Brewer's yeast having low ethanol synthesis and high acetyl-coa synthesis, and use thereof

Abstract

Provided are a brewer's yeast having low ethanol synthesis and high acetyl-CoA synthesis, and a use thereof, belonging to the technical field of biology. In order to reduce the accumulation of ethanol, the main byproduct of brewer's yeast, and increase synthesis of acetyl-CoA, MTH1, MED2 and HXT2 transcription factors are modified, an optimal transcription factor combination for reducing ethanol synthesis is obtained by screening, and then heterologous acetyl-CoA synthesis pathways of different synthesis pathways integrated in the brewer's yeast genome are compared, and a recombinant brewer's yeast strain is provided. The strain can grow in a sterile culture medium using glucose as a carbon source, creating a platform for modifying brewer's yeast by metabolic engineering, to synthesize high-value compounds; in addition, the construction method is simple and easy to use, and has good application prospects.

Description

A brewer's yeast with low ethanol synthesis and high acetyl-CoA synthesis and its application Technical Field

[0001] The invention relates to brewer's yeast with low ethanol synthesis amount and high acetyl-CoA synthesis amount and application thereof, belonging to the field of biotechnology. Background Art

[0002] Saccharomyces cerevisiae, also known as baker's yeast or budding yeast, is the yeast most widely associated with humans. As a food-safe strain, it is used in foods such as bread and steamed buns, as well as in the brewing industry. In recent years, researchers have begun exploring the use of Saccharomyces cerevisiae to produce natural products, such as artemisinic acid and notoginsenosides. Saccharomyces cerevisiae has advantages such as high safety, low pathogenicity, high stress resistance, and low risk of phage contamination, making it an important candidate for genetic engineering. However, its robust ethanol biosynthesis capacity can reduce yields and increase production costs when synthesizing these products. Furthermore, excessive ethanol accumulation can inhibit strain growth, hindering product synthesis. Acetyl-CoA is a key intermediate metabolite in Saccharomyces cerevisiae, used in the synthesis of terpenoids, lipids, and amino acids. Cytosolic acetyl-CoA in Saccharomyces cerevisiae is primarily produced from acetic acid. Inhibiting ethanol synthesis results in a decrease in acetyl-CoA synthesis. Therefore, genetically engineering industrial production strains to reduce ethanol biosynthesis and enhance acetyl-CoA accumulation holds great potential for producing specific products, providing an excellent production platform.

[0003] Currently, the main approaches to reducing ethanol production in Saccharomyces cerevisiae are: knocking out pyruvate decarboxylase, thereby blocking the conversion of pyruvate to acetaldehyde and reducing ethanol production; and altering global transcription factors to mitigate the Crabtree effect, thereby reducing ethanol production. This is achieved by knocking out genes involved in ethanol synthesis and increasing the enzymes involved in converting acetaldehyde to acetate and acetate to acetyl-CoA. However, these approaches to reducing ethanol production in Saccharomyces cerevisiae strains inhibit their growth and result in insufficient acetyl-CoA production, limiting the application of these recombinant strains.

[0004] Summary of the Invention

[0005] To address the above problems, the present invention provides a Saccharomyces cerevisiae strain that reduces ethanol synthesis while increasing acetyl-CoA synthesis. Cre / loxp technology is used to modify transcription factors and perform global metabolic pathway optimization, thereby reducing ethanol synthesis in the Saccharomyces cerevisiae strain and simultaneously expressing a heterologous acetyl-CoA synthesis pathway, thereby achieving low-ethanol and high-acetyl-CoA production. The construction method is simple, easy to use, and has good application prospects.

