An engineered bacterium producing 3-hydroxypropionic acid, its construction method and application

Abstract

This application discloses an engineered bacterium producing 3-hydroxypropionic acid, its construction method, and its application. The construction method includes: introducing the MCRC gene and MCRN gene into a host strain, and modifying the promoter P of the FAS1 gene of the host strain. FAS1 Replace with P HXT1 The MCRC gene encodes a polypeptide consisting of amino acids 1-549 of malonyl-CoA reductase from Chloroflexus aurantiacus; the MCRN gene encodes a polypeptide consisting of a mutant N940V / K1106W / S1114R consisting of amino acids 550-1219 of malonyl-CoA reductase from Chloroflexus aurantiacus; and the host strain is selected from any of the Saccharomyces cerevisiae species.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering and industrial biotechnology, and relates to an engineered bacterium that produces 3-hydroxypropionic acid, its construction method and application. Background Technology

[0002] 3-Hydroxypropionic acid (3HP) is listed by the U.S. Department of Energy as one of the 12 most valuable platform compounds for biomass sources. 3-HP polymerizes to form poly3-hydroxypropionic acid (poly3-HP), a biodegradable plastic that can reduce white pollution. 3-HP can be dehydrated to obtain acrylic acid, an important monomer for synthetic resins with a very fast polymerization rate, used to synthesize resins, coatings, rubber, etc. 3-HP can be dehydrated and nitrified to obtain acrylonitrile, an important monomer for synthetic fibers, synthetic rubber, and synthetic resins. Acrylonitrile fiber, or acrylic fiber, is produced from acrylonitrile (Chen et al. Metab. Eng., 2014, 22, 104-109). Traditional 3-HP synthesis methods are chemical synthesis methods, mainly involving the reaction of 3-hydroxypropionitrile in sodium hydroxide solution. Chemical methods require large amounts of solvents, which is not environmentally friendly.

[0003] 3-HP biosynthesis is gaining increasing attention due to its use of inexpensive raw materials, mild reaction conditions, and environmental friendliness. Currently, biosynthetic routes primarily utilize biomass-derived sugars or glycerol as raw materials, synthesized through fermentation using microbial cells as reactors. *Saccharomyces cerevisiae* is an important eukaryotic model microorganism with a clear genetic background, relatively easy metabolic engineering, strong robustness, and good acid resistance, making it suitable for the industrial production of organic acids. However, current literature reports that *Saccharomyces cerevisiae* synthesis of 3-HP generally suffers from low yields and low conversion rates. For example, the highest yield from shake-flask fermentation is 2 g / L (Hellgren et al. Metab. Eng., 2020, 62, 150-160), and the highest yield from fed-batch fermentation in fermenters is 18.1 g / L (Tong et al. Green Chem. 2021, 23, 4502-4509). Summary of the Invention

[0004] This application constructs a yeast cell factory from a global metabolic perspective to achieve efficient 3-HP synthesis. First, the 3-HP synthase gene is optimized to improve its biocatalytic efficiency, and the neutral carbon metabolic pathway is enhanced to provide the precursor acetyl-CoA and cofactor NADPH. This results in a 3-HP yield of 4.4 g / L in shake flasks and 56.5 g / L in fed-batch fermentation of *Saccharomyces cerevisiae*. The same strategy was then applied to another yeast strain, and diploid strains were obtained by crossbreeding different strains, achieving a 3-HP yield of 70.2 g / L in fed-batch fermentation.

[0005] According to one aspect of this application, a method for constructing an engineered bacterium that produces 3-hydroxypropionic acid is provided. This method involves introducing the MCRC gene and MCRN gene into a Saccharomyces cerevisiae host strain and replacing the promoter of the FAS1 gene in the host strain. The resulting strain uses glucose as a substrate and synthesizes 3-hydroxypropionic acid with high efficiency.

[0006] A method for constructing an engineered bacterium that produces 3-hydroxypropionic acid, the method comprising:

[0007] The MCRC and MCRN genes were introduced into the host strain, and the promoter P of the FAS1 gene of the host strain was also introduced. FAS1 Replace with P HXT1 ;

[0008] The polypeptide encoded by the MCRC gene is amino acid 1-549 of malonyl-CoA reductase derived from Chloroflexus aurantiacus.

[0009] The polypeptide encoded by the MCRN gene is a mutant N940V / K1106W / S1114R, consisting of 550-1219 amino acids of malonyl-CoA reductase from Chloroflexus aurantiacus.

[0010] The host strain is selected from any of the Saccharomyces cerevisiae.

[0011] Optionally, the nucleotide sequence of the MCRC gene is shown in SEQ ID NO: 2;

[0012] The nucleotide sequence of the MCRN gene is shown in SEQ ID NO: 1.

[0013] Optionally, the introduction of the MCRC gene and MCRN gene into the host strain is selected from any one of (a) to (c):

[0014] (a) DNA fragment P GAL7 -MCRN-T DIT1 Integration into the XI-1 site of the host strain; containing P GAL1,10 -MCRC-T TDH2 The free plasmid was transferred into the host strain;

[0015] (b) DNA fragment P GAL7 -MCRN-T DIT1 Integration into the XI-1 site of the host strain; containing P GAL1,10 -MCRC-T TDH2 The free plasmid was transferred into the host strain; the DNA fragment P was transferred into the host strain. TDH3 -MCRN-T FBA1 -TDIT1 -MCRC-P TDH3 It integrates into the XII-3 site of the host strain;

[0016] (c) DNA fragment P GAL7 -MCRN-T DIT1 Integration into the XI-1 site of the host strain; the DNA fragment P TDH3 -MCRN-T FBA1 -T DIT1 -MCRC-P TDH3 Integration into the XII-3 site of the host strain; the DNA fragment T FBA1 -MCRN-P GAL1,10 -MCRC-T DIT1 It integrates into the XII-5 site of the host strain.

[0017] Optionally, the construction method further includes enhancing the central carbon metabolism pathway:

[0018] The genes MmACL, RtME, 'MDH3, CTP1, MPC1, MPC3, AnACLa, AnACLb, RtCIT1, IDP2, YHM2, GND1, TKL1, TAL1, and ZWF1 were introduced into the host strain.

[0019] The promoter of the PYC1 gene of the host strain was changed from P PYC1 Replace with P TEF ;

[0020] The promoter of the PGI1 gene of the host strain was changed from P PGI1 Replace with P COX9 ;

[0021] The promoter of the ACC1 gene of the host strain was changed from P ACC1 Replace with P TEF ;

[0022] The promoter of the IDH2 gene of the host strain was changed from P IDH2 Replace with P GSY1 .

[0023] Optionally, the construction method further includes enhancing the central carbon metabolism pathway:

[0024] DNA fragment P TPI -MmACL-T FBA1 -T CYC1 -RtME-P TDH3 -P tHXT7 -'MDH3-T TDH2 -T ADH1 -CTP1-P PGK1It integrates into the HIS3 site of the host strain;

[0025] The promoter of the PYC1 gene of the host strain was changed from P PYC1 Replace with P TEF ;

[0026] DNA fragment P TPI1 -MPC1-T MPC1 -T DIT1 -MPC3-P PGK1 It integrates into the X-4 site of the host strain;

[0027] DNA fragment T CYC1 -AnACLa-P GAL1,10 -AnACLb-T ADH1 It integrates into the X-2 site of the host strain;

[0028] DNA fragment P TPI1 -RtCIT1-T FBA1 -T CYC1 -IDP2-P TDH3 -P TEF1 -YHM2-T GAL1 It integrates into the GAL1, GAL7, and GAL10 gene loci of the host strain, that is, it integrates the DNA fragment while knocking out the three genes GAL1, GAL7, and GAL10.

[0029] DNA fragment P COX9 -T CYC1 -GND1-P TDH3 -P tHXT7 -TKL1-T TDH2 -T ADH1 -TAL1-P PGK1 -P TEF1 -ZWF1-T ZWF1 P integrated into the host strain PGI1 The site, namely the integration of GND1, TKL1, TAL1, and ZWF1 genes, simultaneously removes the PGI1 promoter from P PGI1 Replace with P COX9 ;

[0030] The ACC1 gene promoter of the host strain was changed by P ACC1 Replace with P TEF ;

[0031] The IDH2 gene promoter of the host strain was changed by P IDH2 Replace with P GSY1 .

[0032] Optionally, the integration of DNA fragments into the host strain or the replacement of the promoter of a gene in the host strain can be achieved using CRISPR / Cas9 technology.

[0033] Optionally, the construction method further includes reintroducing the URA3 gene.

[0034] Optionally, the construction method further includes cell fusion to form a diploid.

[0035] Optionally, the host strain is selected from either Saccharomyces cerevisiae CEN.PK 113-11C or Saccharomyces cerevisiae CEN.PK 110-10C.

[0036] According to one aspect of this application, an engineered bacterium producing 3-hydroxypropionic acid is provided, constructed according to the construction method described above.

[0037] According to one aspect of this application, an engineered bacterium producing 3-hydroxypropionic acid, constructed according to the above-described construction method, is provided, and the application of the above-described engineered bacterium producing 3-hydroxypropionic acid in the preparation of 3-hydroxypropionic acid is provided.

[0038] As one implementation scheme, this invention discloses several engineered Saccharomyces cerevisiae strains producing 3-hydroxypropionic acid (3-HP) and their applications. The engineered Saccharomyces cerevisiae strain is derived from the wild-type strain CEN.PK 113-11C (a type). By optimizing the expression of the malonyl-CoA reductase gene and enhancing the central carbon metabolism pathway, an engineered strain capable of efficiently synthesizing 3-HP was obtained, achieving yields of 4.4 g / L in shake-flask fermentation and 56.5 g / L in fed-batch fermentation in a fermenter. The same strategy was employed in Saccharomyces cerevisiae CEN.PK 110-10C (α type), and then two engineered strains with different morphologies were crossbred to obtain a diploid engineered strain, achieving a yield of 70.2 g / L in fed-batch fermentation in a fermenter.

