A method for improving the conversion rate of ethanol to acetyl coenzyme a in yeast, an engineered bacterium and its application in the production of 3-hydroxypropionic acid

Abstract

The present application relates to 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. The engineered bacterium lacks pfk1 and pfk2 genes; the engineered bacterium also overexpresses mutACC1, MCRC, MCRN, Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1 and the fused CaGAPDH and Pos5 genes. The present application systematically combs and modifies the metabolic pathway of ethanol to acetyl-CoA in Saccharomyces cerevisiae, effectively improves the conversion rate of ethanol to acetyl-CoA, and through adaptive laboratory evolution, the intracellular reducing power and energy balance are remodeled, realizing the efficient and stable synthesis of 3-hydroxypropionic acid in Saccharomyces cerevisiae. In addition, the present application remolds the central metabolism of yeast, providing a new idea for efficient conversion of low-carbon compounds using synthetic biology methods.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a method for improving the conversion rate of ethanol to acetyl-CoA in yeast, an engineered strain, and its application in the production of 3-hydroxypropionic acid. Background Technology

[0002] The instability of the production performance of engineered strains during scale-up production has always been a key problem hindering industrial fermentation production. To achieve overproduction of the target product, it is usually necessary to artificially introduce exogenous metabolic pathways and enhance related endogenous metabolic pathways in engineered bacteria. However, these modifications inevitably place growth and metabolic stress on the cells. During industrial scale-up production, cells tend to limit their ability to overproduce the target product through various metabolic interactions to alleviate metabolic stress, promote cell growth, and consequently weaken or lose productive traits.

[0003] Orthogonalization in cell factory design refers to engineering modifications that minimize or eliminate the influence of the intracellular environment on the function of a specific molecule or gene pathway. Orthogonalization reduces the number of interaction points between the metabolic pathway and the cellular chassis metabolism, allowing the production pathway to function as a relatively independent module, minimizing interference from normal cellular growth and metabolism, thus maintaining stable production performance. Furthermore, orthogonalization can reduce bypass metabolism from substrate to precursor, thereby maximizing conversion efficiency.

[0004] 3-Hydroxypropionic acid (3-HP) is a high-value chemical with significant applications in medical, personal care, coatings, adhesives, textiles, food, and animal feed industries. With accelerating industrialization, the market demand for 3-HP is increasing daily; it is estimated that the global market demand for 3-HP will reach US$10 billion by 2025. Synthesis technology is a crucial factor restricting the large-scale application of 3-HP. 3-HP synthesis technologies include two main categories: chemical synthesis and biosynthesis. Chemical synthesis is currently the mainstream production process, but it suffers from problems such as complex processes and severe pollution. In recent years, 3-HP synthesis technology based on engineered microorganisms has become a focus of research in universities both domestically and internationally. However, the production of 3-HP using biosynthesis still faces challenges such as low strain production efficiency and poor stability.

[0005] In conclusion, developing efficient and stable engineered strains remains one of the urgent problems to be solved in the field of 3-HP biosynthesis. Summary of the Invention

[0006] In view of the shortcomings of existing technologies and practical needs, this invention provides a method for improving the conversion rate of ethanol to acetyl-CoA in yeast, an engineered strain and its application in the production of 3-hydroxypropionic acid, in order to obtain an engineered strain that can produce 3-HP efficiently and stably.

[0007] To achieve this objective, the present invention adopts the following technical solution:

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

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

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

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

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

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

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

[0015] In this invention, based on the engineered bacteria that stably synthesize 3-HP, the efficient conversion pathway from 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 improve the yield of 3-HP.

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

[0017] It is understood that this invention is based on a systematic review and modification of the metabolic pathway from ethanol to acetyl-CoA by Saccharomyces cerevisiae. Yeast strains with similar genomes and metabolic networks are theoretically applicable to this invention, and optionally include Saccharomyces cerevisiae IMX581, etc.

[0018] Optionally, the nucleic acid sequence of the mutACC1 gene is shown in SEQ ID No. 1, the nucleic acid sequence of the MCRC gene is shown in SEQ ID No. 2, the nucleic acid sequence of the MCRN gene is shown in SEQ ID No. 3, the nucleic acid sequence of the CaGAPDH gene is shown in SEQ ID No. 4, and the nucleic acid sequence of the ADA gene is shown in 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 engineered strain described in the first aspect, which stably produces 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources, the method comprising:

[0030] Starting with yeast strains, 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 modification strategy designed in this invention, gene knockout or overexpression methods commonly used in the field are also applicable to this invention.

