CN118146966A - Recombinant strain for producing 3-hydroxy propionic acid by using methanol, construction method and application thereof - Google Patents
Recombinant strain for producing 3-hydroxy propionic acid by using methanol, construction method and application thereof Download PDFInfo
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- CN118146966A CN118146966A CN202211550061.6A CN202211550061A CN118146966A CN 118146966 A CN118146966 A CN 118146966A CN 202211550061 A CN202211550061 A CN 202211550061A CN 118146966 A CN118146966 A CN 118146966A
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- pichia pastoris
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- 125000000830 polyketide group Chemical group 0.000 description 1
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- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
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Abstract
The invention belongs to the application fields of microbial genetic engineering and metabolic engineering, and in particular relates to a recombinant Pichia pastoris strain for producing 3-hydroxy propionic acid by utilizing methanol, a construction method and application thereof. The malonyl-CoA reductase gene CaMCR derived from the orange green flexor is expressed in Pichia pastoris, so that the biosynthesis of 3-hydroxy propionic acid is realized. On this basis, the yield of 3-hydroxypropionic acid is further improved by enhancing intracellular NADPH supply and enhancing methanol metabolism. The invention further provides a construction method of the recombinant Pichia pastoris strain for producing 3-hydroxy propionic acid, which comprises the following steps: the synthesis of 3-hydroxypropionic acid can also be achieved by fusion expression of the benzoic acid decarboxylase gene MdlCKpO derived from Pseudomonas putida and 3-hydroxyisobutyrate dehydrogenase MmsBKpO in Pichia pastoris. According to the invention, the efficient biosynthesis of 3-hydroxy propionic acid is realized in Pichia pastoris by taking methanol as the only carbon source for the first time, the green conversion path of methanol is expanded, and the application potential of Pichia pastoris in the field of microbial cell factories is explored.
Description
Technical Field
The invention belongs to the application fields of microbial genetic engineering and metabolic engineering, and in particular relates to a recombinant Pichia pastoris strain for producing 3-hydroxy propionic acid by utilizing methanol, a construction method and application thereof.
Background
3-Hydroxy propionic acid (3-HP, CAS 503-66-2) is a multifunctional molecule, can be directly used as an additive or preservative for food and feed industry, can be polymerized into poly-3-HP or a copolymer containing 3-HP, and particularly can be used as a substitute of fossil fuel derived plastics, and has very wide application prospect. In addition, 3-HP can also be converted into many other chemicals, such as acrylic acid, methacrylic acid esters, propiolactone, malonic acid, polyesters and acrylamides (J.Biotechnol., 2017, 259:140-147). Acrylic acid is an important monomer of synthetic resin, has very high polymerization speed, and can be used for synthesizing resin, paint, rubber and the like. At present, the main production mode of the acrylic acid is a chemical synthesis method, such as acrylonitrile hydrolysis, propylene oxidation and the like, but the methods all require high-temperature and high-pressure conditions, so that the production cost is increased. Recently, a synthetic route for preparing acrylonitrile from 3-HP through one-step dehydration has been reported, the yield reaches more than 90%, and a more efficient production route is provided for acrylonitrile synthesis (science, 2017,358 (6368):1307-1310).
At present, the 3-HP synthesis mainly comprises a chemical synthesis method and a biological synthesis method, wherein the chemical method mainly comprises the step of adding 3-hydroxy propionitrile into sodium hydroxide solution for reaction, a large amount of solvent is needed for the chemical synthesis method, and the biological synthesis method can utilize cheap raw materials, has mild reaction conditions, is green and environment-friendly, and is attracting more attention. Currently, there are three main biosynthetic routes: the glycerol pathway, the malonyl-CoA pathway and the beta-alanine pathway. The glycerol pathway is mainly found in some bacteria such as klebsiella pneumoniae, and is mainly prepared from glycerol by dehydration with a dehydratase Gdh to give 3-hydroxypropionaldehyde, followed by catalysis with an aldehyde dehydrogenase Adh to give 3-HP (sci.rep., 2016, 6:26932). The malonyl-CoA pathway uses malonyl-CoA as a precursor and is catalyzed by malonyl-CoA reductase (Mcr) for two-step reduction reactions (PLoS.One., 2013,8 (9): e75554; metab.Eng.,2016, 34:104-111). The beta-alanine pathway is mainly produced from aspartic acid by aspartic acid-1-decarboxylase (Adc) to beta-alanine, which is catalyzed by beta-alanine aminotransferase (Baat) and malonic semialdehyde reductase (Bsr) (microb.cell.face., 2015, 15:53.). Among these three routes, the glycerol route is expensive because of the requirement of coenzyme VB12 for Gdh, and is not suitable for industrial production. The beta-alanine pathway is relatively complex, and beta-alanine is also a precursor of vitamin B5, and the pathway has a large number of byproducts, thus affecting the product yield. malonyl-CoA reductase (Mcr) in the malonyl-CoA pathway is a bifunctional enzyme that can be divided into two subunits, the N-and C-terminus, mcrC first reduces malonyl-CoA to malonate semialdehyde, mcrN then reduces malonate semialdehyde to 3-HP, both of which require NADPH to participate (Metab. Eng.,2016, 34:104-111). Because of the use of central metabolic intermediates as precursors, this pathway may also be a competing pathway, resulting in an impaired 3-HP yield, e.g.malonyl-CoA is also a precursor for fatty acid synthesis (Biotechnol. Adv.; 2018,36 (4): 1207-1222). Currently, the highest yield of shake flask fermentation using the malonyl-CoA pathway in Saccharomyces cerevisiae is 3.77g/L (catalysis, 2020,10 (2): 203). In contrast, the malonyl-CoA pathway is more suitable for the production of 3-HP, which is shorter, and the regulatory strategies for intermediate metabolites such as pyruvate and acetyl-CoA are more reported (crit. Rev. Biotechnol.,2017,37 (7): 933-941), which are more prone to directing metabolic flux to product synthesis, and thus have higher production potential. Recently, a new 3-HP synthesis pathway has also been reported, in which oxaloacetate is used as a precursor, and is directly converted to 3-HP by catalysis of benzoyl formate decarboxylase Mdlc and 3-hydroxyisobutyrate dehydrogenase Mmsb, and the intermediates are malonate semialdehyde as well as malonyl-CoA. This pathway is thermodynamically (Δg° = -34.7kJ mol -1) more viable than the malonyl-coa pathway (Δg° = -14.5kJ mol -1) (green.chem., 2021,23 (12): 4502-4509).
At present, biomass-derived saccharides or glycerol is mainly used as raw materials in a biosynthesis route, the glycerol is mainly derived from animal and vegetable oils and fats or petrochemical resources (propane), supply and price fluctuation are large, and the use of biomass saccharides in a large amount increases a potential food crisis, so that a new 3-HP production route still needs to be developed. The coal resources in China are relatively rich, particularly the storage amount of the inferior coal is large, the direct combustion pollution is large, and the establishment of a clean route for converting the coal into energy or chemicals is urgent. At present, the process route of preparing methanol from coal and preparing olefin from methanol is developed in China, commercial production is successfully realized, and great contribution is made to clean utilization of coal resources. The "liquid sunlight" route proposed in recent years enables the direct preparation of methanol starting from CO 2 and water (joule., 2018,2 (10): 1925-1949). Methanol is a widely applied chemical raw material, is a biological raw material without grain source, has higher reducing power, has high biosynthesis selectivity and mild condition compared with a chemical route, and can specifically synthesize high-quality liquid fuel or chemicals with complex structures. Therefore, the methanol biorefinery to synthesize 3-HP is expected to realize carbon neutralization production through a liquid sunlight route. Therefore, methanol will become a rich raw material, and establishing a high-efficiency conversion route for methanol is a feasible path for ensuring future energy and chemical demands. The methanol is utilized to be divided into a chemical route and a biological route, and compared with the chemical route, the methanol has the advantages of good biosynthesis selectivity, green process, mild condition and capability of specifically synthesizing high-quality liquid fuel or chemicals with complex structures. The strategy is expected to be another feasible path for high-efficiency conversion of methanol, and promotes clean utilization of coal and one-carbon resources in China (Clomburg et al.science, 2017,355 (6320): aag0804; zhou et al.Nat.energy.,2018,3 (11): 925-935).
However, the biotoxicity of methanol and its metabolite formaldehyde has limited its use as a biological feedstock, and in nature only a few microorganisms can utilize methanol, such as methanol yeast, of which Pichia pastoris (Pichia pastoris) is an prominent representation of methylotrophic yeasts, capable of growing on methanol as the sole carbon source. The advantages of simple and powerful high cell density culture procedures, strictly regulated super-strong promoters, good post-translational modification and secretion capacity, easiness in genetic manipulation and the like make Pichia pastoris become a wide recombinant protein expression platform in academia and industry, and the Pichia pastoris also serves as a chassis microorganism to produce various metabolites such as terpenes, polyketides, fatty acids, derivatives thereof and the like, so that Pichia pastoris has great potential in the production of fine chemicals (Biotechnol. Adv.,2017,35 (6): 681-710). At present, production of 3-HP in Pichia pastoris has been reported using the malonyl-CoA pathway route glycerol as a carbon source, with yields of 1.81g/L and 24.75g/L in shake flasks and fermentors, respectively (Microb. Biotechnol.,2021,14 (4): 1671-1682). Therefore, it is possible to produce 3-HP by introducing exogenous genes required for the production pathway and genetically modifying the exogenous genes by using Pichia pastoris as the chassis microorganism. However, the research on the production of 3-HP by using methanol as a substrate in Pichia pastoris has not been reported. Therefore, the invention aims to construct a high-yield strain for producing 3-HP by converting pichia pastoris methanol, and expand the routes of methanol utilization and 3-HP production.
Disclosure of Invention
The invention aims to provide a recombinant Pichia pastoris strain for producing 3-hydroxy propionic acid by utilizing methanol, a construction method and application thereof.
In order to achieve the above purpose, the invention adopts the technical scheme that:
The construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol comprises the steps of transferring N-terminal and C-terminal of malonyl-CoA reductase gene CaMCR which is respectively expressed or fusion expressed and derived from orange green flexor into Pichia pastoris strain by adopting a genome integration or plasmid expression mode; wherein the Pichia pastoris strain overexpresses the Pichia pastoris derived gene RAD52. Wherein, the N-terminal and the C-terminal of CaMCR are expressed separately and are fused, the preparation modes of the strains are the same, and the expression modes of the enzymes are different.
Further, the method comprises the following steps:
1. The N-terminal and the C-terminal of malonyl-CoA reductase gene CaMCR from the orange green flexor are expressed respectively, the N-terminal is integrated and expressed on the genome, and the C-terminal is transferred to a Pichia pastoris strain by adopting a genome integration or plasmid expression mode, specifically comprising the following steps:
(1) The N-terminal and C-terminal of malonyl-CoA reductase gene CaMCR from orange green flexor are expressed respectively, the N-terminal genome is integrated into Pichia pastoris strain, and the C-terminal gene is transferred into Pichia pastoris strain by adopting a plasmid expression mode, specifically:
Constructing a polypeptide comprising SEQ ID NO:1 (P AOX1、PCAT1 or P GAP) of the different promoters shown in seq id No. CaMCRN, said donor DNA containing the different promoters was integrated into the neutral site PNSI-2 of pichia pastoris strain PC110 (nucleic acids. Res.,2021,49 (13): 7791-7805), i.e. recombinant strains XY01, XY02 and XY11 were obtained.
Constructing a polypeptide comprising SEQ ID NO:2 or expression vector pGCAI-P AOX1-MCRC-TDAS1 or expression vector pGCAI-P CAT1-MCRC-TDAS1 of the nucleotide sequence of CaMCRC shown in fig. 2; the expression vector pGCAI-P AOX1-MCRC-TDAS1 or the expression vector pGCAI-P CAT1-MCRC-TDAS1 is introduced into Pichia pastoris strains XY01, XY02 and XY11, and recombinant strains XY 18-XY 23 are obtained.
Taking the integration of malonyl-coa reductase gene N-terminus CaMCRN from green-flex orange as an example, the gene editing of pichia pastoris of the present invention is mainly based on an autonomously constructed CRISPR/Cas9 system. Firstly, constructing a donor DNA fragment, respectively amplifying 1000bp sequences, promoters P AOX1、PCAT1 and P GAP, a terminator T FBP1 fragment and a CaMCRN fragment at the upstream and downstream of PNSI-2, and obtaining the complete donor DNA fragment by an overlap extension PCR method; second, transformation, through the electric transformation mode, the gRNA expression vector pPICZ-Cas9-gRNA-PNSI-2 and donor DNA are transformed into Pichia pastoris PC110 (nucleic acids Res.,2021,49 (13): 7791-7805.), standing culture is carried out for 3 days at 30 ℃ on a YPD flat plate containing zeocin, the transformant is cultured overnight through a liquid culture medium of YPD-zeocin +, PCR verification is carried out, and the correct transformant is preserved by a bacterial strain after plasmid loss or the next experiment is carried out. The following methods for integrating genes into the genome are similar.
C-terminal expression vectors were constructed, for example, by introducing expression vectors pGCAI-P AOX1-MCRC-TDAS1 and pGCAI-P CAT1-MCRC-TDAS1. First, construct a polypeptide having SEQ ID NO:2, and expression vectors pGCAI-P AOX1-MCRC-TDAS1 and pGCAI-P CAT1-MCRC-TDAS1, respectively amplifying promoters P AOX1 and P CAT1, terminator T DAS1 fragment and CaMCRC fragment, obtaining complete expression cassette fragment by overlap extension PCR method, and then connecting to vector pGCAI by seamless cloning method (bioresource. Bioprocess, 2022,9,58); secondly, transforming, namely transforming the expression vectors pGCAI-P AOX1-MCRC-TDAS1 and pGCAI-P CAT1-MCRC-TDAS1 into pichia pastoris XY01, XY02 and XY11 by an electric transformation mode, standing and culturing for 3 days at the temperature of 30 ℃ of an SD flat plate, culturing the transformant overnight by a liquid culture medium of SD, carrying out PCR verification, and preserving the correct transformant or carrying out the next experiment to obtain the recombinant strain XY 18-XY 23. The plasmid was constructed as follows, and the method of introducing the plasmid was similar.
(3) The malonyl-coa reductase gene C-terminal CaMCRC and malonyl-coa reductase gene N-terminal CaMCRN derived from green-flex orange are respectively expressed integrated into pichia pastoris strain, wherein the pichia pastoris strain is integrated with protein Cas9, and gene RAD52 derived from pichia pastoris is overexpressed. The method comprises the following steps:
Constructing a polypeptide comprising SEQ ID NO:2, and integrating the donor DNA of CaMCRC into the neutral site PNSIV-16 of pichia pastoris strain PC111 (nucleic acids. Res.,2021,49 (13): 7791-7805), to obtain recombinant strain XY30.
Further, the construction method further comprises: constructing a polypeptide having the sequence of SEQ ID NO:1, a donor DNA containing CaMCRN different promoters (P FGH1 and P FLD1) at different sites (PNSII-3, PNSIII-2 and PNSIII-6) is introduced into Pichia pastoris strain XY30, and the N-terminal CaMCRN of malonyl-CoA reductase gene from Rhizoctonia aurantiaca is integrated at neutral site PNSII-3 or PNSIII-2 or PNSIII-6, thereby obtaining recombinant strains XY33, XY34, XY35, XY43 and XY44.
