Method for improving conversion rate of ethanol to acetyl coenzyme A in saccharomycetes, engineering bacteria and application of engineering bacteria in production of 3-hydracrylic acid

Abstract

The invention relates to a method for improving the conversion rate of ethanol to acetyl coenzyme A in saccharomycetes, engineering bacteria and application of the engineering bacteria in production of 3-hydracrylic acid. Pfk1 and pfk2 genes are deleted from the engineering bacteria; the engineering bacterium is also used for overexpression of mutACC1, MCRC, MCRN, Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1, and fused CaGAPDH and Pos5 genes. The invention further discloses a preparation method of the engineering bacterium. According to the method, a metabolic pathway from ethanol to acetyl coenzyme A is systematically combed and modified in the saccharomyces cerevisiae, so that the conversion rate from ethanol to acetyl coenzyme A is effectively improved, and the reducing power and energy balance in cells are evolved and remodeled through an adaptive laboratory, so that efficient and stable synthesis of 3-hydracrylic acid in the saccharomyces cerevisiae is realized. In addition, by remodeling yeast central metabolism, a new thought is provided for efficient conversion of low-carbon compounds by means of synthetic biology.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a method for improving the conversion rate of ethanol to acetyl coenzyme A in yeast, an engineering bacterium and an application of the same in producing 3-hydroxypropionic acid. Background Art

[0002] The instability of the production performance of engineered strains during the scale-up process has always been a key issue hindering industrial fermentation production. In order to achieve overproduction of the target product, it is usually necessary to artificially introduce exogenous metabolic pathways into the engineered bacteria and strengthen related endogenous metabolic pathways. However, these modifications inevitably cause growth and metabolic stress on the cells. During the industrial scale-up process, cells tend to limit the ability to overproduce the target product through various metabolic interactions to relieve metabolic stress and promote cell growth, which in turn leads to the weakening or loss of production traits.

[0003] Orthogonalization in cell factory design refers to the process of engineering a specific molecule or gene circuit so that the function is not affected by the intracellular environment and does not exert unnecessary influence on the environment. Orthogonalization can minimize the interaction nodes between the production metabolic pathway and the cell chassis metabolism, so that the production pathway is a relatively independent module, which minimizes the interference of the normal cell growth metabolism, thereby maintaining the stability of production performance. In addition, through orthogonalization, the bypass metabolism from substrate to precursor can also be reduced, so that the conversion rate can be maximized.

[0004] 3-Hydroxypropionic acid (3-HP) is a high value-added chemical with great application value in the fields of medicine, personal care, coatings, adhesives, textiles, food, feed, etc. With the acceleration of industrialization, the market demand for 3-HP is increasing. It is estimated that in 2025, the global market demand for 3-hydroxypropionic acid will reach 10 billion US dollars. Synthesis technology is an important factor restricting the large-scale application of 3-HP. 3-HP synthesis technology includes two major categories: chemical synthesis and biosynthesis. Among them, chemical synthesis is the current mainstream production process, but chemical synthesis has problems such as complex process and serious pollution. In recent years, 3-HP synthesis technology based on engineered microorganisms has become the focus of scientific research in universities at home and abroad, but the production of 3-HP by biosynthesis still has problems such as low strain production efficiency and poor stability.

[0005] In summary, how to develop efficient and stable engineered strains is still one of the urgent problems to be solved in the field of 3-HP biosynthesis. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for improving the conversion rate of ethanol to acetyl-CoA in yeast, an engineered bacterium and its application in the production of 3-hydroxypropionic acid, in order to obtain an engineered strain for efficient and stable production of 3-HP.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a yeast engineering bacterium that utilizes glucose and ethanol as dual carbon sources to stably produce 3-hydroxypropionic acid, wherein the engineering bacterium lacks pfk1 and pfk2 genes; the engineering bacterium also overexpresses mutACC1, MCRC, MCRN, Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1 and fused CaGAPDH and Pos5 genes.