[0032] Thirdly, the present invention provides the application of the yeast engineered strain described in the first aspect, which utilizes glucose and ethanol as dual carbon sources to stably produce 3-hydroxypropionic acid, in the production of 3-HP.

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

[0034] The engineered yeast strain described in the first aspect, which stably produces 3-HP using glucose and ethanol as dual carbon sources, is cultured, and the bacterial culture is collected for product separation and purification to obtain the 3-HP.

[0035] Based on the engineered bacteria constructed in this invention, 3-HP can be produced efficiently using culture and purification methods commonly used in the field.

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

[0037] This invention systematically streamlines and modifies the metabolic pathway from ethanol to acetyl-CoA in *Saccharomyces cerevisiae*, effectively improving the conversion rate of ethanol to 3-HP. Furthermore, through adaptive laboratory evolution, it reshapes intracellular reducing power and energy balance, achieving stable synthesis of 3-HP in *Saccharomyces cerevisiae*. In addition, by reshaping yeast central metabolism, this invention provides a new approach for the efficient conversion of low-carbon compounds using synthetic biology techniques. Attached Figure Description

[0038] Figure 1 A schematic diagram of the metabolic pathway modification of engineered Saccharomyces cerevisiae strains.

[0039] Figure 2 HPLC chromatograms for the detection of 3-HP standard and engineered bacterial fermentation broth.

[0040] Figure 3 The graph shows the statistical results of the yield of 3-HP and the growth of engineered Saccharomyces cerevisiae strains after modification. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely 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 is determined by the claims.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0043] This invention provides a method for strain modification based on orthogonalization of metabolic pathways to improve the conversion efficiency of ethanol to acetyl-CoA and its downstream metabolites in yeast cells, a recombinant yeast strain constructed using this 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 recombinant yeast HP01 with the glycolysis pathway interrupted;

[0045] (2) The gene expression cassettes of the related enzyme genes Zwf1 (GenBank accession number: YNL241C), Gnd1 (GenBank accession number: YHR183W), Tal1 (GenBank accession number: YLR354C), Rki1 (GenBank accession number: YOR095C), Rpe1 (GenBank accession number: YJL121C) and Tkl1 (GenBank accession number: YPR074C) on the pentose phosphate pathway 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 Clostridium acetobutylicum CaGAPDH gene (SEQ ID No.4) fused with 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 on the glyoxylic acid cycle were knocked out to obtain strain HP04;

[0048] (5) The expression cassette of the acetaldehyde dehydrogenase ADA gene (SEQ ID No. 5) of Dickeya zeae was integrated 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 strain HP07 was obtained by knocking out the acetyl-CoA synthase genes acs1 (GenBank accession number: YAL054C) and acs2 (GenBank accession number: YLR153C) and the acetyl-CoA hydrolase gene ach1 (GenBank accession number: YBL015W).

[0051] (8) The strain HP08 was obtained by knocking out 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).

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

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

[0054] Example 1

[0055] This embodiment constructs a recombinant genetically engineered strain that produces 3-HP through fermentation using glucose and ethanol as dual carbon sources.

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

[0057] Following 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.), yeast pfk1 and pfk2 cleavage gRNA primers were designed. These 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 using a clean-up kit. Precisely cleaved plasmids were then 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 a template, the plasmid backbone was amplified with primer CRISPR plasmid back F&R (5'-gatcatttatctttcactgcggagaag-3'). 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 2μm fragment and plasmid backbone were assembled using the Gibson Assembly method to construct the cleavage plasmid at the specified site in this application; the ligation product was transformed into E. coli DH5α to obtain the transformation product; the transformation product was plated on LB solid medium (containing a final concentration of 100 mg / L ampicillin), and after obtaining single clones, it was cultured in shake flasks at 37℃ and 220 rpm for 10 h, and then the plasmid was extracted for sequencing verification. If the verification was correct, the pfk1 and pfk2 knockout plasmids were obtained.

