Construction method of saccharomyces cerevisiae mutant with high yield of 3-hydracrylic acid

Abstract

The invention discloses a construction method of a saccharomyces cerevisiae mutant with high yield of 3-hydracrylic acid, and belongs to the technical field of biology. SKN7 gene, PGD1 gene, STB3 gene, YAP1 gene, RIM101 gene, CIN5 gene, FHL1 gene, HAA1 gene, CRZ1 gene, NRG1 gene or PDR1 gene of saccharomyces cerevisiae are mutated and screened to obtain a saccharomyces cerevisiae mutant strain with high yield of malonyl-coenzyme A, and then 3-hydracrylic acid production plasmid is introduced into the obtained saccharomyces cerevisiae mutant strain to obtain the saccharomyces cerevisiae mutant with high yield of 3-hydracrylic acid. Compared with control bacteria, the yield of 3-hydracrylic acid of the saccharomyces cerevisiae mutant is remarkably increased, a technical basis is provided for directional transformation of saccharomyces cerevisiae engineering strains, applicable strains can be provided for carbon fiber production, and industrial production of 3-hydracrylic acid is promoted.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for constructing a Saccharomyces cerevisiae mutant that produces high levels of 3-hydroxypropionic acid. Background Technology

[0002] 3-Hydroxypropionic acid (3-HP) is an isomer of lactic acid, containing a hydroxyl group at one end and a carboxyl group at the other. It ranked third among the 12 most important platform chemicals updated in 2004, and the U.S. Department of Energy listed 3-HP as one of the most promising chemical products of the 21st century. As a hydroxyl-functionalized carboxylic acid, 3-HP is a promising platform chemical with diverse applications, including the production of adhesives, plastic packaging, fibers, detergents, and resins. It can also be converted into various valuable products such as acrylic acid, acrylates, and 1,3-propanediol. The production of 3-HP mainly consists of two methods: chemical and biological. However, chemical methods are not eco-friendly due to the high cost of initial materials and the generation of toxic substances during the production process. Therefore, eco-friendly biological methods have attracted the attention of researchers. Bacteria can be used to ferment sugars into organic acids, but under large-scale organic acid production conditions, bacteria exhibit certain drawbacks: as organic acids are produced, the acidity of the fermentation medium becomes increasingly stronger. However, most bacteria capable of producing organic acids perform poorly in strongly acidic environments, resulting in slow acid production or even bacterial death, making large-scale biological production of 3-HP economically unfeasible.

[0003] In recent years, yeast has been widely used in industrial production to produce organic acids from glucose. Compared with bacteria, yeast has a high tolerance to organic acids; its activity is not significantly inhibited even under highly acidic conditions, making it a more suitable host for organic acid production. In particular, yeast is routinely and widely used in the production of spirits and industrial ethanol. Yeast can be cultured on a large scale and is not contaminated by bacteriophages, making it more suitable for use in the chemical industry than bacteria. Therefore, the development of yeast as a gene-editing vector has become a popular research direction.

[0004] Global transcription factors can specifically regulate the transcriptional level of thousands of genes in the cellular genome. By mutating global transcription factors, the transcription of some genes can be turned off or enhanced, thereby obtaining strains with specific target phenotypes. In 2012, Zhang et al. (Chong H, Geng H, Zhang H, et al. Enhancing…) E. Escherichia coliIsobutanol tolerance through engineering its global transcription factor cAMP receptor protein (CRP) [J]. Biotechnology and bioengineering, 2014, 111(4): 700-708.) Random mutations of the global transcription factor cAMP receptor protein (CRP) yielded *E. coli* strains capable of tolerating 1.2% (v / v) butanol. Therefore, by mutating global transcription factors, the metabolism of microorganisms can be optimized, thereby obtaining an ideal cell phenotype. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for constructing a high-yield 3-hydroxypropionic acid (3-hydroxypropionic acid) mutant of Saccharomyces cerevisiae. This invention utilizes existing global transcription regulators to genetically modify Saccharomyces cerevisiae, thereby obtaining a high-yield 3-hydroxypropionic acid (3-hydroxypropionic acid) mutant strain of Saccharomyces cerevisiae.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for constructing a *Saccharomyces cerevisiae* mutant that produces high levels of 3-hydroxypropionic acid, comprising the following steps: (1) The brewing yeast SKN7 Gene, PGD1 Gene, STB3 Gene, YAP1 Gene, RIM101 Gene, CIN5 Gene, FHL1 Gene, HAA1 Gene, CRZ1 Gene, NRG1 Gene or PDR1 Gene mutations were performed, and a high-producing malonyl-CoA mutant strain of Saccharomyces cerevisiae was obtained through screening. (2) Introduce the 3-hydroxypropionic acid production plasmid into the Saccharomyces cerevisiae mutant strain obtained in step (1) to obtain a Saccharomyces cerevisiae mutant that produces high levels of 3-hydroxypropionic acid.