[0009] In the present invention, based on the metabolic pathway orthogonalization strategy, a strain modification method is designed to improve the conversion efficiency of ethanol to acetyl-CoA and its downstream metabolites in yeast cells, an engineered yeast strain for producing 3-HP is constructed, and the stable synthesis of 3-HP in yeast is achieved, providing a new idea for the biological production of 3-HP.

[0010] Preferably, the engineered bacteria also lack cit2 and icl1 genes.

[0011] Preferably, the engineered bacteria also overexpresses the ADA gene.

[0012] Preferably, the engineered bacteria also lack ald2, ald3, ald4, ald5 and ald6 genes.

[0013] Preferably, the engineered bacteria also lack acs1, acs2 and ach1 genes.

[0014] Preferably, the engineered bacteria also lack yat1, yat2, crc1 and cat2 genes.

[0015] In the present invention, based on the constructed engineered bacteria that stably synthesize 3-HP, the efficient conversion pathway of ethanol to 3-HP is further modified, including at least one of the following: (1) knocking out the cit2 and icl1 genes; (2) overexpressing the ADA gene; (3) knocking out the ald2, ald3, ald4, ald5 and ald6 genes; (4) knocking out the acs1, acs2 and ach1 genes; (5) knocking out the yat1 and yat2, crc1 and cat2 genes; all of which can further increase the yield of 3-HP.

[0016] Preferably, the starting strain of the engineered bacteria includes Saccharomyces cerevisiae.

[0017] It can be understood that the present invention is based on the systematic sorting and transformation of the metabolic pathway of ethanol to acetyl-CoA by Saccharomyces cerevisiae. Yeast species with similar genomes and metabolic networks are theoretically suitable for the present invention, optionally including Saccharomyces cerevisiae IMX581 and the like.

[0018] Optionally, the mutACC1 gene nucleic acid sequence is shown as SEQ ID No.1, the MCRC gene nucleic acid sequence is shown as SEQ ID No.2, the MCRN gene nucleic acid sequence is shown as SEQ ID No.3, the CaGAPDH gene nucleic acid sequence is shown as SEQ ID No.4, and the ADA gene nucleic acid sequence is shown as SEQ ID No.5.

[0019] SEQ ID No.1 (mutACC1):

[0020]

[0021] SEQ ID No.2:(MCRC)

[0022]

[0023] SEQ ID No.3:(MCRN)

[0024]

[0025] SEQ ID No.4:(CaGAPDH)

[0026]

[0027] SEQ ID No.5:(ADA)

[0028]

[0029] In a second aspect, the present invention provides a method for preparing the yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources as described in the first aspect, the method comprising:

[0030] Using yeast as the starting strain, the pfk1 and pfk2 genes were knocked out, and the mutACC1, MCRC, MCRN, Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1 and fused CaGAPDH and Pos5 genes were overexpressed.

[0031] It is understood that based on the transformation strategy designed by the present invention, the gene knockout or overexpression methods commonly used in the art are applicable to the present invention.

[0032] In a third aspect, the present invention provides the use of the yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources as described in the first aspect in the production of 3-HP.

[0033] In a fourth aspect, the present invention provides a method for producing 3-HP, the method comprising:

[0034] The yeast engineered bacteria that utilizes glucose and ethanol as dual carbon sources to stably produce 3-HP as described in the first aspect are cultured, and the bacterial liquid is collected for product separation and purification to obtain the 3-HP.

[0035] The engineered bacteria constructed based on the present invention can achieve efficient production of 3-HP by adopting the cultivation and purification methods commonly used in the art.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention systematically combs and transforms the metabolic pathway from ethanol to acetyl-CoA in Saccharomyces cerevisiae, effectively improving the conversion rate of ethanol to 3-HP, and reshapes the reducing power and energy balance in the cell through adaptive laboratory evolution, thus achieving the stable synthesis of 3-HP in Saccharomyces cerevisiae. In addition, by reshaping the central metabolism of yeast, the present invention provides a new idea for the efficient conversion of low-carbon compounds using synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of metabolic pathway modification in engineered Saccharomyces cerevisiae strains.

