CN115386503B - 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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CN115386503B
CN115386503B CN202210506230.XA CN202210506230A CN115386503B CN 115386503 B CN115386503 B CN 115386503B CN 202210506230 A CN202210506230 A CN 202210506230A CN 115386503 B CN115386503 B CN 115386503B
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saccharomyces cerevisiae
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ethyl crotonate
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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 high-yield ethyl crotonate saccharomyces cerevisiae strain and application thereof. Through over-expressing 3-hydroxy butyryl coenzyme A dehydrogenase Hbd, 3-hydroxy butyryl coenzyme A dehydratase Crt and alcohol acyl transferase AAT in the original strain, 1 strain of yeast strain Ck-HCVL with the capacity of producing ethyl crotonate is obtained, and compared with the original strain which does not produce ethyl crotonate, the improved strain ethyl crotonate yield reaches 58.6+/-6.19 mg/L. After the Erg10 and the two copies of AAT genes are over-expressed, the strain Ck-HC-DVL-E is obtained, the yield of ethyl crotonate reaches 122.99 +/-6.55 mg/L, and compared with the Ck-HCVL strain, the yield is improved by 109.9%, unexpected technical effects are obtained, and a solution idea is provided for producing ethyl crotonate by utilizing microorganisms.

Description

High-yield ethyl crotonate saccharomyces cerevisiae strain and construction method and application thereof
Technical field:
the invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and in particular relates to a high-yield ethyl crotonate saccharomyces cerevisiae strain, a construction method and application thereof.
The background technology is as follows:
ethyl crotonate has strong sour and burnt fragrance and fruit fragrance, and rum and ether fragrance. The natural products are in apples, papaya, strawberries, mangoes, rum, grape wine, cocoa and the like, can be applied to edible essence formulas, and are mainly used for preparing fruit wine essence. In white spirit, grape wine and other beverage wines, the ester substance is the most important one of various trace components, and can improve the aroma intensity and the aroma quality of the wine. Among these esters, acetate is one of the most abundant, and short-medium-chain fatty acid ethyl esters are key flavor components in alcoholic beverages and are the main carriers of alcoholic liquor aroma.
The industrial ethyl crotonate is used as an aryl precursor in cosmetics, shampoos, detergents and cleaners, can be used as an organic synthesis intermediate and a solvent, has wide application in the field of medicine synthesis, and is mainly produced by esterifying crotonic acid and ethanol in the presence of concentrated sulfuric acid by a chemical method, so that no research on producing ethyl crotonate by utilizing microbial fermentation exists at present.
In Saccharomyces cerevisiae, there are mainly three pathways for the synthesis of short medium chain acyl-CoA, the intracellular fatty acid synthesis pathway from the head, the exogenous fatty acid absorption activation pathway and the fatty acid degradation (beta oxidation) pathway, and the regulation of these production pathways is very complex, so that the ability to produce short medium chain fatty acid ethyl ester is low. The saccharomyces cerevisiae produces little ethyl crotonate, and if a crotonyl-coenzyme A synthesis path is constructed in the saccharomyces cerevisiae and a high-efficiency alcohol acyl transferase capable of synthesizing corresponding ethyl ester from acyl-coenzyme A and ethanol is introduced, the saccharomyces cerevisiae strain producing ethyl crotonate can be constructed.
Disclosure of Invention
The first aim of the invention is to solve the problem that the saccharomyces cerevisiae does not synthesize ethyl crotonate in the production of wine, and provide a construction method of an ethyl crotonate producing saccharomyces cerevisiae strain. Specifically, a crotonyl-coenzyme A generating path is constructed in the saccharomyces cerevisiae to enable the saccharomyces cerevisiae to generate the crotonyl-coenzyme A; and then, on the basis, a high-efficiency alcohol acyl transferase is introduced into the saccharomyces cerevisiae, so that a complete ethyl crotonate pathway is constructed, and the production of ethyl crotonate is realized. Further, the pre-substances are increased by over-expressing the endogenous acetyl-CoA acyltransferase gene, and the alcohol acyltransferase gene in the ethyl crotonate production path is subjected to double-copy expression, so that the ethyl crotonate yield 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 original strain, through heterogenous overexpression of a 3-hydroxybutyryl-CoA dehydrogenase gene Hbd and a 3-hydroxybutyryl-CoA dehydratase gene Crt, heterogenous overexpression of an alcohol acyl transferase gene AAT and simultaneous overexpression of an endogenous acetyl-CoA acyl transferase gene Erg 10.