[0007] Based on the above technical solution, further, the brewing yeast mentioned in step (1) is CEN.PK113-5D brewing yeast.

[0008] Based on the above technical solution, the mutation method described in step (1) further includes mutation of global transcription regulators or site-directed mutation.

[0009] Based on the above technical solution, further, the mutation mentioned in step (1) can be one or more of the following combinations: SKN7 The corresponding amino acid sequence change after gene mutation is: K394R; PGD1 The corresponding changes in the amino acid sequence after gene mutation are: V243D, S123S, or V243E; STB3 The corresponding amino acid sequence changes after gene mutation are: L52Y or E96I; YAP1 The corresponding changes in the amino acid sequence after gene mutation are: Q549F, G618E, or L643S; RIM101 The corresponding amino acid sequence change after gene mutation is: Y620W; CIN5 The corresponding amino acid sequence change after gene mutation is: K270A; FHL1 The corresponding amino acid sequence change after gene mutation is: G349N; HAA1 The corresponding amino acid sequence change after gene mutation is: R19K; CRZ1 The corresponding amino acid sequence change after gene mutation is: Y582S; The corresponding amino acid sequence change after NRG1 gene mutation is: R224H; PDR1 The corresponding amino acid sequence change after gene mutation is: T820C.

[0010] Based on the above technical solution, further, the backbone of the 3-HP production plasmid in step (2) is the pCRCT plasmid. The target gene in the plasmid includes the C-terminal gene fragment of malonyl-CoA reductase that converts malonyl-CoA into malonyl hemialdehyde and the N-terminal gene fragment of malonyl-CoA reductase that converts malonyl hemialdehyde into 3-hydroxypropionic acid. The C-terminal gene fragment is started by TDH3p and terminated by CYC1t, and the N-terminal gene fragment is started by PGK1p and terminated by ADH2t.

[0011] Based on the above technical solution, the nucleotide sequence of the C-terminal gene fragment of malonyl-CoA reductase is shown in SEQ ID NO.12, and the nucleotide sequence of the N-terminal gene fragment of malonyl-CoA reductase is shown in SEQ ID NO.13.

[0012] Secondly, the present invention provides a high-yield 3-hydroxypropionic acid-producing Saccharomyces cerevisiae mutant obtained by the above-described construction method.

[0013] Thirdly, the present invention provides a method for producing 3-hydroxypropionic acid by fermentation of the above-mentioned Saccharomyces cerevisiae mutant, comprising the following steps: inoculating the Saccharomyces cerevisiae mutant into a fermentation medium, fermenting at 25~35℃ and 100~300 rpm for 60~200 h, removing the cells, and separating and obtaining 3-hydroxypropionic acid.

[0014] Based on the above technical solution, further, the fermentation medium comprises: 7~8 g / L (NH4)2SO4, 13~15 g / L KH2PO4, 0.3~0.8 g / L MgSO4·7H2O, 15~25 g / L glucose, 0.5~1.5‰ vitamin solution, 0.5~1.5‰ trace metal element solution, pH adjusted to 5.5~7; the vitamin solution comprises: 13~18 g / L EDTA, 4~5 g / L ZnSO4∙7H2O, 0.2~0.4 g / L CoCl2∙6H2O, 0.5~1.5 g / L MnCl2∙4H2O, 0.2~0.4 g / L CuSO4∙5H2O, 4~5 g / L CaCl2∙2H2O, 2~4 g / L FeSO4∙7H2O, 0.3~0.5 g / L The composition of the trace metal element solution is as follows: NaMoO4∙2H2O, 0.5~1.5 g / L H3BO4, 0.05~0.15 g / L KI; the composition of the trace metal element solution is: 0.03~0.08 g / L biotin, 0.5~1.5 g / L calcium pantothenate, 0.5~1.5 g / L nicotinic acid, 20~30 g / L inositol, 0.5~1.5 g / L thiamine hydrochloride, 0.5~1.5 g / L pyridoxine hydrochloride, 0.1~0.3 g / L para-aminobenzoic acid.

