416d site of the Ck-HCVL-E strain, and the strain Ck-HC-DVL-E with high-yield crotonic acid ethyl ester is obtained.
Preferably, the gene VLAAT is expressed in two copies.
More preferably, the heterologous expression of the two copies of the glucose acyltransferase gene VLAAT is achieved by integration at the 416d site.
The genes YCR011C, YBR128C, YHR W, LPP and 416d are both from saccharomyces cerevisiae.
Preferably, the construction method of the saccharomyces cerevisiae gene engineering strain for high yield of the ethyl crotonate comprises the following steps:
(1) Taking a haploid of a saccharomyces cerevisiae strain as an initial strain, taking a gene YCR011C, YBR C as an integration site, and respectively connecting an upstream homology arm 011U500/128U500 of the gene YCR011C, YBR C, a TDH3p-Hbd-TDH1t/CCW12p-Crt-SSA1t fragment and a downstream homology arm 011D500/128D500 of the gene YCR011C, YBR C in sequence through fusion PCR, and inserting a Cas9-011C-128C plasmid into the integration site through lithium acetate conversion to obtain a recombinant strain Ck-HC;
(2) Meanwhile, by taking the gene YHR142W as an integration site, sequentially fusing and connecting an upstream homologous arm 142U500 of the gene YHR142W, a PGK1p-VLAAT-ENO2t fragment and a downstream homologous arm 142D500 of the gene YHR142W, and inserting the fused and connected upstream homologous arm, PGK1p-VLAAT-ENO2t fragment and the downstream homologous arm 142D500 of the gene YHR142W into the integration site through lithium acetate transformation to obtain a recombinant strain Ck-HCVL;
preferably, in the step (1), the haploid of the saccharomyces cerevisiae strain is an alpha type haploid.
More preferably, the recombinant strain obtained by the construction method is subjected to the following experiment:
(1) Taking Ck-HCVL as an initial strain, taking LPP1 as an integration site, sequentially fusing an upstream homologous arm LPP1U500 of the gene LPP1, a TEF1p-Erg10-ADH1t fragment and a downstream homologous arm LPP1D500 of the gene LPP1, connecting with a Cas9-LPP1 plasmid through PCR, and inserting the plasmids into the integration site through lithium acetate conversion to obtain a recombinant strain Ck-HCVL-E;
(2) Taking Ck-HCVL-E as an initial strain, taking 416D as an integration site, connecting an upstream homology arm 416U500 of a gene 416D, a PGK1p-VLAAT-ENO2t fragment and a downstream homology arm 416D500 of the gene 416D with a Cas9-416D plasmid through fusion PCR in sequence, and inserting the fragments into the integration site through lithium acetate transformation to obtain a recombinant strain Ck-HC-DVL-E;
the third purpose of the invention is to provide a fermentation method of the saccharomyces cerevisiae gene engineering strain for high yield of the crotonic acid ethyl ester.
Preferably, the fermentation steps of the saccharomyces cerevisiae gene engineering strain are as follows:
after the saccharomyces cerevisiae gene engineering bacteria are activated in two stages, inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 8-12%, and standing and fermenting for 80-86h at the temperature of 28-30 ℃.
And weighing for 1 time every 12h in the later period of fermentation, and determining that the fermentation is finished when the weight loss of the two times is less than 0.5 g.
Preferably, the corn thick mash fermentation medium consists of: 300-320g/L of corn flour, 180mL of water, 104U/L of high-temperature resistant alpha-amylase (3-4) x, 90-100U/L of saccharifying enzyme and 10-20U/L of acid protease; 5.5-5.6ml/L of nutrient salt and the balance of water. The nutrient salt comprises the following components: mgSO (MgSO) 4 140-160g/L、KH 2 PO 4 70-80g/L of urea, 80-85g/L of urea and the balance of water.