Preferably, the gene VLAAT is expressed in single copy or in double copy.
Preferably, the gene Hbd of 3-hydroxybutyryl-CoA dehydrogenase (3-hydroxybutyryl CoA dehydrogenase) is derived from Clostridium kluyveri DSM 555.
More preferably, the 3-hydroxybutyryl-coa dehydrogenase Gene Hbd has a Gene ID of: 5394457 the nucleotide sequence after Saccharomyces cerevisiae codon optimization is as shown in SEQ ID NO in the nucleotide sequence table: 1.
Preferably, the 3-hydroxybutyryl-CoA dehydratase gene (3-hydroxybutyryl CoA dehydratase) Crt is Clostridium kluyveri DSM 555.
More preferably, the 3-hydroxybutyryl-coa dehydratase Gene Crt has a Gene ID of: 5391750 the nucleotide sequence after Saccharomyces cerevisiae codon optimization is as shown in SEQ ID NO in the nucleotide sequence table: 2.
Preferably, the alcohol acetyl transferase gene (alcohol acyltransferase) AAT is derived from grape (NCBI published under the name Vitis labrusca×vitis vinifera).
More preferably, the alcohol acetyl transferase gene AAT, whose GenBank is: KX963771.1, the nucleotide sequence of the saccharomyces cerevisiae after codon optimization is shown as SEQ ID NO in a nucleotide sequence table: 3.
Preferably, the acetyl-CoA acylase (acetyl-CoA acetyltransferase) Erg10 gene is derived from Saccharomyces cerevisiae.
More preferably, the acetyl-coa acyltransferase Gene Erg10 has a Gene ID of: 856079, the nucleotide sequence is shown as SEQ ID NO: 4.
Preferably, the starting yeast strain is saccharomyces cerevisiae (Saccharomyces cerevisiae) cic 32315;
the heterologous overexpression of the 3-hydroxybutyryl-CoA dehydrogenase gene Hbd is aimed at synthesizing 3-hydroxybutyryl-CoA from acetoacetyl-CoA.
The heterologous overexpression of the 3-hydroxybutyryl-CoA dehydratase gene Crt aims at synthesizing crotonyl-CoA from 3-hydroxybutyryl-CoA.
The heterologous over-expression alcohol acyl transferase gene AAT aims at introducing alcohol acyl transferase so as to construct a complete ethyl crotonate pathway and realize the production of ethyl crotonate.
The over-expression of acetyl-CoA acyltransferase gene Erg10 aims at synthesizing the acetoacetyl-CoA from the acetyl-CoA.
The alcohol acyl transferase gene AAT in the pair pathway is expressed in double copies, and because the alcohol acyl transferase catalyzes the key last step of crotonate ethyl synthesis in the crotonyl-CoA synthesis pathway, the generation of crotonate ethyl is possibly limited, so that the alcohol acyl transferase gene is subjected to double copies, and the yield of crotonate ethyl of saccharomyces cerevisiae is obviously improved.
The over-expression of the Erg10 gene of the endogenous gene is that the production of ethyl crotonate is possibly limited due to the synthesis of an acyl-CoA precursor substance of the Erg10 catalyst in the synthesis path of crotonyl-CoA, so that the over-expression of the Erg10 gene is carried out, thereby obviously improving the yield of ethyl crotonate of Saccharomyces cerevisiae.
The second object of the present invention is to provide a method for constructing the above-mentioned Saccharomyces cerevisiae genetic engineering strain producing ethyl crotonate by using Crispr gene editing technology, wherein first 3-hydroxybutyryl-CoA dehydrogenase (Hbd) and 3-hydroxybutyryl-CoA dehydratase (Crt) are heterologously expressed at YCR011C, YBR C site in Saccharomyces cerevisiae to construct a yeast strain Ck-HC having a crotonyl-CoA production pathway; secondly, carrying out heterologous integration expression on the grape alcohol acyl transferase VLAAT at the YHR142W site of the strain to obtain a yeast strain Ck-HCVL for producing ethyl crotonate; thirdly, an acetyl coenzyme A acylase gene Erg10 is overexpressed at the LPP1 locus of the Ck-HCVL, so that the acetyl coenzyme A is synthesized by the acetyl coenzyme A to obtain a strain Ck-HCVL-E; finally, expression of double copies of VLAAT is carried out at 416d locus of Ck-HCVL-E strain to obtain strain Ck-HC-DVL-E with high ethyl crotonate yield.