2. The N-terminal and C-terminal of malonyl-CoA reductase gene CaMCR from green-droxiella aurantiaca are fusion expressed and transferred to a Pichia pastoris strain by adopting a mode of genome integration or plasmid expression, wherein the Pichia pastoris strain overexpresses a gene RAD52 from Pichia pastoris. The method comprises the following steps:
(1) The N-terminal or C-terminal of malonyl-CoA reductase gene CaMCR derived from green-droxiella aurantiaca is fusion expressed and transferred to Pichia pastoris strain by adopting a genome integration mode, and the method specifically comprises the following steps:
constructing a polypeptide comprising SEQ ID NO:1 and SEQ ID NO:2 or the different promoters P AOX1 and P DAS2 of CaMCRCN or CaMCRNC, respectively, and respectively introducing the donor DNA into neutral sites PNSI-2 or PNSIV-16 of Pichia pastoris strains in an integrated manner, thereby obtaining recombinant strains XY09, XY10, XY27 and XY41.
(2) The N-terminal or C-terminal of malonyl-CoA reductase gene CaMCR from orange green flexor is fusion expressed and transferred to Pichia pastoris strain by adopting a plasmid expression mode, and the method specifically comprises the following steps:
Constructing a polypeptide comprising SEQ ID NO:1 and SEQ ID NO:2 or pGCAI-P DAS2-MCRNC-TFBP1, and introducing said expression vector pGCAI-P AOX1-MCRNC-TFBP1 or expression vector pGCAI-P DAS2-MCRNC-TDAS1 into Pichia pastoris strain to obtain recombinant strains XY37 and XY38.
A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol, wherein the construction method is utilized to obtain a strain, and then the strain XY45 after the strain expresses CAS9 at a neutral site PNSI-1 is used as a host strain, and NADH kinase is overexpressed at a neutral site PNSIII-6 of the strain, so that the recombinant strain is obtained; wherein NADH kinase derived from Pichia pastoris endogenous (encoded by gene UTR 1) or NADH kinase derived from Saccharomyces cerevisiae (obtained by amplification of Saccharomyces cerevisiae CENPK-113 genome) encoded by gene ScUTR1 or ScYEF is overexpressed.
The UTR1 gene has the sequence shown in SEQ ID NO:3, a nucleotide sequence shown in figure 3;
The ScUTR gene has the sequence shown as SEQ ID NO:4, a nucleotide sequence shown in seq id no;
The ScYEF gene has the sequence shown as SEQ ID NO:5, a nucleotide sequence shown in seq id no;
A construction method of a recombinant strain for producing 3-hydroxy propionic acid by utilizing methanol, wherein the strain obtained by the construction method is further used as a host strain of XY45 after CAS9 is expressed at a neutral site PNSI-1, and fructose-1, 6-bisphosphatase, D-ribulose-5-phosphate 3-epimerase or dihydroxyacetone synthase are overexpressed; wherein, fructose-1, 6-bisphosphatase is obtained by encoding a gene FBP 1; d-ribulose-5-phosphate 3-epimerase is encoded by the genes RPE1-1 and RPE1-2 or is encoded by the gene OpRPE; dihydroxyacetone synthase is obtained by encoding gene DAS 2.
The FBP1 gene is integrated into a recombinant strain parent chromosome PNSI-13 site;
the RPE1-1, RPE1-2, opRPE or DAS2 gene is integrated into the recombinant strain chromosome PNSI-12 site;
the FBP1 gene has the sequence shown in SEQ ID NO:6, integrated at PNSI-13 positions;
The RPE1-1 gene has the sequence shown in SEQ ID NO:7, integrated at PNSI-12 positions;
The RPE1-2 gene has the sequence shown in SEQ ID NO:8, and is integrated at PNSI-12 positions;
the OpRPE gene has the sequence shown in SEQ ID NO:9, integrated at PNSI-12 positions;
the DAS2 gene has the sequence as shown in SEQ ID NO:10, and is integrated at PNSI-12 positions.
Further, the method comprises the following steps:
(1) Overexpression of the Gene FBP1
A donor DNA containing FBP1 was constructed. Further, the sequence (the nucleotide sequence of which is shown as SEQ ID No. 6) and the function of FBP1 in Pichia pastoris were identified.
(2) Overexpression of the genes RPE1-1 and RPE1-2 and OpRPE
A donor DNA containing RPE1-1 or RPE1-2 or OpRPE was constructed. Further, the sequences of RPE1-1 and RPE1-2 in Pichia pastoris (the nucleotide sequences of which are shown as SEQ ID No. 7 and SEQ ID No. 8) and their functions were identified. Gene OpRPE was obtained by amplification of Hansenula polymorpha genome (the sequence of which is shown as nucleotide sequence SEQ ID No: 9).
(3) Overexpression of gene DAS2
A donor DNA containing DAS2 was constructed. Further, the sequence (the nucleotide sequence of which is shown as SEQ ID No. 10) and the function of DAS2 in Pichia pastoris were identified.
A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol takes a strain with high fatty acid yield as a host, and supplements FAA1 or FAA2 genes in the strain, and simultaneously expresses malonyl-CoA reductase gene CaMCRNC from orange green flexor; the fatty acid-producing strain is PC124 (PNAS., 119 (29): e 2201711119).
The method comprises the following steps:
First, construct sgRNA expression vector pCAI-gFAA t, wherein 20bp targeting sequence is SEQ ID No:11, and constructing an sgRNA expression vector of pCAI-gFAA t, wherein a 20bp targeting sequence is SEQ ID No:12, and a nucleotide sequence shown in seq id no. Second, construct a polypeptide having SEQ ID NO:1 and SEQ ID NO:2 and a donor DNA of a FAA1 gene, introducing the donor DNA into a Pichia pastoris strain, and integrating a malonyl-CoA reductase gene CaMCR from Rhizopus orange at the FAA1 site, while supplementing the FAA1 gene.
Further, a nucleic acid sequence having SEQ ID NO:1 and SEQ ID NO:2 and a donor DNA of a FAA2 gene, introducing the donor DNA into a pichia pastoris strain, integrating another copy of malonyl-coa reductase gene CaMCR from green-orange flexor at the FAA2 site, while supplementing the FAA2 gene.
A construction method of a recombinant strain for producing 3-hydroxy propionic acid by utilizing methanol comprises the steps of obtaining a promoter P FLD1 of a bifunctional alcohol dehydrogenase gene FLD1 and a formaldehyde dehydrogenase gene FLD1 of a weakened strain in the recombinant strain by the back-filling, and replacing the promoter with the promoter P TEF1、PPEX5 or P PMP20.
The method comprises the following steps:
First, construct sgRNA expression vector pCAI-gFLD p, wherein 20bp targeting sequence is SEQ ID No: 13. Second, a donor DNA containing P TEF1、PPEX5 or P PMP20 was constructed, and the donor DNA was introduced into Pichia pastoris strain to attenuate P FLD1.
A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol comprises the step of supplementing an HIS4 gene into the strain obtained after weakening a promoter P FLD1, so as to obtain the recombinant strain.
The fermentation treatment by using the obtained strain comprises the following steps:
Shaking and feeding and fermenting: inoculating Pichia pastoris FXY17H in a basic salt culture medium with 10g/L methanol as a carbon source, and performing a 3-HP feed fermentation experiment. The parameters were set to an initial inoculation OD 600 of 0.5 and the fermentation conditions were as follows: the shake flask was filled with 50mL/250mL of liquid at 220rpm at 30℃and when the methanol concentration in the fermentation system was below 1g/L, 630. Mu.L of methanol and 315. Mu.L of 10X base medium were added, and the pH of the broth was adjusted to 5.6 using 4M KOH solution every 24 hours. The shaking flask feed supplement fermentation can finally obtain 13.0g/L yield,
And (3) feeding and fermenting a bioreactor: inoculating Pichia pastoris FXY17H in a basic salt culture medium with 10g/L methanol as a carbon source, and performing a 3-HP feed fermentation experiment. The parameters were set to an initial inoculation OD600 of 0.5 and the fermentation conditions were as follows: performed in a 1.0L DasGip Parallel Bioreactors System (DasGip) bioreactor. Temperature, agitation, aeration, and pH were monitored and controlled using DasGip Control 4.0.0 systems. The liquid loading amount is 300mL/1L, the temperature is set to 30 ℃, the initial stirring rotating speed is set to 400rpm, the regulating interval is set to 400-800 rpm according to the Dissolved Oxygen (DO) level, the initial ventilation amount is set to 36sL/h, then the DO level is automatically regulated in the interval of 36-48 sL/h according to the DO level change, the DO level is kept above 30%, 4M KOH and 2M HCl are automatically added through a peristaltic pump to keep the pH value of a fermentation system at about 5.6, 600g/L of methanol and 10 times of basic component culture medium are added when 1-3 g/L of methanol is remained in the fermentation system, the methanol supplementing rate is 0.5-0.75 mL/h (1-1.5 g/L and regulated according to the concentration of the residual methanol), and meanwhile, the concentration of the methanol in the fermentation system is kept below 20g/L. The 10 x basal medium was fed at half the rate of methanol solution. The bioreactor fed-batch fermentation can finally obtain the yield of 48.2 g/L.
A construction method of a recombinant strain for producing 3-hydroxy propionic acid by utilizing methanol comprises the step of over-expressing a benzoic acid decarboxylase gene MdlCKpO and a 3-hydroxy isobutyric acid dehydrogenase gene MmsBKpO of pseudomonas putida (Pseudomonas putida) in a Pichia pastoris strain; wherein the pichia pastoris strain integrates the protein Cas9 and overexpresses the gene RAD52 derived from pichia pastoris.
Further, the recombinant Pichia pastoris strain is fused to express the benzoic acid decarboxylase encoded by MdlC gene or MdlC2 gene and the 3-hydroxyisobutyrate dehydrogenase encoded by MmsB gene, comprising the codon-optimized MdlC1KpO1-MmsBKpO1 and MdlC2KpO1-MmsBKpO1 and MdlC2KpO2-MmsBKpO2 for gene expression, preferably integration to the PNSI-2 locus.
The specific flow is as follows:
Construction of a vector comprising the sequence SEQ ID No:14 or MdlC 1. 1KpO1-MmsBKpO1 as set forth in SEQ id no:15 and MdlC, kpO, 1-MmsBKpO1 as set forth in SEQ ID No:16, mdlC 2. 2KpO2-MmsBKpO 2. The donor DNA was introduced into Pichia pastoris strain, and the benzoic acid decarboxylase gene MdlCKpO and the 3-hydroxyisobutyrate dehydrogenase gene MmsBKpO from Pseudomonas putida were integrated at neutral site PNSI-2.
Recombinant Pichia pastoris strain for producing 3-hydroxy propionic acid constructed according to the method
The use of said strain in the production of 3-HP by fermentation culture of said strain with methanol as the sole carbon source.
The method comprises the following steps:
(1) Activating the strain, picking 3 single colonies from YPD streak plates in Delft-G10 culture medium, shaking and culturing at 220rpm and 30 ℃ for no more than 20 hours; fermentation inoculation, at initial OD 600 =0.2 (Delft-M10 medium). The liquid loading amount was 20mL/100mL conical flask, 220rpm, and fermentation was performed at 30 ℃. Site-directed sampling was used for biomass (expressed as absorbance at 600 nm) and 3-HP production analysis.
(2) Shaking and feeding and fermenting: the fermentation was performed at 30℃with an initial OD 600 =0.5, a liquid loading of 50mL/250mL Erlenmeyer flask, 220 rpm. Site-directed sampling was used for biomass (expressed as absorbance at 600 nm) and 3-HP production analysis.
(3) And (3) feeding and fermenting a bioreactor: according to the initial OD 600 =0.5, the liquid loading amount is 300mL/1L bioreactor, the initial stirring rotating speed is set to 400rpm, the adjusting interval is set to 400-800 rpm according to the Dissolved Oxygen (DO) level, the initial ventilation amount is set to 36sL/h, and then the DO level is automatically adjusted in the interval of 36-48 sL/h according to the DO level change, so that the DO level is kept above 30%. Site-directed sampling was used for biomass (expressed as absorbance at 600 nm) and 3-HP production analysis.
The application has the beneficial effects that:
1) The application provides a construction method of a recombinant Pichia pastoris strain for producing 3-HP, which is characterized in that the N end and the C end of malonyl-CoA reductase gene CaMCR from orange green flexor are expressed on genome separately, and 581mg/L of yield can be achieved in 10g/L of methanol culture medium. CaMCR was expressed directly in fusion, with a yield of up to 1.5g/L in 10g/L methanol medium. Subsequent overexpression of UTR1 can increase the yield by 13%. Finally, overexpression of DAS2 can increase yield by 8%.
2) The application provides a construction method of a recombinant Pichia pastoris strain for producing 3-HP, which is characterized in that FAA1 and FAA2 are supplemented in a strain with high fatty acid yield, and CaMCR is integrated, so that 2.1g/L of the recombinant Pichia pastoris strain can be achieved in a 10g/L methanol culture medium. And after weakening P FLD1, the 3-HP yield can be increased by 7%, and can reach 2.2g/L in 10g/L methanol culture medium.
3) The application provides a production process optimization strategy of a recombinant Pichia pastoris strain with high 3-HP yield, and 13.0 g/L3-HP can be produced by fed-batch fermentation in a 250mL shake flask. Feed fermentation in a 1L bioreactor can produce 48.2 g/L3-HP.
4) The application also provides a construction method of the 3-HP synthesized recombinant Pichia pastoris strain, and the 3-HP yield is 325mg/L in 10g/L methanol culture medium through chromosome integration MdlC1KpO1-MmsBKpO1, mdlC2KpO1-MmsBKpO1 and MdlC2KpO1-MmsBKpO 2.
5) The invention realizes the efficient biosynthesis from the pichia pastoris methanol to the 3-HP for the first time, expands the green methanol conversion path, and explores the application potential of the pichia pastoris in the field of microbial cell factories.
Drawings
FIG. 1 shows a schematic diagram of the 3-HP synthesis pathway construction.
FIG. 2 shows the fermentation of strains constructed by CaMCR separate expression, wherein the N-terminus of a is CaMCR, expressed integrally on the genome and the C-terminus is expressed using a plasmid. b is the fermentation condition of the strain obtained by integrating and expressing the N end and the C end of CaMCR on the genome.
FIG. 3 shows the fermentation of a strain constructed by CaMCR fusion expression, wherein a is the fermentation of a strain obtained by fusion expression of the N-terminal and C-terminal of CaMCR on the genome, and b is the fermentation of a strain obtained by fusion expression of the N-terminal and C-terminal of CaMCR using a plasmid.
FIG. 4 shows the effect of overexpressing genes UTR1, scUTR1, scYEF1, FBP1, RPE-1-1, RPE1-1-2, opRPE and DAS2 on 3-HP production, where a is the fermentation of the strain resulting from the case after overexpressing UTR1, scUTR1 and ScYEF, b is the fermentation of the strain resulting from the case after overexpressing FBP1, RPE1-2 and OpRPE, and c is the fermentation of the strain resulting from the case after overexpressing DAS 2.
FIG. 5 shows the effect of 3-HP production after CaMCR expression in the high-fatty acid-producing strain and of the effect of P FLD1 attenuation on 3-HP production, where a is the fermentation of the strain obtained after simultaneous expression CaMCR of FAA1 and FAA2 in the high-fatty acid-producing strain PC124 and b is the fermentation of the strain obtained after P FLD1 attenuation in FXY 04.
FIG. 6 shows the production process optimization of high yield 3-HP strains, including shake flask fed-batch fermentation and bioreactor fed-batch fermentation, where a is the shake flask fed-batch fermentation and b is the bioreactor fed-batch fermentation.
FIG. 7 shows the fermentation of strains constructed by MdlC, kpO, 1-MmsBKpO, mdlC, 2, kpO, 1-MmsBKpO, and MdlC, 2, kpO, mmsBKpO2 expression, where a is the final OD 600 value after fermentation of the strain and b is the 3-HP yield of the strain.
FIG. 8 shows a liquid chromatogram of the synthesis of 3-HP product from recombinant Pichia pastoris.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, all experimental methods used are conventional and all materials, reagents and the like are commercially available from biological or chemical companies.