[0069] Using the *Saccharomyces cerevisiae* genome as a template, pfk1 and pfk2 repair fragments were obtained by PCR. The obtained repair fragments and precisely cut plasmids were then transformed into *Saccharomyces cerevisiae* cells. The specific steps are as follows: Fresh *Saccharomyces cerevisiae* IMX581 clones were picked and cultured overnight in 1 mL YPD medium (Yeast Extract, Peptone, Dextrose). An appropriate amount of the bacterial culture was then transferred to 20 mL YPD medium to initiate OD. 600 =0.1, 30℃, 200rpm incubation until OD 600 =0.6, centrifuge at 3000×g to remove the culture medium, resuspend the cell pellet in 1 mL of sterile water, centrifuge at 3000×g to remove the supernatant, add 1 mL of 0.1M lithium acetate to resuspend, centrifuge at 3000×g to remove the supernatant, add 200 μL of 0.1M lithium acetate to obtain competent yeast cells. The obtained yeast competent cells were used to construct recombinant Saccharomyces cerevisiae by lithium acetate / polyethylene glycol transformation. The transformation system is as follows: polyethylene glycol 3500 (50% w / v), 120 μL; lithium acetate (1.0M), 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 the yeast engineered strain HP01 was obtained by transformation.

[0070] (2) Enhance the expression of 6 genes in the PP pathway

[0071] Furthermore, taking the construction method of yeast engineered strain HP01 as an example, using the genome of Saccharomyces cerevisiae as a template, the PCR products of expression cassettes of glucose hexaphosphate dehydrogenase Zwf1, 6-phosphate gluconate 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. Using CRISPR / Cas9 gene editing technology, the expression cassettes of the above six enzymes were integrated into the genome of yeast engineered strain HP01 to obtain strain HP02.

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

[0073] Furthermore, taking the construction method of yeast engineered strain HP01 as an example, the expression cassette of 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 CaGAPDH gene (SEQ ID No.4) of Clostridium acetobutylicum fused with endogenous NADH kinase ScPOS5 was integrated into the genome of strain HP02 to obtain strain HP03.

[0074] Example 2

[0075] This embodiment describes the modification of an efficient conversion pathway from ethanol to 3-HP in Saccharomyces cerevisiae.

[0076] (1) Disruption of the glyoxylic acid cycle

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

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

[0079] Furthermore, taking the construction method of yeast engineered strain HP01 as an example, the acetaldehyde 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, the repair fragments of the acetaldehyde dehydrogenase genes ald2, ald3, ald4, ald5 and ald6 were obtained by PCR. Using CRISPR / Cas9 gene editing technology, the above 5 genes were knocked out in strain HP05 to obtain strain HP06.

[0081] Furthermore, using the genome of Saccharomyces cerevisiae as a template, the repair fragments of the acetyl-CoA synthase genes acs1 and acs2, and the acetyl-CoA hydrolase gene ach1 were obtained by PCR. Using CRISPR / Cas9 gene editing technology, the above three genes were knocked out in strain HP06 to obtain strain HP07.

[0082] (3) Disrupt 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. Using CRISPR / Cas9 gene editing technology, these four genes were knocked out in strain HP07 to obtain strain HP08. Subsequently, the modified Saccharomyces cerevisiae underwent adaptive evolution to obtain the evolved strain HP09. After adaptive evolution, the growth rate of the engineered strain recovered, and the yield of 3-HP was significantly increased.

[0084] Example 3

[0085] This embodiment describes the preparation of product 3-HP.

[0086] To evaluate the ability of the modified strain to synthesize 3-hydroxypropionic acid, the starting strain and the modified strain were shake-flask fermented separately. The specific steps are as follows: Fresh yeast clones were picked and transferred to 1 mL of YPGE medium (yeast-rich medium with 2% (w / v) glycerol and 2% (w / v) ethanol as carbon sources), cultured overnight, and an appropriate amount of the bacterial culture was transferred to 15 mL of DelftDE (yeast-inorganic salt medium with 0.5% or 1% glucose and 2% ethanol as carbon sources) to initiate OD. 600 =0.5, 30℃, 200rpm incubation. After fermentation to the plateau phase, the bacterial culture was collected, centrifuged at 12000rpm for 1min at 4℃, and the supernatant was collected. After filtration through a 0.22μm filter membrane, liquid chromatography analysis was performed. The liquid chromatography analysis method is as follows: an Aminex HPX87H column (1300×7.8mm, particle size 9μm) was used, the column temperature was 50℃, and the injection volume was 5μL. The mobile phase was deionized water containing 5mM sulfuric acid, and the flow rate was 0.6mL / min. The HPLC chromatograms of 3-HP standard and HP03 strain fermentation broth detection (exemplary results for HP03 strain are shown below) are as follows. Figure 2 As shown. The method for calculating 3-HP yield includes: constructing a standard curve using standards, and detecting and quantifying 3-HP in the fermentation broth.