Preferably, the gene VLAAT is expressed in double copies.
More preferably, the dual copy heterologous expression of the VLAAT gene is achieved by integration into the 416d locus.
The genes YCR011C, YBR, 128C, YHR, 142W, LPP and 416d are both derived from Saccharomyces cerevisiae.
Preferably, the construction method of the saccharomyces cerevisiae genetic engineering strain for high-yield crotonic acid ethyl ester comprises the following steps:
(1) Taking a haploid of a saccharomyces cerevisiae strain as a starting strain, taking a gene YCR011C, YBR C as an integration site, and inserting 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, YBR128C into the integration site through lithium acetate conversion after the connection of fusion PCR and Cas9-011C-128C plasmid in sequence to obtain a recombinant strain Ck-HC;
(2) Meanwhile, taking the gene YHR142W as an integration site, sequentially fusing and connecting an upstream homology arm 142U500 of the gene YHR142W, a PGK1p-VLAAT-ENO2t fragment and a downstream homology arm 142D500 of the gene YHR142W with a Cas9-142W plasmid, and inserting the plasmid 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 and LPP1 as an integration site, sequentially fusing and PCR connecting an upstream homology arm LPP1U500 of the gene LPP1, a TEF1p-Erg10-ADH1t fragment and a downstream homology arm LPP1D500 of the gene LPP1 with a Cas9-LPP1 plasmid, and inserting the fusion products into the integration site through lithium acetate transformation to obtain a recombinant strain Ck-HCVL-E;
(2) Taking Ck-HCVL-E as an initial strain and 416D as an integration site, sequentially 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 by fusion PCR, and inserting the fusion PCR connection with a Cas9-416D plasmid into the integration site by lithium acetate transformation to obtain a recombinant strain Ck-HC-DVL-E;
the third object of the invention is to provide a fermentation method of the Saccharomyces cerevisiae genetic engineering strain for high yield of ethyl crotonate.
Preferably, the fermentation steps of the saccharomyces cerevisiae genetically engineered strain are as follows:
after two-stage activation, the Saccharomyces cerevisiae genetic engineering bacteria are inoculated into a fermentation culture medium according to the inoculum size of 8-12%, and are kept stand and fermented for 80-86h at 28-30 ℃.
And weighing 1 time every 12 hours in the later fermentation period, and determining that the fermentation is finished when the weight loss is less than 0.5g twice.
Preferably, the corn thick mash fermentation medium comprises the following components: 300-320g/L corn flour, 180mL water, 3-4 high temperature resistant alpha-amylase (3-4) multiplied by 104U/L, 90-100U/L saccharifying enzyme and 10-20U/L acid proteinase; nutrient salt 5.5-5.6ml/L and water for the rest. The nutrient salt comprises the following components: mgSO (MgSO) 4 140-160g/L、KH 2 PO 4 70-80g/L, 80-85g/L urea and the balance of water.
Preferably, the two-stage activation conditions of the saccharomyces cerevisiae are: firstly, inoculating saccharomyces cerevisiae genetic engineering bacteria into a primary seed culture medium, standing and culturing for 24 hours at 28-30 ℃ to obtain primary seed liquid, inoculating the primary seed liquid into a secondary seed culture medium according to an inoculum size of 8-10%, and standing and culturing at 28-30 ℃ to the later stage of a logarithmic phase, namely 15-18 hours, to obtain secondary seed liquid.
More preferably, the primary seed medium composition is: 80-85g/L corn flour, and the addition amount of high temperature resistant alpha amylase is about (0.5-1.0) x 10 4 U/L, saccharifying enzyme about 30-35U/L, water for the rest, and sugar degree of 8 degrees BX.
More preferably, the secondary seed medium consists of: corn flour about 120-130g/L, and high temperature resistant alpha amylase added amount about (1.0-2.0) x 10 4 U/L, saccharifying enzyme about 45-55U/L, water for the rest, and sugar degree of 12 degrees BX.
The beneficial effects are that:
1. the technical content of the invention provides a new way for producing ethyl crotonate in saccharomyces cerevisiae, and a saccharomyces cerevisiae strain for producing ethyl crotonate in high yield is constructed by introducing an exogenous crotonyl-coenzyme A synthesis way and introducing a high-efficiency alcohol acyl transferase capable of synthesizing corresponding ethyl ester from acyl-coenzyme A and ethanol. Compared with wild saccharomyces cerevisiae incapable of producing ethyl crotonate, the strain has the ethyl crotonate yield reaching 122.99 +/-6.55 mg/L, lays a theoretical foundation for producing ethyl crotonate by saccharomyces cerevisiae, and 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 byproducts and energy consumption by microbial fermentation, and also ensures that the ethyl crotonate is not limited to chemical production.