Preferably, the two-stage activation conditions of the saccharomyces cerevisiae are as follows: firstly, saccharomyces cerevisiae gene engineering bacteria are inoculated into a primary seed culture medium, static culture is carried out for 24 hours at the temperature of 28-30 ℃ to obtain a primary seed liquid, the primary seed liquid is inoculated into a secondary seed culture medium according to the inoculation amount of 8-10%, and static culture is carried out at the temperature of 28-30 ℃ until the later stage of logarithmic phase, namely 15-18 hours, so as to obtain a secondary seed liquid.
More preferably, the primary seed medium consists of: 80-85g/L corn flour, and the addition amount of high-temperature resistant alpha amylase is about (0.5-1.0) multiplied by 10 4 U/L, 30-35U/L saccharifying enzyme, water for the rest, and 8 ° BX sugar degree.
More preferably, the secondary seed medium consists of: corn flour of 120-130g/L, and high temperature resistant alpha amylase of 1.0-2.0 times 10 4 U/L, saccharifying enzyme about 45-55U/L, balance water, and sugar degree of 12 ° BX.
Has the advantages that:
1. the technical content of the invention provides a new way for producing the crotonic acid ethyl ester in the saccharomyces cerevisiae, and a saccharomyces cerevisiae strain capable of producing the crotonic acid ethyl ester at high yield is constructed by introducing an exogenous crotonyl coenzyme A synthesis way and introducing high-efficiency alcohol acyltransferase capable of synthesizing corresponding ethyl ester from acyl coenzyme A and ethanol. Compared with wild saccharomyces cerevisiae which can not produce the crotonic acid ethyl ester, the yield of the crotonic acid ethyl ester of the strain reaches 122.99 +/-6.55 mg/L, theoretical basis is laid for the saccharomyces cerevisiae to produce the crotonic acid ethyl ester, and the strain has wide application prospect.
2. The saccharomyces cerevisiae strain for producing the ethyl crotonate is realized on the premise of keeping excellent fermentation performance, provides a solution for producing the ethyl crotonate in brewed wine, reduces the consumption of byproducts and energy by microbial fermentation for producing ester, and also ensures that the ethyl crotonate is not limited to chemical production.
Drawings
FIG. 1 is a metabolic diagram of the synthetic pathway of ethyl crotonate constructed in Saccharomyces cerevisiae;
FIG. 2 is a schematic diagram of the construction process of the recombinant plasmids Yep352-TT-Hbd (a), yep352-CS-Crt (b), yep352-PE-VLAAT (c) and Yep352-TA-Erg10 (d);
FIG. 3 is a diagram showing the confirmation electrophoresis of the construction of recombinant plasmids Yep352-TT-Hbd, yep352-CS-Crt, yep352-PE-VLAAT, yep352-TA-Erg10;
(a) In the figure, lane 1 is the Yep352-TT plasmid; lane 2 is Yep352-TT-CkHbd; lane 3 is the CkHbd gene fragment; lane 4 is 10000bp DNA Ladder Marker; (b) lane 1 of the figure is Yep352-CS plasmid; lane 2 is Yep352-CS-CkCrt; lane 3 is the CkCrt gene fragment; lane 4 is 10000bp DNA Ladder Marker; (ii) a (c) lane 1 of the figure is Yep352-PE plasmid; lane 2 is Yep352-PE-VLAAT; lane 3 is a VLAAT gene fragment; lane 4 is 10000bp DNA Ladder Marker; (d) lane 1 of the figure is Yep352-TA plasmid; lane 2 is Yep352-TA-Erg10; lane 3 is the Erg10 gene fragment; lane 4 is 10000bp DNA Ladder Marker.