[0006] The first object of the present invention is to provide a low ethanol synthesis amount of cerevisiae yeast, the low ethanol synthesis amount of cerevisiae yeast expressing

[0007] Transcription factor mutant MTH1 A81D&I85S ;or

[0008] Transcription factor mutant MTH1 A81D 、MED2 *432Y and HXT2 W466* ;or

[0009] Transcription factor mutant MTH1 I85S 、MED2 *432Y and HXT2 W466* ;or

[0010] Transcription factor mutant MTH1 A81D&I85S 、MED2 *432Y and HXT2 W466* ;

[0011] Among them, MTH1 A81D MTH1 was obtained by mutating the 81st alanine of the amino acid sequence shown in SEQ ID NO.18 to aspartic acid. I85S MTH1 was obtained by mutating the 85th isoleucine of the amino acid sequence shown in SEQ ID NO.18 to serine. A81D&I85S MED2 was obtained by mutating the 81st alanine of the amino acid sequence shown in SEQ ID NO. 18 to aspartic acid and the 85th isoleucine to serine. *432Y HXT2 was obtained by replacing the 432nd stop codon of the amino acid sequence shown in SEQ ID NO.19 with tyrosine. W466* The protein is obtained by replacing the tryptophan at position 466 of the amino acid sequence shown in SEQ ID NO. 20 with a stop codon.

[0012] Furthermore, the transcription factor mutant MTH1 A81D 、MTH1 I85S 、MTH1 A81D&I85S 、MED2 *432Y 、HXT2 W466* The nucleotide sequences are shown in SEQ ID NO.1-5 respectively.

[0013] Furthermore, Saccharomyces cerevisiae CEN PK2-1C MATa; ura3-52; trp1-289; leu2-3,112; his3-Δ1; MAL2-8C; SUC2 was used as the host strain.

[0014] Furthermore, the transcription factor mutant MTH1 A81D 、MTH1 I85S or MTH1 A81D&I85S Integration into the MTH1 locus, transforming the transcription factor mutant MED2 *432Y Integration into the MED2 locus, transforming the transcription factor mutant HXT2 W466* Integrated into the HXT2 locus.

[0015] Furthermore, the transcription factor mutant was integrated into the Saccharomyces cerevisiae genome using the Cre / loxp method. The specific construction method includes the following steps:

[0016] 1) Select the defective amino acid tag and the constructed transcription factor mutant plasmid, design PCR primers so that the overlapping region of adjacent fragments of the gene expression cassette reaches 40 to 100 bp, and construct MTH1 A81D ,MTH1 I85S ,MTH1 A81D&I85S ,MED2 *432Y ,HXT2 W466* Transcription factor mutants express integration cassettes;

[0017] 2) The transcription factor mutant expression integration frames were integrated into the MTH1, MED2 and HXT2 sites of Saccharomyces cerevisiae using the Cre / loxp method.

[0018] The second object of the present invention is to provide a recombinant Saccharomyces cerevisiae with low ethanol synthesis and high acetyl-CoA synthesis, wherein the recombinant Saccharomyces cerevisiae with low ethanol synthesis is used as the starting strain, and the recombinant Saccharomyces cerevisiae with low ethanol synthesis is heterologously expressed.

[0019] The gene encoding pyruvate oxidase, po, and the gene encoding phosphotransacetylase, pta; or

[0020] The genes encoding phosphoenolpyruvate carboxylase (ppc), malate thiokinase large subunit (mtkA), malate thiokinase small subunit (mtkB), malyl-CoA lyase (mcl), and hydroxypyruvate reductase (hprA) were also included.

[0021] Furthermore, the nucleotide sequence of the po gene is shown in SEQ ID NO.10, the nucleotide sequence of the pta gene is shown in SEQ ID NO.12, the nucleotide sequence of the ppc gene is shown in SEQ ID NO.13, the nucleotide sequence of the mtkA gene is shown in SEQ ID NO.14, the nucleotide sequence of the mtkB gene is shown in SEQ ID NO.15, the nucleotide sequence of the mcl gene is shown in SEQ ID NO.16, and the nucleotide sequence of the hprA gene is shown in SEQ ID NO.17.

[0022] Furthermore, the po gene and the pta gene were integrated into the 1021 site; the ppc gene, the mtkA gene, the mtkB gene, the mcl gene and the hprA gene were integrated into the PCK1 site.

[0023] Furthermore, the gene po is driven by the promoter P TDH3 Regulates expression, gene pta is driven by promoter P GPD Regulates expression, gene ppc is driven by promoter P TEF1 Regulates expression of gene mtkA by promoter P GPD Regulates expression of gene mtkB by promoter P TDH3 Regulates expression, gene mcl is driven by promoter P ADH1 Regulates expression of gene hprA by promoter P SED1 Regulate expression.