Drawings
FIG. 1 is a metabolic diagram of the ethyl crotonate synthesis pathway constructed in Saccharomyces cerevisiae;
FIG. 2 is a schematic diagram of the construction flow of recombinant plasmids Yep352-TT-Hbd (a), yep352-CS-Crt (b), yep352-PE-VLAAT (c), yep352-TA-Erg10 (d);
FIG. 3 is a verification electrophoresis pattern of recombinant plasmids Yep352-TT-Hbd, yep352-CS-Crt, yep352-PE-VLAAT, yep352-TA-Erg10;
(a) Lane 1 in the figure 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) FIG. 1 shows the Yep352-CS plasmid; lane 2 is Yep352-CS-CkCrt; lane 3 is the CkCrt gene fragment; lane 4 is 10000bp DNA Ladder Marker; the method comprises the steps of carrying out a first treatment on the surface of the (c) FIG. 1 shows the Yep352-PE plasmid; lane 2 is Yep352-PE-VLAAT; lane 3 is the VLAAT gene fragment; lane 4 is 10000bp DNA Ladder Marker; (d) FIG. 1 shows the 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 the fusion junction of the upper and lower homology arms of the integration site with the plasmid fragment of the expression cassette containing CkHbd, ckCrt, VLAAT, erg gene;
(a) Lane 1 is the TT-CkHbd fragment; lane 2 is the 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 the YBR128C-U gene fragment; lane 8 is the YBR128C-D gene fragment; lane 10 is 5000bp DNA Ladder Marker; (b) lane 1 in 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) FIG. 1 shows a fragment of TA-Erg10; lane 2 is the LPP1-TA-Erg10 fragment; lane 3 is the LPP1-U gene fragment; lane 4 is the LPP1-D gene fragment; lane 5 is 5000bp DNA Ladder Marker; (d) lane 1 in the figure is a PE-VLAAT fragment; lane 2 is the 416-PE-VLAAT fragment; lane 3 is the 416d-U gene fragment; lane 4 is the 416D-D gene fragment; lane 5 is 5000bp DNA Ladder Marker.
FIG. 5 is a schematic diagram of the construction of a recombinant Saccharomyces cerevisiae strain overexpressing the CkHbd and CkCrt genes at position YCR011C, YBR C;
FIG. 6 is a schematic diagram of the construction of a recombinant Saccharomyces cerevisiae strain overexpressing the VLAAT gene at position YHR 142W;
FIG. 7 is a schematic diagram of the construction of a recombinant Saccharomyces cerevisiae strain overexpressing the Erg10 gene at position LPP 1;
FIG. 8 is a schematic diagram of the construction of a recombinant Saccharomyces cerevisiae with double-copy of the VLAAT gene at position 416 d;
FIG. 9 is a verification electrophoretogram of each strain construction;
(a) Lane 1 is a validated fragment using the recombinant strain genome as template and 011Cyz-F/R as primer; lane 2 is a validated fragment using the recombinant strain genome as template, 011-TT-CkHbd-F/R as primer; lane 3 is a validated fragment using recombinant strain genome as template, 128Cyz-F/R as primer; lane 4 is a validated fragment using 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 validated fragment using the recombinant strain genome as template, 142Wyz-F/R as primer; lane 2 is a validated fragment using the recombinant strain genome as template and 142-PE-VLAAT-F/R as primer; lane 3 is 5000bp DNA Ladder Marker; (c) Lane 1 is a verification fragment using recombinant strain genome as template and LPP1-yz-F/R as primer; lane 2 is a verification fragment using recombinant strain genome as a template and LPP1-TA-Erg10-F/R as a primer; lane 3 is 5000bp DNA Ladder Marker; (d) Lane 1 is a validated fragment using the recombinant strain genome as template, 416dyz-F/R as primer; lane 2 is a validated fragment using the recombinant strain genome as template and 416-PE-VLAAT-F/R as primer; lane 3 is 5000bp DNA Ladder Marker.
FIG. 10 is a graph showing ethyl crotonate yield results for a parent strain and a modified strain of Saccharomyces cerevisiae at various stages.
The specific embodiment is as follows:
the invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
The Saccharomyces cerevisiae used in the present invention may be any source of Saccharomyces cerevisiae strain, and the yeast strains used in the examples below are all alpha haploids (AY 14 alpha) of Saccharomyces cerevisiae CICC32315.