FIG. 4 is an electrophoretogram of fusion of the upper and lower homology arms of the integration site with a plasmid fragment containing the expression cassette of the genes CkHbd, ckCrt, VLAAT, and Erg10;
(a) In the figure, lane 1 shows a TT-CkHbd fragment; lane 2 is 011-TT-CkHbd fragment; lane 3 is the YCR011C-U gene fragment; lane 4 is the YCR011C-D gene fragment; lane 5 is 5000bp DNA Ladder Marker; lane 6 is the CS-CkCrt fragment; lane 7 is a 128-CS-CkCrt fragment; lane 8 is a YBR128C-U gene fragment; lane 8 is a YBR128C-D gene fragment; lane 10 is 5000bp DNA Ladder Marker; (b) lane 1 of the figure is a PE-VLAAT fragment; lane 2 is the 142-PE-VLAAT fragment; lane 3 is the YHR142W-U gene fragment; lane 4 is the YHR142W-D gene fragment; lane 5 is 5000bp DNA Ladder Marker; (c) in lane 1, a TA-Erg10 fragment; lane 2 is LPP1-TA-Erg10 fragment; lane 3 is an LPP1-U gene fragment; lane 4 is an LPP1-D gene fragment; lane 5 is 5000bp DNA Ladder Marker; (d) lane 1 of the figure is a PE-VLAAT fragment; lane 2 is the 416-PE-VLAAT fragment; lane 3 is 416d-U gene fragment; lane 4 is 416D-D gene fragment; lane 5 is 5000bp DNA Ladder Marker.
FIG. 5 is a schematic diagram of a recombinant Saccharomyces cerevisiae strain construction overexpressing the CkHbd and CkCrt genes at position YCR011C, YBR C;
FIG. 6 is a schematic diagram showing the construction of a recombinant Saccharomyces cerevisiae strain overexpressing VLAAT gene at the locus YHR 142W;
FIG. 7 is a schematic diagram of the construction of a recombinant Saccharomyces cerevisiae strain overexpressing the Erg10 gene at the site LPP 1;
FIG. 8 is a schematic diagram of a recombinant Saccharomyces cerevisiae construct with double copies of VLAAT gene at position 416 d;
FIG. 9 is a diagram showing the confirmation of the construction of each strain;
(a) Lane 1 is a verification fragment with the genome of the recombinant strain as a template and 011Cyz-F/R as a primer; lane 2 is a verification fragment with the genome of the recombinant strain as a template and 011-TT-CkHbd-F/R as a primer; lane 3 is a verification fragment with the recombinant strain genome as template and 128Cyz-F/R as primer; lane 4 is a verification fragment with the recombinant strain genome as template and 128-CS-CkCrt-F/R as primer; lane 5 is 5000bp DNA Ladder Marker; (b) Lane 1 is a verified fragment using the recombinant strain genome as template and 142Wyz-F/R as primers; lane 2 is a verified fragment using the genome of the recombinant strain as a template and 142-PE-VLAAT-F/R as a primer; lane 3 is 5000bp DNA Ladder Marker; (c) Lane 1 is a verification fragment using the recombinant strain genome as a template and LPP1-yz-F/R as a primer; lane 2 is a verified fragment using the recombinant strain genome as template and LPP1-TA-Erg10-F/R as primer; lane 3 is 5000bp DNA Ladder Marker; (d) Lane 1 is a verified fragment using the genome of the recombinant strain as a template and 416dyz-F/R as primers; lane 2 is a verification fragment using the genome of the recombinant strain as a template and 416-PE-VLAAT-F/R as a primer; lane 3 is 5000bp DNA Ladder Marker.
FIG. 10 is a graph showing the results of the production of ethyl crotonate by the parent strain and the saccharomyces cerevisiae engineered strain at each stage.
The specific implementation mode is as follows:
the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The Saccharomyces cerevisiae used in the present invention can be any Saccharomyces cerevisiae strain, and the yeast strains used in the following examples are all alpha haploids (AY 14 alpha) of Saccharomyces cerevisiae CICC32315.