[0024] Furthermore, the gene po is terminated by the terminator T ATP5 Regulates expression, gene pta is controlled by terminator T CYC1 Regulates expression, gene ppc is controlled by terminator T ADH1 Regulates expression, gene mtkA is controlled by terminator T CYC1 Regulates expression of the gene mtkB by the terminator T ATP5 Regulates expression, gene mcl is controlled by terminator T TDH3 Regulates expression, gene hprA is controlled by terminator T PGK1 Regulate expression.

[0025] Furthermore, the heterologous acetyl-CoA replenishment pathway was integrated into the Saccharomyces cerevisiae genome using Cre / loxp technology. The specific construction method includes the following steps:

[0026] 1) Select defective amino acid tag, heterologous acetyl-CoA complement pathway gene, constitutive promoter P TEF1 , P GPD , P TDH3 , P ADH1 or P SED1 , terminator T ADH1 , T CYC1 , T ATP5 , T TDH3 or T PGK1 , design PCR primers so that the overlapping regions of adjacent fragments of the gene expression cassette reach 40 to 100 bp, and construct the gene expression integration cassette of PO-PTA or PPC-MtkA-MtkB-McL-HPRA.

[0027] 2) The acetyl-CoA expression integration frame was integrated into the Saccharomyces cerevisiae 1021 or PCK1 gene locus by the Cre / loxp method.

[0028] The third object of the present invention is to provide the use of the above-mentioned low ethanol synthesis yeast or the recombinant ethanol synthesis yeast with low ethanol synthesis and high acetyl-CoA synthesis in biosynthesis.

[0029] Furthermore, the amount of ethanol synthesis can be reduced in biosynthesis, the growth inhibition of the strain caused by ethanol accumulation can be alleviated, and the synthesis of related products can be promoted. At the same time, the carbon conversion rate and biomass yield of brewer's yeast can be improved, and the ability of brewer's yeast to synthesize high-value compounds can be enhanced.

[0030] The fourth object of the present invention is to provide a method for producing acetyl-CoA or its metabolites, comprising the steps of fermentation production using the above-mentioned low ethanol synthesis yeast or recombinant yeast.

[0031] Furthermore, acetyl-CoA metabolites include but are not limited to fatty acids, ketone bodies, cholesterol, 3-hydroxypropionic acid, squalene, and the like.

[0032] Furthermore, fermentation production is carried out using glucose as a substrate.

[0033] Furthermore, the recombinant strain was cultured at 28-32°C, 180-260rpm for 16h-24h to obtain seed liquid, which was transferred into fermentation medium at an inoculation rate of 2-5%, and fermented with aeration at pH = 6.0-8.0, 180-260rpm, 28-32°C.

[0034] The present invention provides a transcription factor mutant MTH1 A81D&I85S , obtained by mutating the 81st alanine of the amino acid sequence shown in SEQ ID NO.18 to aspartic acid and the 85th isoleucine to serine.

[0035] The present invention also provides a method for encoding the above-mentioned transcription factor mutant MTH1 A81D&I85S Genes containing the above-mentioned transcription factor mutant MTH1 A81D&I85S The recombinant plasmid or host cell can be selected from Saccharomyces cerevisiae. The recombinant Saccharomyces cerevisiae constructed can not only significantly reduce the amount of ethanol synthesis, but also promote the growth of the host bacteria, and has great potential in biosynthesis.

[0036] The present invention also claims to protect the above-mentioned transcription factor mutant MTH1 A81D&I85S Application in reducing ethanol synthesis of strains.

[0037] Beneficial effects of the present invention:

[0038] The present invention uses the Cre / loxp method to modify the MTH1, MED2, and HXT2 transcription factors. By mutating or combining different transcription factors, the optimal transcription factor combination for reducing ethanol synthesis is screened. The resulting recombinant strain significantly reduces ethanol accumulation during fermentation production, facilitating the elimination of ethanol growth inhibition. Furthermore, the present invention compares heterologous acetyl-CoA synthesis pathways from different synthetic pathways integrated into the Saccharomyces cerevisiae genome, ultimately integrating and expressing the optimal combination of transcription factors and acetyl-CoA synthesis pathways to obtain a Saccharomyces cerevisiae strain with low ethanol synthesis and high acetyl-CoA accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows the ethanol production of recombinant strains containing transcription factor mutants.