Firstly, carrying out heterologous over-expression on 3-hydroxybutyryl-CoA dehydrogenase (Hbd) and 3-hydroxybutyryl-CoA dehydratase (Crt) from Clostridium kluyveri DSM 555 in saccharomyces cerevisiae to construct a yeast strain Ck-HC with a crotonyl-CoA generating pathway; secondly, carrying out heterologous integration strong expression on the grape alcohol acyl transferase VLAAT in the strain to obtain a yeast strain Ck-HCVL for producing ethyl crotonate; then, over-expressing Erg10 on the basis of the strain Ck-HCVL to obtain the strain Ck-HCVL-E; finally, double copy VLAAT expression is carried out on the basis of Ck-HCVL-E strain, and the strain Ck-HC-DVL-E with high ethyl crotonate yield is obtained.
Example 1: construction of ethyl crotonate-producing Saccharomyces cerevisiae Strain
The starting strain used in this example was Saccharomyces cerevisiae CICC32315. The E.coli DH 5. Alpha. Was purchased from Takara. The YPD medium is a general complete medium, and the solid medium contains 2% of imported agar powder.
The following primers were designed based on the respective gene sequences and the integrative plasmid sequences in NCBI Genebank, as shown in Table 1.
TABLE 1 primers
The PCR amplification system used in this example is shown in Table 2.
TABLE 2
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 plasmid
The construction flow of plasmids Yep352-TT-Hbd, yep352-CS-Crt, yep352-PE-VLAAT and Yep352-TA-Erg10 was constructed on the basis of Yep352-TDH3p-TDH1t, yep352-CCW12p-SSA1t, yep352-PGK1p-ENO2t and Yep352-TEF1p-ADH1 t.
Codon optimized gene synthesis was performed with reference to the gene sequences of 3-hydroxybutyryl-CoA dehydrogenase (Hbd) and 3-hydroxybutyryl-CoA dehydratase (Crt) in Clostridium kluyveri DSM 555, which were obtained by the query in NCBI, and the alcohol acyltransferase (VLAAT) in grape. PCR amplification using primer pair TT-Hbd-F/R (SEQ ID NO: 5/6) to obtain a Hbd fragment of 849 bp; PCR amplification using primer pair CS-Crt-F/R (SEQ ID NO: 7/8) to obtain 734bp CkCrt fragment; PCR amplification using primer pair PE-AAT-F/R (SEQ ID NO: 9/10) to obtain 1350bp VLAAT fragment; the vector plasmid with the target gene obtained by gene synthesis is used as a template, and target gene fragments CkHbd, ckCrt, VLAAT are obtained by PCR amplification.
The construction process is described with Yep352-TDH3p-CkHbd-TDH1t as an example. The plasmid was linearized by cleavage with restriction enzyme XhoI from the multiple cloning site intermediate TDH3p and TDH1t, and the gene fragment of interest CkHbd obtained by amplification in the previous step was then purified with NorprazidII One Step Cloning KitInserted between the ends of the linearized plasmid, the plasmid of interest was obtained, and the verification result is shown in FIG. 3.
(2) Construction of ethyl crotonate-producing Yeast Strain
The AY14 alpha is taken as an original strain, YCR011C, YBR C, YHR W is selected as an integration site, 3-hydroxybutyryl-CoA dehydrogenase (Hbd), 3-hydroxybutyryl-CoA dehydratase (Crt) gene CkHbd (Crt) and an alcohol acyl transferase gene VLAAT are respectively and overexpressed to construct an improved strain Ck-HCVL.
Using genomic DNA of AY 14. Alpha. As a template, upper homology arms YCR011C-U, YBR C-U at YCR011C, YBR C were obtained by PCR amplification using primer pairs 011CU500-F/R (SEQ ID NO: 17/18) and 128CU500-F/R (SEQ ID NO: 21/22), and lower homology arms YCR011C-D, YBR C-D at YCR011C, YBR128C were obtained by PCR amplification using primer pairs 011CD500-F/R (SEQ ID NO: 19/20) and 128CD500-F/R (SEQ ID NO: 23/24). The recombinant plasmid constructed above 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 fragments of TDH3p-CkHbd-TDH1t and CCW12p-CkCrt-SSA1 t. Fusion PCR the upper and lower homology arms of the integration site YCR011C, YBR C are respectively connected with TDH3p-CkHbd-TDH1t and CCW12p-CkCrt-SSA1t fragments, namely 011U-TDH3p-CkHbd-TDH1t-011D and 128U-CCW12p-CkCrt-SSA1t-128D, and agarose gel electrophoresis results are shown in FIG. 4-a.