The invention expresses the malonyl-CoA reductase gene CaMCR from the orange green flexor in pichia pastoris, thereby realizing the biosynthesis of 3-hydroxy propionic acid. On this basis, the yield of 3-hydroxypropionic acid is further improved by enhancing intracellular NADPH supply and enhancing methanol metabolism. The invention also provides another construction method of the recombinant Pichia pastoris strain for producing 3-hydroxy propionic acid, which comprises the following steps: and (3) supplementing FAA1 and FAA2 in the Pichia pastoris strain with high fatty acid yield, and integrating malonyl-CoA reductase gene CaMCR to quickly switch the intracellular metabolic flow from fatty acid synthesis to biosynthesis of 3-hydroxy propionic acid. On the basis, the yield of 3-hydroxy propionic acid is further improved by optimizing the gene copy number. The invention further provides a construction method of the recombinant Pichia pastoris strain for producing 3-hydroxy propionic acid, which comprises the following steps: the synthesis of 3-hydroxypropionic acid can also be achieved by fusion expression of the benzoic acid decarboxylase gene MdlCKpO derived from Pseudomonas putida and 3-hydroxyisobutyrate dehydrogenase MmsBKpO in Pichia pastoris. According to the invention, the efficient biosynthesis of 3-hydroxy propionic acid is realized in Pichia pastoris by taking methanol as the only carbon source for the first time, the green conversion path of methanol is expanded, and the application potential of Pichia pastoris in the field of microbial cell factories is explored.
In examples 1 to 7 below, the production of 3-HP by fermentation culture using methanol as a substrate is described, specifically:
(1) Activating the strain, picking 3 single colonies from YPD streak plates in Delft-G10 culture medium, shaking and culturing at 220rpm and 30 ℃ for no more than 20 hours; fermentation inoculation, at initial OD 600 =0.2 (Delft-M10 medium). The liquid loading amount was 20mL/100mL conical flask, 220rpm, and fermentation was performed at 30 ℃. Site-directed sampling was used for biomass (expressed as absorbance at 600 nm) and 3-HP production analysis.
(2) Shaking and feeding and fermenting: the fermentation was performed at 30℃with an initial OD 600 =0.5, a liquid loading of 50mL/250mL Erlenmeyer flask, 220 rpm. Site-directed sampling was used for biomass (expressed as absorbance at 600 nm) and 3-HP production analysis.
(3) And (3) feeding and fermenting a bioreactor: according to the initial OD 600 =0.5, the liquid loading amount is 300mL/1L bioreactor, the initial stirring rotating speed is set to 400rpm, the adjusting interval is set to 400-800 rpm according to the Dissolved Oxygen (DO) level, the initial ventilation amount is set to 36sL/h, and then the DO level is automatically adjusted in the interval of 36-48 sL/h according to the DO level change, so that the DO level is kept above 30%. Site-directed sampling was used for biomass (expressed as absorbance at 600 nm) and 3-HP production analysis.
Example 1
Construction of Pichia pastoris Strain producing 3-HP by expression CaMCR
The synthesis route of 3-HP is shown in figure 1, 3-HP is synthesized by malonyl-CoA through malonyl-CoA reductase Mcr, and 3-HP can be synthesized only by integrating malonyl-CoA reductase gene CaMCR from orange green flexor in Pichia pastoris strain. CaMCR expression mode is that N end and C end are expressed separately, caMCRC is divided into plasmid expression and genome integration expression, and CaMCRN is expressed on genome only. The other is CaMCR expressed on the genome or on a plasmid in a fusion expression manner.
(1) CaMCRN and CaMCRC are expressed separately:
① Genome expression CaMCRN, plasmid expression CaMCRC to obtain recombinant strain:
First CaMCRN integration of the Donor DNA was constructed, PCR was performed with a stack of primers AOX1p-MCRN-F (AGATCA AAAAACAACTAATTATTCGAAACGATGTCTGGAACCGGTAGGTTG) or CAT1p-MCR N-F (TTGCTCTAGTCAAGACTTACAATTAAAATGATGTCTGGAACCGGTAGGTTGG) or GAPp-MCRN-F (TTCAATCAATTGAACAACTATCAAAACACAATGTCTGGAACCG GTAGGTTG), respectively, with primers MCRN-R (TCAGATGTTAGCTGGAATATTAAGGGTAATCT CG) (template: synthetic CaMCRN plasmid; the system comprises: 5 XSF Buffer 10. Mu.L, dNTP 1. Mu.L, template 50ng, upper and lower primers 2. Mu.L, DNA polymerase 1. Mu.L, H 2 O33. Mu.L, respectively. PCR conditions: 95℃for 3min,95℃for 10s,56℃for 30s,72℃for 51s, and then to the second step, 34 times, 72℃for 10min and 18℃for unlimited time. ) The CaMCRC sequences were amplified, and then the homologous arm region sequences of 1000bp each were obtained upstream and downstream of Pichia pastoris PNSI-2 by the same PCR amplification method, followed by overlap extension of PC R (first round: the system comprises: 5 XPS Buffer 5. Mu.L, dNTP 3. Mu.L, 5 fragments were added in an amount of 1:3:5:3:1, 0.5. Mu.L of DNA polymerase, and H 2 O to 25. Mu.L. PCR conditions: 95℃for 3min,98℃for 10s,58℃for 15s,72℃for 5min, followed by 15 cycles to the second step, 72℃for 10min,18℃for an unlimited time. A second wheel: and (3) a template: first round PCR products. The system comprises: 5 XSF Buffer 10. Mu.L, dNTP 1. Mu.L, template 2. Mu.L, upper and lower primers 2. Mu.L, DNA polymerase 1. Mu.L, H 2 O32. Mu.L, respectively. PCR conditions: 95℃for 3min,95℃for 10s,56℃for 30s,72℃for 2min for 30s, and then to the second step, 34 times, 72℃for 10min and 18℃for unlimited time. ) The complete donor DNA was obtained by gene fusion with promoters P AOX1、PCAT1 and P GAP, terminator T AOX1 and homology arm CaMCRN.
The donor DNA was used in subsequent electrotransformation experiments to obtain the gene editing effect, starting strain PC110 (Nuclei c. Acids. Res.,2021,49 (13): 7791-7805.) and thus Pichia pastoris recombinant strains XY01 (P AOX1-CaMCRN)、XY02(PCAT1 -CaMCRN) and XY11 (P GAP -CaMCRN) expressing the Mcrn protein were obtained.
The electric conversion process comprises the following steps:
1) Selecting a monoclonal in 5mL YPD culture medium (50 mL centrifuge tube) in the evening of the first day (16:00-18:00), and culturing at 220rpm and 30 ℃ overnight for 12-16 h;
2) Transferring the seed strain cultured overnight in the morning to 20mL YPD culture medium (100 mL shake flask) to ensure that the initial OD 600 is 0.15-0.2, and culturing for 4-6 hours to ensure that the OD 600 reaches 0.8-1;
3) Taking 10mL of bacterial liquid, centrifuging for 5min at 500g, collecting cells, removing supernatant, and re-suspending by 1.8mL BED S+0.2mL 1M Dithiothreitol (DTT);
4) Incubating at 30deg.C and 100rpm for 5min;
5) Cells were collected by centrifugation at 500g for 5 min.
6) Resuspension of cells with 400 mu L BEDS (without DTT)
7) Pichia pastoris competent cells were aliquoted into 1.5mL centrifuge tubes (80. Mu.L per tube).
8) Pichia pastoris competent cells can be stored at-80 ℃. (competent cells can be kept for 6 months, and use is not recommended when the transformation efficiency experiment is studied)
9) G418 as resistance to 600ng DNA fragment and 300ng gRNA plasmid, zeocin as resistance to 1 μg DNA fragment and 500ng gRNA plasmid, the mixed cells and DNA were transferred to a cuvette and incubated on ice for 2min.
10 Electric shock parameters): the Bio-rad electroreformer uses the fungal Pic mode.
11 Immediately after clicking, suspension with medium:
Auxotroph: resuspended in 1mL of precooled 1M sorbitol and then spread on selection medium (YNB, 2% glucose+1M sorbitol).
Plasmids based on G418 or zeocin were used: resuspended in 0.5mL 1M sorbitol+0.5 mL YPD and incubated for 1h at 30 ℃. . After 500G centrifugation for 5min to remove supernatant, 200. Mu.L of the water was added to resuspend the strain on G418 or zeocin resistant YPD plates.
12 Culturing in a 30 ℃ incubator for 2-4 days until the transformant appears.
Vector plasmids pGCAI-P AOX1-MCRC-TDAS1 and pGCAI-P CAT1-MCRC-TDAS1 were subsequently constructed. The first step: the primers AOX1p-MCRC-F (AGATCAAAAAACAACTAATTATTCGAAACGATGTCTGC TACTACTGGTGCTAGG) or CAT1p-MCRC-F (TTGCTCTAGTCAAGACTTACAATTAA AATGATGTCTGCTACTACTGGTGCTAGG) and DAS1t-MCRC-R (CTCCTAACTAA AACTGTAAAGACTTCCCGTTCAGACAGTGATAGCCCTACCTCTATG), respectively, a CaMCRC sequence was obtained by PCR amplification using the above primers according to the PCR conditions described in the above procedure, followed by obtaining a promoter fragment P AOX1 or P CAT1 by the same PCR amplification method described above using the primers SpeI-AO X1P-F (GGAAGTAAGATGACACTAGTAGATCTAACATCCAAAGACGAAAGGTTG) and AOX1P-R (CGTTTCGAATAATTAGTTGTTTTTTGATCTTCTCAA), and the primers SpeI-CA T1P-F (GGAAGTAAGATGACACTAGTTAATCGAACTCCGAATGCGGTTCTCCTG) and CAT1P-R (CATTTTAATTGTAAGTCTTGACTAGAGCAAGTG), the terminator fragment T DAS1 was obtained by PCR amplification using the primers DAS1T-F (ACGGGAAGTCTT TACAGTTTTAGTTAGG) and SacII-DAS1T-R (GATTTTACTACCGCCCGCGGGACCCTT GTGACTGACACTTTGG) as well. Gene expression cassettes P AOX1-MCRC-TDAS1 and P CAT1-MCRC-TDAS1 were obtained by gene fusion with the promoter P AOX1 or P CAT1, the terminator T DAS1 and the CaMCRC by overlap extension PCR according to the PCR conditions described in the above steps.
And a second step of: pGCAI (bioresource. Bioprocess.,2022,9,58.) plasmids were digested for 2h with the endonucleases Sac II and Spe I to obtain the backbone of the plasmid.
And a third step of: the expression cassette fragments and the plasmid skeletons are respectively connected by a seamless cloning mode, then are respectively transformed into escherichia coli DH5 alpha, are coated on a flat plate of LB+Amp, and are placed in a 37 ℃ incubator for overnight culture.
Fourth step: the next day 8 single colonies were picked from the plate and sequenced to obtain the correct plasmid strain.
Fifth step: extracting the plasmid. The plasmids were transformed into Pichia pastoris by the above-described electrotransformation process to obtain strains expressing CaMCRC of the plasmids, the starting strains were XY01, XY02 and XY11, thus obtaining recombinant Pichia pastoris strains XY18(PAOX1-CaMCRN+Episomal-PAOX1-CaM CRC)、XY19(PAOX1-CaMCRN+Episomal-PCAT1-CaMCRC)、XY20(PCAT1-CaMCRN+Epi somal-PAOX1-CaMCRC)、XY21(PCAT1-CaMCRN+Episomal-PCAT1-CaMCRC)、XY22(PGA P-CaMCRN+Episomal-PAOX1-CaMCRC) and XY23 (P GAP-CaMCRN+Episomal-PCAT1 -CaMCR C) expressing Mcrn and Mcrc proteins for 3-HP, and then fermentation culture was performed using methanol as a substrate, followed by fermentation culture using methanol as a substrate, whereby XY23 produced 521 mg/L3-HP at the highest (see FIG. 2 a).
② Genomic expression CaMCRN and CaMCRC to obtain recombinant strains:
First, caMCRC integrated Donor DNA was constructed, caMCRC sequences were obtained by PCR amplification using a pair of primers DAS2P-MCRC-F (TCACTCT TATCAAACTATCAAACATCAAAAATGTCTGCTACTACTGGTGCTAG) and AOX1T-MCR C-R (CAGGCAAATGGCATTCTGACATCCTCTTGATCAGACAGTGATAGCCCTACCTC TAT) according to the PCR conditions described in the above steps, and then homologous arm region sequences of 1000bp each upstream and downstream of pichia pastoris PNSIV-16 were obtained by the same manner as by PCR amplification, and complete Donor DNA was obtained by fusion with promoter P DAS2, terminator T AOX1 and homology arm and CaMCRC genes by overlap extension PCR according to the PCR conditions described in the above steps. The donor DNA was used in the subsequent electrotransformation experiments to obtain the gene editing effect, and the starting strain was PC111 (nucleic acids. Res.,2021,49 (13): 7791-7805.), thereby obtaining Pichia pastoris recombinant strain XY30 (PIV-16-P DAS2 -CaMCRC) expressing Mcrc protein.
Following construction CaMCRN of an integrated Donor DNA, the Ca MCRN sequence was amplified by PCR using a pair of primers FGH1p-MCRN-F (CCTCGCT GTAGCGTTCATTCCATCTTTCTAATGTCTGGAACCGGTAGGTTG) or FLD1p-MCRN-F (TGCTTGTTCATACAATTCTTGATATTCACAATGTCTGGAACCGGTAGGTTG) with MCRN-R (TCAGATGTTAGCTGGAATATTAAGGGTAATCTCG), respectively. Then, the homologous arm region sequences of 1000bp respectively on the upstream and downstream of Pichia pastoris PNSII-3 or PNSII I-2 or PNSIII-6 are obtained by the same PCR amplification method, and the complete donor DNA is obtained by overlapping extension PCR and fusion of the promoter P FGH1 or P FLD1, the terminator T FBP1 and the homologous arm and CaMCRN genes under the PCR conditions described in the steps.
The donor DNA was used in the subsequent electrotransformation experiments to obtain the gene editing effect, the starting strain was XY30, thus obtaining recombinant Pichia pastoris strains XY33(PIV-16-PDAS2-CaMCRC+PII-3::PFGH1-CaMCRN)、XY34(PIV-16-PDAS2-CaMCRC+PII-3::PFLD1-CaMCRN)、XY35(PIV-16-PDAS2-CaMCRC+PIII-2::PFLD1-CaMCRN)、XY43(PIV-16-PDAS2-CaMCRC+PII I-6::PFGH1-CaMCRN) and XY44 (PIV-16-P DAS2-CaMCRC+PIII-6::PFLD1 -CaMCRN) expressing Mcrc and Mcrn proteins, and then fermentation culture was performed using methanol as a substrate, with XY34 producing 581 mg/L3-HP at the highest (see FIG. 2 b).
(2) CaMCR fusion expression on genome to obtain recombinant strain:
① When CaMCRCN integrating Donor DNA is constructed, a pair of primers AOX1p-MCRC-F (AGATCAAAAAACAACTAATTATTCGAAACGATGTCTGCTACTACTGGTGCTAGG) and MCRC-R (GACAGTGATAGCCCTACCTCTATGAATC) are used, and the MCRC sequence is obtained by PCR amplification according to the PCR conditions described in the steps by using the primers;
a pair of primers MCRC-MCRN-F (AGGATTCATAGAGGTAGGGCTATCACTGTCTC TGGAACCGGTAGGTTGGC) and MCRN-R (TCAGATGTTAGCTGGAATATTAAGGGT AATCTCG) was used, and the CaMCRN sequence was obtained by PCR amplification using the above primers according to the PCR conditions described in the above steps.
Subsequently, the homologous arm region sequences of 1000bp each on the upstream and downstream of Pichia pastoris PNSI-2 were obtained by PCR amplification according to the same method as described above, and then subjected to overlap extension PCR and fusion with the promoter P AOX1, terminator T FBP1 and homologous arm and CaMCRC and CaMCRN genes to obtain the complete donor DNA.