[0015] Based on the above technical solution, the inoculation amount is further 1-10% of the fermentation medium volume.

[0016] Compared with the prior art, the present invention has the following beneficial effects: When using glucose as the carbon source for shake-flask fermentation for 144 h, the 3-HP yield of the SKN7 (K394R) mutant strain reached 13.6 g / L, an increase of 26% compared to the control strain; the PGD1 (S123S) mutant strain reached 14.4 g / L, an increase of 33% compared to the control strain; the YAP1 (Q549F) mutant strain reached 13.3 g / L, an increase of 23% compared to the control strain; the RIM101 (Y620W) mutant strain reached 13.3 g / L, an increase of 23% compared to the control strain; the CIN5 (K270A) mutant strain reached 12.1 g / L, an increase of 12% compared to the control strain; the STB3 (E96I) mutant strain reached 13.5 g / L, an increase of 25% compared to the control strain; and the FHL1 (G349N) mutant strain reached 11.1 g / L. The 3-HP yield of the YAP1(G618E) mutant strain reached 11.3 g / L, an increase of 4% compared to the control strain; the 3-HP yield of the YAP1(L643S) mutant strain reached 12.6 g / L, an increase of 17% compared to the control strain; the 3-HP yield of the PGD1(V243E) mutant strain reached 11.6 g / L, an increase of 8% compared to the control strain; the 3-HP yield of the HAA1(R19K) mutant strain reached 13.4 g / L, an increase of 24% compared to the control strain; the 3-HP yield of the CRZ1(Y582S) mutant strain reached 12.2 g / L, an increase of 13% compared to the control strain; and the 3-HP yield of the NRG1(R224H) mutant strain reached 13.1 g / L. The 3-HP yield of the STB3(L52Y) mutant strain reached 12.4 g / L, an increase of 15% compared to the control strain; the 3-HP yield of the PDR1(T820C) mutant strain reached 12.9 g / L, an increase of 19% compared to the control strain; and the 3-HP yield of the PGD1(V243D) mutant strain reached 11.3 g / L, an increase of 5% compared to the control strain. In summary, the Saccharomyces cerevisiae mutant strains constructed in this invention can produce high levels of 3-HP. Attached Figure Description

[0017] Figure 1 The liquid chromatograms are of the 3-HP standard (A) and the fermentation broth sample (B).

[0018] Figure 2 This is a comparison of the fluorescence values ​​of the mutant strain and the negative control strain.

[0019] Figure 3 A spectrum of plasmids produced for 3-HP.

[0020] Figure 4 This is a comparison of the 3-HP production results of the mutant strain and the negative control strain after 144 h of shake-flask fermentation. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0022] Example 1 The pJfapO-fapR plasmid was introduced into the *Saccharomyces cerevisiae* strain CEN.PK113-5D. The pJfapO-fapR plasmid was provided by Professor Hou Jin of Shandong University, with the reference being "Screening Phosphorylation Site Mutations in Yeast Acetyl-CoA Carboxylase Using Malonyl-CoA Sensor to Improve Malonyl-CoA-Derived Product". The pJfapO-fapR plasmid backbone is the pCRCT plasmid, specifically composed of plasmids derived from *Bacillus subtilis* (…). Bacillus subtilis Codon-optimized fapR The TEFI-GALI heterozygous promoter was obtained by fusing the upstream activating region (203 bp) of the TEF1 promoter with the core region (147 bp) of the GAL1 promoter. fapO The sequence was inserted 7 bp downstream of the TATA box of the GAL1 promoter and replaced the original sequence to obtain the GAL1p(7)FapO heterozygous promoter; the green fluorescent protein gene was then fused using fusion PCR. yeGFP The plasmid pJfapO-fapR was obtained by fusing it with the GAL1p(7)fap heterozygous promoter. The plasmid was then transformed into Saccharomyces cerevisiae. When the concentration of malonyl-CoA in Saccharomyces cerevisiae was low, the transcription of GFP was inhibited. When the concentration of malonyl-CoA in Saccharomyces cerevisiae gradually increased, the fluorescence intensity increased with the increase of malonyl-CoA concentration, and gradually approached the level when transcription was not inhibited.