Firstly, 3-hydroxybutyryl-CoA dehydrogenase (Hbd) and 3-hydroxybutyryl-CoA dehydratase (Crt) from Clostridium kluyveri DSM 555 are subjected to heterologous overexpression in Saccharomyces cerevisiae to construct a yeast strain Ck-HC with a crotonyl-CoA generation pathway; secondly, carrying out heterologous integration strong expression on the glucosyl transferase VLAAT in the strain to obtain a yeast strain Ck-HCVL for producing the ethyl crotonate; then, over-expressing Erg10 on the basis of the strain Ck-HCVL to obtain a strain Ck-HCVL-E; finally, expression of double-copy VLAAT is carried out on the basis of the Ck-HCVL-E strain to obtain the strain Ck-HC-DVL-E with high yield of the crotonic acid ethyl ester.
Example 1: construction of Saccharomyces cerevisiae strain for producing ethyl crotonate
The starting strain Saccharomyces cerevisiae CICC32315 used in this example. The E.coli DH 5. Alpha. Was purchased from Takara. The YPD culture medium is a general complete culture medium, and the solid culture medium contains 2% imported agar powder.
Based on the respective gene sequences and integration plasmid sequences in NCBI Genebank, the following primers were designed as shown in Table 1.
TABLE 1 primers
Figure BSA0000272668670000081
Figure BSA0000272668670000091
The PCR amplification system used in this example is shown in Table 2.
TABLE 2
Figure BSA0000272668670000092
The main construction process of the strain is as follows:
(1) Construction of Yep352-TT-Hbd, yep352-CS-Crt, yep352-PE-VLAAT, yep352-TA-Erg10 plasmids
Plasmids Yep352-TDH3p-TDH1t, yep352-CCW12p-SSA1t, yep352-PGK1p-ENO2t and Yep352-TEF1p-ADH1t are taken as basic plasmids to construct Yep352-TT-Hbd, yep352-CS-Crt, yep352-PE-VLAAT and Yep352-TA-Erg10, and the construction process is shown in FIG. 2.
Codon-optimized gene synthesis was performed with reference to the 3-hydroxybutyryl-CoA dehydrogenase (Hbd), 3-hydroxybutyryl-CoA dehydratase (Crt) and alcohol acyltransferase (VLAAT) gene sequences in grape, which were obtained from the query in NCBI of Clostridium kluyveri DSM 555. Obtaining an 849bp Hbd fragment by PCR amplification by using a primer pair TT-Hbd-F/R (SEQ ID NO: 5/6); PCR amplification is carried out by using a primer pair CS-Crt-F/R (SEQ ID NO: 7/8) to obtain 734bp CkCrt fragment; PCR amplification was performed using the primer pair PE-AAT-F/R (SEQ ID NO: 9/10) to obtain a 1350bp VLAAT fragment; and respectively carrying out PCR amplification by using a vector plasmid with a target gene obtained by gene synthesis as a template to obtain target gene fragments CkHbd, ckCrt and VLAAT.
The construction process is illustrated by taking Yep352-TDH3p-CkHbd-TDH1t as an example. Cutting a polyclonal site between TDH3p and TDH1t by using restriction enzyme XhoI, linearizing the plasmid, and then using Novozam for the target gene segment CkHbd amplified in the previous step
Figure BSA0000272668670000101
II One Step Cloning Kit was inserted between both ends of the linearized plasmid to obtain the objective plasmid, and the results of the verification are shown in FIG. 3.
(2) Construction of yeast strain producing ethyl crotonate
The AY14 alpha is used as an initial strain, YCR011C, YBR C, YHR W is selected as an integration site, and 3-hydroxybutyryl-CoA dehydrogenase (Hbd), 3-hydroxybutyryl-CoA dehydratase (Crt) gene CkHbd (Crt) and alcohol acyl transferase gene VLAAT are respectively overexpressed to construct a modified strain Ck-HCVL.