[0039] As one implementation, this application provides a 3-HP-producing Saccharomyces cerevisiae genetically engineered strain, which optimizes the expression of malonyl-CoA reductase (MCR) gene derived from Chloroflexus aurantiacus, and divides the MCR that catalyzes 3-HP synthesis into N-terminus and C-terminus; and achieves stable intracellular gene expression by replacing free plasmids through genome integration.

[0040] As one implementation scheme, this application provides a genetically engineered Saccharomyces cerevisiae strain that produces 3-HP, which enhances the central carbon metabolism pathway and directs more carbon metabolism toward the synthesis of 3-HP.

[0041] As one implementation, this application provides a diploid engineered strain of Saccharomyces cerevisiae that produces 3-HP. The 3-HP synthesis strategy of type α Saccharomyces cerevisiae (optimized MCR expression and enhanced central carbon metabolism pathway) is replicated into wild-type type α Saccharomyces cerevisiae strains, and the diploid strain is obtained by mating two yeasts with different morphologies.

[0042] As one implementation method, the construction method provided in this application optimizes the expression of the rate-limiting enzyme MCR in the 3-HP synthesis pathway by splitting MCR into an N-terminus (malonyl-CoA reductase, MCRN, 1-549 amino acids) and a C-terminus (malonyl hemialdehyde reductase, MCRC, 550-1219 amino acids), and using a mutant (N940V / K1106W / S1114R) for MCRC to improve enzyme catalytic efficiency;

[0043] As one implementation scheme, the construction method provided in this application utilizes genome integration instead of free plasmid expression of the MCR gene, integrating P into the XI-1 site of *Saccharomyces cerevisiae*. GAL7 Promoter expression MCRN(P) GAL7 -MCRN), in XII-3 integrated P TDH3 -MCRC+P TDH3 -MCRN, integrating MCRC-P at site XII-5 GAL1,10 -MCRC, thereby replacing the expression of MCRC in plasmid pYX8;

[0044] As one implementation method, the construction method provided in this application enhances the central metabolic pathway to increase the supply of precursor acetyl-CoA and cofactor NADPH, including overexpression of the ATP-dependent citrate lyase gene MmACL derived from mice (Mus musculus) and NADP derived from Rhodospuridium toruloides. + The following genes are mentioned: malate synthase RtME and citrate synthase 1 RtCIT1; malate dehydrogenase 'MDH3' (removing peroxisome signal peptide); citrate transporter CTP1; pyruvate carboxylase 1 PYC1; pyruvate transporter 1 and 3 genes MPC1 and MPC3; ATP-dependent citrate lyase a and b genes AnACLa and AnACLb from Aspergillus niger; and NADP. + The following genes were selected: citrate-dependent isocitrate dehydrogenase 2 (IDP2), citrate / α-ketoglutarate transporter (YHM2), glucose-6-phosphate dehydrogenase (ZWF1), gluconeate-6-phosphate dehydrogenase (GND1), ketone transoxidase (TKL1), and aldehyde transoxidase (TAL1). A strong promoter P was used. GSY1 Replace the IDH2 promoter to enhance mitochondrial NAD+ Expression of the isocitrate dehydrogenase gene IDH2 using the weak promoter P COX9 Replacing the PGI1 promoter weakens the expression of the phosphoglucose isomerase gene PGI1, using a strong promoter P. TEF1 Replace the PYC1 promoter itself to enhance the expression of the pyruvate carboxylase gene PYC1; use the strong promoter P TEF1 Replace the ACC1 promoter to enhance the expression of the acetyl-CoA carboxylase gene ACC1;

[0045] As one implementation method, the construction method provided in this application integrates and expresses the MCR gene (as in claim 5) and the central carbon metabolism pathway gene (as in claim 6) in a wild-type strain of α-type Saccharomyces cerevisiae, and the resulting engineered strain is fused with the α-type engineered strain to obtain a diploid strain.

[0046] The beneficial effects that can be obtained from this application include:

[0047] (1) The method for constructing engineered bacteria that produce 3-hydroxypropionic acid provided in this application optimizes the 3-HP synthase gene and improves its biocatalytic efficiency.

[0048] (2) The method for constructing engineered bacteria that produce 3-hydroxypropionic acid provided in this application strengthens the central carbon metabolism pathway to provide precursor acetyl-CoA and cofactor NADPH, thereby increasing the yield of 3-HP in Saccharomyces cerevisiae.

[0049] (3) The method for constructing engineered bacteria that produce 3-hydroxypropionic acid provided in this application obtains diploids by mating engineered bacteria of different types, which further improves the yield of 3-HP. Attached Figure Description

[0050] Figure 1 A schematic diagram illustrating the metabolic engineering strategy that promotes the synthesis of 3-HP in Saccharomyces cerevisiae is shown.

[0051] Figure 2 This demonstrates how enhanced central carbon metabolism increases 3-HP production;

[0052] Figure 3 This demonstrates how optimized MCR expression improves 3-HP yield;

[0053] Figure 4 The results of fed-batch fermentation in the SH14 fermenter are shown; where A represents the yield of 3-HP during fed-batch fermentation in the fermenter; and B represents the plasmid loss during fed-batch fermentation in the fermenter.

[0054] Figure 5 The results show the yield of 3-HP in the fed-batch fermentation of the SH18 fermenter;

[0055] Figure 6The results show the yield of 3-HP fermentation in a fed-batch fermenter of diploid SH70 fermenter. Detailed Implementation

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0057] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0058] Saccharomyces cerevisiae CEN.PK 113-11C, CEN.PK 110-10C, and S288C can be purchased from Euroscarf GmbH in Germany.

[0059] The schematic diagram of the metabolic engineering strategy used in this application to promote the synthesis of 3-HP in Saccharomyces cerevisiae is shown below. Figure 1 As shown.

[0060] The technical problem to be solved by this invention is to overcome the difficulties of low efficiency and metabolic mismatch of the rate-limiting enzyme MCR in the biosynthesis of 3-HP in current engineered yeast strains. This invention aims to increase the biosynthesis efficiency of 3-HP by optimizing MCR expression and enhancing global carbon metabolism, and provides several 3-HP engineered yeast strains and their applications.

[0061] In a first aspect, the present invention provides a method for constructing a 3-HP-producing Saccharomyces cerevisiae strain, which can significantly increase the yield of 3-HP.

[0062] The construction method includes: MCR expression optimization and enhancement of neutral carbon metabolism pathway.

[0063] In one specific implementation plan, a CRISPR / Cas9 system is first constructed, and a complete homologous recombination fragment containing the upstream and downstream homologous arms of the integration site XI-5 and the promoter P is obtained through fusion PCR. TEF1 Termination of sub-T CYC1The Cas9 gene and the selection marker resistance KanMX gene were amplified using the CEN.PK113-11C genome (purchased from Euroscarf, Germany) as templates. The fragments were then fused into homologous recombination fragments using overlap extension PCR (OE-PCR) (the fusion fragment construction process is described in Zhou et al. J Am Chem Soc 2012, 134:3234-3241). 500 ng of the homologous recombination fragment was transformed into the *Saccharomyces cerevisiae* starting strain CEN.PK113-11C to obtain the *Saccharomyces cerevisiae* strain SH01 expressing the Cas9 protein. Then, MCRN was integrated into the XI-1 site of the genome. The MCRN gene (nucleotide sequence as shown in SEQ ID NO: 1) was amplified using the gene synthesis plasmid as a template. The upstream and downstream homologous arms XI-1up and XI-1dw, and the promoter P... GAL7 and Termination T DIT2 Obtained by amplification using the CEN.PK113-11C genome as a template, XI-1up and P GAL7 MCRN gene, T DIT1 The XI-1dw plasmid was fused with donor DNA using OE-PCR and transformed into Saccharomyces cerevisiae SH01(MATa;MAL2-8c;SUC2;his3Δ1;ura3-52;XI-5::P) along with the gRNA plasmid. TEF1 -Cas9-T CYC1 The transformants were plated onto SD+His selection plates and incubated at 30℃ for 2–3 days. After culturing in liquid YPD medium, the transformants were verified as correct by colony PCR. They were then plated onto plates containing 5-fluoroorotic acid for plasmid loss. The plasmid-loss-free strains were stored for later use, yielding the engineered strain SH20. Other genome editing procedures for *Saccharomyces cerevisiae* described below followed a similar procedure. (P is replaced.) FAS1 The promoter is a glucose-responsive promoter P. HXT1 To reduce competition between fatty acid synthesis and 3-HP synthesis for the precursor malonyl-CoA and cofactor NADPH, the FAS1 promoter (500 bp) was amplified with 500 bp upstream and downstream homologous arms and the promoter P... HXT1 Donor DNA was obtained through OE-PCR fusion and transformed into SH20 along with FAS1 pgRNA, successfully yielding SH21. To achieve high expression of MCRC, the MCRC gene (nucleotide sequence as shown in SEQ ID NO: 2) was amplified using the pYX312 plasmid (preserved in the laboratory) as a backbone and the gene synthesis plasmid as a template. The promoter P... GAL1,10 and Termination T TDH2 The MCRC gene and P were amplified using the CEN.PK113-11C genome as a template. GAL1,10 and TTDH2 The donor DNA was fused using OE-PCR and seamlessly ligated with the pYX312 backbone to form a circular plasmid, which was then transformed into competent E. coli cells. Sequencing confirmed the correctness of the obtained plasmid pYX8. The plasmid was then transformed into Saccharomyces cerevisiae to obtain engineered strain SH22. The strain was fermented in shake flasks with 20 g / L glucose as the carbon source. HPLC analysis showed that the 3-HP yield reached 410 mg / L.

[0064] Furthermore, to enhance the central carbon metabolism pathway, expression cassettes (Table 1) were constructed using different combinations of promoters and terminators for the central metabolism-related enzyme genes with the help of the CRISPR / Cas9 system and integrated into different sites of the genome to obtain the engineered bacterium SH03 with enhanced central carbon metabolism pathway.

[0065] Table 1.