[0023] The chemical transformation method for Saccharomyces cerevisiae plasmids is as follows: (1) Acquisition of Saccharomyces cerevisiae CEN.PK113-5D: Saccharomyces cerevisiae were inoculated into 5 mL of YPD liquid medium (10 g / L yeast extract, 20 g / L protein, 20 g / L glucose, pH 5.5) and cultured at 30℃ and 200 rpm for 12-16 h; approximately 700 μL of the culture was transferred to 50 mL of YPD liquid medium and cultured at 30℃ and 200 rpm for 3-5 h (OD). 600(Value 0.6-0.8), centrifuge to collect the precipitate, wash twice with sterile water to obtain competent bacterial cells; (2) Transform pJfapO-fapR plasmid into Saccharomyces cerevisiae CEN.PK113-5D competent cells: Mix Saccharomyces cerevisiae CEN.PK113-5D competent cells, 5 μL of 10 mg / mL salmon sperm DNA, 240 μL of 0.5 g / mL polyethylene glycol (PEG) aqueous solution (PEG molecular weight 3350 Da), 35 μL of 1.0 M lithium acetate solution, 70 μL of water and plasmid (plasmid concentration approximately 1-5 μg), pipette to mix well, incubate at 30℃ for 30 min, then heat shock at 42℃ for 30 min; centrifuge at 3000×g for 15 s, remove the supernatant, add 200 μL of ddH2O, gently pipette to mix well, spread on SC-LEU plates, and incubate at 30℃ for 2-3 days.

[0024] Example 2 The global transcription factor library was imported into the *Saccharomyces cerevisiae* strain obtained in Example 1. The sites in the global transcription factor library were domains and residues identified from protein sequences, as well as residues or regions of interest selected from literature using directed evolution methods. The computational information for these domains and residues was obtained from the Yeast Genome Database (SGD), SUPERFAMILY, PROSITE, Gene3D, SMART, Pfam, and PDB. The global transcription factor library plasmid consisted of CRISPR guide sequences (Cas9 protein and scaffold series) at mutation sites and a homologous recombination template. The plasmid backbone was the *Saccharomyces cerevisiae* pCRCT plasmid, with the reference being "Transcriptional regulatory networks involved in C3-C4 alcohol stress response and tolerance in yeast."

[0025] After the global transcription factor library was introduced into the Saccharomyces cerevisiae obtained in Example 1 using the aforementioned transformation method, it was plated on SC-LEU-URA plates for screening. The plates were observed using a gel imaging system, and fluorescent positive clones were obtained through screening. Subsequently, these positive clones were subjected to first-generation sequencing (Sanger sequencing) to finally identify and obtain the desired target mutant strain.

[0026] This study found that by introducing global transcriptional regulators, the effects of these regulators on *Saccharomyces cerevisiae* were significantly improved. SKN7 Gene, PGD1 Gene, STB3 Gene, YAP1 Gene,RIM101 Gene, CIN5 Gene, FHL1 Gene, HAA1 Gene, CRZ1 Gene, NRG1 Gene, PDR1 Mutating the gene can significantly improve the ability of Saccharomyces cerevisiae to produce 3-HP.