The donor DNA was used in a subsequent electrotransformation experiment to obtain the gene editing effect, the starting strain was PC110 (nucleic acids. Res.,. 2021,49 (13): 7791-7805.) and thus a recombinant Pichia pastoris strain XY09 (PI-2:: P AOX1 -CaMCRCN) was obtained expressing Mcr protein to produce 3-HP, which can produce 1.2 g/L3-HP (see FIG. 3 a).
② When CaMCRNC is constructed to integrate Donor DNA, a CaMCRN sequence is obtained by PCR amplification with MCRN-R1 (GATGTTAGCTGGAATATTAAGGGTAATCTCG) using a pair of primers AOX1p-MCRN-F (AGATCAAAAAACAACTAATTATTCGAAACGATGTCTGGAACCGGTAGGTTG) or DAS2p-MCRN-F (TCACTCTTATCAAACTATCAAACATCAAAAATGTCTGGAACCG GTAGGTTG), respectively;
CaMCRC sequence was obtained by PCR amplification using a pair of primers MCRN-MCRC-F (GAGATTACCCTTAATATTCCAGCTAACATCTCT GCTACTACTGGTGCTAGGAGTG) and FBP1t-MCRC-R (AAATCTCGGAAACAGTGC CAATCGAACGCATCAGACAGTGATAGCCCTACCTCT) according to the PCR conditions described in the above procedure. Then, the homologous arm region sequences of 1000bp respectively on the upstream and downstream of Pichia pastoris PNSI-2 and PNSIV-16 were obtained by the same PCR amplification method, and the complete donor DNA was obtained by fusion of the overlapping extension PCR with the promoter P AOX1 or P DAS2, the terminator T FBP1 and the homologous arm and CaMCRN and CaMCRC by the PCR conditions described in the above steps.
The donor DNA was used in subsequent electrotransformation experiments to obtain the gene editing effect, the starting strain was PC110 (nucleic acids. Res.; 2021,49 (13): 7791-7805.), from which recombinant Pichia pastoris strains XY10 (PI-2:: P AOX1-CaMCRNC)、XY27(PI-2::PDAS2 -CaMCRNC) and XY41 (PIV-16:: P AOX1 -CaMCRNC) were obtained expressing the Mcr protein to produce 3-HP, and subsequently fermentation cultures were performed using methanol as substrate, with XY10 producing up to 1.5 g/L3-HP (see FIG. 3 a).
(3) CaMCRNC fusion expression on plasmid:
When constructing a plasmid, the first step: using CaMCRNC donor DNA of (2) as a template, a pair of primers SpeI-AOX1P-F (GGAAGTAAGATGACACTAGTAGATCTAACATCCAAAGACGAAA GGTTG) and SacII-FBP1t-R (GATTTTACTACCGCCCGCGGCGCGGAACCTTACTTTT CTATTATCCC) was used to obtain an expression cassette fragment of P AOX1-CaMCRNC-TFBP1 or P DAS2-CaMCRNC-TFBP1 by PCR amplification using the above primers according to the PCR conditions described in the above steps.
And a second step of: pGCAI (bioresource. Bioprocess.,2022,9,58.) plasmids were digested for 2h with the endonucleases Sac II and Spe I to obtain the backbone of the plasmid.
And a third step of: the expression cassette fragments are respectively connected with plasmid skeletons obtained after enzyme digestion in a seamless cloning mode, then are respectively transformed into escherichia coli DH5 alpha, are coated on a flat plate of LB+Amp, and are placed in a 37 ℃ incubator for overnight culture.
Fourth step: the next day 8 single colonies were picked from the plate and sequenced to obtain the correct plasmid strain.
Fifth step: plasmids were extracted and transformed into pichia pastoris by electrotransformation to give a strain expressing CaMCRNC of the plasmid, the starting strain being PC110 (nucleic acids. Res.,2021,49 (13): 7791-7805), whereby pichia pastoris recombinant strains XY37 (P AOX1 -CaMCRNC) and XY38 (P DAS2 -CaMCRNC) expressing Mcr protein for 3-HP production were obtained, and the empty plasmid was transformed to give strain XY36. Then, by fermentation culture using methanol as a substrate, XY37 can produce 803 mg/L3-HP at the highest (see FIG. 3 b).
Example 2
Effect of increasing coenzyme NADPH supply on 3-HP Synthesis
Construction of an integrated Donor DNA, first, a pair of primers GAPp-UTR1-F (TTCAATCAATTGAACAACTATCAAAACACAATGTCTTCTTTGAAGCCGCACAA) and CAT1t-UTR1-R (AATCTTTAATTAATAATAAATATAGTTAGCTCATTTGGAAGCGCG GTCTAAAC) were used; or GAPp-ScUTR1-F (TTCAATCAATTGAACAACTATCAAAACACAAT GAAGGAGAATGACATGAATAATGGCG) and CAT1t-ScUTR1-R (AATCTTTAATTAATA ATAAATATAGTTAGCTTATACTGAAAACCTTGCTTGAGAAGGTTTTG); or GAPp-ScYEF1-F (TTCAATCAATTGAACAACTATCAAAACACAATGAAAACTGATAG ATTACTGATTAACGCTTCC) and CAT1T-ScYEF1-R (AATCTTTAATTAATAATAAATAT AGTTAGCTTAGATTGCAAAATGAGCCTGACGAG) are subjected to PCR amplification according to the PCR conditions described in the steps to obtain UTR1, scUTR1 and ScYEF1 sequences respectively, then homologous arm region sequences of 1000bp respectively on the upstream and downstream of Pichia pastoris PNSIII-6 are obtained in the same manner of PCR amplification, and the PCR conditions described in the steps are subjected to overlap extension PCR and fused with a promoter P GAP, a terminator T CAT1 and homologous arms and UTR1, scUTR1 and ScYEF1 to obtain complete donor DNA.
The donor DNA was used in the subsequent electrotransformation experiments to obtain the gene editing effect, the starting strain was XY45 (strain XY10 integrated with CAS9 gene at PNSI-2 locus), thus obtaining recombinant Pichia pastoris strains XY81 (UTR 1), XY83 (ScUTR 1) and XY85 (ScYEF 1) expressing Utr protein to produce 3-HP, and then fermentation culture was performed using methanol as substrate, wherein the 3-HP yield of XY81 was increased by 13% compared to XY45, reaching 1.5g/L in 10g/L methanol medium (see FIG. 4 a).
Example 3
Effect of methanol utilization pathway on 3-HP Synthesis
The strain XY45 with the CAS9 gene integrated at PNSI-2 site is obtained by the construction method as a host bacterium to overexpress fructose-1, 6-bisphosphatase, D-ribulose-5-phosphate 3-epimerase or dihydroxyacetone synthase;
overexpression of FBP1, RPE1-2 and OpRPE genes improves methanol utilization capacity of pichia pastoris, and specifically comprises the following steps:
when the integrated Donor DNA is constructed,
(1) The primer pair FBP1-FDH1P-F (CAAATACCTCCAACATCACCCACTTA AACAATGTCCAATAACACCACCCAAAACTTG) and FBP1-R (CTAGAGATTCACAGA TTGGATCTTAATTTTTTTTGTTAG) are used for PCR amplification to obtain FBP1 fragments according to the PCR conditions described in the steps, then the homologous arm region sequences of 1000bp on the upstream and downstream sides of Pichia pastoris PNSI-13 are obtained in the same way through PCR amplification, and the PCR conditions described in the steps are used for fusion with the promoter P FDH1 terminator T FBP1 and the homologous arm and FBP1 through overlap extension PCR to obtain the complete donor DNA.
(2) First, a pair of primers RPE1-CAT1p-F (CACTTGCTCTAGTCAAGACTTACAATT AAAATGATGGTCAAACCTGTTATCGCTCC) and RPE1-1-R (CAAGGCAACGGCCCTA GTGA) were used to obtain a fragment of RPE1-1-T RPE1-1 by PCR amplification according to the PCR conditions described in the above steps, and the primer RPE2-CAT1p-F was used
(TGCTCTAGTCAAGACTTACAATTAAAATGATGGTTAAAACAATTATTGCT CCTTCAATC) and RPE1-2-R (TTTTTCGAATAGCTAGGTGATATGAAGGAAAGGTA) were amplified by PCR according to the PCR conditions described in the above procedure to obtain the RPE1-2-T RPE1-2 fragment, and then the homologous arm region sequences of 1000bp each upstream and downstream of Pichia pastoris PNSI-12 were obtained by the same PCR amplification method, and the PCR conditions described in the above procedure were used to obtain the complete donor DNA by overlapping extension PCR with the promoter P CAT1 and the homology arm and the RPE1-1 and RPE1-2 fusion.
Or using the primers OpRPE-CAT1P-F (CTTGCTCTAGTCAAGACTTACAATTAAAATGA TGGTGAAACCAATTATTGCTCCCTC) and OpRPE-ADH2T-R (CATTACATAAGACGTA TACAAACTATTCGGCTCACTCAAGAAGTCCGCGGG) to obtain OpRPE fragments by PCR amplification according to the PCR conditions described in the steps, then obtaining homologous arm region sequences of 1000bp on the upstream and downstream of Pichia pastoris PNSI-12 by the same PCR amplification method, and fusing the homologous arm region sequences with the promoter P CAT1, the terminator T ADH2 and the homologous arm and OpRPE by overlap extension PCR according to the PCR conditions described in the steps to obtain the complete donor DNA.
The different donor DNAs obtained above were used in the subsequent electrotransformation experiments to obtain the gene editing effect, with XY45 as the starting strain, thereby obtaining recombinant Pichia pastoris strains XY70 (FBP 1), XY76 (RPE 1-1), XY77 (RPE 1-2), XY78 (OpRPE) that overexpress Fbp1 and Rpe proteins to produce 3-HP. Then, the fermentation culture was performed using methanol as a substrate, and the yield of 3-HP was not significantly changed (see FIG. 4 b).
The methanol utilization capacity of pichia pastoris is improved by over-expressing DAS2 gene, which comprises the following steps:
when the integrated Donor DNA is constructed,
(1) First, a pair of primers DAS2P-F (ATTACTGTTTTGGGCAATCCTGTTGATAAG) and AOX1t-DAS2-R (CAGGCAAATGGCATTCTGACATCCTCTTGATTATAGTTTGTC GTGCTTTGGTTTTCCC) were used to obtain the P DAS2 -DAS2 sequence by PCR amplification under the PCR conditions described in the above steps. Then the homologous arm region sequences of 1000bp respectively on the upstream and downstream of Pichia pastoris PNSI-12 are obtained by the same PCR amplification method, and the complete donor DNA is obtained by overlapping extension PCR and fusion of the terminator T AOX1, the homologous arm and DAS2 under the PCR conditions described in the steps.
(2) A pair of primers CAT1p-DAS2-F (TTGCTCTAGTCAAGACTTACAATTAAAAT GATGGCTAGAATTCCAAAAGCAGTATCG) and AOX1t-DAS2-R (CAGGCAAATGGC ATTCTGACATCCTCTTGATTATAGTTTGTCGTGCTTTGGTTTTCCC) were used to obtain the DAS2 sequence by PCR amplification according to the PCR conditions described in the above steps. Then the homologous arm region sequences of 1000bp respectively on the upstream and downstream of Pichia pastoris PNSI-12 are obtained by the same PCR amplification method, and the complete donor DNA is obtained by fusion of the overlapping extension PCR with the promoter P CAT1, the terminator T AOX1, the homologous arm and the DAS2 by the PCR conditions described in the steps.
The different donor DNAs obtained above were used in the subsequent electrotransformation experiments to obtain the gene editing effect, with the starting strain XY45, thus obtaining the Pichia pastoris recombinant strains XY100 (P DAS2 -DAS 2) and XY101 (P CAT1 -DAS 2) that overexpress the Das protein to produce 3-HP. Fermentation culture was then performed using methanol as substrate, wherein the 3-HP yield of XY100 was increased by 8% compared to XY45, reaching 1.4g/L in10 g/L methanol medium (see FIG. 4 c).
Example 4
Production of 3-HP by using high fatty acid-producing Pichia pastoris strain as chassis cell expression CaMCR
The common precursor for fatty acid synthesis and 3-HP synthesis is malonyl-CoA, and the metabolic stream of synthetic fatty acids can be directed to the synthesis of 3-HP after the high-fatty acid-producing strain PC124 (PNAS, 119 (29): e 2201711119) is supplemented with FAA1 and FAA2 further in the high-fatty acid-producing strain. The method comprises the following steps:
Construction of a sgRNA expression vector of pCAI-gFAA t, wherein a 20bp targeting sequence is SEQ ID No:11, using a pair of primers gFAA T-F (GAAGCTATGAATGAAAAGCAGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAG) and gFAA T-R (TGCTTTTCATTCATAGCTT CGACGAGCTTACTCGTTTCGTCC), a gFAA T plasmid sequence was obtained by PCR (template: pCAI plasmid (nucleic. Acids. Res.; 2021,49 (13): 7791-7805.); system: 5 XSF Buffer 10. Mu.L, dNTP 1. Mu.L, template 50ng, upper and lower primers 2. Mu.L, DNA polymerase 1. Mu.L, H 2 O33. Mu.L, PCR conditions: 95℃3min,95℃10s,56℃30s,72℃1min55s, followed by cycling to the second step, 25 cycles, 72℃10min,18℃unlimited time.), followed by digestion with DpnI enzyme (system: dpnI 1. Mu.L, 10 XT Buffer 2. Mu.L, DNA. Mu.g, H 2 O to 20. Mu.L) 37℃for 1H, followed by transfer of the plasmid to E.coli DH 5. Mu.C, 5. Alpha.; 5. And subsequent single-day overnight measurement of the second step, and single-phase amplification of the plasmid sequence was obtained by taking the correct strain of Ka. In addition, a sgRNA expression vector is constructed to be pCAI-gFAA t, wherein a 20bp targeting sequence is SEQ ID No:12, using a pair of primers gFAA t-F (AAAAATGAAATAAAAAACAGGTTTTAGAGCTA)
GAAATAGCAAGTTAAAATAAGGCTAG) and gFAA t-R (CTGTTTTTTATTTCATTTTT GACGAGCTTACTCGTTTCGTCC) were amplified by PCR according to the PCR conditions described in the above procedure to obtain gFAA t plasmid sequences, then the plasmids were transferred to E.coli DH 5. Alpha. And incubated overnight in an incubator at 37℃and 4 single clones were picked up the next day into LB+Kana and sequenced to obtain the correct plasmid strain.
The Donor DNA of the quick-repair FAA1 and CaMCR is constructed and integrated, a pair of primers FAA1-HRUP-F1 (AAACACGCGTACAAAGAAAACTTCAAGAAA) and FAA1-CAT1T-R (AATCTTTAA TTAATAATAAATATAGTTAGCTCAACTGTTTTGCCTATATACTTCATCGACAC) are firstly used for obtaining a FAA1 sequence through PCR amplification according to the PCR conditions described in the steps, then the homologous arm region sequence of 1000bp upstream of the FAA1 of Pichia pastoris is obtained in a PCR amplification mode, and the Donor DNA of the quick-repair part of the FAA1 gene is obtained through overlapping extension PCR, fusion of the terminator T FBP1、TCAT1 and the homologous arm and the FAA1 according to the PCR conditions described in the steps.