Using AY14 alpha genomic DNA as a template, the upper homology arm YCR011C-U, YBR C-U at YCR011C, YBR C was obtained by PCR amplification using primer pair 011CU500-F/R (SEQ ID NO: 17/18), 128CU500-F/R (SEQ ID NO: 21/22), and the lower homology arm YCR011C-D, YBR C-D at YCR011C, YBR C was obtained by PCR amplification using primer pair 011CD500-F/R (SEQ ID NO: 19/20), 128CD500-F/R (SEQ ID NO: 23/24). The constructed recombinant plasmid is used as a template, and a primer pair 011-TT-Hbd-F/R (SEQ ID NO: 11/12) and 128-CS-Crt-F/R (SEQ ID NO: 13/14) are used for PCR amplification to obtain target gene segments of TDH3p-CkHbd-TDH1t and CCW12p-CkCrt-SSA1 t. Fusion PCR was performed by connecting the upper and lower homologous arms of the integration site YCR011C, YBR C to TDH3p-CkHbd-TDH1t and CCW12p-CkCrt-SSA1t fragments, i.e., 011U-TDH3p-CkHbd-TDH1t-011D and 128U-CCW12p-CkCrt-SSA1t-128D, respectively, and the agarose gel electrophoresis results are shown in FIG. 4-a.
The gene segments obtained by the method are converted by a saccharomyces cerevisiae lithium acetate method: 011U-TDH3p-CkHbd-TDH1t-011D, 128U-CCW12p-CkCrt-SSA1t-128D and Cas9-011C-128C (containing KAN resistance markers) are transformed into saccharomyces cerevisiae strain AY14 alpha in a plasmid mode. Over-expression CkHbd and CkCrt modified bacteria Ck-HC are obtained, and the conversion process is shown in figure 5.
Then, a Ck-HC starting strain is selected, YHR142W is selected as an integration site, and an alcohol acyltransferase gene VLAAT is overexpressed to construct a modified strain Ck-HCVL. The upper homology arm 142U at YHR142W was obtained by PCR amplification using the genomic DNA of AY 14. Alpha. As a template, the primer set 142WU500-F/R (SFQ ID NO: 25/26), and the lower homology arm 142D at YHR142W was obtained by PCR amplification using the primer set 142D500-F/R (SEQ ID NO: 27/28), and the results of agarose gel electrophoresis are shown in FIG. 4-b. The recombinant plasmid Yep352-PE-VLAAT constructed above is used as a template, and a primer pair 142-PE-AAT-F/R (SEQ ID NO: 15/16) is used for PCR amplification to obtain a PGK1p-VLAAT-ENO2t target gene fragment. Fusion PCR the upper and lower homologous arms of the integration site YHR142W were ligated to the PGK1p-VLAAT-ENO2t fragment, i.e., 142U-PGK1p-VLAAT-ENO2t-142D, and the transformation procedure is shown in FIG. 6.
The gene segments obtained by the method are converted by a saccharomyces cerevisiae lithium acetate method: 142U-PGK1p-VLAAT-ENO2t-142D and Cas9-142W are transformed into starting bacteria Ck-HC. And obtaining over-expressed CkHbd, ckCrt and VLAAT modified bacteria Ck-HCVL.
And respectively designing verification primers according to gene sequences at two ends of the integration site of the saccharomyces cerevisiae AY14a, and performing PCR amplification by using a genome of a haploid transformant with better growth as a template to verify a recon. The sizes of the obtained bands are about 2760bp, 2717bp and 3290bp verified by primer pairs 011Cyz500-F/R (SEQ ID NO: 29/30), 128Cyz500-F/R (SEQ ID NO: 31/32) and 142Wyz500-F/R (SEQ ID NO: 33/34), and are consistent with the expected sizes. The verification results are shown in FIGS. 9-a, b.