[0027] This embodiment will respectively contain yeast SKN7 Gene( SKN7 The changes in the amino acid sequence corresponding to the gene mutation are: K394R). PGD1 Gene( PGD1 The gene mutation corresponds to the amino acid sequence changes: V243D, S123S, or V243E. STB3 Gene( STB3 The gene mutation corresponds to the amino acid sequence change: L52Y or E96I. YAP1 Gene( YAP1 The gene mutation corresponds to the amino acid sequence changes of: Q549F, G618E, or L643S. RIM101 Gene( RIM101 The gene mutation corresponds to the change in the amino acid sequence: Y620W. CIN5 Gene( CIN5 The changes in the amino acid sequence corresponding to the gene mutation are: K270A). FHL1 Gene( FHL1 The changes in the amino acid sequence corresponding to the gene mutation are: G349N. HAA1 Gene( HAA1 The changes in the amino acid sequence corresponding to the gene mutation are: R19K). CRZ1 Gene( CRZ1 The changes in the amino acid sequence corresponding to the gene mutation are: Y582S). NRG1 The gene (the amino acid sequence change corresponding to the NRG1 gene mutation is: R224H) PDR1 Gene( PDR1 The changes in the amino acid sequence corresponding to the gene mutation mode are as follows: T820C) was mutated, and a total of 16 mutant strains of Saccharomyces cerevisiae were obtained (only one gene site was mutated in each mutant strain).

[0028] SKN7 The nucleotide sequence of the gene is shown in SEQ ID NO.1. SKN7 The AAA mutation at positions 1180-1182 of the gene is transformed into AGA. PGD1 The nucleotide sequence of the gene is shown in SEQ ID NO.2. PGD1The three gene mutations are: GTC at positions 727-729 is mutated to GAT; AGT at positions 367-369 is mutated to TCT; and GTC at positions 727-729 is mutated to GAA. STB3 The nucleotide sequence of the gene is shown in SEQ ID NO.3. STB3 The two gene mutations are: TTA at positions 154-156 is mutated to TAT; and GAA at positions 286-288 is mutated to ATT. YAP1 The nucleotide sequence of the gene is shown in SEQ ID NO.4. YAP1 The three gene mutations are: CAA at positions 1645-1647 mutates to TTT; GGT at positions 1852-1854 mutates to GAA; and TTA at positions 1927-1929 mutates to TCT. RIM101 The nucleotide sequence of the gene is shown in SEQ ID NO.5. RIM101 The TAT mutation at positions 1858-1860 of the gene is replaced by TGG. CIN5 The nucleotide sequence of the gene is shown in SEQ ID NO.6. CIN5 The AAA mutation at positions 808-810 of the gene is GCT. FHL1 The nucleotide sequence of the gene is shown in SEQ ID NO.7. FHL1 The GGT at positions 1045-1047 of the gene is mutated to AAT. HAA1 The nucleotide sequence of the gene is shown in SEQ ID NO.8. HAA1 The AGA mutation at positions 55-57 of the gene is changed to AAA. CRZ1 The nucleotide sequence of the gene is shown in SEQ ID NO.9. CRZ1 The TAT mutation at positions 1744-1746 of the gene is transformed into TCT. NRG1 The nucleotide sequence of the gene is shown in SEQ ID NO.10. NRG1 The AGA mutation at positions 670-672 of the gene is replaced by CAT. PDR1 The nucleotide sequence of the gene is shown in SEQ ID NO.11. PDR1 The ACT mutation at positions 2458-2460 of the gene is replaced by TGT.

[0029] Screening of high-malonyl-CoA-producing *Saccharomyces cerevisiae* strains: The presence of increased malonyl-CoA was verified using a microplate reader. Sixteen mutant *Saccharomyces cerevisiae* strains were inoculated into 96-well microplates containing 150 μL of SC-LEU-URA medium and incubated overnight at 30°C and 600 rpm. Each strain was performed in triplicate, with wild-type *Saccharomyces cerevisiae* CEN.PK113-5D serving as a control. After 24 hours, the OD value of the mutant strains at 600 nm was measured using a microplate reader, and fluorescence was detected under excitation conditions of 485 nm and emission conditions of 535 nm. Cellular fluorescence intensity (au) was used to represent the fluorescence level; higher fluorescence intensity indicated a higher malonyl-CoA content in the *Saccharomyces cerevisiae*.

[0030] The results are as follows Figure 2 The fluorescence intensity of most mutant strains was much greater than that of the control strains, with the fluorescence intensity concentrated at 4.0 × 10⁻⁶. 7 ~1.0×10 8 Among them, PGD1 (S123S) had the highest fluorescence intensity, while FHL1 (G349N) had the lowest fluorescence intensity.