Then, a pair of primers CAT1T-FBP1T-F (GATGATCGAAACATCATCAGAAAACTAAA CCGCGGAACCTTACTTTTCTATTATCCCTA) and AOX1P-F (AGATCTAACATCCAAA GACGAAAGGTTG) were used to obtain an expression cassette P AOX1-CaMCRNC-TFBP1 of CaMCRNC by PCR amplification under the PCR conditions described in the above steps, and then a homologous arm region sequence of 1000bp downstream of Pichia pastoris FAA1 was obtained by the same PCR amplification method, and a donor DNA of CaMCR gene integration portion was obtained by fusion of the PCR conditions described in the above steps with terminator T CAT1 and homologous arm and CaMCRNC expression cassette by overlap extension PCR. In transformation experiments, two parts of donor DNA were simultaneously introduced into Pichia pastoris by electrotransformation with pCAI-gFAA t plasmid for in vivo recombination, starting strain PC124 (PNAS., 119 (29): e 2201711119), whereby anaplerotic FAA1 was obtained and integrated CaMCRNC to obtain recombinant strain FXY01, and then fermentation culture with methanol as substrate was performed to obtain 3-HP production which was 13% higher than that of wild-type strain XY45, reaching 1.7g/L in 10g/L methanol medium (see FIG. 5 a).
When constructing the Donor DNA for supplementing FAA2 and integrating CaMCR, firstly, a pair of primers FAA2-F (ATGTCACATCTCAAAAAGATCCAGGATAGG) and FAA2-GAPt-R (GAATTTCAGCT ATTTCACATACAAATCGATCTACATCTTAGTCTCCCTCAGAAGACTC) are used for obtaining a FAA2 sequence through PCR amplification according to the PCR conditions described in the steps, then, the homologous arm region sequence 1000bp upstream of Pichia pastoris FAA2 is obtained through the same PCR amplification mode, and the Donor DNA of the FAA2 gene supplementing part is obtained through fusion of overlap extension PCR with a terminator T GAP、TADH2, a homologous arm and FAA2 according to the PCR conditions described in the steps.
Then, a pair of primers ADH2T-MCRC-R (ATTACATAAGACGTATACAAACTATTCGG CTCAGACAGTGATAGCCCTACCTCTATG) and AOX1P-MCRN-F (AGATCAAAAAAC AACTAATTATTCGAAACGATGTCTGGAACCGGTAGGTTG) or DAS2P-MCRN-F (TCACTCTTATCAAACTATCAAACATCAAAAATGTCTGGAACCGGTAGGTTG) are used for carrying out PCR amplification according to the PCR conditions described in the steps to obtain CaMCRNC sequences, then the homologous arm region sequences 1000bp downstream of Pichia pastoris FAA2 are obtained in a PCR amplification mode, and the overlapping extension PCR is carried out according to the PCR conditions described in the steps to fuse with the promoter P AOX1 or P DAS2, the terminator T GAP、TADH2 and the homologous arm and CaMCRNC to obtain CaMCRNC gene integration to obtain P AOX1-MCRNC-TADH2 and P DAS2-MCRNC-TADH2 donor DNA. In transformation experiments, two portions of donor DNA were simultaneously electrotransformed with pCAI-gFAA t plasmid into Pichia pastoris for in vivo recombination purposes, starting with FXY01, whereby recombinant Pichia pastoris strains FXY03 (P AOX1) and FXY04 (P DAS2) for 3-HP production were obtained, complementing FAA2 and integrating another copy of CaMCRNC. Then, the fermentation culture is carried out by taking methanol as a substrate, so that the yield of the produced 3-HP can be improved by 29 percent relative to FXY01, and the yield reaches 2.1g/L in 10g/L methanol culture medium (see FIG. 5 a).
Example 5
Effect of P FLD1 promoter attenuation
Construction of a sgRNA expression vector of pCAI-gFLD p, wherein a 20bp targeting sequence is SEQ ID No:13, and using a pair of primers gFLD p-F (TATGCATGATTACTACACAAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT) and gFLD p-R (TTGTGTAGTAATCATG CATAGACGAGCTTACTCGTTTCGTCCT), a gFLD p plasmid sequence was obtained by PCR amplification according to the PCR conditions described in the above steps, followed by transferring the plasmid to E.coli DH 5. Alpha. And culturing overnight in an incubator at 37℃and picking up 4 single clones the next day into LB+Kana, and sequencing to obtain the correct plasmid strain.
When constructing an integrated Donor DNA, the P TEF1 sequence was first obtained by PCR amplification using a pair of primers TEF1-F (ATAACTGTCGCCTCTTTTAT CTGCC) and TEF1P-R (GTTGGCGAATAACTAAAATGTATGTAGTGAGAATAAAAG) according to the PCR conditions described in the above procedure. Or using a pair of primers PEX5P-F (TCCAAACCAAACGGTCTAGCAAAA) and PEX5P-R (TACGATTAGTTAGATGGTTG GGTTGAGAATAG) to obtain the P PEX5 sequence by PCR amplification according to the PCR conditions described in the above steps. Or using a pair of primers pPMP-F (TTCTGGAGTGTCAAAACAGTAGTGATAAAAGGCTATG) and pPMP-R (CTTAGATTTTTTTTTTTGCTTGGTGGGATTCCTTC) to obtain the P PMP20 sequence by PCR amplification according to the PCR conditions described in the above steps. Then the homologous arm region sequences of 1000bp respectively at the upstream and downstream of the Pichia pastoris P FLD1 are obtained by the same PCR amplification method, and the complete donor DNA with P FLD1 replaced by P TEF1、PPEX5、PPMP20 is obtained by fusion of the homologous arm and P TEF1、PPEX5、PPMP20 by overlapping extension PCR under the PCR conditions described in the steps.
The obtained donor DNA was used in the subsequent electrotransformation experiments to obtain the gene editing effect, the starting strain was FXY04, and P FLD1 -attenuated Pichia pastoris recombinant strains FXY17 (P FLD1::PTEF1)、FXY18(PFLD1::PPEX5) and FXY19 (P FLD1::PPMP20) were obtained. Then, by using methanol as a substrate for fermentation culture, the 3-HP yield of FXY17 can be improved by 7% relative to FXY04, and 2.2g/L of the FXY17 is achieved in 10g/L of methanol culture medium (see FIG. 5 b).
Example 6
Production process optimization of high-yield 3-HP strain
FXY17H is obtained after the HIS4 gene is supplemented by FXY17 of the high-yield 3-HP strain, and the HIS4 supplement Donor is obtained by PCR amplification using primers HIS4P2-F (GAGGATCTCCTGATGACTGACTCACTG) and HIS4T2-R (GATCTATCGAATCTAA ATGTAAGTTAAAATCTCTAAATAATTAAATAAGT) according to the PCR conditions described in the above steps. The obtained donor DNA is used for the subsequent electrotransformation experiment to obtain the gene editing effect, and the initial strain is FXY17, so that the Pichia pastoris recombinant strain FXY17H of the anaplerosis HIS4 is obtained. And then, fermenting and culturing the recombinant strain by using methanol as a substrate, and optimizing the production process, wherein the process comprises feeding and fermenting in a shake flask and a bioreactor.
Shaking and feeding and fermenting: inoculating Pichia pastoris FXY17H in a basic salt culture medium with 10g/L methanol as a carbon source, and performing a 3-HP feed fermentation experiment. The parameters were set to an initial inoculation OD 600 of 0.5 and the fermentation conditions were as follows: the shake flask was filled with 50mL/250mL of liquid at 220rpm at 30℃and when the methanol concentration in the fermentation system was below 1g/L, 630. Mu.L of methanol and 315. Mu.L of 10X base medium were added, and the pH of the broth was adjusted to 5.6 using 4M KOH solution every 24 hours. The shake flask fed-batch fermentation finally gives a yield of 13.0g/L (see FIG. 6 a).
And (3) feeding and fermenting a bioreactor: inoculating Pichia pastoris FXY17H in a basic salt culture medium with 10g/L methanol as a carbon source, and performing a 3-HP feed fermentation experiment. The parameters were set to an initial inoculation OD 600 of 0.5 and the fermentation conditions were as follows: performed in a 1.0L DasGip Parallel Bioreactors System (DasGip) bioreactor. Temperature, agitation, aeration, and pH were monitored and controlled using DasGip Control 4.0.0 systems. The liquid loading amount is 300mL/1L, the temperature is set to 30 ℃, the initial stirring rotating speed is set to 400rpm, the regulating interval is set to 400-800 rpm according to the Dissolved Oxygen (DO) level, the initial ventilation amount is set to 36sL/h, then the DO level is automatically regulated in the interval of 36-48 sL/h according to the DO level change, the DO level is kept above 30%, 4M KOH and 2M HCl are automatically added through a peristaltic pump to keep the pH value of a fermentation system at about 5.6, 600g/L of methanol and 10 times of basic component culture medium are added when 1-3 g/L of methanol is remained in the fermentation system, the methanol supplementing rate is 0.5-0.75 mL/h (1-1.5 g/L and regulated according to the concentration of the residual methanol), and meanwhile, the concentration of the methanol in the fermentation system is kept below 20g/L. The 10 x basal medium was fed at half the rate of methanol solution. The bioreactor feed fermentation eventually gave a yield of 48.2g/L (see FIG. 6 b).
Example 7
Construction of Pichia pastoris Strain producing 3-HP by fusion expression MdlC and MmsB
When the integrated Donor DNA is constructed, first, a pair of primers AOX1p-MdlC1-F (AGATCAAAAAACAACTAATTATTCGAAACGATGGCTTCTGTTCATGGTACTACTT ATG) and MmsB-FL3-MdlC1-R (ACCAATAAAAGCAATTCTAGAACCACCACCTTTA ACAGGAGAAACAGTAGAAACTTCAATCAAAAC) were used to amplify MdlC1KpO1 sequence by PCR according to the PCR conditions described in the above steps, then, a MmsBKpO sequence was amplified by PCR using a pair of primers FL3-MmsB-F (GGTGGTGGTTCTAGAATTGCTTTTATTGGTTTGGGTAATATGGG) and FBP1t-MmsB-R (AAATCTCGGAAACAGTGCCAATCGAACGCATTAATCTTTTTTTCT ATAACCTTCAACAATAGCAGAAAAATC) according to the PCR conditions described in the above steps. Then the homologous arm region sequences of 1000bp respectively at the upstream and downstream of Pichia pastoris PNSI-2 are obtained by the same PCR amplification method, and then the complete MdlC1KpO-MmsBKpO1 gene expression donor DNA is obtained by overlapping extension PCR and fusion with the promoter P AOX1, the terminator T FBP1 and the homologous arm and MdlC1KpO1 and MmsBKpO 1. Or a pair of primers AOX1p-MdlC2-F (AGATCAAAAAACAACTAATTATTCGAAACGATGAAAACTGTTCATGGTGCTACTT ATGATATTTTG) and MmsB-FL3-MdlC2-R (ACCAATAAAAGCAATTCTAGAACCAC CACCTGGTTCAATAGTTTGAGTTGGAACTTCAATC) are used for PCR amplification to obtain MdlC2KpO1 sequence according to the PCR conditions described in the above steps, and then carrying out overlap extension PCR and fusing with a promoter P AOX1, a terminator T FBP1, a homology arm, mdlC2KpO1 and MmsBKpO1 to obtain the complete MdlC2KpO1-MmsBKpO1 gene expression donor DNA. Or a pair of primers AOX1p-MdlC2-KpO2-F (AGATCAAAAAACAACTAATTATTCGAAACGATGAAAACTGTTCACGGTGCTACT TAC) and MmsB-KpO2-FL3-MdlC2-KpO2-R (ACCAATAAAAGCAATTCTAGAACCA CCACCTGGTTCAATAGTTTGAGTTGGAACCTC) are used for PCR amplification to obtain MdlC2KpO2 sequence according to the PCR conditions described in the above steps, then uses a pair of primers FL3-MmsB-KpO2-F (GGTGGTGGTTCTAGAATTGCTTTTATTGGTTTGGGTAACATG) and FBP1t-MmsB-KpO2-R (AAATCTCGGAAACAGTGCCAATCGAACGCATTAATCCTTC TTTCTATAACCTTCAACAATAGCGG) to make PCR amplification to obtain MmsBKpO sequence, then makes overlap extension PCR and promoter P AOX1, The terminator T FBP1 and the homology arms and MdlC2KpO2 and MmsBKpO2 are fused to obtain complete MdlC2KpO2-MmsBKpO2 gene expression donor DNA.
The donor DNA was used in the subsequent electrotransformation experiments to obtain the gene editing effect, the starting strain was PC111B (PNAS., 119 (29): e 2201711119), and thus recombinant Pichia pastoris strains OXY15, OXY16 and OXY18 expressing Mdlc protein and Mmsb were obtained, and then fermentation culture was performed using methanol as a substrate, wherein the 3-HP yield of OXY15 was the highest, and it was possible to achieve 325mg/L (see FIG. 7).
While the application has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the scope of the application, and that the application is not limited to the specific embodiments, but is intended to cover all modifications and variations of the application as fall within the scope of the application, e.g., 3-HP can be produced by other methanolic yeasts (e.g., hansenula, candida boidinii, etc.), without departing from the scope of the application.