[0087] Results of cell biomass and 3-HP yield are as follows: Figure 3 As shown, by Figure 3 It is known that by knocking out the pfk1 and pfk2 genes in Saccharomyces cerevisiae and simultaneously overexpressing Zwf1, Gnd1, Tal1, Rki1, Rpe1, Tkl1, mutACC1, MCRC, MCRN, and the fused CaGAPDH and Pos5 genes, a recombinant strain (HP03) that stably produces 3-HP using glucose and ethanol as dual carbon sources can be successfully constructed. Furthermore, by adopting optimization strategies, 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 these can further increase the yield of 3-HP.

[0088] In summary, this invention effectively improved the conversion rate of ethanol to 3-HP by systematically streamlining and modifying the metabolic pathway from ethanol to acetyl-CoA in Saccharomyces cerevisiae. Furthermore, through adaptive laboratory evolution to reshape intracellular reducing power and energy balance, it achieved stable synthesis of 3-HP in Saccharomyces cerevisiae. In addition, by reshaping yeast central metabolism, this invention provides a new approach for the efficient conversion of low-carbon compounds using synthetic biology techniques.

[0089] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A yeast engineering strain for stable production of 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources, characterized in that, The engineering bacteria take Saccharomyces cerevisiae IMX581 as a starting strain, and knock out pfk1 and pfk2 genes; The engineered bacteria also overexpressed mutACC1 , MCRC , MCRN , Zwf1 , Gnd1 , Tal1 , Rki1 , Rpe1 , Tkl1 And integration CaGAPDH and Pos5 Gene; The mutACC1 The gene nucleic acid sequence is shown in SEQ ID No.

1. MCRC The gene nucleic acid sequence is shown in SEQ ID No.

2. MCRN The gene nucleic acid sequence is shown in SEQ ID No.

3. Zwf1 The gene's accession number is YNL241C. Gnd1 The gene's accession number is YHR183W. Tal1 The gene's accession number is YLR354C. Rki1 The gene's accession number is YOR095C. Rpe1 The gene accession number is YJL121C and Tkl1 The gene's accession number is YPR074C. CaGAPD The H gene nucleic acid sequence is shown in SEQ ID No.

4. Pos5 The gene's accession number is YPL188W.

2. The yeast engineering bacteria for stable production of 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 stable production of 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to claim 2, characterized in that, The engineered bacteria also overexpress a gene of the nucleotide sequence as shown in SEQ ID NO. 5 ADA 5.

4. The yeast engineering bacteria for stable production of 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to claim 3, characterized in that, The engineered bacteria also lack ald2 , ald3 , ald4 , ald5 and ald6 genes.

5. The yeast engineering bacteria for stable production of 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to claim 4, characterized in that, The engineered bacteria also lack acs1 、 acs2 and ach1 genes.

6. The yeast engineering bacteria for stable production of 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to claim 5, characterized in that, The engineered bacteria also lack yat1 , yat2 , crc1 and cat2 Gene.

7. A method for preparing the engineered yeast strain for stable production of 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources according to any one of claims 1-6, characterized in that, The method includes: Using Saccharomyces cerevisiae IMX581 as the starting strain, knockout pfk1 and pfk2 Genes, and overexpression mutACC1 , MCRC , MCRN , Zwf1 , Gnd1 , Tal1 , Rki1 , Rpe1 , Tkl1 And integration CaGAPDH and Pos5 Gene; The mutACC1 The gene nucleic acid sequence is shown in SEQ ID No.

1. MCRC The gene nucleic acid sequence is shown in SEQ ID No.

2. MCRN The gene nucleic acid sequence is shown in SEQ ID No.

3. Zwf1 The gene's accession number is YNL241C. Gnd1 The gene's accession number is YHR183W. Tal1 The gene's accession number is YLR354C. Rki1 The gene's accession number is YOR095C. Rpe1 The gene accession number is YJL121C and Tkl1 The gene's accession number is YPR074C. CaGAPD The H gene nucleic acid sequence is shown in SEQ ID No.

4. Pos5 The gene's accession number is YPL188W.

8. The application of the yeast engineered strain according to any one of claims 1-6, which stably produces 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources, in the production of 3-hydroxypropionic acid.

9. A method for producing 3-hydroxypropionic acid, characterized by, The method includes: The yeast strain according to any one of claims 1-6 that stably produces 3-hydroxypropionic acid using glucose and ethanol as dual carbon sources is cultured, and the bacterial culture is collected for product separation and purification to obtain the 3-hydroxypropionic acid.

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