[0031] Example 3 The mutant strain from Example 2 was passaged multiple times in SC-LEU medium (5 g / L ammonium sulfate, 0.7 g / L leucine-deficient medium, 1.7 g / L amino-free yeast nitrogen source, 20 g / L glucose) until the aforementioned library editing plasmid was lost. Then, a 3-HP production plasmid (such as...) was introduced. Figure 3 As shown, the backbone of the 3-HP production plasmid is the pCRCT plasmid, in which the key gene, bifunctional malonyl-CoA reductase (MCR), is produced by *Flexobacter orangeii* (…). Chloroflexus aurantiacus ) coming, will MCR The gene was split into C-terminal and N-terminal sequences and constructed into the nonsense region of the pCRCT plasmid (containing a 2μ origin of replication and a URA3 selector) using Gibson ligation. The C-terminus is initiated by TDH3p and terminated by CYC1t, and its function is to convert malonyl-CoA to malonyl hemialdehyde. The N-terminus is initiated by PGK1p and terminated by ADH2t, and its function is to convert malonyl hemialdehyde to 3-HP. Mutations were made at three sites at the C-terminus (N940V, K1106W, and S1114R) to increase enzyme activity, thus obtaining a high-yield 3-HP mutant. MCR The specific sequence after the C-terminal mutation is shown in SEQ ID NO.12. MCR The specific N-terminal sequence is shown in SEQ ID NO.13. Screening of high-yielding 3-HP Saccharomyces cerevisiae strains: Fermentation experiments were conducted to verify whether the strains improved the yield of 3-HP. The fermentation medium consisted of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 1‰ vitamin solution, and 1‰ trace metal element solution, with the pH adjusted to 6 using sodium hydroxide solution. The vitamin solution contained: 15 g / L EDTA, 4.5 g / L ZnSO4·7H2O, 0.3 g / L CoCl2·6H2O, 1 g / L MnCl2·4H2O, 0.3 g / L CuSO4·5H2O, 4.5 g / L CaCl2·2H2O, 3 g / L FeSO4·7H2O, 0.4 g / L NaMoO4·2H2O, 1 g / L H3BO4, and 0.1 g / L KI. The trace metal element solution contained: 0.05 g / L biotin, 1 g / L... Calcium pantothenate, 1 g / L nicotinic acid, 25 g / L inositol, 1 g / L thiamine hydrochloride, 1 g / L pyridoxine hydrochloride, 0.2 g / L para-aminobenzoic acid.

[0032] The shake-flask fermentation conditions for the production of 3-HP by Saccharomyces cerevisiae were as follows: culture medium volume 25 mL / 250 mL, initial pH 6-7, and initial OD of the Saccharomyces cerevisiae mutant in the fermentation medium was controlled. 600 =0.1, temperature 30℃, speed 200 rpm, time 144 h.

[0033] After fermentation, 1 mL of bacterial culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm for 5 min to remove bacterial cells. The supernatant was sterilized by membrane filtration and transferred to a liquid chromatography vial. Qualitative and quantitative analysis was performed using high-performance liquid chromatography (HPLC). The yield of 3-HP was measured at 210 nm using an HPLC system equipped with RID and UV detectors. 0.5 mM H2SO4 aqueous solution was used as the eluent and an Aminex HPX-87H (300 × 7.8 mm) column (Bio-Rad) was used. The column temperature was 65℃, the flow rate was 0.4 mL / min, and the injection volume was 20 μL. The external standard method was used for quantification of 3-HP. After detecting the fermentation products, the data were analyzed.

[0034] The results are as follows Figure 4 Compared with the control strain, the 3-HP yield of the Saccharomyces cerevisiae mutant was significantly improved. Compared with the existing technology, the Saccharomyces cerevisiae mutant constructed by this invention can produce high 3-HP, which not only provides a technical basis for the targeted modification of Saccharomyces cerevisiae engineered strains, but also provides suitable strains for carbon fiber production, thus promoting the industrial production of 3-HP.

Claims

1. A method for constructing a *Saccharomyces cerevisiae* mutant with high 3-hydroxypropionic acid production, characterized in that, Includes the following steps: (1) The brewing yeast SKN7 Gene, PGD1 Gene, STB3 Gene, YAP1 Gene, RIM101 Gene, CIN5 Gene, FHL1 Gene, HAA1 Gene, CRZ1 Gene, NRG1 Gene or PDR1 Gene mutations were performed, and a high-producing malonyl-CoA mutant strain of Saccharomyces cerevisiae was obtained through screening. (2) Introduce the 3-hydroxypropionic acid production plasmid into the Saccharomyces cerevisiae mutant strain obtained in step (1) to obtain a Saccharomyces cerevisiae mutant that produces high levels of 3-hydroxypropionic acid.