SEQ ID NO：1(CaMCRN)：
ATGTCTGGAACCGGTAGGTTGGCCGGTAAGATCGCCCTTATTACCGGTGGAGCCGGTAACATCG
GATCTGAGTTGACTAGGAGGTTCCTTGCCGAAGGAGCCACCGTCATTATCAGTGGTAGGAATAG
AGCCAAACTTACCGCCTTGGCCGAGAGAATGCAAGCTGAGGCTGGAGTTCCAGCTAAGAGGAT
CGACCTTGAGGTCATGGATGGAAGTGACCCAGTTGCTGTTAGGGCTGGTATCGAAGCTATCGTT
GCCAGACACGGTCAGATCGACATCTTGGTCAACAACGCTGGTTCTGCTGGAGCTCAGAGAAGG
TTGGCCGAAATCCCACTTACCGAAGCCGAGCTTGGTCCCGGAGCTGAGGAGACCTTGCACGCT
AGTATCGCCAACCTTTTGGGAATGGGTTGGCACCTTATGAGGATCGCTGCCCCACACATGCCAG
TCGGATCTGCCGTCATCAATGTTTCTACTATTTTTTCTAGGGCTGAATATTACGGAAGGATCCCAT
ACGTCACTCCAAAGGCCGCTTTGAATGCCTTGAGTCAACTTGCTGCTAGAGAATTGGGTGCCA
GAGGAATTAGAGTCAACACCATCTTCCCCGGACCAATCGAGTCTGATAGGATTAGAACCGTCTT
CCAGAGGATGGATCAATTGAAGGGTAGGCCAGAAGGTGACACCGCCCATCATTTCTTGAACAC
CATGAGACTTTGCAGAGCCAATGACCAAGGAGCTTTGGAGAGGAGGTTTCCAAGTGTCGGTGA
TGTCGCTGACGCTGCTGTCTTCTTGGCTAGTGCCGAGTCTGCTGCCTTGTCTGGTGAGACCATT
GAGGTTACCCACGGAATGGAGCTTCCAGCTTGCTCTGAGACCTCTTTGTTGGCTAGAACCGATT
TGAGGACCATCGATGCCTCTGGAAGGACCACCCTTATCTGTGCTGGTGACCAGATTGAGGAGG
TCATGGCCTTGACCGGTATGTTGAGGACTTGCGGATCTGAGGTCATCATCGGTTTTAGAAGTGC
TGCTGCTTTGGCCCAGTTCGAGCAAGCCGTTAATGAGAGTAGGAGGTTGGCTGGTGCCGACTT
CACCCCTCCAATTGCCTTGCCATTGGACCCAAGGGACCCAGCTACTATCGACGCTGTCTTTGATT
GGGCTGGAGAGAACACTGGTGGTATCCATGCTGCCGTTATCTTGCCAGCTACCAGTCACGAACC
AGCTCCTTGTGTTATCGAAGTCGACGATGAGAGGGTCTTGAACTTCTTGGCCGACGAAATCACC
GGTACCATTGTCATCGCCTCTAGACTTGCTAGATACTGGCAGAGTCAGAGATTGACTCCCGGAG
CTAGGGCCAGAGGTCCTAGGGTCATCTTCCTTAGTAACGGAGCCGACCAAAACGGAAACGTCT
ACGGTAGGATCCAGTCTGCCGCCATCGGACAATTGATTAGAGTCTGGAGACATGAGGCCGAATT
GGACTATCAGAGAGCCTCTGCTGCTGGAGACCATGTTTTGCCACCAGTTTGGGCCAACCAGATC
GTTAGATTCGCCAATAGAAGTCTTGAAGGTCTTGAGTTCGCTTGTGCTTGGACCGCTCAGCTTC
TTCACAGTCAGAGGCACATCAACGAGATTACCCTTAATATTCCAGCTAACATCTGASEQ ID NO：2(CaMCRC)：
ATGTCTGCTACTACTGGTGCTAGGAGTGCTAGTGTCGGATGGGCCGAAAGTCTTATCGGTTTGC
ACCTTGGTAAGGTCGCCTTGATCACCGGAGGATCTGCTGGTATCGGAGGACAGATCGGAAGATT。
GCTTGCCCTTAGTGGAGCTAGAGTCATGTTGGCTGCCAGAGATAGGCACAAGTTGGAACAGAT
GCAAGCTATGATCCAGAGTGAACTTGCCGAAGTCGGATACACCGACGTCGAGGATAGAGTCCA
CATTGCCCCCGGATGTGACGTTAGTTCTGAGGCTCAGCTTGCCGATCTTGTCGAGAGAACCTTG
TCTGCCTTCGGAACCGTCGACTACCTTATCAACAATGCCGGTATCGCCGGTGTTGAAGAGATGG
TCATCGACATGCCAGTTGAAGGTTGGAGGCACACCTTGTTCGCCAACTTGATCAGTAACTACTC
TCTTATGAGGAAGTTGGCCCCACTTATGAAGAAGCAAGGATCTGGTTACATCTTGAATGTCTCTA
GTTATTTCGGTGGAGAAAAAGACGCCGCCATCCCATACCCTAACAGAGCTGACTACGCTGTCTC
TAAAGCCGGTCAAAGAGCCATGGCTGAGGTCTTTGCTAGGTTCCTTGGACCAGAGATCCAGAT
CAACGCCATCGCTCCCGGACCAGTTGAAGGAGACAGATTGAGAGGTACCGGTGAAAGGCCCG
GACTTTTTGCTAGGAGAGCTAGACTTATTTTGGAGAACAAGAGGTTGAATGAGTTGCACGCCG
CTTTGATTGCCGCTGCTAGAACCGATGAAAGGAGTATGCACGAGTTGGTCGAGTTGTTGCTTCC
AAACGACGTCGCTGCCTTGGAGCAAAACCCAGCTGCCCCTACTGCTCTTAGGGAGTTGGCTAG
AAGATTCAGATCTGAAGGTGATCCAGCTGCTTCTTCTTCTAGTGCCCTTTTGAATAGATCTATCG
CTGCCAAGCTTCTTGCCAGACTTCACAACGGAGGATACGTCTTGCCAGCTGACATCTTTGCCAA
CTTGCCAAACCCACCAGATCCTTTCTTCACTAGAGCTCAGATTGATAGAGAGGCCAGAAAGGTT
AGAGATGGAATTATGGGAATGTTGTACCTTCAGAGGATGCCAACCGAGTTCGATGTTGCCATGG
CCACCGTTTACTACTTGGCCGATAGAGTCGTCAGTGGAGAGACTTTCCACCCATCTGGTGGTCT
TAGGTACGAAAGAACCCCAACCGGTGGAGAATTGTTCGGATTGCCATCTCCAGAGAGATTGGC
CGAACTTGTCGGAAGTACCGTCTACCTTATCGGAGAGCACCTTACCGAGCACTTGAACCTTCTT
GCTAGAGCCTACTTGGAAAGGTATGGAGCTAGACAAGTCGTCATGATCGTTGAGACCGAGACC
GGAGCCGAGACCATGAGGAGGCTTCTTCATGATCACGTCGAAGCCGGTAGGTTGATGACTATTG
TCGCCGGAGACCAAATCGAGGCCGCCATCGACCAAGCCATTACCAGATATGGTAGGCCCGGAC
CAGTCGTCTGCACCCCTTTCAGACCTTTGCCAACCGTTCCTTTGGTCGGAAGGAAGGATTCTGA
CTGGAGTACCGTCTTGAGTGAGGCCGAGTTCGCTGAGCTTTGTGAGCACCAGCTTACCCACCA
CTTTAGAGTTGCTAGGTGGATCGCTTTGTCTGATGGTGCCAGACTTGCCTTGGTTACCCCAGAA
ACCACCGCCACCTCTACTACCGAGCAGTTCGCCCTTGCCAACTTCATCAAAACCACCCTTCACG
CTTTCACCGCCACCATTGGTGTTGAAAGTGAGAGGACCGCCCAGAGAATCCTTATCAACCAAG
TCGACCTTACTAGAAGGGCTAGAGCTGAAGAACCTAGAGACCCACACGAGAGGCAGCAAGAA
TTGGAAAGGTTCATCGAGGCCGTTCTTTTGGTCACTGCCCCTCTTCCACCAGAAGCTGATACTA
GATATGCCGGAAGGATTCATAGAGGTAGGGCTATCACTGTCTGA
SEQ ID NO：3(UTR1)：
ATGTCTTCTTTGAAGCCGCACAAGGGAGAAGATCCTGAACCTTCTATTTTGGATAATACCTCTACGGAGCCCAAGCATAGATATGAGTCACTCCCTAAGCTTCCTATATTGCACAACAGTTTGTACTGTAAGGATAACCCAAGAGAATTGCCATCAGACAAGGAGATCAGTGGCCTTCAACGCAAGCCGTCAAAACTAAAGTCGAAGCTTTCAGACGAGATTAAGCACCTCTCACCTTCGCTGAAAAGTGTGAAGTCCCATGCACAGTTAGCTAAGACGGCGAACGGAGTTAGATCACTGGCTAGAAACTTGAACAAGGCCACCATCAAGCTCAATGTTCGGTCGGTGATGATCATCACAAAAGCCCGTGACAATTCACTTATATACCTGACAAAGGAGTTGACAGAGTGGCTGCTGAGAAGAGAGCCTCACATGGACATTTATGTTGACCATCACCTTGAGAAATCTAGAAGATTCGATCCAAAATCAATATGGCAGGAGATACCAACAGCGCAGAAACACCTCAAGTTTTGGAACAAGGCTTTGATTAGGGACTGTCCGGATATGTTTGACTTGGTTATCACTCTTGGGGGTGATGGGACTGTATTGTACGCATCTACACTGTTTCAAAGAGTGGTCCCACCTGTTCTTTCATTTTCCCTTGGTTCGCTGGGTTTTTTGACTAACTTTGCATTTGAAGACTTTGCTTCCATTTTGACCGATGTTCTGGAGAATGGTGTGCGCACCAACTTGCGTATGAGATTTACCTGTCGAGCTCATAAAGAAAATGGTGAATTGATGTGCGAGCAGCAGGCCCTCAATGAGCTGACTGTTGATAGAGGCCCTTCACCTTGGGTATCGATGCTTGAGTTATACGGAGATGGTTCTTTGTTGACCGTAGCCCAAGCCGACGGCCTCATAATAGCTACACCAACGGGGTCTACCGCATATTCTCTCAGCGCAGGTGGATCTCTAGTACACCCCAGTGTTTCTGCTATTAGTGTTACACCTATTTGCCCTCACACCCTTTCCTTTCGACCTATTCTTTTGCCAGACTCAATGACTTTACGCATAAAGGTTCCTGCAAGATCAAGATCCACGGCATGGGCCTCCTTTGATGGTAGGTCACGCGTGGAGCTTCTCAAAGGGTATTATGTAACTGTGGCCGCCTCACCCTTCCCATTTCCAACGGTGAGGTCGTCCAAGAATGAATATTTCGACAGCGTGAGCAGGGTCTTAAATTGGAACAGTCGAGAGGAGCAAAAATCATTTGTACACCTGTTGAGCGACAAGAATAAGGAATCTTACAATCAATATCATTCAGGGAGAACTGCTGCTGGGAATATACGTTCTGGCCAGAGTAGAATGAACAGCATCGGCAACCCACAAGAAGAGTCCGAACAGACGAACATGAACGTAGGCCGTCATGCCTACAATAGAGTGACAGACTCGCTTAGACAAGTTCAATTAGAGAGAAGACACAGTAATACTTTTGAAATTGACTACAACGATAGTTCAAGCTCCGATGATAACAATAATCAACTGCCTGGAGAAGCATCTGATTCTACCATCGAGTTAGAAGACAATGCTGGTGACAACTATTTTGTGCCTCTTCCTGGTGAAGGAATCAACACTCCAGTGCCACAACCTTTCTTATCCAATGATTCAAACGTGCACTCAATGTCTAGTAGTAGGGTTAGCCCACTACACGACGGTTTAGACCGCGCTTCCAAATGA
SEQ ID NO：4(ScUTR1)：
ATGAAGGAGAATGACATGAATAATGGCGTAGATAAATGGGTAAATGAGGAAGATGGTCGAAATGATCATCATAACAACAATAATAACTTGATGAAGAAGGCCATGATGAACAATGAGCAAATTGATAGAACTCAGGATATCGACAACGCCAAAGAAATGTTGAGGAAAATATCAAGTGAAAGCAGCTCGCGCAGAAGCTCCCTGTTGAATAAAGATTCATCTCTCGTGAACGGCAATGCAAACAGTGGCGGTGGTACGAGCATTAACGGAACAAGAGGAAGTTCTAAGAGTAGTAATACACACTTTCAGTATGCCTCCACGGCGTATGGTGTAAGAATGTTGAGTAAAGATATATCTAATACCAAAGTGGAACTGGATGTGGAAAATTTGATGATTGTTACGAAACTCAACGATGTCTCACTGTATTTCTTAACAAGAGAGTTGGTAGAATGGGTTTTGGTACATTTTCCACGTGTGACTGTTTATGTGGATTCCGAATTGAAAAACAGCAAAAAATTTGCCGCTGGCGAGTTATGTGAAGATAGTAAATGTAGAGAATCAAGGATCAAGTATTGGACAAAGGATTTCATCAGGGAACATGATGTTTTCTTCGATTTGGTAGTGACTTTGGGTGGCGACGGTACTGTTCTTTTTGTAAGTTCCATTTTTCAGAGACATGTACCACCCGTTATGTCGTTTTCATTAGGGTCTCTAGGATTTTTAACAAATTTTAAGTTTGAACATTTCAGGGAGGATTTACCTCGGATTATGAATCATAAAATCAAGACAAATTTACGGTTGAGGTTGGAGTGCACAATTTATCGTAGACACCGCCCTGAAGTAGACCCAAACACGGGGAAGAAAATATGTGTGGTGGAAAAACTAAGCACACACCACATTTTGAACGAAGTGACCATCGATCGTGGTCCAAGTCCTTTCCTATCCATGTTAGAATTGTATGGTGACGGCTCATTAATGACCGTTGCGCAGGCGGACGGACTGATTGCTGCTACTCCGACTGGGTCCACGGCCTATTCTTTGAGTGCAGGTGGGTCATTGGTATGCCCAACCGTCAATGCAATCGCTTTAACACCCATTTGTCCACATGCATTGAGTTTCAGACCCATCATCTTACCAGAAAGTATAAATTTAAAAGTGAAAGTCTCGATGAAGTCAAGGGCTCCAGCATGGGCGGCTTTTGATGGGAAAGATAGAATTGAATTGCAAAAAGGTGATTTTATAACCATATGCGCCAGCCCATATGCTTTTCCAACCGTGGAAGCCTCGCCCGATGAGTTTATTAACAGTATCAGTCGACAACTAAACTGGAATGTGAGGGAACAACAAAAGTCCTTTACGCATATTTTGTCCCAAAAGAACCAAGAAAAATATGCACATGAGGCGAACAAAGTCAGAAATCAAGCAGAACCTTTAGAGGTAATAAGAGATAAATACTCTCTGGAAGCAGACGCTACTAAGGAAAACAACAACGGAAGCGATGATGAGAGCGACGATGAGAGTGTAAACTGCGAAGCTTGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAA
SEQ ID NO：5(ScYEF1)：
ATGAAAACTGATAGATTACTGATTAACGCTTCCCCGGAGACATGTACCAAGGGAGATGCTGAGATGGATACTATGGATACTATTGGCAGAATGACATCAGTTAAAGTTTTAGCGGAAGGCAAGGTATTAAGCAATTTCGAAGAACCGGGCTTAATGAGGTGCGCTTATCATGATGCAAAAAACTGGGTCAGAAGATTATCGAGCGAAACAATTGTCGGTGAGGACACGAGTAATTTATACCCATTTTATGTTGATACTGCATACGATGTAAGGCGTTTGAGAAAGGATATTATAAATGCTAAGGTTGACTTGCAGGTTGAAAACCTGATCATAATCTGCAATATTAATGATATTTCCACTGTATTTCTCATGAGAGAAGTGGTGGAATGGATCTTACGCAATTTCCATTCAATGACTGTATATGTACAAGATATTTTTGAAAAGTCAACTCAGTTTGCTGTTGGTGACCTCTGCAAAGACAGCAATTGCAGTAAAAACAGAGTAAAGTATTGGTCAAAAGAATTTGTTGAAAAACACGATTCATTCTTTGACTTGATGATTACACTAGGGGGTGATGGAACTGTCCTTTTTGCTTCATCTATATTCACGAAAGATGTTCCGCCGATTGTTCCATTTGCCCTTGGATCATTAGGATTTCTAACAAATTTTGAATTTCAAAATTTCAAAGAAACATTGAAACATATCTTAACAGATGAGGTTCGTATTAATTTACGAATGAGGTTGCAATGCAAACTCTACCGTAGAAATAAACCAGAAATTGATGCCGCAACTGGGAGAAAAATATGTTACATCGATTTCATCTCCGAACATCACGTATTGAACGAAGTAACCATAGATAGAGGTCCAGCTCCTTGTTTATCCCTATTAGAACTCTATGGAAACGACTCACTAATGACTAAGGTTCAGGGAGATGGATTGATTGTTGCCACGCCTACGGGATCCACGGCATACTCATTGAGTGCAGGAGGCTCTTTAATATCGCCAAGCGTAAATGCCATAGCGGTGACGCCTATATGTCCTCATACTTTGAGCTTTAGGCCTATAATTTTACCAGATAGCATGGAATTAAAAGTTAGAGTAGATATGAACTCAAGAGGGACGTCGTGGGTGAATTTTGACGGAAAAGATAGAGTTGAATTGAAACAGGGTGACTATGTTGTGATAACTGCAAGCCCCTATTCGGTACCGACTATCGAGTCATCTGCCAGTGAATTTTTTGAAAGTATCAGTAAAAATCTTAATTGGAATGACCGCGAAGAGCAGAAGCCATTTGCACATATTCTCTCGCCAAAAAATCAAGAAAAATATAGATTAGACTCATCGAAAAATGGAAACGACACCATAAGTAATCCCCTCGAGAGTTCATGCATAAGCTCAGATGCACAAGATGAGGAGAGGAAATCCGTAACGGAAACAGAAACAGAAATAGTTGTTGAACGGACTCGTCAGGCTCATTTTGCAATCTAA