[0040] Figure 2 shows the growth of recombinant strains of transcription factor mutants.

[0041] Figure 3 shows the acetyl-CoA production of recombinant strains with different heterologous acetyl-CoA synthesis pathways.

[0042] FIG4 shows the ethanol production in the recombinant strain.

[0043] FIG5 shows the production of fatty acids in the recombinant strains. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0045] The materials and methods involved in the following examples are as follows:

[0046] (1) Sequence information:

[0047] Transcription factor mutant MTH1 A81D ,MTH1 I85S ,MTH1 A81D&I85S ,MED2 *432Y ,HXT2 W466* The nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

[0048] The transcription factor mutants were expressed by gene integration using the Cre / loxp system as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. The transcription factor mutant expression cassettes were integrated into the MTH1, MED2 and HXT2 sites of Saccharomyces cerevisiae.

[0049] The gene sequences related to the acetyl-CoA replenishment pathway are: XPK shown in SEQ ID NO.9, PO shown in SEQ ID NO.10, XSFPK shown in SEQ ID NO.11, PTA shown in SEQ ID NO.12, PPC shown in SEQ ID NO.13, MtkA shown in SEQ ID NO.14, MtkB shown in SEQ ID NO.15, McL shown in SEQ ID NO.16, and HPRA shown in SEQ ID NO.17.

[0050] The amino acid sequence of the wild type of MTH1 is shown in SEQ ID NO. 18. A81D Refers to the mutation of alanine at position 81 of the sequence shown in SEQ ID NO.18 to aspartic acid, MTH1 I85S Refers to the mutation of isoleucine at position 85 to serine, MTH1 A81D&I85S Refers to the mutation of alanine at position 81 to aspartic acid and isoleucine at position 85 to serine.

[0051] The amino acid sequence of the wild type of MED2 is shown in SEQ ID NO. 19. MED2 *432Y It refers to replacing the 432nd stop codon in the sequence shown in SEQ ID NO. 19 with tyrosine.

[0052] The amino acid sequence of the wild type HXT2 is shown in SEQ ID NO. 20. W466* It refers to replacing the tryptophan at position 466 of the sequence shown in SEQ ID NO. 20 with a stop codon.

[0053] (2) Method

[0054] All recombinant Saccharomyces cerevisiae strains in the present invention were fermented in the following manner: the recombinant Saccharomyces cerevisiae strains were streaked onto amino nitrogen source-free plates (lacking amino acids corresponding to the defective type) and cultured at 30° C. until a large number of colonies grew.

[0055] Fatty acid gas chromatography-mass spectrometry (GC-MS) detection method: Shimadzu (GCMS-QP2010SE), SH-Rtx-Wax column, He mobile phase, column temperature 100°C, injection volume 1 μL, split ratio 20:1, flow rate 1.0 ml / min.

[0056] Ethanol HPLC detection method: Agilent (1260 Infinity II), organic acid column, mobile phase: 5 mM H2SO4, column temperature: 55°C, differential detector temperature: 40°C, injection volume: 10 μL, flow rate: 0.6 ml / min.

[0057] Acetyl-CoA high-performance liquid chromatography detection method: ZORBAX Eclipse Plus C18 column (250 m × 4.6 mm × 5 μm); mobile phase: 0.2 mol / L sodium phosphate buffer (A) 90% and acetonitrile (B) 10%; flow rate 1 mL / min; column temperature 25°C; UV detection wavelength 254 nm; injection volume 10 μL.