2. The construction method according to claim 1, characterized in that, The brewing yeast mentioned in step (1) is CEN.PK113-5D brewing yeast.

3. The construction method according to claim 1, characterized in that, The mutation methods described in step (1) include mutations of global transcriptional regulators or site-directed mutations.

4. The construction method according to claim 1, characterized in that, The mutation described in step (1) is one or more of the following combinations: SKN7 The corresponding amino acid sequence change after gene mutation is: K394R; PGD1 The corresponding changes in the amino acid sequence after gene mutation are: V243D, S123S, or V243E; STB3 The corresponding amino acid sequence changes after gene mutation are: L52Y or E96I; YAP1 The corresponding changes in the amino acid sequence after gene mutation are: Q549F, G618E, or L643S; RIM101 The corresponding amino acid sequence change after gene mutation is: Y620W; CIN5 The corresponding amino acid sequence change after gene mutation is: K270A; FHL1 The corresponding amino acid sequence change after gene mutation is: G349N; HAA1 The corresponding amino acid sequence change after gene mutation is: R19K; CRZ1 The corresponding amino acid sequence change after gene mutation is: Y582S; The corresponding amino acid sequence change after NRG1 gene mutation is: R224H; PDR1 The corresponding amino acid sequence change after gene mutation is: T820C.

5. The construction method according to claim 1, characterized in that, The backbone of the 3-HP production plasmid described in step (2) is the pCRCT plasmid. The target genes in the plasmid include the C-terminal gene fragment of malonyl-CoA reductase that converts malonyl-CoA to malonyl hemialdehyde and the N-terminal gene fragment of malonyl-CoA reductase that converts malonyl hemialdehyde to 3-hydroxypropionic acid. The C-terminal gene fragment is started by TDH3p and terminated by CYC1t, and the N-terminal gene fragment is started by PGK1p and terminated by ADH2t.

6. The Saccharomyces cerevisiae mutant with high 3-hydroxypropionic acid production obtained by the construction method according to any one of claims 1-5.

7. The method for producing 3-hydroxypropionic acid by fermentation of a *Saccharomyces cerevisiae* mutant according to claim 6, characterized in that, The process includes the following steps: inoculating the described Saccharomyces cerevisiae mutant into a fermentation medium, fermenting at 25-35℃ and 100-300 rpm for 60-200 h, removing the cells, and separating 3-hydroxypropionic acid.

8. The method according to claim 7, characterized in that, The fermentation medium consists of: 7-8 g / L (NH4)2SO4, 13-15 g / L KH2PO4, 0.3-0.8 g / L MgSO4·7H2O, 15-25 g / L glucose, 0.5-1.5‰ vitamin solution, and 0.5-1.5‰ trace metal element solution, with the pH adjusted to 5.5-7. The vitamin solution consists of: 13-18 g / L EDTA, 4-5 g / L ZnSO4·7H2O, 0.2-0.4 g / L CoCl2·6H2O, 0.5-1.5 g / L MnCl2·4H2O, 0.2-0.4 g / L CuSO4·5H2O, 4-5 g / L CaCl2·2H2O, 2-4 g / L FeSO4·7H2O, and 0.3-0.5 g / L... The composition of the trace metal element solution is as follows: NaMoO4∙2H2O, 0.5~1.5 g / L H3BO4, 0.05~0.15 g / L KI; the composition of the trace metal element solution is: 0.03~0.08 g / L biotin, 0.5~1.5 g / L calcium pantothenate, 0.5~1.5 g / L nicotinic acid, 20~30 g / L inositol, 0.5~1.5 g / L thiamine hydrochloride, 0.5~1.5 g / L pyridoxine hydrochloride, 0.1~0.3 g / L p-aminobenzoic acid.

9. The method according to claim 7, characterized in that, The inoculum volume is 1-10% of the fermentation medium volume.

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