SEQ ID NO：6(FBP1)：
ATGTCCAATAACACCACCCAAAACTTGGCTGAACAGAAGGGTATTCAGACCGACTTGGTCACTCTGACTCGTTTCATCTTGGACGAGCAGAAGAAATCTGCCCCAAACGCTACCGGTGAGCTGACTTTGCTTCTGAACTCCCTGCAATTTGCCTTCAAATTCATTGCTCACACCATCAGAAGATCCGAATTGGTCAACTTAATTGGATTGGCCGGTGTGACCAACGCCACTGGTGACGATCAGAAAAAGCTGGATGTTATTGGAGATGAGATCTTCATCAATGCCATGAAGGGATCCGGTAACGTTAAGTTACTTGTTTCCGAAGAGCAAGAAGACCTTATTGTGTTTGAGTCCTCCAAGGGAAACTATGCTGTTGTCTGCGACCCAATTGACGGTTCATCCAACTTGGACGCTGGTGTCTCAGTTGGTACCATTTTTGGTGTATACAAACTGTTGCCAGGCTCAGCTGGTTCTATTAAGGATGTTTTGAGATCAGGAACAGAGATGGTGGCTGCCGGTTACACCATGTACGGTGCATCTTCTCACTTGATGCTTACTACCGGAAATGGAGTCAACGGTTTCACCTTGGACACCGACTTGGGAGAGTTCATTCTCACGTATCCATCCTTAAAAATTCCGCACACCAGGGCTATCTACTCTATCAATGAAGGTAACTCGCACTACTGGACCGATGGAGTCAATGAGTACATTGCTTCTTTGAAGAAACCCCAAGCAAACGGAAAACCATACAGTGCCCGTTATATCGGATCAATGGTTGCTGATGTCCACAGAACTCTGTTATATGGAGGTATCTTTGGCTATCCAGCAGACTCCAAGTCTAAGTCCGGTAAACTGAGAGTCTTGTACGAGTGTTTCCCAATGGCCCTCCTGTTGGAGCAGGCTGGTGGTGAGGCAGTCAACGATAAGGGTGAGAGAATTCTAAACTTGGAGCCAAAACAAGTGCATGAGAGATCTGGTATCTGGTTGGGTTCCAAGGGAGAAGTAGAGAGATTACTACCCTATCTAACAAAAAAAATTAAGATCCAATCTGTGAATCTCTAG
SEQ ID NO：7(RPE1-1)：
ATGGTCAAACCTGTTATCGCTCCCTCAATCCTGTCGGCAGACTTTTCAAACTTGGGATGTGAATGTCACAAGATGTTTAAGCTACGGGCTGACTGGGTCCACATTGACGTCATGGACGGCCATTTCGTCCCTAATTTGACCATGGGTCCTCCAATTATCTCGTCCCTTAGAAAAGCTGTTCCGAGGGGAGAGAAAGATGGACAGACGACACACTTCTTTGACTGCCATATGATGGTGGCCAATCCCGAACAATGGGTCCCAGAGGTTGCAAAGGCAGGTGGTGATCAGTACACTTTTCACTACGAAGCAACTAAGGATCCAGTGAAGTTAGTGGAACTTATCAAGAGCCATGGACTGAAAGCTGCATGTGCTATCAAGCCCGGAACATCAGTCGATGTCTTGTACCCTCTAGCAGACAAGCTTGACATGGCTCTGGTAATGACAGTTGAGCCAGGGTTTGGTGGACAGAAGTTTATGGCTGATATGATGCCCAAAGTTGAAGCATTGCGGGCCAAATTTCCAAACTTAGATATTCAGGTTGACGGTGGTCTTGGAAAGGAAACCATAGGAGTAGCAGCCGATGCTGGGGCAAACGTAATTGTTGCTGGATCTTCTGTTTTTGGTGCTAAAGACCCCGGTGAGGTTATCCAATTTTTGCGTGACACTGTCCAAGATAGTCTCAAAAAGAAAGGTCTTTTAGATGAATAG
SEQ ID NO：8(RPE1-2)：
ATGGTTAAAACAATTATTGCTCCTTCAATCCTGTCGGCAGATTTTTCAAACTTGGGATGTGAATGTCACAAGATGTTTAAGCTACGGGCTGACTGGGTCCACATTGACGTCATGGACGGCCATTTCGTCCCTAATTTGACCATGGGTCCTCCAATTATCTCGTCCCTTAGAAAAGCTGTTCCGAGGGGAGAGAAAGATGGACAGACGACACACTTCTTTGACTGCCATATGATGGTGGCCAATCCCGAACAATGGGTCCCAGAGGTTGCAAAGGCAGGTGGTGATCAGTACACTTTTCACTACGAAGCAACTAAGGATCCAGTGAAGTTAGTGGAACTTATCAAGAGCCATGGACTGAAAGCTGCATGTGCTATCAAGCCCGGAACATCAGTCGATGTCTTGTACCCTCTAGCAGACAAGCTTGACATGGCTCTGGTAATGACAGTTGAGCCAGGGTTTGGTGGACAGAAGTTTATGGCTGATATGATGCCCAAAGTTGAAGCATTGCGGGCCAAATTTCCAAACTTAGATATTCAGGTCGATGGTGGTCTTGGAAAGGAGACCATTGGAGTAGCAGCTGATGCTGGGGCAAACGTAATTGTTGCTGGATCTTCTGTTTTTGGTGCTAAAGACCCCGGTGAGGTTATCCGATATCTTCGTGATACGGTAGAAAATGCTCAGAAAAAAGCTAAAGCCAAACCGAAACCCAATTTATGA
SEQ ID NO：9(OpRPE)：
ATGGTGAAACCAATTATTGCTCCCTCTGTTCTGGCAGGTGACTTTGCGCAGCTCGGCTGCGAGTGCCACCGCATGTTTAAGTGCAACGCCGATTGGGTCCATTTGGACGTCATGGATGGCCATTTCGTCCCTAATCTCACCATGGGTCCGCCTATAATTTCCAGCCTGAGAAAGGCTGTTCCGAGAGACGAGAGCCAGCCGGGGAAATTCTTCTTCGACTGTCATATGATGGTCGCCAACCCTGAGCAGTGGGTTCCTGAGATTGCAGATGCTGGCGGCGACCAGTATACGTTCCACTACGAAGCAACCAAGGACCCTGTCGGACTTTGCAAGCTGATCAAAAAGCATGGCATGAAGGCCGCATGCGCCATCAAGCCGGGCACACCGGTCGACGTGCTCTATCCACTCGCAGAGCACCTGGACATGGCCCTTGTGATGACGGTCGAGCCAGGCTTCGGAGGCCAGTCGTTCATGGCTGACATGATGCCCAAGGTTGAGACTCTGCGTGCCAAGTACCCGCATCTGAACATCCAGGTTGACGGAGGACTTGGTCCCAAAACCATCCCGGCTGCTGCCAAGGCGGGAGCCAACGTCATTGTTGCCGGCACGTCTGTGTTCAAGGCAGAGAAACCGGAGGAAGTTATCCAGCTGCTCAGAGATTACGTGTCGAAAGAACTGGAGTCCCGCGGACTTCTTGAGTGA
SEQ ID NO：10(DAS2)：
ATGGCTAGAATTCCAAAAGCAGTATCGACACAAGATGACATTCATGAATTGGTCATCAAAACCTTCCGTTGTTACGTTCTCGACTTAGTCGAACAGTATGGTGGTGGTCACCCTGGTTCTGCCATGGGTATGGTCGCCATTGGTATCGCTCTGTGGAAGTACCAGATGAAGTACGCTCCAAATGATCCAGACTACTTCAACAGAGATCGTTTTGTCTTGTCAAACGGTCACGTCTGTCTGTTCCAATACTTGTTCCAGCACTTAACTGGTTTGAAGGAGATGACTGTCAAGCAACTTCAATCTTACCACTCTTCCGATTATCACTCATTGACTCCTGGACACCCTGAAATTGAGAACCCTGCTGTTGAGGTTACCACTGGTCCCCTGGGACAAGGTATCTCTAACGCTGTCGGTATGGCCATTGGTTCAAAGAACCTGGCCGCTACTTACAACAGACCTGGCTTCCCTGTCGTTGACAACACTATCTATGCTATTGTTGGTGATGCTTGTTTGCAAGAGGGACCTGCTTTGGAATCGATTTCCTTAGCTGGTCACTTGGCCTTGGACAACCTTATTGTGATCTACGACAACAACCAGGTTTGTTGTGATGGTTCCGTCGATGTTAACAACACCGAAGACATTTCTGCTAAGTTTAGAGCTCAGAACTGGAATGTCATTGAAGTCGAGAATGGTTCTAGAGATGTTGCTACCCTTGTCAAGGCCATCGAATGGGCCAAGGCTGAGAATGAGAGACCAACTCTGATCAACGTTAGAACTGAAATTGGACAGGATTCTGCTTTCGGTAACCACCACGCTGCTCACGGTTCTGCTCTTGGTGAGGAAGGTATCCGGGAGTTGAAGGCCAAGTACGGTTTCGATGTCGCTAGAAAGTTCTGGTTCCCACAGGAGGTCTATGATTTCTTTGCTGAAAAACCAGCCGAGGGTGATCAACTAGTTGCTAACTGGAAGAAACTTTTGGATGAGTACGTTAAGAACTATCCTCAAGAAGGTGAGGAATTAAAGGCCCGTATTAGAGGTGAACTTCCAAAGAACTGGAAGAGTTTCATTCCACAGGACAAACCAACCGAGCCAACTGCTACCAGAACCTCTGCTAGAGAAATTGTTAGATCTCTGGGACAAAACCTTCCTCAGGTTATTGCTGGTTCTGGTGACTTGTCCGTGTCCATTCTTTTGAACTGGGGAGGAGTTAAGTACTTCTTCAACCCTAAGTTACAAACTTTCTGTGGATTGGGTGGTGACTACTCTGGTAGATATATTGAGTTTGGTATCAGAGAACACTCTATGTGTGCTATTGCCAATGGTTTGGCTGCATACAACAAGGGTACTTTCTTGCCTATTACCTCAACTTTCTACATGTTCTACCTGTATGCAGCACCTGCCTTGCGTATGGCTGCACTTCAAGAGTTGAAAGCAATTCACATTGCTACACACGACTCCATCGGAGCTGGTGAAGATGGTCCAACGCACCAGCCTATTGCTTTGTCTTCATTATTCAGAGCTATGCCCAACTTCTACTACATTAGACCAGCCGATGCTACCGAGGTTGCAGCTCTGTTTGAAGTAGCTGTTGAGCTCGAGCACTCCACCTTGTTCTCTCTGTCCAGACACGAGGTTGAGCAATACCCAGGTAAGACTTCGGCTGAGGGAGCCAAAAGAGGTGGTTACGTCGTTGAAGACTGTGAGGGTAAGCCAGACGTCCAATTAATTGGTGCTGGTTCCGAATTGGAGTTTGCCGTCAAAACTGCTCGTTTGCTAAGACAACAGAAGGGATGGAAGGTCAGAGTTCTGTCATTCCCATGTCAGAGACTGTTTGACCAACAATCCCTGGCATACAGACGTTCTGTCCTTAGAAGAGGAGAGGTTCCAACTGTCGTTGTTGAGGCCTATGTCGCATACGGATGGGAGAGATACGCCACTGCTGGTTACACCATGAACACCTTTGGTAAGTCTCTTCCTGTTGAGGATGTCTACAAATACTTCGGATACACTCCTGAGAAGATTGGTGAGAAGGTTGCTGCATACGTCAACTCTATTAAGGCTAGTCCTCAAATCCTTTACGAATTCACCGATTTGAAGGGAAAACCAAAGCACGACAAACTATAA
SEQ ID NO：11(gFAA1t)：GAAGCTATGAATGAAAAGCA
SEQ ID NO：12(gFAA2t)：AAAAATGAAATAAAAAACAG
SEQ ID NO：13(gFLD1p)：TATGCATGATTACTACACAA
SEQ ID NO：14(MdlC1KpO1-MmsBKpO1)：
ATGGCTTCTGTTCATGGTACTACTTATGAATTGTTGAGAAGACAAGGTATTGATACTGTTTTTGGTAATCCTGGTTCTAACGAATTGCCATTTTTGAAAGATTTTCCTGAAGATTTTAGATATATTTTGGCTTTACAAGAGGCTTGTGTTGTTGGTATTGCTGATGGTTATGCTCAAGCTTCTAGAAAACCTGCTTTTATTAATTTGCATTCTGCTGCTGGTACTGGTAATGCTATGGGTGCTTTGTCTAATGCTTGGAATTCTCATTCTCCATTGATTGTTACTGCTGGTCAACAAACTAGAGCTATGATTGGTGTTGAAGCTTTGTTGACTAATGTTGATGCTGCTAATTTGCCTAGACCATTGGTTAAATGGTCTTATGAACCTGCTTCTGCTGCTGAAGTTCCACATGCTATGTCTAGAGCTATTCATATGGCTTCTATGGCTCCACAAGGTCCTGTTTATTTGTCTGTTCCATATGATGATTGGGATAAAGATGCTGATCCACAATCTCATCATTTGTTTGATAGACATGTTTCTTCATCTGTTAGATTGAATGATCAAGATTTGGATATTTTGGTTAAAGCTTTGAATTCTGCTTCTAATCCTGCTATTGTTTTGGGTCCTGATGTTGATGCAGCTAATGCAAATGCTGATTGTGTTATGTTGGCTGAAAGATTGAAAGCTCCTGTTTGGGTTGCTCCATCTGCTCCAAGATGTCCATTTCCAACTAGACATCCATGTTTTAGAGGTTTGATGCCTGCTGGTATTGCTGCTATTTCTCAATTGTTGGAAGGTCATGATGTTGTTTTGGTTATTGGTGCTCCTGTTTTTAGATATCATCAATATGATCCTGGTCAATATTTGAAACCTGGTACTAGATTGATTTCTGTTACTTGTGATCCATTGGAAGCTGCTAGAGCTCCAATGGGTGATGCTATTGTTGCTGATATTGGTGCTATGGCTTCTGCTTTGGCTAATTTGGTTGAAGAATCTTCTAGACAATTGCCAACTGCTGCTCCTGAACCTGCTAAAGTTGATCAAGATGCTGGTAGATTGCATCCTGAAACTGTTTTTGATACTTTGAATGATATGGCTCCTGAAAATGCTATTTATTTGAATGAATCTACTTCTACTACTGCTCAAATGTGGCAAAGATTGAATATGAGAAACCCTGGTTCTTATTACTTTTGTGCTGCTGGTGGTTTGGGTTTTGCTTTGCCTGCTGCTATTGGTGTTCAATTGGCTGAACCTGAAAGACAAGTTATTGCTGTTATTGGTGATGGTTCTGCTAATTATTCTATTTCTGCTTTGTGGACTGCTGCTCAATATAATATTCCAACTATTTTTGTTATTATGAATAATGGTACTTATGGTGCTTTGAGATGGTTTGCTGGTGTTTTGGAAGCTGAAAATGTTCCTGGTTTGGATGTTCCTGGTATTGATTTTAGAGCTTTGGCTAAAGGTTATGGTGTTCAAGCTTTGAAAGCTGATAATTTGGAACAATTGAAAGGTTCTTTGCAAGAAGCTTTGTCTGCTAAAGGTCCTGTTTTGATTGAAGTTTCTACTGTTTCTCCTGTTAAAGGTGGTGGTTCTAGAATTGCTTTTATTGGTTTGGGTAATATGGGTGCTCCAATGGCTAGAAATTTGATTAAAGCTGGTCATCAATTGAATTTGTTTGATTTGAATAAAGCTGTTTTGGCTGAATTAGCTGAGTTGGGTGGTCAAATTTCTCCATCTCCAAAAGATGCAGCTGCTAATTCTGAATTGGTTATTACTATGTTGCCTGCTGCAGCTCATGTTAGATCTGTTTATTTGAATGAAGATGGTGTTTTGGCTGGTATTAGGGCTGGTACTCCAACTGTTGATTGTTCTACTATTGATCCACAAACTGCTAGAGATGTTTCTAAAGCTGCAGCTGCAAAAGGAGTTGATTTGGGTGATGCTCCTGTTTCTGGTGGTACTGGTGGTGCTGCAGCTGGTACTTTGACTTTTATGGTTGGTGCTTCTGCTGAATTGTTTGCTACTTTGAAACCTGTTTTGGAACAAATGGGTAGAAATATTGTTCATTGTGGTGAAGTTGGTACTGGTCAAATTGCTAAAATTTGTAATAATTTGTTATTGGGTATTTCTATGATTGGTGTTTCTGAAGCTATGGCTTTGGGTAATGCTTTGGGTATTGATACTAAAGTTTTGGCTGGTATCATCAATTCTTCTACTGGTAGATGTTGGTCTTCTGATACTTATAATCCATGGCCTGGTATTATTGAAACTGCTCCTGCTTCTAGAGGTTATACTGGTGGTTTTGGTGCTGAATTGATGTTGAAAGATTTGGGTTTGGCTACTGAAGCTGCTAGACAAGCTCATCAACCTGTTATTTTGGGTGCTGTTGCTCAACAATTGTATCAAGCTATGTCTTTGAGAGGTGAAGGTGGTAAAGATTTTTCTGCTATTGTTGAAGGTTATAGAAAAAAAGATTAA
SEQ ID NO：15(MdlC2KpO1-MmsBKpO1)：