(3) Overexpression of the Erg10 Gene
The genomic DNA of AY14 alpha is used as a template, a primer pair T-Erg10-A-F/R (SEQ ID NO: 35/36) is used for obtaining an Erg10 gene fragment through PCR amplification, yep352-TEF1p-ADH1T is selected as a gene expression cassette, a plasmid containing the Erg10 is constructed, the method is the same as the above, the Yep352-TEF1p-Erg10-ADH1T is obtained, and the construction flow is shown in FIG. 2. The constructed recombinant plasmid is used as a template, a primer pair LPP1-TA-Erg10-F/R (SEQ ID NO: 37/38) is used for obtaining a TEF1p-Erg10-ADH1t fragment through PCR amplification, the genomic DNA of AY14 alpha is used as a template, a primer pair LPP1500U-F/R (SEQ ID NO: 39/40) and LPP1500D-F/R (SEQ ID NO: 41/42) are used for obtaining a fragment of LPP1U, LPP D through PCR amplification, fusion PCR is used for fusing the upper and lower homologous arms of LPP1 and the TA-Erg10 fragment to obtain LPP1U-TEF1p-Erg10-ADH1t-LPP1D, and the agarose gel electrophoresis result is shown in a picture 4-c.
The plasmid LPP1U-TEF1p-Erg10-ADH1t-LPP1D, cas-LPP 1 is integrated into Ck-HCVL through lithium acetate transformation to obtain the engineered bacterium Ck-HCVL-E over-expressing Erg10, and the transformation process is shown in FIG. 7.
And selecting a transformant dot plate with better growth vigor, and extracting a genome for verification after bacterial sludge grows out. The size of the resulting band was 2858bp, as verified by the primer pair LPP1yz500-F/R (SEQ ID NO: 43/44), respectively. The verification results are shown in FIG. 9-c.
(4) Double copy VLAAT gene
The fragments 416U and 416D were obtained by PCR amplification using genomic DNA of AY 14. Alpha. As a template and the primer pair 416dU500-F/R (SEQ ID NO: 47/48) and 416dD500-F/R (SEQ ID NO: 49/50). The recombinant plasmid Yep352-PE-VLAAT constructed above is used as a template, and a primer pair 416-PE-AAT-F/R (SEQ ID NO: 45/46) is used for PCR amplification to obtain a PGK1p-VLAAT-ENO2t target gene fragment. Fusion PCR 416D upper and lower homologous arms and PE-VLAAT fragment fusion to obtain 416U-PGK1p-VLAAT-ENO2t-416D, agarose gel electrophoresis results are shown in FIG. 4-D.
The fragment 416U-PGK1p-VLAAT-ENO2t-416D, cas-416 d plasmid obtained by the PCR is simultaneously transformed into the recombinant strain Ck-HCVL-E by a lithium acetate transformation method, and the Saccharomyces cerevisiae recombinant strain Ck-HC-DVL-E is obtained after intracellular integration, wherein the transformation process is shown in figure 8.
And (4) selecting a transformant dot plate with better growth vigor, and extracting a genome for verification after bacterial sludge grows out. The size of the resulting band was 3281bp, identical to the expected size, as verified by primer pair 416dyz-F/R (SEQ ID NO: 51/52), respectively. The verification results are shown in FIG. 9-d.