[0039] Figure 2 This is the HPLC spectrum of 3-HP standard and engineered bacteria fermentation broth.

[0040] Figure 3 This is a statistical diagram of 3-HP production and growth of engineered yeast after modification. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0042] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0043] The present invention provides a strain modification method for improving the conversion efficiency of ethanol to acetyl-CoA and its downstream metabolites in yeast cells based on orthogonalization of metabolic pathways, a recombinant yeast strain constructed using the strategy, and its application in the production of 3-hydroxypropionic acid. The specific modification strategy includes:

[0044] (1) The phosphofructokinase genes pfk1 (GenBank accession number: YGR240C) and pfk2 (GenBank accession number: YMR205C) of wild-type Saccharomyces cerevisiae were knocked out to obtain the recombinant yeast HP01 in which the glycolysis pathway was cut off;

[0045] (2) The gene expression cassettes of the pentose phosphate pathway-related enzyme genes Zwf1 (GenBank Accession No.: YNL241C), Gnd1 (GenBank Accession No.: YHR183W), Tal1 (GenBank Accession No.: YLR354C), Rki1 (GenBank Accession No.: YOR095C), Rpe1 (GenBank Accession No.: YJL121C) and Tkl1 (GenBank Accession No.: YPR074C) were integrated into the genome of strain HP01 to obtain strain HP02;

[0046] (3) The 3-HP synthase mutant mutACC1 (SEQ ID No. 1), the malonyl-CoA reductase MCRC gene (SEQ ID No. 2) and MCRN gene (SEQ ID No. 3) of Chloroflexus aurantiacus, and the expression cassette of the CaGAPDH gene (SEQ ID No. 4) of Clostridium acetobutylicum fused with the endogenous NADH kinase Pos5 (GenBank accession number: YPL188W) were integrated into the genome of strain HP02 to obtain strain HP03;

[0047] (4) The cit2 (GenBank accession number: YCR005C) and icl1 (GenBank accession number: YER065C) genes in the glyoxylate cycle were knocked out to obtain strain HP04;

[0048] (5) Integrating the expression cassette of the acetaldehyde dehydrogenase ADA gene (SEQ ID No. 5) of Dickeya zeae into the genome of strain HP04 to obtain strain HP05;

[0049] (6) The acetaldehyde dehydrogenase genes ald2 (GenBank accession number: YMR170C), ald3 (GenBank accession number: YMR169C), ald4 (GenBank accession number: YOR374W), ald5 (GenBank accession number: YER073W), and ald6 (GenBank accession number: YPL061W) were knocked out to obtain strain HP06;

[0050] (7) The acetyl-CoA synthetase genes acs1 (GenBank accession number: YAL054C) and acs2 (GenBank accession number: YLR153C) and the acetyl-CoA hydrolase gene ach1 (GenBank accession number: YBL015W) were knocked out to obtain strain HP07;

[0051] (8) The acetylcarnitine transferase genes yat1 (GenBank accession number: YAR035W) and yat2 (GenBank accession number: YER024W), the mitochondrial carnitine transporter gene crc1 (GenBank accession number: YOR100C), and the acetyl-CoA carnitine transferase cat2 (GenBank accession number: YML042W) were knocked out to obtain strain HP08.

[0052] (9) The HP08 strain was subjected to adaptive evolution to obtain the evolved strain HP09. After adaptive evolution, the growth rate of the engineered bacteria was restored and the 3-HP production was significantly improved.

[0053] The schematic diagram of the metabolic pathway modification of the corresponding engineered Saccharomyces cerevisiae strain is as follows Figure 1 As shown, 3-HP was stably produced using glucose and ethanol as dual carbon sources.

[0054] Example 1

[0055] This example constructs a recombinant genetic engineering strain that produces 3-HP using glucose and ethanol as dual carbon sources.