ATGAAAACTGTTCATGGTGCTACTTATGATATTTTGAGACAACATGGTTTGACTACTATTTTTGGTAATCCTGGTTCTAATGAATTGCCATTTTTGAAAGGTTTTCCTGAAGATTTTAGATATATTTTGGGTTTGCATGAAGGTGCTGTTGTTGGTATGGCTGATGGTTATGCTTTGGCTTCTGGTCAACCAACTTTTGTTAATTTGCATGCTGCAGCTGGTACTGGTAATGGTATGGGTGCTTTGACTAATGCTTGGTATTCTCATTCTCCATTGGTTATTACTGCTGGTCAACAAGTTAGATCTATGATTGGTGTTGAAGCTATGTTGGCTAATGTTGATGCTGCTCAATTGCCAAAACCATTGGTTAAATGGTCTCATGAACCTGCTACTGCTCAAGATGTTCCAAGAGCTTTGTCTCAAGCTATTCATACTGCTAATTTGCCACCAAGAGGTCCTGTTTATGTTTCTATTCCATATGATGATTGGGCTTGTGAAGCTCCATCTGGTGTTGAACATTTGGCTAGAAGACAAGTTTCTTCTGCTGGTTTGCCATCTCCTGCTCAATTGCAACATTTGTGTGAAAGATTGGCTGCAGCTAGAAATCCTGTTTTGGTTTTGGGTCCTGATGTTGATGGATCTGCAGCTAATGGTTTGGCTGTTCAATTGGCTGAAAAATTGAGAATGCCTGCTTGGGTTGCTCCATCTGCTTCTAGATGTCCATTTCCAACTAGACATGCTTGTTTTAGAGGTGTTTTGCCTGCTGCAATTGCTGGTATTTCTCATAATTTGGCTGGTCATGATTTGATTTTGGTTGTTGGTGCTCCTGTTTTTAGATATCATCAATTTGCTCCTGGTAATTATTTGCCTGCTGGTTGTGAATTGTTGCATTTGACTTGTGATCCTGGTGAAGCTGCTAGAGCTCCAATGGGTGATGCTTTGGTTGGTGATATTGCTTTGACTTTGGAAGCTGTTTTGGATGGTGTTCCACAATCTGTTAGACAAATGCCAACTGCTTTACCTGCTGCTGAACCTGTTGCTGATGATGGTGGTTTGTTGAGACCTGAAACTGTTTTTGATTTGTTGAATGCTTTGGCTCCAAAAGATGCTATTTATGTTAAAGAATCTACTTCTACTGTTGGTGCTTTTTGGAGAAGAGTTGAAATGAGAGAACCTGGTTCTTATTTTTTTCCTGCTGCTGGTGGTTTAGGTTTTGGATTGCCTGCTGCTGTTGGTGTCCAATTGGCTTCTCCTGGTAGACAAGTTATTGGTGTTATTGGTGATGGTTCTGCTAATTATGGTATTACTGCTTTGTGGACTGCTGCTCAATATAATATTCCTGTTGTTTTTATTATTTTGAAAAATGGTACTTATGGTGCTTTGAGATGGTTTGCTGATGTTTTGGATGTTAATGATGCTCCTGGTTTGGATGTTCCTGGTTTGGATTTTTGCGCTATTGCTAGAGGTTATGGTGTTCAAGCTGTTCATGCTGCTACTGGTTCTGCTTTTGCTCAAGCTTTGAGAGAAGCTTTGGAATCTGATAGACCTGTTTTGATTGAAGTTCCAACTCAAACTATTGAACCAGGTGGTGGTTCTAGAATTGCTTTTATTGGTTTGGGTAATATGGGTGCTCCAATGGCTAGAAATTTGATTAAAGCTGGTCATCAATTGAATTTGTTTGATTTGAATAAAGCTGTTTTGGCTGAATTAGCTGAGTTGGGTGGTCAAATTTCTCCATCTCCAAAAGATGCAGCTGCTAATTCTGAATTGGTTATTACTATGTTGCCTGCTGCAGCTCATGTTAGATCTGTTTATTTGAATGAAGATGGTGTTTTGGCTGGTATTAGGGCTGGTACTCCAACTGTTGATTGTTCTACTATTGATCCACAAACTGCTAGAGATGTTTCTAAAGCTGCAGCTGCAAAAGGAGTTGATTTGGGTGATGCTCCTGTTTCTGGTGGTACTGGTGGTGCTGCAGCTGGTACTTTGACTTTTATGGTTGGTGCTTCTGCTGAATTGTTTGCTACTTTGAAACCTGTTTTGGAACAAATGGGTAGAAATATTGTTCATTGTGGTGAAGTTGGTACTGGTCAAATTGCTAAAATTTGTAATAATTTGTTATTGGGTATTTCTATGATTGGTGTTTCTGAAGCTATGGCTTTGGGTAATGCTTTGGGTATTGATACTAAAGTTTTGGCTGGTATCATCAATTCTTCTACTGGTAGATGTTGGTCTTCTGATACTTATAATCCATGGCCTGGTATTATTGAAACTGCTCCTGCTTCTAGAGGTTATACTGGTGGTTTTGGTGCTGAATTGATGTTGAAAGATTTGGGTTTGGCTACTGAAGCTGCTAGACAAGCTCATCAACCTGTTATTTTGGGTGCTGTTGCTCAACAATTGTATCAAGCTATGTCTTTGAGAGGTGAAGGTGGTAAAGATTTTTCTGCTATTGTTGAAGGTTATAGAAAAAAAGATTAA
SEQ ID NO：16(MdlC2KpO2-MmsBKpO2)：
ATGAAAACTGTTCACGGTGCTACTTACGATATTTTGAGACAACATGGTTTGACTACTATTTTTGGTAACCCAGGTTCTAACGAATTGCCATTTTTGAAGGGTTTTCCAGAAGATTTCAGATACATTTTGGGTTTGCATGAAGGTGCTGTTGTTGGTATGGCTGATGGTTATGCTTTGGCTTCTGGTCAACCAACTTTCGTTAACCTGCATGCTGCTGCTGGTACTGGTAATGGTATGGGTGCTTTGACCAATGCTTGGTACTCTCATTCTCCATTGGTTATTACTGCTGGTCAGCAAGTTAGATCTATGATTGGTGTTGAGGCTATGTTGGCTAACGTTGATGCTGCCCAATTGCCAAAACCATTGGTTAAATGGTCTCATGAGCCAGCTACTGCACAAGATGTTCCTAGAGCTTTGTCTCAAGCCATTCACACTGCTAACTTGCCTCCAAGAGGTCCAGTTTACGTTTCTATTCCATACGATGATTGGGCCTGTGAGGCTCCATCTGGTGTTGAGCATTTGGCCAGAAGGCAAGTTTCTTCTGCTGGTTTGCCATCTCCTGCTCAATTGCAACATTTGTGTGAAAGATTGGCTGCTGCTAGAAACCCAGTTTTGGTTTTGGGTCCAGATGTTGATGGTTCTGCTGCTAACGGTTTGGCTGTTCAATTGGCTGAAAAGTTGAGAATGCCTGCTTGGGTCGCTCCATCTGCTTCTAGATGTCCTTTCCCAACTAGACATGCTTGTTTTAGAGGTGTTTTGCCAGCTGCTATTGCTGGTATTTCTCATAATTTGGCTGGACATGATTTGATTTTGGTTGTTGGTGCTCCAGTTTTTAGATACCATCAATTTGCTCCTGGAAACTACTTGCCAGCTGGTTGTGAATTGTTGCATCTGACTTGTGATCCTGGTGAGGCTGCTAGAGCTCCAATGGGTGACGCTTTAGTTGGAGATATTGCTTTGACTTTGGAGGCCGTTCTGGATGGTGTTCCACAATCCGTGAGACAAATGCCAACTGCTTTGCCTGCTGCTGAGCCAGTTGCTGATGACGGTGGTTTGTTGAGACCTGAGACTGTTTTTGACTTGTTGAACGCCTTGGCTCCTAAGGATGCTATCTACGTTAAGGAGTCTACTTCTACTGTTGGTGCTTTTTGGAGAAGAGTTGAGATGAGAGAACCAGGTTCTTATTTCTTTCCAGCTGCTGGTGGACTGGGATTCGGTTTGCCAGCAGCTGTTGGTGTTCAATTGGCTTCTCCAGGTAGACAAGTTATTGGTGTCATCGGAGATGGATCTGCTAACTATGGTATTACTGCTTTGTGGACTGCTGCCCAATACAACATTCCAGTTGTTTTTATTATTTTGAAGAACGGTACTTACGGAGCTTTGAGATGGTTTGCTGATGTTCTTGATGTTAACGATGCACCTGGTCTTGATGTTCCTGGTTTAGATTTTTGTGCTATTGCTAGAGGTTACGGTGTTCAAGCTGTTCATGCTGCTACTGGTTCAGCTTTTGCTCAAGCTTTGAGAGAAGCTTTGGAATCTGATAGACCAGTTTTGATTGAGGTTCCAACTCAAACTATTGAACCAGGTGGTGGTTCTAGAATTGCTTTTATTGGTTTGGGTAACATGGGTGCTCCAATGGCTAGAAACTTGATTAAGGCTGGTCATCAATTGAACTTGTTTGATTTGAACAAGGCTGTTTTGGCTGAATTGGCTGAATTGGGTGGTCAAATTTCCCCATCTCCTAAGGATGCTGCTGCCAACTCTGAGTTGGTTATTACTATGTTGCCAGCTGCTGCTCATGTTAGATCTGTTTACTTGAACGAAGATGGTGTTTTGGCTGGTATTAGAGCTGGTACTCCAACTGTTGATTGTTCTACTATTGATCCACAAACTGCTAGAGATGTTTCTAAGGCTGCTGCTGCTAAGGGTGTTGATTTGGGAGATGCTCCAGTTTCTGGTGGAACTGGTGGTGCAGCTGCTGGAACTTTAACTTTTATGGTTGGAGCTAGTGCTGAATTGTTTGCTACTTTGAAGCCTGTTTTGGAACAAATGGGTAGAAACATTGTCCATTGTGGTGAAGTTGGTACTGGTCAAATTGCTAAGATTTGTAACAATTTGTTGTTGGGTATTTCTATGATTGGAGTTTCTGAAGCTATGGCTTTGGGTAACGCTTTGGGTATTGATACTAAGGTTTTGGCTGGTATTATTAACAGTTCTACTGGTAGATGTTGGTCATCTGATACTTACAACCCATGGCCAGGTATTATTGAAACTGCTCCAGCTTCCAGAGGTTACACTGGTGGTTTTGGAGCCGAGTTGATGTTGAAGGATTTGGGTTTGGCTACTGAAGCTGCTAGACAAGCTCATCAACCAGTTATTTTGGGTGCTGTTGCTCAACAATTGTACCAAGCTATGTCTTTGAGAGGTGAAGGTGGTAAAGATTTTTCCGCTATTGTTGAAGGTTATAGAAAGAAGGATTAA
Claims (10)
1. A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol is characterized by comprising the following steps: the strain is characterized in that the N end and the C end of malonyl-CoA reductase gene CaMCR which are respectively expressed or fusion expressed and derived from orange green flexor are transferred to a Pichia pastoris strain by adopting a genome integration or plasmid expression mode; wherein pichia pastoris overexpresses the pichia pastoris derived gene RAD52.
2. A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol is characterized by comprising the following steps: the strain is recombinant strain obtained by over-expressing NADH kinase at neutral site PNSIII-6 of the recombinant strain obtained in the above claim 1; wherein, the NADH kinase is NADH kinase encoded by gene UTR1, NADH kinase encoded by gene ScUTR1 or NADH kinase encoded by ScYEF.
3. A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol is characterized by comprising the following steps: the strain is a recombinant strain obtained in the above claim 1 over-expressing fructose-1, 6-bisphosphatase, D-ribulose-5-phosphate 3-epimerase or dihydroxyacetone synthase; wherein, fructose-1, 6-bisphosphatase is obtained by encoding a gene FBP 1; d-ribulose-5-phosphate 3-epimerase is encoded by the genes RPE1-1 and RPE1-2 or is encoded by the gene OpRPE; dihydroxyacetone synthase is obtained by encoding gene DAS 2.
4. The method for constructing a recombinant strain for producing 3-hydroxypropionic acid using methanol as claimed in claim 3, wherein: the FBP1 gene is integrated into a chromosome PNSI-13 locus of the recombinant strain of Pichia pastoris obtained in claim 1;
integration of the RPE1-1, RPE1-2, opRPE or DAS2 genes into the PNSI-12 locus of the chromosome of the recombinant strain obtained in claim 1;
the FBP1 gene has the sequence shown in SEQ ID NO:6, a nucleotide sequence shown in seq id no;
The RPE1-1 gene has the sequence shown in SEQ ID NO: 7;
The RPE1-2 gene has the sequence shown in SEQ ID NO:8, a nucleotide sequence shown in seq id no;
The OpRPE gene has the sequence shown in SEQ ID NO: 9;
the DAS2 gene has the sequence as shown in SEQ ID NO:10, and a nucleotide sequence shown in seq id no.
5. A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol is characterized by comprising the following steps: the strain with high fatty acid yield is taken as a host, FAA1 or FAA2 genes are supplemented back in the strain, and malonyl-CoA reductase gene CaMCRNC from orange green flexor is expressed simultaneously; the strain with high fatty acid yield is PC124.
6. A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol is characterized by comprising the following steps: the method of claim 5 further comprising supplementing FAA1 and FAA2 to obtain the promoter P FLD1 of the bifunctional alcohol dehydrogenase and formaldehyde dehydrogenase gene FLD1 of the attenuated strain itself in the recombinant strain, and replacing the promoter with the promoter P TEF1、PPEX5 or P PMP20.
7. A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol is characterized by comprising the following steps: and (3) the strain obtained after weakening the promoter P FLD1 is subjected to HIS4 gene complementation, so that a recombinant strain is obtained.
8. A construction method of a recombinant strain for producing 3-hydroxy propionic acid by using methanol is characterized by comprising the following steps: over-expressing a benzoic acid decarboxylase gene MdlCKpO and a 3-hydroxyisobutyrate dehydrogenase gene MmsBKpO of pseudomonas putida in a pichia pastoris strain; wherein the pichia pastoris strain integrates the protein Cas9 and overexpresses the gene RAD52 derived from pichia pastoris.
9. A recombinant pichia pastoris strain that produces 3-hydroxypropionic acid constructed according to the method of any one of claims 1-8.
10. Use of a strain according to claim 9, characterized in that: the strain is applied to the fermentation culture of the strain by taking methanol as the sole carbon source to produce 3-hydroxy propionic acid.
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