[0058] Example 1 Construction of a recombinant Saccharomyces cerevisiae strain with low ethanol production

[0059] This example constructs MTH1 A81D ,MTH1 I85S ,MTH1 A81D&I85S ,MED2 *432Y ,HXT2 W466* Transcription factor mutant plasmid. According to the overlapping derivative PCR primer design method, primers were designed to make the overlap region of adjacent fragments of the gene expression cassette reach 40 to 100 bp. S. cerevisiae CEN PK2-1C MATa; ura3-52; trp1-289; leu2-3,112; his3-Δ1; MAL2-8C; SUC2 was used as strain 1. Primers were designed to amplify the upstream and downstream homology arms of the gene integration cassette on both sides of the MTH1 site on the chromosome of Saccharomyces cerevisiae CEN PK2-1C. Using the transcription factor mutant MTH1 A81D Using the plasmid as a template, primers were designed to amplify the mutant transcription factor. PCR was performed using the plasmid pMHyLp-LEU as a template (shown in SEQ ID NO.6) to amplify the defective expression cassette fragment. The gene integration cassette was then obtained by overlap extension PCR. The obtained gene expression cassette was transformed into Saccharomyces cerevisiae to obtain strain 2. The same steps were used to transform MTH1 I85S and MTH1 A81D&I85S Strain 3 and strain 4 were obtained. Primers were designed to amplify the upstream and downstream homology arms of the gene integration frame on both sides of the MED2 site on the chromosome of Saccharomyces cerevisiae CEN PK2-1C. *432Y Plasmid was used as template and primers were designed to amplify mutant transcription factors. PCR was performed using plasmid pMHyLp-HIS as template (as shown in SEQ ID NO.7) to amplify the defective expression cassette fragment and obtain the gene integration cassette by overlap extension PCR. The obtained gene expression cassette was transformed into Saccharomyces cerevisiae to obtain strain 5. Primers were designed to amplify the upstream and downstream homology arms of the gene integration cassette on both sides of the HXT2 site on the chromosome of Saccharomyces cerevisiae CEN PK2-1C. W466*Plasmid was used as template to design primers to amplify the mutant transcription factor. PCR was performed using plasmid pMHyLp-TRP as template (as shown in SEQ ID NO.8) to amplify the defective expression cassette fragment, and the gene integration cassette was obtained by overlap extension PCR. The obtained gene expression cassette was transformed into Saccharomyces cerevisiae to obtain strain 6. By transforming the transcription factor mutant MTH1 A81D and HXT2 W466* Strain 7 was obtained by combining the transcription factor mutant MTH1 I85S and HXT2 W466* Strain 8 was obtained by combining the transcription factor mutant MTH1 A81D&I85S and HXT2 W466* Strain 9 was obtained by combining the transcription factor mutant MED2 *432Y and HXT2 W466* Strain 10 was obtained by combining the transcription factor mutant MTH1 A81D , MED2 *432Y and HXT2 W466* Strain 11 was obtained by combining the transcription factor mutant MTH1 I85S , MED2 *432Y and HXT2 W466* Strain 12 was obtained by combining the transcription factor mutant MTH1 A81D&I85S , MED2 *432Y and HXT2 W466* The strain 13 was obtained by combination.

[0060] Ethanol production was measured in strains 1-13 over 96 hours of fermentation (see Figure 1). Results showed that the ethanol production trends varied among the 13 constructed transcription factor mutants, but that ethanol production decreased somewhat in the double and triple mutation combinations, particularly in the triple mutation combination, where ethanol production decreased by approximately 50%. In all of the above processes, the constructed gene integration cassettes were transformed into competent Saccharomyces cerevisiae cells using the lithium acetate method. Colonies were selected for PCR verification, and a subset of PCR-positive transformants were selected for sequencing verification.

[0061] The growth of recombinant strains introduced with different transcription factor mutants was also measured, and the results are shown in Figure 2. It can be seen that the MTH1 mutant significantly promoted the growth of the strain, but the growth of the strain under MED2 and HXT2 conditions and the combination conditions did not change significantly compared with the wild-type strain.

[0062] Example 2 Construction of a high-yield acetyl-CoA recombinant yeast strain and detection of acetyl-CoA synthesis