[0066]

[0067]

[0068] Based on the background bacterium SH03 with enhanced neutral carbon metabolism, MCRN was integrated into the pYX8 free plasmid to express MCRC, and the FAS1 promoter was replaced with the glucose-responsive promoter P. HXT1 The engineered strain SH11 was obtained, and the yield of 3-HP reached 1.4 g / L during shake-flask fermentation, indicating that strengthening the neutral carbon metabolism pathway can divert more carbon metabolism to 3-HP synthesis.

[0069] Since MCR is the rate-limiting enzyme in 3-HP synthesis, it integrates P, controlled by a constitutive promoter, based on SH11. TDH3 -MCRN+P TDH3 -MCRC strain SH14 was obtained, and the yield was 2.4 g / L in shake flask fermentation. Fed-batch fermentation in a fermenter reached a yield of 5.8 g / L after 24 hours, but no further increase was observed. Plasmid loss was found during dilution plating. Therefore, to ensure stable gene expression, MCRC-P was integrated with a double-copy genome. GAL1,10 Using MCRC instead of plasmid expression, strain SH17h was obtained, and the yield of 3-HP reached 3.6 g / L during shake-flask fermentation. Replenishment of the URA3 gene as a selection marker yielded strain SH18, which achieved a 3-HP yield of 4.4 g / L during shake-flask fermentation and 56.5 g / L during fed-batch fermentation in a fermenter.

[0070] In a second aspect, the present invention provides a method for constructing a diploid strain of Saccharomyces cerevisiae that produces 3-HP, thereby further improving the yield of 3-HP.

[0071] The construction method includes: expressing optimized MCR in CEN.PK 110-10C (α-type Saccharomyces cerevisiae, purchased from Euroscarf GmbH, Germany), enhancing the central carbon metabolism pathway, and obtaining diploid engineered strains by crossbreeding MATa and MATα.

[0072] Using the CRISPR / Cas9 system, the Cas9 protein gene was integrated into the XI-5 site of *Saccharomyces cerevisiae* CEN.PK 110-10C to obtain SHα01(MATa; MAL2-8c; SUC2; ura3-52; XI-5::P). TEF1 -Cas9-T CYC1 Then P is integrated at site XI-1. GAL7 -MCRN-T DIT1 In XII-3 integration P TDH3 -MCRN-T FBA1 -T DIT1 -MCRC-P TDH3 T is integrated at site XII-5 FBA1 -MCRN-P GAL1,10 -MCRC-T DIT1 Replace P FAS1 P is a glucose-responsive promoter HXT1 To enhance the central carbon metabolism pathway (same as Table 1), the above operations were performed as above to obtain the engineered strain SHα23.

[0073] The HIS3 gene in SH17h and the LYS3 gene in SHα23 were knocked out, respectively. The specific implementation plan was as follows: gRNA expression plasmids targeting the HIS3 and LYS3 genes were constructed. Then, 300 bp upstream and downstream of the HIS3 and LYS3 genes, as well as the ORF frames of the HIS3 and LYS3 genes, were amplified, and donor DNA was obtained by OE-PCR. Subsequently, the gRNA expression plasmids and donor DNA (500 ng each) were chemically transformed into *Saccharomyces cerevisiae* SH17h and SHα23, respectively. The transformed strains were plated on screening plates SD+His and SD+Lys and incubated statically at 30℃ for 2–3 days. After the transformants were cultured in liquid YPD medium and verified by colony PCR, they were plated on plates containing 5-fluoroorotic acid for plasmid loss. The strains after plasmid loss were named SH17 and SHα24, respectively. Furthermore, the engineered bacteria SH17 and SHα24 were cultured separately in YPD medium, and then 0.5 mL of each bacterial culture was mixed and inoculated into 20 mL of YPD medium and cultured for 48 h. The fermentation broth was centrifuged, washed twice with water, appropriately diluted, and spread on SD+URA3 solid medium. The single colonies that grew were diploids, named SH70. The 3-HP yield reached 70.2 g / L in the fed-batch fermentation of the fermenter.

[0074] Example 1: Optimization of MCR expression + replacement of the FAS1 promoter with a glucose-responsive promoter P HXT1 (Engineering bacteria SH22)

[0075] The malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus (NCBI accession number WP_012258473.1) was split into an N-terminus (MCRN, 1-549) and a C-terminus (MCRC, 550-1219). The MCRC was improved by using a high-efficiency mutant (N940V / K1106W / S1114R). The MCRC and MCRN genes were codon optimized and synthesized according to the codon preference of Saccharomyces cerevisiae. The nucleotide sequence of the optimized MCRN is shown in SEQ ID NO: 1, and the nucleotide sequence of the optimized MCRC is shown in SEQ ID NO: 2.

[0076] MCRN is integrated into the XI-1 site of the genome. The MCRN gene is amplified using a gene synthesis plasmid as a template. The upstream and downstream homologous arms XI-1up and XI-1dw, and the promoter P are also included. GAL7 and Termination T DIT1 Obtained by amplification using the CEN.PK113-11C genome as a template, XI-1up and P GAL7 MCRN gene, T DIT1 The donor DNA was fused with XI-1dw using OE-PCR: XI-1up-P GAL7 -MCRN-T DIT1 -XI-1dw, where the sequence of XI-1up is as shown in SEQ ID NO: 3, and the sequence of XI-1dw is as shown in SEQ ID NO: 4; using pROS10 (purchased by Addgene) as a template, the guide RNA plasmid backbone was amplified using primer 6005 (GATCATTTATCTTTCACTGCGGAGAAG, SEQ ID NO: 21), primer P1 (GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATC, SEQ ID NO: 22) and XI-1gRNA1 (AAACTTCTCCGCAGTGAAAGATAAATGATC TATCATCGCCGAGAAATTTG GTTTTAGAGCTAGAAATAG, SEQ ID NO: 23, where the underlined portion is the guide RNA sequence of XI-1gRNA1) amplification fragment 1, primer P2 and XI-1gRNA2 (AAACTTCTCCGCAGTGAAAGATAAATGATC TCTCGGAGGATTCTTTGCACFragment 2 (GTTTTAGAGCTAGAAATAG, SEQ ID NO: 24, where the underlined part is the guide RNA sequence of XI-1gRNA2) was amplified. These three fragments were ligated into XI-1gRNA plasmid using Novizan one-step ligase (the subsequent guide RNA plasmid construction process is the same, only the 20bp guide RNA sequence is listed). All three fragments were then transformed into Saccharomyces cerevisiae SH01 (MATa; MAL2-8c; SUC2; his3Δ1; ura3-52; XI-5::P). TEF1 -Cas9-T CYC1 This strain is an integration of XI-5::P based on CEN.PK113-11C. TEF1 -Cas9-T CYC1 The transformed strains were obtained, plated onto SD+His selection plates, and incubated at 30°C for 2–3 days. After being cultured in liquid YPD medium, the transformants were verified to be correct by colony PCR. They were then plated onto plates containing 5-fluoroorotic acid for plasmid loss. The strains after plasmid loss were stored for later use. Other genome editing work of Saccharomyces cerevisiae in the following text followed a similar procedure.

[0077] Using the CEN.PK 113-11C genome as a template, the glucose-responsive promoter P was amplified by PCR. HXT1 (Located 500bp upstream of the HXT1 gene) and P FAS (Located 500bp upstream of the FAS1 gene) 300bp upstream as P FAS1 up and downstream 300bp as P FAS1 dw, the donor DNA fragment P was obtained by OE-PCR fusion. FAS1 up-P HXT1 -P FAS1 dw, transform the DNA fragment together with the FAS1 pgRNA (guide RNA sequence: CTTTTTTTCCATACAATTCA, SEQ ID NO: 35) plasmid into the host strain from the previous step, and change the promoter of the FAS1 gene from P FAS1 Replace with P HXT1 It was named SH21.

[0078] To achieve high expression of MCRC, the MCRC gene was amplified using the pYX312 plasmid as a backbone and the gene synthesis plasmid as a template. The promoter P... GAL1,10 and Termination T TDH2 The MCRC gene and P were amplified using the CEN.PK113-11C genome as a template. GAL1,10 and T TDH2 The donor DNA fragment P was obtained by fusion using the OE-PCR method. GAL1,10 -MCRC-TTDH2 The pYX312 backbone was amplified using primers pYX312-F:CTTAATTTCGAATAAACACACATAAACAAACAAAGTTACTCCGCAACGCTTTTCTGAAC (SEQ ID NO: 25) and pYX312-R:CCCCGGGGTCGACCTCGAGTATAGTTTTTTCTCCTTGACGTTAAAGTATAG (SEQ ID NO: 26). GAL1,10 -MCRC-T TDH2 The pYX312 backbone was seamlessly cloned into a circular plasmid and transformed into competent E. coli cells. Sequencing confirmed the correctness of the pYX8, which was then transformed into Saccharomyces cerevisiae SH21. The transformed cells were plated on SD+His selection plates and incubated at 30°C for 2–3 days. The transformed cells were then cultured in liquid YPD medium to obtain engineered bacteria SH22.