[0056] (1) Knockout of the phosphofructokinase genes pfk1 and pfk2;

[0057] The gRNA primers for yeast pfk1 and pfk2 cutting were designed according to the method described in the reference (MIKKELSEN, Michael Dalgaard, et al. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in aversatile yeast expression platform. Metabolic engineering, 2012, 14.2: 104-111.), and the primers were used for PCR amplification of 2 μm fragments. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified by a clean-up kit, and the precise cutting plasmid was constructed. The reaction system was as follows: pROS10 plasmid template, 40 ng; gRNA primer 1 (10 μM), 2 μL; gRNA primer 2 (10 μM), 2 μL; Max DNA Polymerase, 25 μL; total volume (add ddH2O) to 50 μL.

[0058] gRNA Primers:

[0059] pfk1_up sgRNA F&R:

[0060] 5'-gaaagataaatgatcTTTGGAAAGAATCTGTGAAAgttttagagctagaaatagcaagt-3';

[0061] pfk1_dw sgRNA F&R:

[0062] 5'-gaaagataaatgatcGTGGCTGGAATCAAACACATgttttagagctagaaatagcaagt-3';

[0063] pfk2_up sgRNA F&R:

[0064] 5'-gaaagataaatgatcTTTTTCGTTAACAGCAATCAAgttttagagctagaaatagcaagt-3';

[0065] pfk2_dw sgRNA F&R:

[0066] 5'-gaaagataaatgatcTAGCTGGTATCAAGACCATTgttttagagctagaaatagcaagt-3'.

[0067] Using pROS10 plasmid as template, the plasmid backbone was amplified with primers CRISPR plasmid back F&R (5'-gatcatttatctttcactgcggagaag-3'), and the reaction system was as follows: pROS10 plasmid template, 40 ng; CRISPR plasmid back F&R (10 μM), 2 μL; Max DNA Polymerase, 25 μL; total volume (add ddH2O) to 50 μL.

[0068] The 2 μm fragment and the plasmid backbone were assembled by the Gibson Assembly method to construct a plasmid with a designated site cut in this application; the ligation product was transformed into E. coli DH5α to obtain a transformation product; the transformation product was spread on LB solid medium (containing ampicillin at a final concentration of 100 mg / L), and after obtaining a single clone, the plasmid was extracted and sequenced after being cultured in a shake flask at 37° C. and 220 rpm for 10 hours. If the verification is correct, the pfk1 and pfk2 knockout plasmids are obtained;

[0069] Using the Saccharomyces cerevisiae genome as a template, the repair fragments of pfk1 and pfk2 were obtained by PCR. The repair fragments and the precise cutting plasmids were transferred into Saccharomyces cerevisiae cells. The specific steps are as follows: fresh Saccharomyces cerevisiae IMX581 was cloned into 1mL YPD medium (Yeast Extract, Peptone, Dextrose), cultured overnight, and an appropriate amount of bacterial solution was transferred to 20mL YPD to make the starting OD 600 =0.1, 30°C, 200 rpm, culture until OD 600 =0.6, centrifuge at 3000×g to remove the culture medium, resuspend the cell pellet with 1 mL of sterile water, centrifuge at 3000×g to remove the supernatant, add 1 mL of 0.1 M lithium acetate to resuspend, centrifuge at 3000×g to remove the supernatant, add 200 μL of 0.1 M lithium acetate to obtain brewer's cell competent cells, and construct recombinant brewer's yeast by lithium acetate / polyethylene glycol transformation method. The transformation system is as follows: polyethylene glycol 3500 (50% w / v), 120 μL; lithium acetate (1.0 M), 18 μL; salmon sperm DNA (2.0 mg / mL), 25 μL; repair fragment, 1-2 μg; knockout plasmid, 1-2 μg; total volume (add ddH2O) to 180 μL, and transform to obtain yeast engineering strain HP01;