[0063] The XPK and PTA genes were codon-optimized according to the codon preference of Saccharomyces cerevisiae and fully synthesized (the gene sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 12, respectively). Using the overlapping derivatization PCR primer design method, primers were designed to achieve an overlap of 40 to 100 bp between adjacent fragments of the gene expression cassette. Primers were designed to amplify the upstream and downstream homology arms of the gene integration cassette on both sides of the Saccharomyces cerevisiae CEN PK2-1C chromosome 1021 site, and the promoter P TEF1 , P GPD , P TDH3 , P ADH1 and P SED1 , terminator T ADH1 , T CYC1 , T ATP5 , T TDH3 and T PGK1 . Using the fully synthesized XPK and PTA plasmids as templates, primers were designed to amplify the XPK and PTA genes. PCR was performed using the plasmid pMHyLp-LEU as a template (as shown in SEQ ID NO.6) to amplify the defective expression cassette fragment, and the gene integration cassette was obtained by overlap extension PCR. The obtained gene expression cassette was transformed into Saccharomyces cerevisiae to obtain strain A-1. Similarly, using the fully synthesized PO and PTA plasmids (the gene sequences are shown in SEQ ID NO.10 and SEQ ID NO.12, respectively) as templates, and using the XFSPK and PTA plasmids (the gene sequences are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively) as templates, strains A-2 to A-3 were constructed respectively according to the above method. According to the codon preference of Saccharomyces cerevisiae, the PPC, MtkA, MtkB, McL and HPRA genes were codon optimized and fully synthesized (the gene sequences are shown in SEQ ID NO.13-17, respectively). According to the overlapping derivative PCR primer design method, primers were designed to make the overlapping region of adjacent fragments of gene expression cassette reach 40-100bp. Primers were designed to amplify the upstream and downstream homology arms of the gene integration cassette on both sides of the PCK1 site of Saccharomyces cerevisiae CEN PK2-1C chromosome, promoter P TEF1 , P GPD , P TDH3 , P ADH1 and P SED1 , terminator T ADH1 , T CYC1 , T ATP5 , T TDH3 and T PGK1. Using the fully synthesized PPC, MtkA, MtkB, McL and HPRA plasmids as templates, primers were designed to amplify the PPC, MtkA, MtkB, McL and HPRA genes. PCR was performed using the plasmid pMHyLp-LEU as a template (as shown in SEQ ID NO.6), and a defective expression cassette fragment was amplified, and the gene integration cassette was obtained by overlap extension PCR. The obtained gene expression cassette was transferred into Saccharomyces cerevisiae to obtain strain A-4. In the above process, the constructed gene integration cassette was transformed into Saccharomyces cerevisiae competent cells by the lithium acetate transformation method, colonies were picked for PCR verification, and some PCR-correct transformants were selected for sequencing verification.

[0064] The acetyl-CoA content in different recombinant strains was detected according to the following method: 10 mL of fermentation broth was centrifuged at 8,000 r / min and 4°C for 10 min, washed twice with 1 mL of 0.25 mol / L, pH 5, phosphate buffer saline (PBS), centrifuged at 8,000 r / min and 4°C for 10 min, the supernatant was removed, and the solution was resuspended with 1 mL of 6% perchloric acid. 0.3 mol / L potassium carbonate solution was added dropwise for salt precipitation (vortexing was performed during the addition process to mix it thoroughly), the pH was adjusted to 3.0, and potassium perchlorate (KClO4) crystals were removed by centrifugation at 12,000 r / min and 4°C for 10 min.

[0065] The results are shown in Figure 3. Compared with the wild-type strain, the acetyl-CoA content in the recombinant strains A-2 and A-4 increased significantly during the logarithmic growth phase.

[0066] Example 3 Construction of a strain synthesizing high acetyl-CoA from low ethanol

[0067] Based on the recombinant strains (11, 12, and 13) with the lowest ethanol production in Example 1, the optimal acetyl-CoA synthesis pathway was expressed. Using the overlapping derivatization PCR primer design method, primers were designed to achieve a 40-100 bp overlap between adjacent fragments of the gene expression cassette. Primers were designed to amplify the upstream and downstream homology arms of the gene integration cassette on both sides of the PCK1 locus on the Saccharomyces cerevisiae CEN PK2-1C chromosome. The promoter P TEF1 , P GPD , P TDH3 , P ADH1 and P SED1 , terminator T ADH1 , T CYC1 , T ATP5 , T TDH3 and T PGK1. Using the fully synthesized PPC, MtkA, MtkB, McL and HPRA plasmids as templates, primers were designed to amplify the PPC, MtkA, MtkB, McL and HPRA genes. PCR was performed using the plasmid pMHyLp-LEU as a template (as shown in SEQ ID NO.6), and a defective expression cassette fragment was amplified, and the gene integration cassette was obtained by overlapping extension PCR. The obtained gene expression cassette was transferred into bacteria 11, 12 and 13 to obtain strains 14-16. In the above process, the constructed gene integration cassette was transformed into Saccharomyces cerevisiae competent cells by the lithium acetate transformation method, colonies were picked for PCR verification, and some PCR-correct transformants were selected for sequencing verification.