The gene fragment obtained above was transformed by Saccharomyces cerevisiae lithium acetate transformation: 011U-TDH3p-CkHbd-TDH1t-011D, 128U-CCW12p-CkCrt-SSA1t-128D and Cas9-011C-128C (containing KAN resistance markers) are transformed into Saccharomyces cerevisiae AY14 alpha by texturing. The transformed strain Ck-HC of the overexpression CkHbd and CkCrt is obtained, and the transformation process is shown in FIG. 5.
And then, selecting YHR142W as an integration site by using a Ck-HC starting strain, and constructing an improved strain Ck-HCVL by over-expressing an alcohol acyl transferase gene VLAAT. Using the genomic DNA of AY 14. Alpha. As a template, the upper homology arm 142U at YHR142W was obtained by PCR amplification using the primer pair 142WU500-F/R (SFQ ID NO: 25/26), the lower homology arm 142D at YHR142W was obtained by PCR amplification using the primer pair 142D500-F/R (SEQ ID NO: 27/28), and the agarose gel electrophoresis results are shown in FIG. 4-b. The recombinant plasmid Yep352-PE-VLAAT constructed in the above way 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 homology arms of integration site YHR142W were ligated to the PGK1p-VLAAT-ENO2t fragment, i.e., 142U-PGK1p-VLAAT-ENO2t-142D, and the transformation procedure was as shown in FIG. 6.
The gene fragment obtained above was transformed by Saccharomyces cerevisiae lithium acetate transformation: the 142U-PGK1p-VLAAT-ENO2t-142D and Cas9-142W plasmids were transformed into the starting strain Ck-HC. Obtaining the overexpression CkHbd, ckCrt, VLAAT modified bacterium Ck-HCVL.
And respectively designing verification primers according to the gene sequences at two ends of the Saccharomyces cerevisiae AY14a integration site, and performing PCR amplification by taking the genome of the well-grown haploid transformant as a template to verify the recombinant. The sizes of the obtained bands are verified to be about 2760bp, 2717bp and 3290bp by using primer pairs 011Cyz500-F/R (SEQ ID NO: 29/30), 128Cyz500-F/R (SEQ ID NO: 31/32) and 142Wyz-F/R (SEQ ID NO: 33/34), and the sizes are consistent with expected sizes. The verification results are shown in fig. 9-a and b.
(3) Overexpression of the Erg10 Gene
The genome 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 carrying out PCR amplification to obtain a gene fragment of Erg10, yep352-TEF1p-ADH1T is selected as a gene expression cassette, a plasmid containing Erg10 is constructed, the method is the same as above, and Yep352-TEF1p-Erg10-ADH1T is obtained, and the construction flow is shown in figure 2. The recombinant plasmid constructed as described above was used as a template, the primer set LPP1-TA-Erg10-F/R (SEQ ID NO: 37/38) was used to obtain a TEF1p-Erg10-ADH1t fragment by PCR amplification, the genomic DNA of AY 14. Alpha. Was used as a template, the primer set LPP1500U-F/R (SEQ ID NO: 39/40) and LPP1500D-F/R (SEQ ID NO: 41/42) were used to obtain a fragment of LPP1U, LPP D by PCR amplification, and the fusion PCR was performed to fuse the LPP1 upper and lower homology arms with the TA-Erg10 fragment to obtain LPP1U-TEF1p-Erg10-ADH1t-LPP1D, and the agarose gel electrophoresis results are shown in FIG. 4-c.
The modified strain Ck-HCVL-E over-expressing Erg10 is obtained by integrating the LPP1U-TEF1p-Erg10-ADH1t-LPP1D, cas-LPP 1 plasmid into Ck-HCVL through lithium acetate transformation, and the transformation process is shown in FIG. 7.
And selecting a transformant spot plate with better growth vigor, growing bacterial sludge, and carrying out genome verification. The size of the obtained band was 2858bp by verification with the primer pair LPP1yz500-F/R (SEQ ID NO: 43/44), respectively. The verification result is shown in FIG. 9-c.