Example 2: thick mash fermentation experiment of corn raw material of starting strain and improved strain
Carrying out corn raw material thick mash fermentation experiments on the parent strain AY14 alpha and the recombinant strains Ck-HCVL, ck-HCVL-E, ck-HC-DVL-E, wherein a fermentation process route diagram: corn flour → soaking → liquefying → saccharifying → cooling → inoculating bacteria → fermenting → distilling wine → measuring index;
respectively selecting a torula yeast cell, respectively inoculating into test tubes containing 5mL of primary seed culture medium, standing and culturing at 30 ℃ for 24h, inoculating into a 150mL triangular flask containing 45mL of secondary seed culture medium according to the inoculum size of 10%, standing and culturing at 30 ℃ for 16h to the later stage of logarithmic phase, inoculating into a fermentation culture medium according to the inoculum size of 10%, and standing and fermenting at 30 ℃. Weighing for 1 time every 12h, and finishing fermentation when the weight loss of two times is less than 0.5g, namely finishing fermentation for 84-96h. After the fermentation is finished, 100mL of mash is taken, 100mL of water is added, and 100mL of wine sample is distilled out. Determination of CO 2 The results of the fermentation performance indexes such as cumulative discharge amount, alcohol content and residual reducing sugar are shown in Table 3. Wherein, the first-level seed culture medium comprises the following components: 82g/L of corn flour and about 1.0 multiplied by 10 of high-temperature resistant alpha amylase 4 U/L, the activity of the saccharifying enzyme is about 32U/L, the balance is water, and the sugar degree is 8 degrees BX. The secondary seed culture medium comprises the following components: about 125g/L of corn flour and about 1.5 multiplied by 10 of high-temperature resistant alpha amylase 4 U/L, the activity of the saccharifying enzyme is about 50U/L, the balance is water, and the sugar degree is 12 degrees BX. The fermentation medium consists of: 315g/L corn flour and 3.5 multiplied by 10 high temperature resistant alpha amylase 4 U/L, 95U/L of saccharifying enzyme, 15U/L of acid protease, 5.5-5.6mL/L of nutrient salt solution and the balance of water; the nutrient salt solution consists of: mgSO (MgSO) 4 150g/L，KH 2 PO 4 75g/L of urea, 81g/L of urea and the balance of water, filtering and storing at 4 ℃.
The treatment process conditions of the fermentation medium are as follows:
soaking conditions are as follows: soaking corn flour at 60-70 deg.c for 20min; liquefaction conditions: adding high-temperature resistant alpha amylase at the temperature of 85-90 ℃ according to the proportion, and liquefying for 90min; saccharification conditions are as follows: adding saccharifying enzyme and nutrient salt solution at 55-60 ℃, saccharifying for 20min, adding acid protease, and reacting for 20min at 30 ℃ to obtain the fermentation culture medium.
TABLE 3 comparison of fermentation Performance of parent strains and recombinant strains
Figure BSA0000272668670000131
Note: the data shown are the average of the results of three replicates.
From Table 3, it can be seen that the alcohol content and residual sugar content after fermentation of the recombinant strains Ck-HCVL, ck-HCVL-E and Ck-HC-DVL-E are not significantly different from those of the original strain AY14 alpha, thereby indicating that the growth and fermentation performance of the modified strains are not significantly changed.
The yield of ethyl crotonate was determined from 100mL of the final wine samples obtained in the fermentation experiments of corn mash from the above recombinant strains Ck-HCVL, ck-HCVL-E and Ck-HC-DVL-E with the parent strain (AY 14. Alpha.).
The measuring method comprises the following steps: setting the conditions of the gas chromatograph, namely GC conditions: chromatographic column Agilent CP-WAX (50 m × 250 μm × 0.25 μm) with high purity nitrogen (> 99.999%) as carrier gas; the column flow rate was 1mL/min; the temperature of a sample inlet is 250 ℃; the detector temperature was 148.8 ℃; temperature programming: the initial temperature is 35 ℃, the temperature is kept for 1min, the temperature is increased to 70 ℃ at 3 ℃/min, the temperature is kept for 15min, the temperature is increased to 190 ℃ at 3.5 ℃/min, and the temperature is kept for 22min; the sample injection volume is 1 mu L; split-flow sample injection is carried out, and the split-flow ratio is 30: 1. The measured yields of Ck-HCVL, ck-HCVL-E and Ck-HC-DVL-E from the parent strain (AY 14. Alpha.) for ethyl crotonate are shown in Table 4.
TABLE 4 ester yields (in mg/L) of parent and recombinant strains
Figure BSA0000272668670000141
Note: the data shown are the average of the results of three replicates.