[0068] Example 4 Recombinant Saccharomyces cerevisiae fermentation

[0069] Pick a single colony to the seed culture medium (select YPD sterile medium with glucose as the carbon source as the seed culture medium), and culture it at 30°C and 220 rpm for 16 to 24 hours until the cells reach the logarithmic growth phase.

[0070] Inoculate the seed culture into a fermentation medium (sterile YPD medium with glucose as the carbon source) at an initial inoculum concentration of 1-3% and incubate at 30°C and 220 rpm for 72 hours. After 72 hours, remove the supernatant from the fermentation broth and centrifuge the yeast cells, then lyophilize them for subsequent analysis.

[0071] Example 5 Detection of ethanol synthesis and fatty acid synthesis by recombinant Saccharomyces cerevisiae strains

[0072] Ethanol and fatty acids from all recombinant Saccharomyces cerevisiae strains of the present invention were extracted using the following method: After fermentation, the fermentation broth was centrifuged at 12,000 rpm for 5 minutes. The supernatant was aspirated using a 1 ml syringe and filtered through a 0.22 μm aqueous filter to remove impurities. Liquid chromatography was then used to detect the supernatant.

[0073] Pentadecanoic acid was added to the freeze-dried cells as an internal standard. Methanol:chloroform and glass beads were added for oscillation. The oscillation was then performed and the resulting solution was transferred to a new volumetric flask. 1.5 ml of methanol / chloroform solvent was added for extraction. This step was repeated twice. The resulting lipid extracts were combined and 1 ml of chloroform and 3 ml of aqueous solution were added to the combined solution. The sample was vigorously shaken and centrifuged at 10,000 rpm for 5 minutes. The upper layer was discarded and the lower organic phase was transferred to a new glass tube. 1 ml of sulfuric acid:methanol was added and the mixture was heated in a 95°C water bath for 90 minutes. After removal and cooling, n-hexane and NaCl aqueous solution were added and the supernatant was removed by centrifugation. GC-MS analysis was then performed (Note: The total content of C16:0, C16:1, C18:0, and C18:1 was used for the fatty acid content).

[0074] The ethanol and fatty acid yields of the recombinant strains after fermentation are shown in Figures 4-5. Compared to the starting strains, the ethanol yield of strains 14-16 decreased by over 66%, while the fatty acid yield increased by approximately 75%.

[0075] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A low ethanol synthesis yeast, characterized in that: The low ethanol synthesis yeast expressed Transcription factor mutant MTH1 A81D&I85S ;or Transcription factor mutant MTH1 A81D 、MED2 *432Y and HXT2 W466* ;or Transcription factor mutant MTH1 I85S 、MED2 *432Y and HXT2 W466* ;or Transcription factor mutant MTH1 A81D&I85S 、MED2 *432Y and HXT2 W466* ; Among them, MTH1 A81D MTH1 was obtained by mutating the 81st alanine of the amino acid sequence shown in SEQ ID NO.18 to aspartic acid. I85S MTH1 was obtained by mutating the 85th isoleucine in the amino acid sequence shown in SEQ ID NO.18 to serine. A81D&I85S MED2 was obtained by mutating the 81st alanine of the amino acid sequence shown in SEQ ID NO.18 to aspartic acid and the 85th isoleucine to serine. *432Y HXT2 was obtained by replacing the 432nd stop codon of the amino acid sequence shown in SEQ ID NO.19 with tyrosine. W466* The protein is obtained by replacing the tryptophan at position 466 of the amino acid sequence shown in SEQ ID NO. 20 with a stop codon.

2. The low ethanol synthesis yeast according to claim 1, characterized in that: Transcription factor mutant MTH1 A81D 、MTH1 I85S 、MTH1 A81D&I85S 、MED2 *432Y 、HXT2 W466* The nucleotide sequences are shown in SEQ ID NO.1-5 respectively.