(4) Double copy VLAAT gene
The genomic DNA of AY 14. Alpha. Was used as a template, and the primer pairs 416dU500-F/R (SEQ ID NO: 47/48) and 416dD500-F/R (SEQ ID NO: 49/50) were used for PCR amplification to obtain fragments 416U and 416D. The recombinant plasmid Yep352-PE-VLAAT constructed in the above way 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 the 416D upper and lower homology arms and the PE-VLAAT fragment were fused to obtain 416U-PGK1p-VLAAT-ENO2t-416D, and agarose gel electrophoresis results are shown in FIG. 4-D.
The fragment 416U-PGK1p-VLAAT-ENO2t-416D, cas-416 d obtained by the PCR is simultaneously transformed into a recombinant strain Ck-HCVL-E by a lithium acetate transformation method, and the recombinant strain Ck-HC-DVL-E of the saccharomyces cerevisiae is obtained after intracellular integration, and the transformation process is shown in figure 8.
And selecting a transformant spot plate with better growth vigor, growing bacterial sludge, and carrying out genome verification. The sizes of the obtained bands were 3281bp and were consistent with the expected sizes, as confirmed by primer pairs 416dyz-F/R (SEQ ID NO: 51/52), respectively. The results of the verification are shown in FIG. 9-d.
Example 2: corn raw material thick mash fermentation experiment of original strain and modified strain
Carrying out a corn raw material thick mash fermentation experiment on parent strain AY14 alpha and recombinant strains Ck-HCVL, ck-HCVL-E, ck-HC-DVL-E, and carrying out a fermentation process route diagram: corn flour, soaking, liquefying, saccharifying, cooling, inoculating bacteria, fermenting, steaming wine, and measuring indexes;
respectively picking up one ring of yeast cells, respectively inoculating into test tubes filled with 5mL of primary seed culture medium, standing at 30deg.C for 24h, inoculating into 150mL triangular flask filled with 45mL of secondary seed culture medium according to 10% of inoculum size, standing at 30deg.C for 16h to the late stage of logarithmic phase, inoculating into fermentation culture medium according to 10% of inoculum size, and standing at 30deg.C for fermentation. Weighing for 1 time every 12h, and when the weight loss of the two times is less than 0.5g, ending the fermentation, namely, ending the fermentation for 84-96h. After fermentation, 100mL of mash is taken, 100mL of water is added, and 100mL of wine sample is distilled out. Determination of CO 2 The fermentation performance indexes such as the accumulated discharge amount, the alcohol content and the residual reducing sugar are shown in Table 3. Wherein, the first seed culture medium comprises the following components: 82g/L corn flour, and high temperature resistant alpha amylase with addition amount of about 1.0X10 4 U/L, saccharifying enzyme activity is about 32U/L, the balance is water, and the sugar degree is 8 degrees BX. The secondary seed culture medium consists of: corn flour about 125g/L, and high temperature resistant alpha amylase added in an amount of about 1.5X10 4 U/L, saccharifying enzyme activity 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 with 3.5X10 high temperature resistant alpha amylase 4 U/L, saccharifying enzyme 95U/L, acid proteinase 15U/L, nutrient salt solution 5.5-5.6mL/L, and water for the rest; the nutrient salt solution comprises the following components: mgSO (MgSO) 4 150g/L，KH 2 PO 4 75g/L, 81g/L of urea and the balance of water, and preserving at 4 ℃ after filtering.
Treatment process conditions of the fermentation medium:
soaking conditions: soaking corn flour at 60-70 deg.c for 20min; liquefaction conditions: adding high temperature resistant alpha amylase according to the proportion at 85-90 ℃ and liquefying for 90min; saccharification conditions: adding saccharifying enzyme and nutrient salt solution at 55-60 ℃, saccharifying for 20min, adding acid protease, and reacting for 20min at 30 ℃ to obtain a fermentation medium.
TABLE 3 comparison of fermentation Performance of parent and recombinant strains
Note that: the data shown are the average of three parallel test results.
As can be seen from Table 3, the recombinant strains Ck-HCVL, ck-HCVL-E and Ck-HC-DVL-E were not significantly different in alcohol content and residual sugar content from the starting strain AY 14. Alpha. After fermentation, thus indicating that the growth and fermentation properties of the modified strains were not significantly changed.
The ethyl crotonate yield was measured using 100mL of wine obtained from the corn raw material thick mash fermentation experiments of the recombinant strains Ck-HCVL, ck-HCVL-E and Ck-HC-DVL-E and the parent strain (AY 14. Alpha.).