[0070] (2) Enhanced expression of six genes in the PP pathway

[0071] Furthermore, taking the construction method of yeast engineering strain HP01 as an example, the genome of Saccharomyces cerevisiae was used as a template, and PCR products of the expression cassettes of glucose-6-phosphate dehydrogenase Zwf1, 6-phosphogluconate dehydrogenase Gnd1, transaldolase Tal1, ribose-5-phosphate isomerase Rki1, D-ribulose-5-phosphate-3-epimerase Rpe1 and transketolase Tkl1 in the pentose phosphate pathway were obtained by PCR, and the expression cassettes of the above six enzymes were integrated into the genome of yeast engineering strain HP01 by using CRISPR / Cas9 gene editing technology to obtain strain HP02;

[0072] (3) Construction of 3-HP synthesis pathway

[0073] Furthermore, taking the construction method of the yeast engineering strain HP01 as an example, the 3-HP synthase mutant mutACC1 (SEQ ID No. 1), the malonyl-CoA reductase MCRC gene (SEQ ID No. 2) and the MCRN gene (SEQ ID No. 3) of Chloroflexus aurantiacus, and the CaGAPDH gene (SEQ ID No. 4) of Clostridium acetobutylicum fused with the endogenous NADH kinase ScPOS5 were integrated into the genome of strain HP02 to obtain strain HP03.

[0074] Example 2

[0075] This example is about the transformation of the efficient conversion pathway of ethanol to 3-HP in Saccharomyces cerevisiae.

[0076] (1) Disconnection of the glyoxylate cycle

[0077] Taking the construction method of yeast engineering strain HP01 as an example, the genome of Saccharomyces cerevisiae was used as a template, and the repair fragments of citrate synthase cit2 and isocitrate lyase icl1 in the glyoxylate cycle were obtained by PCR. CRISPR / Cas9 gene editing technology was used to knock out the citrate synthase cit2 and isocitrate lyase icl1 genes in strain HP03 to obtain strain HP04.

[0078] (2) Modification of the metabolic pathway from ethanol to acetyl-CoA

[0079] Furthermore, taking the construction method of yeast engineering strain HP01 as an example, the aldehyde dehydrogenase ADA gene expression cassette (SEQ ID No. 5) of Dickeyazeae was integrated into the genome of strain HP04 to obtain strain HP05;

[0080] Furthermore, using the genome of Saccharomyces cerevisiae as a template, repair fragments of aldehyde dehydrogenase genes ald2, ald3, ald4, ald5 and ald6 were obtained by PCR, and the above five genes in strain HP05 were knocked out by CRISPR / Cas9 gene editing technology to obtain strain HP06;

[0081] Furthermore, using the genome of Saccharomyces cerevisiae as a template, PCR was used to obtain the repair fragments of the acetyl-CoA synthetase genes acs1 and acs2, and the acetyl-CoA hydrolase gene ach1, and the CRISPR / Cas9 gene editing technology was used to knock out the above three genes in strain HP06 to obtain strain HP07;

[0082] (3) Cutting off the carnitine shuttle pathway of acetyl-CoA

[0083] Furthermore, taking the construction method of yeast engineered strain HP01 as an example, repair fragments of acetylcarnitine transferase genes yat1 and yat2, mitochondrial carnitine transporter gene crc1, and acetyl-CoA carnitine transferase cat2 were obtained by PCR, and the above four genes in strain HP07 were knocked out using CRISPR / Cas9 gene editing technology to obtain strain HP08. Then, the modified brewer's yeast was adaptively evolved to obtain the evolved strain HP09. After adaptive evolution, the growth rate of the engineered bacteria was restored and the 3-HP yield was significantly improved.

[0084] Example 3

[0085] This example is used to prepare the product 3-HP.