[0079] Example 2: Optimization of MCR expression + replacement of the FAS1 promoter with a glucose-responsive promoter P HXT1 +Enhancement of the central carbon metabolism pathway (engineered bacteria SH11)

[0080] Optimizing the central carbon metabolism pathway in Saccharomyces cerevisiae to enhance the supply of precursor malonyl-CoA and cofactor NADPH, the specific process is as follows: Using the CEN.PK113-11C genome as a template, the promoter P was amplified by PCR. TPI P PGK1 P TDH3 P tHXT7 and Termination T CYC1 T TDH2 T ADH1 T FBA1 The homologous arm sequences His3up (300 bp upstream of the HIS3 gene) and His3dw (300 bp downstream of the HIS3 gene) were used as templates for gene synthesis plasmids. MmACL (SEQ ID NO: 5), RtME (SEQ ID NO: 6), and 'MDH3 (SEQ ID NO: 7) were amplified by PCR, and the donor DNA fragment HIS3up-P was obtained by OE-PCR fusion. TPI -MmACL-T FBA1 -T CYC1 -RtME-P TDH3 -P tHXT7 -'MDH3-T TDH2 -T ADH1 -CTP1-P PGK1-HIS3dw, the DNA fragment was transformed into SH01 along with the His3gRNA (guideRNA sequence: CATGCTCTGGCCAAGCATTC, SEQ ID NO: 27) plasmid;

[0081] Using the CEN.PK 113-11C genome as a template, promoter P was amplified by PCR. TEF and P PYC1 (Located 500bp upstream of the PYC gene) 300bp upstream as P PYC1 up and downstream 300bp as P PYC1 dw, the donor DNA fragment P was obtained by OE-PCR fusion. PYC1 up-P TEF -P PYC1 dw, transform the DNA fragment together with the PYC1 pgRNA (guide RNA sequence: CGAGGACATGATTGCTATCG, SEQ ID NO: 28) plasmid into the host strain from the previous step, and change the promoter of the PYC1 gene from P PYC1 Replace with P TEF ;

[0082] Using the CEN.PK 113-11C genome as a template, promoter P was amplified by PCR. TPI1 P PGK1 and Termination T MPC1 T DIT1 In addition, X-4up (SEQ ID NO: 8), X-4dw (SEQ ID NO: 9), and genes MPC1 and MPC3 were fused by OE-PCR to obtain the donor DNA fragment X-4up-P. TPI1 -MPC1-T MPC1 -T DIT1 -MPC3-P PGK1 -X-4dw transforms the DNA fragment along with the X-4gRNA (guide RNA sequence: CGCCATTCAAGAGCAGCAAC, SEQ ID NO: 29) plasmid into the host strain from the previous step.

[0083] Using the CEN.PK 113-11C genome as a template, promoter P was amplified by PCR. GAL1,10 and Termination T CYC1 T ADH1X-2up (SEQ ID NO: 10) and X-2dw (SEQ ID NO: 11) were used as templates to amplify the genes AnACLa (NCBI accession number: XM_001396694.2) and AnACLb (NCBI accession number: XM_025594417.1), and the donor DNA fragment X-2up-T was obtained by OE-PCR fusion. CYC1 -AnACLa-P GAL1,10 -AnACLb-T ADH1 -X-2dw, transform the DNA fragment together with the X-2gRNA (guide RNA sequence: CTCTCGAAGTGGTCACGTGC, SEQ ID NO: 30) plasmid into the host strain of the previous step;

[0084] Using the CEN.PK 113-11C genome as a template, promoter P was amplified by PCR. TPI1 P TDH3 P TEF1 and Termination T FBA1 T CYC1 T GAL1 In addition to GAL7up (SEQ ID NO: 12), GAL1dw (SEQ ID NO: 13), and genes IDP2 and YHM2, the gene RtCIT1 was amplified using the genome of *Rhodosporidium toruloides* as a template, and the donor DNA fragment GAL7up-P was obtained by OE-PCR fusion. TPI1 -RtCIT1-T FBA1 -T CYC1 -IDP2-P TDH3 -P TEF1 -YHM2-T GAL1 -GAL1dw, transform the DNA fragment together with GAL10gRNA (guide RNA sequence: TCCCAGAAGAATGTCCCTTA, SEQ ID NO: 31) into the host strain of the previous step, that is, integrate the DNA fragment while knocking out the three genes GAL1, GAL7 and GAL10.

[0085] Using the CEN.PK 113-11C genome as a template, promoter P was amplified by PCR. COX9 (SEQ ID NO: 14), P TDH3 P TDH3 P PGK1 P TEF1 and Termination T CYC1 T TDH2 T ADH1T GAL1 T ZWF1 In addition, the donor DNA fragment PGI1up (SEQ ID NO: 15), PGI1dw (SEQ ID NO: 16), and genes GND1, TKL1, TAL1, and ZWF1 were obtained by OE-PCR fusion. COX9 -T CYC1 -GND1-P TDH3 -P tHXT7 -TKL1-T TDH2 -T ADH1 -TAL1-P PGK1 -P TEF1 -ZWF1-T ZWF1 -PGI1dw transforms the DNA fragment along with the PGI1gRNA (guide RNA sequence: AACAAAAATCACGATCTGGG, SEQ ID NO: 32) plasmid into the host strain from the previous step, integrating the GND1, TKL1, TAL1, and ZWF1 genes while simultaneously changing the PGI1 promoter from P... PGI1 Replace with P COX9 ;

[0086] Using the CEN.PK 113-11C genome as a template, promoter P was amplified by PCR. TEF and P ACC1 (Located 500bp upstream of the ACC1 gene) 300bp upstream as P ACC1 up and downstream 300bp as P ACC1 dw, the donor DNA fragment P was obtained by OE-PCR fusion. ACC1 up-P TEF -P ACC1 dw, transform the DNA fragment together with the ACC1pgRNA (guide RNA sequence: CTTCAGGTAAACTGTACGAA, SEQ ID NO: 33) plasmid into the host strain from the previous step, and change the promoter of the ACC1 gene from P ACC1 Replace with P TEF ;

[0087] Using the CEN.PK 113-11C genome as a template, promoter P was amplified by PCR. GSY1 and P IDH2 (Located 500bp upstream of the IDH2 gene) 300bp upstream as P IDH2 up and downstream 300bp as P IDH2 dw, the donor DNA fragment P was obtained by OE-PCR fusion. IDH2 up-P GSY1 -PIDH2 dw, transform the DNA fragment together with the IDH2 pgRNA (guide RNA sequence: ACAATTTCTTCGCCGAACCT, SEQ ID NO: 34) plasmid into the host strain from the previous step, and change the promoter of the IDH2 gene from P IDH2 Replace with P GSY1 ;

[0088] The strain obtained above is a background bacterium that enhances central carbon metabolism and is named SH03. XI-1up–P is integrated into SH03. GAL7 -MCRN-T DIT1 -XI-1dw, obtained engineered bacteria SH04;

[0089] Using the CEN.PK 113-11C genome as a template, the glucose-responsive promoter P was amplified by PCR. HXT1 (Located 500bp upstream of the HXT1 gene) and P FAS (Located 500bp upstream of the FAS1 gene) 300bp upstream as P FAS1 up and downstream 300bp as P FAS1 dw, the donor DNA fragment P was obtained by OE-PCR fusion. FAS1 up-P HXT1 -P FAS1 dw, transform the DNA fragment together with the FAS1 pgRNA (guide RNA sequence: CTTTTTTTCCATACAATTCA, SEQ ID NO: 35) plasmid into the host strain from the previous step, and change the promoter of the FAS1 gene from P FAS1 Replace with P HXT1 SH09 was obtained, and pYX8 plasmid was transformed to obtain engineered bacteria SH11.

[0090] Example 3: Optimization of MCR expression + replacement of the FAS1 promoter with a glucose-responsive promoter P HXT1 Enhancement of the MCR+ central carbon metabolism pathway controlled by a constitutive promoter (engineered strain SH14)

[0091] Based on the engineered bacterium SH09, the constitutive promoter P was integrated at site XII-3 of the genome. TDH3 The controlled MCR, MCRN, and MCRC genes were amplified using gene synthesis plasmids as templates. The upstream and downstream homologous arms XII-3up (SEQ ID NO: 17) and XII-3dw (SEQ ID NO: 18), and the promoter P were obtained. TDH3 and Termination T FBA1 T DIT1The DNA was amplified using the CEN.PK113-11C genome as a template and fused into donor DNA: XII3up-P using OE-PCR. TDH3 -MCRN-T FBA1 -T DIT1 -MCRC-P TDH3 -XII3dw, together with the XII-3gRNA (guide RNA sequence: ATTAACGAGCAGAAAGTTTT, SEQ ID NO: 36) plasmid, was transformed into SH09 to obtain engineered bacteria SH13. The pYX8 plasmid was then transformed to obtain engineered bacteria SH14.

[0092] Example 4: Optimization of MCR expression + replacement of the FAS1 promoter with a glucose-responsive promoter P HXT1 +Strengthening of the central carbon metabolism pathway controlled by the constitutive promoter +MCRC integration to replace plasmid expression (engineered strain SH17h)

[0093] To address the issue of plasmid loss, the constitutive promoter P was integrated at site XII-5 of the genome of engineered strain SH13. GAL1,10 The controlled double-copy MCRC gene was amplified using a gene synthesis plasmid as a template. The upstream and downstream homologous arms XII-5up (SEQ ID NO: 19) and XII-5dw (SEQ ID NO: 20), and the promoter P... GAL1,10 and Termination T FBA1 T DIT1 The DNA was amplified using the CEN.PK113-11C genome as a template and fused into donor DNA: XII5up-T using OE-PCR. FBA1 -MCRN-P GAL1,10 -MCRC-T DIT1 -XII5dw, together with the XII-5gRNA (guide RNA sequence: CTGATGTAGGCTCCTTAAAT, SEQ ID NO: 37) plasmid, was transformed into SH13 to obtain engineered bacteria SH17h.

[0094] Example 5: Optimization of MCR expression + replacement of the FAS1 promoter with a glucose-responsive promoter P HXT1 +Enhancement of the central carbon metabolism pathway+Completion selection markers HIS3 and URA3 genes (engineered bacterium SH18)

[0095] After the genetic engineering modification is completed, the selection marker is no longer needed. In order to save fermentation costs and promote the normal growth of the strain, the selection marker gene URA3 is reintroduced in situ. Using the Saccharomyces cerevisiae S288C genome as a template, the URA3 gene expression cassette (gene and its upstream and downstream 500bp) is amplified and transformed together with the engineered strain SH17 to obtain the engineered strain SH18.

[0096] Example 6: Diploid Construction (SH70)

[0097] Using the CRISPR / Cas9 system, the Cas9 protein gene was integrated into the XI-5 site of *Saccharomyces cerevisiae* CEN.PK 110-10C to obtain SHα01(MATa; MAL2-8c; SUC2; ura3-52; XI-5::P). TEF1 -Cas9-T CYC1 Then P is integrated at site XI-1. GAL7 -MCRN-T DIT1 In XII-3 integration P TDH3 -MCRN-T FBA1 -T DIT1 -MCRC-P TDH3 T is integrated at site XII-5 FBA1 -MCRN-P GAL1,10 -MCRC-T DIT1 Replace P FAS1 P is a glucose-responsive promoter HXT1 To enhance the central carbon metabolism pathway (same as Table 1), the above operations were performed as above to obtain the engineered strain SHα23.