In tables 3 and 4, AY14 alpha is the original strain, ck-HCVL is the strain over expressing genes CkHbd, ckCrt and VLAAT, and Ck-HCVL-E is the strain over expressing gene Erg10 based on Ck-HCVL; ck-HC-DVL-E is a strain with double copies of VLAAT on the basis of Ck-HCVL-E.
Figure ISA0000272668690000011
Figure ISA0000272668690000021
Figure ISA0000272668690000031
Figure ISA0000272668690000041
Figure ISA0000272668690000051
Figure ISA0000272668690000061
Figure ISA0000272668690000071
Figure ISA0000272668690000081
Figure ISA0000272668690000091
Figure ISA0000272668690000101
Figure ISA0000272668690000111
Figure ISA0000272668690000121

Claims (6)

1. A saccharomyces cerevisiae gene engineering strain for high yield of ethyl crotonate is characterized in that: the strain is constructed by taking saccharomyces cerevisiae as an initial strain and overexpressing acetyl-CoA acyltransferase gene Erg10, 3-hydroxybutyryl-CoA dehydrogenase gene Hbd and 3-hydroxybutyryl-CoA dehydratase gene Crt and alcohol acyltransferase gene AAT;
wherein, the acetyl-CoA acyltransferase gene Erg10 is overexpressed at a saccharomyces cerevisiae LPP1 site, and meanwhile, the 3-hydroxybutyryl-CoA dehydrogenase encoding gene Hbd and the 3-hydroxybutyryl-CoA dehydratase encoding gene Crt are integrated to saccharomyces cerevisiae YCR011C and YBR128C sites; meanwhile, the alcohol acyltransferase gene AAT realizes overexpression by integrating into a saccharomyces cerevisiae YHR142W site.
2. The genetically engineered strain of saccharomyces cerevisiae producing high-yield ethyl crotonate as claimed in claim 1, wherein: the gene AAT is expressed in double copy;
wherein the double copy of AAT is by integration into Saccharomyces cerevisiae 416 d.
3. The genetically engineered strain of saccharomyces cerevisiae producing high-yield ethyl crotonate as claimed in claim 1, wherein: the nucleotide sequence of the acetyl coenzyme A acyltransferase gene Erg10 is shown as SEQ ID NO:4 is shown in the specification; the nucleotide sequence of the 3-hydroxybutyryl-CoA dehydrogenase gene Hbd is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the 3-hydroxybutyryl coenzyme A dehydratase gene Crt is shown as SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the alcohol acyltransferase gene AAT is shown as a nucleotide sequence table SEQ ID NO:3, respectively.
4. The genetically engineered strain of saccharomyces cerevisiae producing high-yield ethyl crotonate as claimed in claim 1, wherein: the starting strain is Saccharomyces cerevisiae CICC32315.
5. The use of the genetically engineered strain of saccharomyces cerevisiae producing high yield of ethyl crotonate claimed in any one of claims 1 to 4 in the fields of fermentation, flavors and fragrances, and pharmaceutical synthesis.
6. The fermentation method of the saccharomyces cerevisiae gene engineering strain for high yield of ethyl butyrate according to claim 5, wherein the fermentation method comprises the following steps: after the saccharomyces cerevisiae gene engineering bacteria are activated in two stages, inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 8-12%, and standing and fermenting for 84-96h at 30 ℃.
The fermentation medium comprises the following components: corn flour 300-320g/L, high-temperature resistant alpha amylase (2-5) x 10 4 U/L, 90-100U/L of saccharifying enzyme, 10-20U/L of acid protease, 5.5-5.6mL/L of nutrient salt solution and the balance of water; the nutrient salt solution consists of: mgSO (MgSO) 4 140-160g/L，KH 2 PO 4 70-80g/L of urea, 80-85g/L of urea and the balance of water.
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