[0086] In order to evaluate the ability of the modified strain to synthesize 3-hydroxypropionic acid, the starting strain and the modified strain were fermented in shake flasks. The specific steps are as follows: pick a fresh yeast monoclone into 1 mL YPGE medium (yeast rich medium with 2% (w / v) glycerol and 2% (w / v) ethanol as carbon source), culture overnight, take an appropriate amount of bacterial liquid and transfer it to 15 mL DelftDE (yeast inorganic salt medium with 0.5% or 1% glucose and 2% ethanol as carbon source), and make the starting OD 600 =0.5, 30°C, 200rpm culture, collect the bacterial liquid after fermentation to the plateau phase, centrifuge at 4°C, 12000rpm for 1min, collect the supernatant, filter through a 0.22μm filter membrane and perform liquid chromatography analysis. The liquid chromatography analysis method is as follows: use Aminex HPX87H column (1300×7.8mm, particle size 9μm) chromatographic column, column temperature 50°C, injection volume 5μL. The mobile phase contains 5mM sulfuric acid in deionized water, and the flow rate is 0.6mL / min. HPLC spectrum of 3-HP standard and strain HP03 fermentation broth detection (exemplary display of the results of HP03 strain) as shown Figure 2 The 3-HP yield calculation method includes: using a standard substance to construct a standard curve, and detecting and quantifying 3-HP in the fermentation broth.

[0087] The results of bacterial biomass and 3-HP production are as follows Figure 3 As shown by Figure 3 It can be seen that by knocking out the pfk1 and pfk2 genes in Saccharomyces cerevisiae and overexpressing the Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1, mutACC1, MCRC, MCRN and fused CaGAPDH and Pos5 genes, a recombinant bacterium (HP03) that stably produces 3-HP using glucose and ethanol as dual carbon sources can be successfully constructed. Furthermore, an optimization strategy is adopted, including at least one of the following: (1) knocking out the cit2 and icl1 genes; (2) overexpressing the ADA gene; (3) knocking out the ald2, ald3, ald4, ald5 and ald6 genes; (4) knocking out the acs1, acs2 and ach1 genes; (5) knocking out the yat1 and yat2, crc1 and cat2 genes; all of which can further increase the yield of 3-HP.

[0088] In summary, the present invention systematically combs and transforms the metabolic pathway from ethanol to acetyl-CoA in Saccharomyces cerevisiae, effectively improves the conversion rate of ethanol to 3-HP, and reshapes the reducing power and energy balance in the cell through adaptive laboratory evolution, thereby achieving the stable synthesis of 3-HP in Saccharomyces cerevisiae. In addition, by reshaping the central metabolism of yeast, the present invention provides a new idea for the efficient conversion of low-carbon compounds using synthetic biology.

[0089] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A yeast engineered bacterium that utilizes glucose and ethanol as dual carbon sources to stably produce 3-hydroxypropionic acid, characterized in that: The engineered bacteria lack pfk1 and pfk2 genes; The engineered bacteria also overexpress mutACC1, MCRC, MCRN, Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1 and fused CaGAPDH and Pos5 genes.

2. The yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to claim 1, characterized in that: The engineered bacteria also lack cit2 and icl1 genes.

3. The yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to claim 1 or 2, characterized in that: The engineered bacteria also overexpress the ADA gene.

4. The yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to any one of claims 1 to 3, characterized in that: The engineered bacteria also lack ald2, ald3, ald4, ald5 and ald6 genes.

5. The yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to any one of claims 1 to 4, characterized in that: The engineered bacteria also lack acs1, acs2 and ach1 genes.

6. The yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to any one of claims 1 to 5, characterized in that: The engineered bacteria also lack yat1, yat2, crc1 and cat2 genes.

7. The yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to any one of claims 1 to 6, characterized in that: The starting strain of the engineering bacteria includes Saccharomyces cerevisiae.

8. A method for preparing the yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to any one of claims 1 to 7, characterized in that: The method comprises: Using yeast as the starting strain, the pfk1 and pfk2 genes were knocked out, and the mutACC1, MCRC, MCRN, Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1 and fused CaGAPDH and Pos5 genes were overexpressed.

9. Use of the yeast engineering bacteria for stably producing 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to any one of claims 1 to 7 in producing 3-hydroxypropionic acid.

10. A method for producing 3-hydroxypropionic acid, characterized in that: The method comprises: Cultivate the yeast engineered bacteria that stably produces 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources as described in any one of claims 1 to 7, collect the bacterial liquid for product separation and purification, and obtain the 3-hydroxypropionic acid.

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