[0098] The HIS3 gene in SH17h and the LYS3 gene in SHα23 were knocked out, respectively. The specific implementation was as follows: sgRNA expression vectors targeting the HIS3 (SEQ ID NO: 27) and LYS3 (guide RNA sequence: CATCAAATTATGATTGAGGA) gene regions were constructed. Then, 300 bp upstream and downstream of the HIS3 and LYS3 genes, as well as the ORF frames of the HIS3 and LYS3 genes, were amplified, and donor DNA was obtained by OE-PCR. Subsequently, the gRNA expression vectors and gene expression cassettes (500 ng each) were chemically transformed into *Saccharomyces cerevisiae* SH17h and SHα23, respectively. The transformed strains were plated on selection plates SD+His and SD+Lys and incubated statically at 30°C for 2–3 days. After culture in liquid YPD medium, the transformants were verified to be correct by colony PCR and plated on plates containing 5-fluoroorotic acid for plasmid loss. The strains after plasmid loss were named SH17 and SHα24, respectively. Furthermore, the engineered bacteria SH17 and SHα24 were cultured separately in YPD medium, and then 0.5 mL of each bacterial culture was mixed and inoculated into 20 mL of YPD medium and cultured for 48 h. The fermentation broth was centrifuged, washed twice with water, appropriately diluted, and spread on SD+URA3 solid medium. The single colonies that grew were diploids and named SH70.

[0099] Example 7: 3-HP shake flask batch fermentation

[0100] The shake-flask fermentation of engineered bacteria used a basic culture medium consisting of (NH4)2SO4 2.5 g / L, KH2PO4 14.4 g / L, MgSO4·7H2O 0.5 g / L, and trace metals (3 mg / L FeSO4·7H2O, 4.5 mg / L ZnSO4·7H2O, 4.5 mg / L CaCl2·2H2O, 1 mg / L MnCl2·4H2O, 0.3 mg / L CoCl2·6H2O, 0.3 mg / L CuSO4·5H2O, 0.4 mg / L Na2MoO4·2H2O, 1 mg / L H3BO3, 0.1 mg / L KI, and 19 mg / L MgSO4·7H2O). The culture medium contained Na₂EDTA·2H₂O, vitamins (0.1 mg / L biotin, 2 mg / L pantothenic acid, 2 mg / L thiamine, 2 mg / L pyridoxine, 2 mg / L nicotinic acid, 0.4 mg / L aminobenzoic acid, 50 mg / L inositol), with 20 g / L glucose added, and the initial pH adjusted to 5.6 with KOH. The activated strain was inoculated into this medium and cultured at 30°C and 220 rpm for 24 h. It was then transferred to 20 mL of fermentation medium per 100 mL shake flask. The initial OD₂O₃ was measured. 600 Fermentation was carried out at 0.2, 30℃, and 220 rpm for 96 h, and samples were taken to determine the biomass OD. 600The yield of 3-HP was determined by HPLC.

[0101] The results showed that enhancing the central carbon metabolism pathway increased 3-HP production by approximately 4 times. Figure 2 ); Integrated constitutive promoter P TDH3 Controlled MCR can increase 3-HP production by approximately 100%, indicating that the constitutive promoter P TDH3 and the inductive promoter P GAL1,10 Using them together ensures that the gene is expressed both before and after glucose consumption, which helps to improve biosynthetic efficiency. Figure 3 Due to severe plasmid loss during fermentation in the upper tank, free plasmids were lost. Figure 4 ), through the bidirectional promoter P GAL1,10 Integrating MCR expression instead of free plasmid expression increased 3-HP yield by approximately 50%, indicating that genome integration ensures stable gene expression. Further addition of the selection marker URA3 ensured normal growth and production even without amino acid additions. Figure 3 The SH18 strain achieved a 3-HP yield of 56.5 g / L during fermentation in a tank. Figure 5 This represents the highest reported yield of 3-HP from glucose sources to date.

[0102] Example 8: Feed-in batch fermentation in a 3-HP fermenter

[0103] The fed-batch fermentation used a 1L DasGip parallel bioreactor system with an initial fermenter volume of 0.4L. Fed-batch fermentation used an initial culture medium ((NH4)2SO4 2.5 g / L, KH2PO4 14.4 g / L, MgSO4·7H2O 0.5 g / L, trace metals (3 mg / L FeSO4·7H2O, 4.5 mg / L ZnSO4·7H2O, 4.5 mg / L CaCl2·2H2O, 1 mg / L MnCl2·4H2O, 0.3 mg / L CoCl2·6H2O, 0.3 mg / L CuSO4·5H2O, 0.4 mg / L Na2MoO4·2H2O, 1 mg / L H3BO3, 0.1 mg / L KI, 19 mg / L Na2EDTA·2H2O), with a glucose concentration of 20 g / L, in a volume of 0.4 L. The inoculum was OD200... 600=0.4, pH 5.6. The feed medium used was 4×D medium ((NH4)2SO4 2.5g / L, KH2PO4 14.4g / L, MgSO4·7H2O 0.5g / L), trace metals (3mg / L FeSO4·7H2O, 4.5mg / L ZnSO4·7H2O, 4.5mg / L CaCl2·2H2O, 1mg / L MnCl2·4H2O, 0.3mg / L CoCl2·6H2O, 0.3mg / L CuSO4·5H2O, 0.4mg / L Na2MoO4·2H2O, 1mg / L H3BO3, 0.1mg / L KI, 19mg / L) The culture medium was prepared using Na₂EDTA·2H₂O and a glucose concentration of 500 g / L. A pulsed feeding method was used: 15 mL of the above-mentioned feed medium was added every 4 hours before 48 hours, and then every 8 hours thereafter. Biomass OD was measured periodically during fermentation. 600 The concentration of ethanol was determined, and the yield of 3-HP was measured by HPLC.

[0104] like Figure 5 As shown, the highest yield of 3-HP fermentation using engineered strain SH18 with fed-batch method was 56.5 g / L. (As...) Figure 6 As shown, the highest yield of 3-HP fermentation using diploid engineered bacteria SH70 with fed-batch method was 70.2 g / L.