The measuring method comprises the following steps: conditions GC conditions of the gas chromatograph were set: column Agilent CP-WAX (50 m x 250 μm x 0.25 μm), carrier gas is high purity nitrogen (> 99.999%); the column flow rate is 1mL/min; the temperature of the sample inlet is 250 ℃; detector temperature 148.8 ℃; programming temperature: the initial temperature is 35 ℃ and kept for 1min, the temperature is increased to 70 ℃ at 3 ℃/min and kept for 15min, and then the temperature is increased to 190 ℃ at 3.5 ℃/min and kept for 22min; the sample injection volume is 1 mu L; and (3) split sample injection, wherein the split ratio is 30:1. The measured yields of Ck-HCVL, ck-HCVL-E and Ck-HC-DVL-E with ethyl crotonate of the parent strain (AY 14. Alpha.) are shown in Table 4.
Table 4 ester yields (unit mg/L) of the parent strain and recombinant strain
Note that: the data shown are the average of three parallel test results.
In tables 3 and 4, AY 14. Alpha. Was the original strain, ck-HCVL was the strain over-expressing gene CkHbd, ckCrt, VLAAT, ck-HCVL-E was the strain over-expressing gene Erg10 on the basis of Ck-HCVL; ck-HC-DVL-E is a strain that double copies VLAAT on the basis of Ck-HCVL-E.

Claims (5)

1. A saccharomyces cerevisiae genetic engineering strain for high yield of ethyl crotonate is characterized in that: the strain is constructed by taking saccharomyces cerevisiae as an original strain, over-expressing an acetyl coenzyme A acylase gene Erg10, over-expressing a 3-hydroxybutyryl coenzyme A dehydrogenase gene Hbd and a 3-hydroxybutyryl coenzyme A dehydratase gene Crt, and over-expressing an alcohol acylase gene AAT;
wherein, the acetyl coenzyme A acylase gene Erg10 is over-expressed at the saccharomyces cerevisiae LPP1 locus, and simultaneously the 3-hydroxybutyryl coenzyme A dehydrogenase encoding gene Hbd and the 3-hydroxybutyryl coenzyme A dehydratase encoding gene Crt are integrated into saccharomyces cerevisiae YCR011C and YBR128C loci; meanwhile, the alcohol acyl transferase gene AAT is integrated into the Saccharomyces cerevisiae YHR142W locus to realize over-expression;
the nucleotide sequence of the acetyl-CoA acyltransferase gene Erg10 is shown as SEQ ID NO in a nucleotide sequence table: 4 is shown in the figure; the nucleotide sequence of the 3-hydroxybutyryl-CoA dehydrogenase gene Hbd is shown as SEQ ID NO in a nucleotide sequence table: 1 is shown in the specification; the nucleotide sequence of the 3 hydroxybutyryl-coenzyme A dehydratase gene Crt is shown as SEQ ID NO in a nucleotide sequence table: 2 is shown in the figure; the nucleotide sequence of the alcohol acyl transferase gene AAT is as shown in a nucleotide sequence table SEQ ID NO: 3.
2. The saccharomyces cerevisiae genetically engineered strain for high yield of ethyl crotonate according to claim 1, wherein the saccharomyces cerevisiae genetically engineered strain is characterized by: the gene AAT is expressed in double copies;
wherein, the double copy of AAT is by integration into the 416d site of Saccharomyces cerevisiae.
3. The saccharomyces cerevisiae genetically engineered strain for high yield of ethyl crotonate according to claim 1, wherein the saccharomyces cerevisiae genetically engineered strain is characterized by: the starting strain is Saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC32315.
4. Use of the saccharomyces cerevisiae genetic engineering strain for high yield of ethyl crotonate according to any one of claims 1-3 in the fields of fermentation, essence and spice and drug synthesis.
5. A method for fermenting a genetically engineered strain of saccharomyces cerevisiae for high production of ethyl crotonate according to any one of claims 1-3, wherein: two-stage activation of Saccharomyces cerevisiae genetically engineered bacteria, inoculating 8-12% seed solution to fermentation medium, standing at 28-30deg.C, and fermenting for 80-86 hr;
the fermentation medium comprises the following components: corn flour 300-320g/L, high temperature resistant alpha amylase (2-5) x 10 4 U/L, saccharifying enzyme 90-100U/L, acid proteinase 10-20U/L, nutrient salt solution 5.5-5.6mL/L, and water in balance; the nutrient salt solution comprises the following components: mgSO (MgSO) 4 140-160g/L，KH 2 PO 4 70-80g/L, 80-85g/L urea and the balance of water.
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