3. The low ethanol synthesis yeast according to claim 1, characterized in that: Saccharomyces cerevisiae CEN PK2-1C MATa; ura3-52; trp1-289; leu2-3,112; his3-Δ1; MAL2-8C; SUC2 was used as the host strain.

4. The low ethanol synthesis yeast according to claim 1, characterized in that: MTH1 transcription factor mutant A81D 、MTH1 I85S or MTH1 A81D&I85S Integration into the MTH1 locus, transforming the transcription factor mutant MED2 *432Y Integration into the MED2 locus transforms the transcription factor mutant HXT2 W466* Integrated into the HXT2 locus.

5. A recombinant Saccharomyces cerevisiae with low ethanol synthesis and high acetyl-CoA synthesis, characterized in that: Using the low ethanol synthesis amount brewer's yeast according to any one of claims 1 to 4 as the starting bacteria, heterologous expression The gene encoding pyruvate oxidase, po, and the gene encoding phosphotransacetylase, pta; or The genes encoding phosphoenolpyruvate carboxylase (ppc), malate thiokinase large subunit (mtkA), malate thiokinase small subunit (mtkB), malyl-CoA lyase (mcl), and hydroxypyruvate reductase (hprA) were also included.

6. The recombinant Saccharomyces cerevisiae according to claim 5, characterized in that: Gene po and gene pta were integrated into the 1021 site; gene ppc, gene mtkA, gene mtkB, gene mcl and gene hprA were integrated into the PCK1 site.

7. The recombinant Saccharomyces cerevisiae according to claim 5, characterized in that: Gene po is mediated by promoter P TDH3 Regulates expression, gene pta is driven by promoter P GPD Regulates expression, gene ppc is regulated by promoter P TEF1 Regulates expression, gene mtkA is driven by promoter P GPD Regulates expression, gene mtkB is driven by promoter P TDH3 Regulates expression, gene mcl is driven by promoter P ADH1 Regulates expression, gene hprA is driven by promoter P SED1 Regulate expression.

8. The recombinant Saccharomyces cerevisiae according to claim 5, characterized in that: The gene po is terminated by the terminator T ATP5 Regulates expression, gene pta is controlled by terminator T CYC1 Regulates expression, gene ppc is controlled by terminator T ADH1 Regulates expression, gene mtkA is controlled by terminator T CYC1 Regulates expression, gene mtkB is controlled by terminator T ATP5 Regulates expression, gene mcl is controlled by terminator T TDH3 Regulates expression, gene hprA is controlled by terminator T PGK1 Regulate expression.

9. Use of the low ethanol synthesis Saccharomyces cerevisiae according to any one of claims 1 to 4 or the recombinant Saccharomyces cerevisiae according to any one of claims 5 to 8 in biosynthesis.

10. A method for producing acetyl-CoA or its metabolites, comprising the step of fermentation production using the low ethanol synthesis Saccharomyces cerevisiae described in any one of claims 1 to 4 or the recombinant Saccharomyces cerevisiae described in any one of claims 5 to 8.

11. The method according to claim 10, characterized in that: The metabolites of acetyl-CoA include one or more of fatty acids, ketone bodies, cholesterol, 3-hydroxypropionic acid, and squalene.

12. The method according to claim 10, characterized in that: Fermentation production is carried out using glucose as substrate.

13. The method according to claim 10, characterized in that: The strain was cultured at 28-32°C and 180-260rpm to obtain seed liquid, which was transferred into fermentation medium and fermented with aeration at pH=6.0-8.0, 180-260rpm and 28-32°C.

14. A transcription factor mutant MTH1 A81D&I85S , characterized in that: The amino acid sequence is obtained by mutating the 81st alanine to aspartic acid and the 85th isoleucine to serine in the amino acid sequence shown in SEQ ID NO.

18.

15. Encoding the transcription factor mutant MTH1 according to claim 14 A81D&I85S or a mutant of the transcription factor MTH1 A81D&I85S recombinant plasmid or host cell.

16. The transcription factor mutant MTH1 according to claim 14 A81D&I85S Or the use of the gene, recombinant plasmid or host cell according to claim 15 in reducing the amount of ethanol synthesis.

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