[0105] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution. sequence list <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> An engineered bacterium producing 3-hydroxypropionic acid, its construction method and application <160> 37 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1650 <212> DNA <213> Artificial Sequence <400> 1 atgtctggaa ctggtaggtt ggccgggaaag atcgctttga tcaccggagg agccggagaat 60 atcggaagtg aattgactag aagattcttg gccgagggag ctactgttat catctctggt 120 aggaaagag ctaagttgac cgccttggct gaagaatgc aagctgaggc cggtgtccca 180 gctaaagga tcgaccttga ggtcatggac ggttctgacc cagttgctgt tagagctggt 240 atcgaagcta ttgttgctag acacggtcag attgacattc ttgtcaataa cgccggttct 300 gccggagccc aaagggatt ggccgagatc ccacttaccg aagctgaatt gggtcccggt 360 gctgaaaaa ccttgcacgc foottcgcc aatcttttgg gaatgggttg gcaccttatg 420 aggattgctg cccctcatat gccagtcgga tctgctgtca tcaacgtcag taccatcttt 480 tctagggccg agtattacgg taggatccca tacgttaccc caaaggccgc cttgaatgct 540 ttgtctcaac ttgccgccag agagttgggt gccagaggta ttagggttaa caccatcttc 600 cccggtccaa tcgagagatga tagaatcaga accgtcttcc aaaggatgga ccagttgaaa 660 ggtaggccag aaggacac cgcccatcat ttcttgaaca ccatgaggct ttgtagagcc 720 aacgaccaag gtgcccttga aaggagattc ccttctgtcg gagatgttgc tgatgctgcc 780 gtttccttg cctctgccga atctgccgcc ctttctggtg agaccatcga ggtcacccat 840 ggtatggagc ttccagcttg tagtgagact agtcttcttg ctagaaccga cttgagaact 900 atcgacgcca gtggagaac caccttgatc tgtgccggag accaaatcga ggaggtcatg 960 gcccttactg gtatgcttag gacttgtgga agtgaggtta tcatcggttt tagaagtgct 1020 gccgctcttg cccagttcga gcaagccgtt aatgagagta ggaggttggc tggagccgac 1080 ttcacccctc ctattgccct tcctttggac ccaagagacc cagccaccat cgatgccgtc 1140 tttgactggg ctggtgagaa tactggtggt attcacgccg ctgttatctt gccagctacc 1200 agtcacgaac cagctccatg cgttattgag gtcgatgacg agagggtctt gaactttttg 1260 gccgacgaaa tcaccggtac catcgtcatt gctttaggt tggctagata ctggcaaagt 1320 cagagactta cccccggtgc tagggctagg ggtcctaggg tcatcttctt gtctaacggt 1380 gccgaccaga acggaaacgt ctacggaagg atccagagtg ccgccatcgg tcagcttatt 1440 agagtctgga gacatgaggc cgaattggac tatcagagag cctctgctgc tggtgaccat 1500 gttttgccac cagtttgggc caaccagatc gttagattcg ccaatagaag tcttgaagga 1560 ttggagttcg cttgtgcttg gactgcccag cttttgcaca gtcagaggca catcaatgag 1620 attaccttga atatcccagc taatatttaa 1650 <210> 2 <211> 2016 <212> DNA <213> Artificial Sequence <400> 2 atgtctgcta ccactggtgc taggtctgct tctgtcggtt gggctgagtc tttgatcggt 60 ttgcaccttg gaaaagtcgc ccttatcacc ggaggatctg ccggtattgg tggtcagatc 120 ggtaggcttt tggctttgag tggagctaga gtcatgttgg ctgctagaga tagacataag 180 cttgagcaga tgcaagccat gatccagtct gagcttgccg aggtcggata cactgacgtc 240 gaggacagag tccatattgc ccccggttgc gacgtcagta gtgaagccca acttgccgac 300 ttggtcgaaa ggaccctttc tgcctttgga accgtcgact atttgatcaa caatgccggt 360 attgccggtg tcgaagaaat ggtcatcgac atgccagtcg agggatggag gcacaccttg 420 tttgccaact tgattagtaa ttattctttg atgaggaagc ttgccccact tatgaagaag 480 caaggttctg gttatattct taatgtcagt tcttatttcg gtggagagaa ggacgccgcc 540 attccatacc caaatagggc tgactacgcc gttagtaagg ctggacagag agccatggct 600 gaagtctttg ctagattctt gggtccagag atccagatca atgccatcgc ccccggtcca 660 gttgagggag atagacttag gggaactggt gaaaggcccg gtcttttcgc taggagggct 720 agacttatcc ttgagaataa gaggcttaac gaacttcacg ccgctcttat cgctgctgcc 780 agaaccgatg agaggagtat gcacgagttg gtcgaattgc ttttgccaaa cgacgttgcc 840 gccttggaac agaatccagc tgccccaact gctcttaggg agttggctag aaggtttaga 900 agtgaaggag atccagccgc ctcttctagt tctgctttgc ttaatagatc tatcgccgcc 960 aagcttttgg ccagacttca caacggtgga tacgttttgc cagccgacat cttcgctaac 1020 ttgccaaacc caccagaccc tttcttcacc agagcccaaa tcgatagaga ggctagaaag 1080 gttagagacg gtatcatggg aatgttgtac ttgcagagga tgccaaccga atttgatgtt 1140 gccatggcta ccgtttacta cttggctgat agggtcgtct ctggtgaaac cttccatcca 1200 agtggaggtt tgaggtacga gaggacccca accggaggag agcttttcgg tcttccttct 1260 ccagagaggt tggccgaatt ggtcggttct accgtctacc ttattggaga gcacttgacc 1320 gaacatttga atttgcttgc tagagcctac cttgaaaggt acggagccag acaagttgtt 1380 atgatcgtcg aaaccgagac tggagctgag accatgagga gacttcttca cgaccacgtt 1440 gaggctggaa gattgatgac catcgtcgct ggagaccaga ttgaggctgc catcgaccaa 1500 gctatcacta gatatggaag acccggtcca gttgtctgta ctcctttcag acctcttcca 1560 accgtccctt tggtcggtag gaaggatagt gactggtcta ccgtcttgag tgaggccgaa 1620 ttcgccgaac tttgcgaaca ccaacttacc caccatttca gagtcgctag atggattgct 1680 cttagtgatg gtgccagact tgctttggtc actccagaaa ccaccgccac cagtaccacc 1740 gaacagttcg ccttggccaa cttcatcaag actaccttgc acgctttcac cgccactatt 1800 ggagttgagt ctgaaaggac cgcccagagg atcttgatca accaagttga ccttaccaga 1860 agggctaggg ccgaggaacc tagagatcct cacgaaaggc agcaagaact tgagaggttc 1920 atcgaggccg tccttttggt tactgctcct ttgcctccag aagccgatac cagatatgct 1980 ggaaggatcc atagaggaag agctatcact gtctaa 2016 <210> 3 <211> 330 <212> DNA <213> Artificial Sequence <400> 3 ggaatagtga cgttgtgatg cggtgagttc ggcggttagg ggaatggtat atgataaaaa 60 acggaaacgt gcttctttaa tttaattgtt taatattgtt gcagatatat aaaaaggggg 120 aaagaaccga agatgtaatt atttttttat cgcctcaacc taaagcaagc aataaggtat 180 aaagatcagg acgtctcgag cgctgatatc taaatttgaa gccacgcaag taactacgta 240 ggtcagaggg gacaaggaat aacacttgac atttttcttt tttctttttt tttctttttt 300 ttttttttgt taatcttggc ttctgtaccg 330 <210> 4 <211> 285 <212> DNA <213> Artificial Sequence <400> 4 cttccacgga ataccaagcc cattgcaatg cgatgttagt ttagtggagt ttcttggcat 60 tggcaaatct ctgctaaatg ctgcgtacag acggaaactc acaccgccgc gaagactggt 120 cagtggcaaa aaaaaaaaaa aattaaaaaa taaaaaataa ctattacgta tgatactgtt 180 tctggtagtt gatatgaggt tggtgttgta tattgtacgt tttaggaaca gggaagtgaa 240 tattatttac tctgctgcac attctggcta ggtcgaagcc ggaac 285 <210> 5 <211> 3306 <212> DNA <213> Artificial Sequence <400> 5 atgtccgcta aagctatttc cgaacaaact ggtaaagaat tattatacaa gtacatttgc 60 accacctcag ccatacaaaa cagattcaag tatgcaagag ttacaccaga taccgactgg 120 gcccatttgt tacaagatca cccttggttg ttatctcaat cattggttgt caaacctgac 180 caattgatta aaagacgtgg taaattgggt ttagtcggtg taaacttgag tttagatggt 240 gttaagtctt ggttgaagcc aagattaggt catgaagcta cagttggtaa agcaaagggt 300 ttcttgaaaa atttcttgat cgaaccattc gtacctcact cacaagctga agaattttac 360 gtttgtatct atgcaactag agaaggtgac tatgtcttgt ttcatcacga aggtggtgtt 420 gacgtcggtg acgttgacgc caaagctcaa aagttgttag taggtgttga tgaaaagtta 480 aacacagaag acatcaagag acatttgttg gtacacgccc cagaagataa aaaggaagtt 540 ttggcttcct ttataagtgg tttgtttaat ttctacgaag atttgtactt cacctacttg 600 gaaattaacc ctttagtagt tactaaggat ggtgtctata tattggactt agctgcaaaa 660 gtagatgcaa ctgccgacta catctgtaag gttaagtggg gtgacattga atttccacct 720 ccattcggta gagaagcata tccagaagaa gcctacattg ctgatttgga cgcaaaatct 780 ggtgcctcat tgaagttaac attgttgaac cctaagggta gaatatggac tatggttgct 840 ggtggtggtg caagtgtcgt atattctgat acaatctgcg acttgggtgg tgttaacgaa 900 ttagctaact acggtgaata ctcaggtgca ccatccgaac aacaaactta tgattacgct 960 aagaccatct tgagtttaat gactagagaa aagcatcctg aaggtaaaat tttgatcatc 1020 ggtggttcta tagcaaactt cactaacgtt gccgctacat tcaagggtat agtcagagct 1080 atcagagatt atcaaggtcc attgaaggaa cacgaagtta caatattcgt cagaagaggt 1140 ggtcctaact accaagaagg tttaagagta atgggtgaag ttggtaaaac tacaggtatc 1200 ccaattcatg tatttggtac tgaaaacac atgactgcca tcgttggtat ggctttaggt 1260 catagaccaa ttcctaatca acctccaaca gcagcccaca ccgccaattt cttgttaaac 1320 1380 gctgatgaag ttgctccagc taagaaagca aaaccagcca tgcctcaaga ctccgttcca 1440 agtcctagat cattgcaagg taaatcagca acattatttt ccagacatac caaagccatt 1500 gtatggggta tgcaaaag agctgttcaa ggcatgttgg atttcgacta tgtttgtagt 1560 agagatgaac catctgtcgc tgcaatggta tatcctttta ccggtgacca taaacaaaag 1620 ttctactggg gtcacaagga aatattaatc ccagttttta aaaacatggc cgatgctatg 1680 aaaaagcatc ctgaagttga tgtattgatt aacttcgctt cattaagatc cgcttatgat 1740 tctactatgg aaaatgaa ctacgcacaa attagaacca tagctatcat tgcagaaggt 1800 ataccagaag cattgactag aaagttaatc aaaaaggccg atcaaaaagg tgtcactata 1860 atcggtccag ctacagtagg tggtataaaa cctggttgtt ttaagatcgg taatactggt 1920 ggcatgttgg ataacatatt ggcatcaaaa ttgtatagac caggttccgt agcttacgtt 1980 tcaagaagcg gtggtatgag taacgaattg aacaacataa tttcaagaac cactgatggt 2040 gtttatgaag gtgtcgctat tggtggtgac agatacccag gttctacttt tatggatcat 2100 gttttgagat atcaagacac acctggtgtc aaaatgatcg ttgtcttagg tgaaataggt 2160 ggtactgaag aatacaaaat ttgcagaggt ataaaggaag gtagattgac aaaaccagta 2220 gtttgttggt gcattggtac ttgtgcaact atgttttctt cagaagttca attcggtcat 2280 gcaggtgcct gcgctaatca agcatctgaa acagcagttg ccaaaaacca agccttaaag 2340 gaagctggtg tttttgtccc tagatcattc gatgaattgg gtgaaatcat tcaatccgta 2400 tatgaagact tagttgccaa gggtgctatt gtcccagctc aagaagtacc tccacctact 2460 gttcctatgg attactcatg ggcaagagaa ttgggtttga tcagaaagcc agctagtttt 2520 atgacctcta tctgtgatga aagaggtcaa gattgatct atgctggtat gcctatcact 2580 gaagtcttca aggaagaat gggtatcggt gtgtattgg gtttgttgtg gttccaaaga 2640 agattaccaa agtactcatg tcaatcata gaatgtgct taatggttac agctgatcat 2700 ggtccagctg tttctggtgc ccacaacc ataatctgcg ctagagcagg taagatttg 2760 gttctctt tgactctgg ttgttact attgtgaca gatttggtgg tgcattgac 2820 gccgctgcaa aaatgttttc aaaggctttc gattccggta taatcccaat ggaatttgtt 2880 aaaaggagggg taattaatc atgggtacg gtcatcgtgt taagtcatt 2940 aaaccctg atatgagagt ccaatattg aaggacttcg taaagcaa cttcccagca 3000 acacctttgt tagattacgc cttagaagtt gaaagatta caacctta aaagccaaat 3060 ttgatcttga acgttgatgg ttttaggt gtcgctttcg tagacatgtt aagaaactgt 3120 ggttcttta ctagagaaga agccgatgaa tatgttgaca ttggtgcttt gatggtata 3180 ttgtcttag gtagatcaat gggttttatt ggtcattact tggatcaaa gagattaaag 3240 caaggtttgt atagacaccc ttgggacgat atttcctacg ttttgcctga acacatgagt 3300 atgtaa 3306 <210> 6 <211> 1638 <212> DNA <213> Artificial Sequence <400> 6 atgcctgctc attttgcccc ttcacaacca ttacaaggtg gtccatcccc ttcacaattg 60 ggtcctaaag aattattgat agaaagagca ttgacaagat tgagatcaat cccaaacgat 120 ttggaaaaat ataccttttt ggccggttta agaggtagaa atcctgatgt cttctacggt 180 ttagtaggtg gtaacatgaa ggaatgttgc ccaattatct atactcctgt tataggttta 240 gcttgtcaaa attggtcctt gatccatcca cctccacctg aaagtgatcc aacaattgac 300 gcattgtatt tgtcttactc agatttgcca aacttacctc aattgatcgg tggtttgaag 360 actagattgc ctcacgatca aatgcaaatc tccgttgtca cagacggtag tagagtattg 420 ggtttgggtg acttgggtgt tggtggtatg ggtatatctc agggtaaatt gtcattatac 480 gttgctgctg gtggtgtcaa tccaaaggcc actttaccta tcgctattga ttttggtact 540 gacaacgaaa cttgttagc tgatccattg tacgttggtc aaagaattag aagattatct 600 caagaaaagt gtttggagtt tatggaagtt ttcatgagat gcatgcatga aaccttccca 660 aatatggtta ttcaacacga agactggcaa actccattgg cttcccttt gttgcataag 720 aacagagatt tgtacccttg tttcaacgat gacattcaag gtactggtgc agtagtttta 780 gcaggtgcca taagagcttt tcacttaaac ggtgttgcat tgaaggatca aaagattttg 840 ttttcggtg ccggttcttc aggtgttggt gtcgctgaaa caatatgcaa gtacttcgaa 900 ttgcaaggca tgtctgaaga cgaagccaaa tcaaagttct ggttggtaga ttcaaagggt 960 ttggttgctc ataatagagg tgacacatta ccatctcaca aaaagtattt ggcaagatca 1020 gaaccagatg cccctaaatt gagaaccttg aaggaagtcg tagaacatgt tcaaccaact 1080 gctttgttag gtttatctac agtcggtggt acttttacaa aggaaatctt ggaagctatg 1140 gcaacttaca ataagagacc aattgtcttt gctttatcaa accctgtagc ccaagctgaa 1200 tgtaccttcg aagaagctgt tgaaggtact gacggtagag tcttgtacgc atccggtagt 1260 ccattcgatc ctgttgaata caagggtaaa agatacgaac caggtcaagg taataacatg 1320 tatatcttcc ctggtttagg tattggtgct atattggcaa gagtctccaa aattccagaa 1380 gaattagtac atgcatccgc ccaaggttta gcagacagtt tgacaccaga agaaaccgcc 1440 agacacttgt tgtaccctga tatcgaaaga attagagaag tttctataaa aatcgctgta 1500 acagttatac aagccgctca aaagttaggt gttgatagaa acgaagaatt gcgtggtaaa 1560 tccagtgcag aaattgaagc ctatgtcaga aaaggtatgt atcacccatt attagaagca 1620 gaacaacaag cacaatga 1638 <210> 7 <211> 1023 <212> DNA <213> Artificial Sequence <400> 7 atggtcaaag tcgcaattct tggcgcttct ggtggcgtgg gacaaccgct atcattactg 60 ctaaaattaa gcccttacgt ttccgagctg gcgttgtacg atatccgagc tgcggaaggc 120 attggtaagg atttatctca catcaacacc aactcaagtt gtgtcggtta tgataaggat 180 agtattgaga acaccttgtc aaatgctcag gtggtgctaa taccggctgg tgttcccaga 240 aagcccggtt taactagaga tgatttgttc aagatgaacg ccggtattgt caaaagcctg 300 gtaaccgctg ttggaaagtt cgcaccaaat gcgaggattt tagtcatttc aaaccctgta 360 aacagtttgg tccctattgc tgtggaaact ttgaagaaaa tgggtaagtt caaacctgga 420 aacgttatgg gtgtgacgaa ccttgacctg gtacgtgcag aaaccttttt ggtagattat 480 ttgatgctaa aaaaccccaa aattggacaa gaacaagaca aaactacaat gcacagaaag 540 gtcactgtta ttgggggtca ttcaggggaa accattatcc caataatcac cgacaaatcg 600 ctggtatttc aacttgataa gcagtacgag cacttcattc atagggtcca gttcggaggt 660 gatgaaattg tcaaagctaa acagggcgcc ggttccgcca cgttgtccat ggcgttcgcg 720 ggggccaagt ttgctgaaga agttttgagg agcttccata atgagaaacc agaaacggag 780 tcactttccg cattcgttta tttaccaggc ttaaaaaacg gtaagaaagc gcagcaatta 840 gttggcgaca actctattga gtatttttcc ttgccaattg ttttgagaaa tggtagcgta 900 gtatccatcg ataccagtgt tctggaaaaa ctgtctccga gagaggaaca actcgttaat 960 actgcggtca aagagctacg caagaatatt gaaaaaggca agagtttcat cctagactct 1020 tga 1023 <210> 8 <211> 301 <212> DNA <213> Artificial Sequence <400> 8 cccaaagcta agagtcccat tttattcttc tatatgtata ttttcgatac tctaaaccac 60 cctacaatgt agccctatac taaatctgct caattttcag cttctacaag tgactcgaga 120 ccacgtggaa agatccaact actccagcac aacgattcaa tataatcgat tgctccactc 180 ataagaggca agaacaagct tcaacttttg gtaagccgcc gtttataaac agggaagatg 240 tcctttgtca agggaggcac agagcatggc caatttggca aattgcaggt ttttctgagt 300 g 301 <210> 9 <211> 302 <212> DNA <213> Artificial Sequence <400> 9 gacatagaaa tgtagatata caggtatttt tctcgataat cgataaaaat ctcgtcgcgc 60 tgaaccaaac ttggtggtta cggagagttt ttctctcatc attactgtct ttcgcattga 120 tttccccttt gaccgataaa atcccttgga ttcataagat taaacaaaga ggtgatcaaa 180 gagaaccctg tgaaagttta tgtttataac cgggcataaa gtgaactaga cactttcaag 240 aagccaacca aagcatgagt aacgaagctt accagcatga tcataccgta aatcctcacc 300 ag 302 <210> 10 <211> 303 <212> DNA <213> Artificial Sequence <400> 10 gagactgtta gttggatatc agtaatgaga cgaaaaagct cgaaatgaat ggatatattc 60 tttttgctac tggcaactgt tgaatattta atgttaaaac aaactaactg aggtatattc 120 gtatctgtat gtacacatat actatataca ggaaaagata agcaagagag aggatatcaa 180 ctacgagagc gatcgattat atatcaaaag ctgtccgctt tgccacccat aatcggcgct 240 tagtttcgga gttcaatcat aattctacca ccttacactc aacttactct ttaactccta 300 tag 303 <210> 11 <211> 301 <212> DNA <213> Artificial Sequence <400> 11 cgtttccttt attggggttt ccgtgtagcc ttcccctgaa tagtgtggga cgttttatga 60 gaagccgtaa gaaataggca aattgagtta tgacaagtag acatgatgcc gcagccttgc 120 ctgactttac gtctccttca tgaataagtt tttctatcga gttcttttcc ttttttcgcc 180 ttaattagct caattaagcc tgtcctcact acttttcttt ttcttatcgg ctttgtgcca 240 cacctaacct tcgaatgctg ttttattccg ttcttacatg ggatggtaat gccttggcga 300 g 301 <210> 12 <211> 303 <212> DNA <213> Artificial Sequence <400> 12 agcaaaactc tatgacccgg attagaaaac tacgaaaaga gggtaataac ataggtgcag 60 gatttccatc gataacgacg ccgacaatga gccttgctgc aacatccaat taggactaat 120 aactatcgta ggaatttcta cgtaataaac ttcaacagag cctaaaattt gaaaataaat 180 aatctagagg ggaaacttaa agaaattcta ttcttgtcaa taaagtggaa atctgtcaga 240 tgtcacagtt tctttatttg tgacacatat tttcaacata aattcaggca ttagtgctgt 300 aag 303 <210> 13 <211> 302 <212> DNA <213> Artificial Sequence <400> 13 ctttgaaaca cagggacaca attcttgata tgctttcaac cgctgcgttt tggataccta 60 ttcttgacat aatatgacta ccattttgtt attgtacgtg gggcagttga cgtcttatca 120 tatgtcaaag tcatttgcga agttcttggc aagttgccaa ctgacgagat gcagtaaaaa 180 gagattgccg tcttgaaact ttttgtcctt ttttttttcc ggggactcta cgagaaccct 240 ttgtcctact gattaatttt gtactgaatt tggacaattc agattttagt agacaagcgc 300 ga 302 <210> 14 <211> 438 <212> DNA <213> Artificial Sequence <400> 14 gctgggcgat cttccttgtg cgtctgttgt ctcacaattg cttgaaggaa gatttcataa 60 gatcatatga gtccctcttt atatgggcaa gtggaattat gtgagtcaaa atccgcgcgt 120 gacccgtaaa gcgttatcag aaggtgcaaa cggtgctatt tagctcataa aaggaatgat 180 tcaagctctt ttggattgta agacaccttt atttagtcca agatcattgc agacccttgt 240 tatggtcttt agcagagtcc tcctctatat ctcttcattt actgcaacct gattggcccg 300 ctaccacgat gcccgctttg ttcctgtggt attaaaagaa tcgatgaaag agactcttat 360 cttcagggaa aattaggacc gagaattaga gcaagcaaga tatttgcaaa ctactaacta 420 caagcgactt acacagac 438 <210> 15 <211> 301 <212> DNA <213> Artificial Sequence <400> 15 ggccaaagtc ttactcgcct ctaaccccac gtaggacaag tgctgagaat aaaatagtct 60 tggcgtagta tgccttcttc agtaatattt tttcttttga aagtactacc cacatccgaa 120 cattgccact tacatagcga tgcaaatagc cgccaggaaa tgccgcatgt tatccactaa 180 gaatagtcaa cattgcatag catccaaata cgtgaaagcg gaccgatctt gagcgaaatg 240 tttagtaccg ttgatgtgta gaagtagtgc cctgaaatac atagcggtgt attatttagc 300 a 301 <210> 16 <211> 301 <212> DNA <213> Artificial Sequence <400> 16 aatcggaacc accaataccg atgttaacaa catcggtgat cttcttaccg gtataaccct 60 tccattcacc agaacgaact tgttcagaga actccttcat gtgcttcaag acagagtcga 120 cttctggagc aacgttgaca ccatcaacgt acattggctt gttagctctg tttctcaatg 180 cgacgtggta gacagcacga tcttcagtgg agttgatgtg ttcacctttg aacatagcat 240 ctctcaaacc ggtgacgtta gcctccttgg ccagttcaat caatgcagca atgatttcat 300 c 301 <210> 17 <211> 525 <212> DNA <213> Artificial Sequence <400> 17 gaaactaacc cgatgggaca attactgatc gattgcatta ttaaaggtga taaataatcc 60 tttgttattt gtgcccctta aaattcatat acactttatg tttaattcga caggtataac 120 agaaagaaac aaggaaaaca atgcaattaa tcattaactg caccaacttt tatatgaata 180 tattgtgcta catgtcttaa ttagtcctct gtgaatgagc atcacaaaga atcacgagga 240 aaagattgat tacttaatat tgataggaat cagccgcgga aatggaaatc ttggtaatat 300 aattttaatg cggcacgctg aaaattgatg gcggcctgct aggagcccta ttgattccgc 360 ggaaataggc ggattattac aaacgttgag cattcagcag gtgcggctgt gccttttcac 420 tttcagctaa gatgtttgca agcaagaatt aacagataag ccgcttcgct agtaacgcaa 480 ctcattgtgt ttttaatgga agctaaggat ggttgaaatt atggg 525 <210> 18 <211> 411 <212> DNA <213> Artificial Sequence <400> 18 ggtgtgatag gctctaggaa cgaatattaa tattgggggg gggaaactgg aatgaaatta 60 aacaaaagca aaaaaatcta aaagaagaaa aaaaggagag agagagagag agagagagag 120 agagagagag agagagagag agagagagag agagagagat gagggttatt gataatataa 180 tataataata aaagagaaga tagtagaaaa ggaagaaaga aaaaaaaaaa ttaagatgta 240 ctttaataaa aagagggaaa ttctaaaaag tgcgactgga attagaagaa tgcaggtatt 300 ttgttttctc tcctatgacg gagaaggtaa caaaatcata aaggtaaaat aacaaattaa 360 ttatgatcaa agagaaatgc cctttgtaaa ccgtggtttg taaatgcgtt g 411 <210> 19 <211> 190 <212> DNA <213> Artificial Sequence <400> 19 gcgaactgcc gtactcgatg ctttatttct cacggtagag cggaagaaca gataggggca 60 gcgtgagaag agttagaaag taaattttta tcacgtctga agtattctta ttcataggaa 120 attttgcaag gttttttagc tcaataacgg gctaagttat ataaggtgtt cacgcgattt 180 tcttgttatg 190 <210> 20 <211> 241 <212> DNA <213> Artificial Sequence <400> 20 ggagttcacc acgtaatgcc tgtttaagac catcagttaa ctctagtatt atttggtctt 60 ggctactggc cgtttgctat tattcaagtc ttttgtgcct tcccgtcggg taagggagtt 120 atttagggat acagaatcta acgaaaacta aatctcaatg attaactcca tttaatcctt 180 ttttgaaagg caaaagaggt cccttgttca cttacaacgt tcttagccaa attcgcttat 240 c 241 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <400> twenty one gatcatttat ctttcactgc ggagaag 27 <210> twenty two <211> 59 <212> DNA <213> Artificial Sequence <400> twenty two gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatc 59 <210> twenty three <211> 69 <212> DNA <213> Artificial Sequence <400> twenty three aaacttctcc gcagtgaaag ataaatgatc tatcatcgcc gagaaatttg gttttagagc 60 tagaaatag 69 <210> twenty four <211> 69 <212> DNA <213> Artificial Sequence <400> twenty four aaacttctcc gcagtgaaag ataaatgatc tctcggagga ttctttgcac gttttagagc 60 tagaaatag 69 <210> 25 <211> 59 <212> DNA <213> Artificial Sequence <400> 25 cttaatttcg aataaacaca cataaacaaa caaagttat ccgcaacgct tttctgaac 59 <210> 26 <211> 51 <212> DNA <213> Artificial Sequence <400> 26 ccccggggtc gacctcgagt atagtttttt ctccttgacg ttaaagtata g 51 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 catgctctgg ccaagcattc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <400> 28 cgaggacatg attgctatcg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 cgccattcaa gagcagcaac 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 ctctcgaagt ggtcacgtgc 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 tcccagaaga atgtccctta 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 aacaaaaatc acgatctggg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 cttcaggtaa actgtacgaa 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 acaatttctt cgccgaacct 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 ctttttttcc atacaattca 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <400> 36 attaacgagc agaaagtttt 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <400> 37 ctgatgtagg ctccttaaat 20

Claims

1. A method for constructing an engineered bacterium that produces 3-hydroxypropionic acid, characterized in that, The construction method includes: Introduced into the host strain MCRC Genes and MCRN Genes, and will the host strain FAS1 gene promoter P FAS1 Replace with P HXT1 ; The MCRN The gene-encoded polypeptide is derived from Chloroflexus aurantiacus The 1-549 amino acids of malonyl-CoA reductase; The MCRC The gene-encoded polypeptide is derived from Chloroflexus aurantiacus The 550-1219 amino acid mutant of malonyl-CoA reductase, N940V / K1106W / S1114R; The host strain is selected from any one of the Saccharomyces cerevisiae; The MCRC The nucleotide sequence of the gene is shown in SEQ ID NO: 2; The MCRN The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The introduction into the host strain MCRC Genes and MCRN The gene is selected from any one of (b) to (c): (b) DNA fragment P GAL7 - MCRN -T DIT1 Integration into the host strain XI-1 Site; containing P GAL1,10 - MCRC - T TDH2 The free plasmid was transferred into the host strain; the DNA fragment P was transferred into the host strain. TDH3 - MCRN- T FBA1 -T DIT1 - MCRC- P TDH3 Integration into the host strain XII-3 site; (c) DNA fragment P GAL7 - MCRN -T DIT1 Integration into the host strain XI-1 Site; DNA fragment P TDH3 - MCRN- T FBA1 -T DIT1 -MCRC- P TDH3 Integration into the host strain XII-3 Site; to place DNA fragment T FBA1 - MCRN- P GAL1,10 - MCRC -T DIT1 Integration into the host strain XII-5 site; The construction method also includes enhancing the central carbon metabolism pathway: Will MmACL, RtME, 'MDH3, CTP1, MPC1, MPC3, AnACLa, AnACLb, RtCIT1, IDP2, YHM2, GND1 , TKL1 , TAL1 and ZWF1 Genes are introduced into the host strain; The construction method also includes enhancing the central carbon metabolism pathway: DNA fragment P TPI -MmACL- T FBA1 -T CYC1 -RtME- P TDH3 - P tHXT7 -'MDH3- T TDH2 - T ADH1 - CTP1 -P PGK1 Integration into the host strain HIS3 site; host strain PYC1 Gene promoters are P PYC1 Replace with P TEF ; DNA fragment P TPI1 -MPC1- T MPC1 - T DIT1 - -MPC3- P PGK1 Integration into the host strain X-4 site; DNA fragment T CYC1 -AnACLa -P GAL1,10 -AnACLb- T ADH1 Integration into the host strain X-2 site; DNA fragment P TPI1 -RtCIT1- T FBA1 - T CYC1 -IDP2- P TDH3 - P TEF1 -YHM2- T GAL1 Integration into the host strain GAL1, GAL7, GAL10 Gene locus, that is, the DNA segment that is integrated and simultaneously knocked out. GAL1 , GAL7 , GAL10 Three genes ； DNA fragment P COX9 -T CYC1 - GND1- P TDH3 - P tHXT7 -TKL1- T TDH2 - T ADH1 - TAL1- P PGK1 - P TEF1 -ZWF1- T ZWF1 P integrated into the host strain PGI1 Site, i.e., integration GND1 , TKL1 , TAL1 and ZWF1 At the same time as genes PGI1 The promoter is P PGI1 Replace with P COX9 ; host strain ACC1 Gene promoters are composed of P ACC1 Replace with P TEF ; host strain IDH2 Gene promoters are composed of P IDH2 Replace with P GSY1 .

2. The construction method according to claim 1, characterized in that, Integrating DNA fragments into host strains or replacing gene promoters in host strains is achieved using CRISPR / Cas9 technology.

3. The construction method according to claim 1, characterized in that, The construction method also includes patching. URA3 Gene.

4. The construction method according to claim 1, characterized in that, The construction method also includes cell fusion to form diploids.

5. The construction method according to claim 1, characterized in that, The host strain is selected from either Saccharomyces cerevisiae CEN.PK113-11C or Saccharomyces cerevisiae CEN.PK110-10C.

6. The engineered bacteria producing 3-hydroxypropionic acid constructed by the construction method according to any one of claims 1 to 5.

7. The application of the engineered bacteria producing 3-hydroxypropionic acid constructed by the construction method according to any one of claims 1 to 5 in the preparation of 3-hydroxypropionic acid.

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