CN115975833B - Saccharomyces cerevisiae recombinant strain for producing vanillin and construction method thereof - Google Patents
Saccharomyces cerevisiae recombinant strain for producing vanillin and construction method thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a saccharomyces cerevisiae recombinant strain for producing vanillin and a construction method thereof, wherein the construction method comprises the steps of knocking out ADH6 gene, ADH7 gene, SFA1 gene, GRE2 gene, HFD1 gene, GRE3 gene, GCY1 gene, YPR1 gene, YDL124W gene, ARI1 gene, AAD3 gene and YDR541C gene of saccharomyces cerevisiae to obtain engineering strains, and enabling the engineering strains to heterologously express 3-dehydroshikimate dehydratase ASBF or AROZ, 2-oxygen-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE to obtain the saccharomyces cerevisiae recombinant strain. The recombinant strain can improve the purity and yield of vanillin, solves the problem that Saccharomyces cerevisiae cannot produce high-purity vanillin, and has wide industrial application prospect.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a saccharomyces cerevisiae recombinant strain for producing vanillin and a construction method thereof.
Background
Vanillin, also known as vanillin, is one of the most widely used seasonings in the world, and is known as "flavor king" and is widely used in the fields of foods, medicines, cosmetics, agriculture, and the like. Vanillin also has antibacterial, antioxidant, antimutagenic, hypolipidemic, anti-stinging and anti-inflammatory activities. In addition, vanillin is an important raw material for preparing medicines such as L-dopa, dopamine, aldehyde amine and the like. As a plant secondary metabolite, natural vanillin is extracted from the seed bulb of orchid. Vanillin consumed annually throughout the world is in excess of 16,000 tons, making it one of the most widely used flavoring additives in foods and beverages. However, due to the slow growth of vanilla, the concentration of vanillin in the mature seed pods is very low, with only a small fraction (about 0.25%) of the total vanillin yield from vanilla. Thus, the market demand for such seasonings is mainly met by chemical synthesis of lignin and/or fossil hydrocarbons, which is also considered an environmentally unfriendly and unsustainable process. In addition, biocatalytic processes can be used to produce, often sold as "natural extracted products", resulting in a price 250 times higher than "chemically synthesized products" vanillin.
There is an increasing demand for the induction of many biotechnology processes for the synthesis of natural vanillin. Phytochemicals such as ferulic acid are the main substrates used in the process for producing natural vanillin. Although in the last decade different microorganisms have been isolated and studied, which are able to convert ferulic acid into vanillin, the high price of ferulic acid limits its use.
The biosynthesis of vanillin from simple carbon sources (such as glucose) is more attractive because it is more readily available and cheaper. In 2009, hansen et al used glucose as an initial substrate, introduced into Schizosaccharomyces Schizosaccharomyces pombe and Saccharomyces cerevisiae Saccharomyces cerevisiae respectively 3-dehydroshikimate dehydratase from Penicillium, bacterial aromatic carboxylic acid reductase (Carboxylic acid reductase, CAR) from Nocarpia genus of Nocarphia and 2-oxo-methyltransferase (O-METHYLTRANSFERASE, OMT) from human homosapiens, and knocked out genes degrading vanillin to obtain vanillin at 0.065g/L and 0.045g/L, respectively. However, most of vanillin synthesized in the fermentation broth is converted into vanillyl alcohol, and the downstream separation cost is high, so that the purity and the yield of vanillin are limited. Therefore, there is a need to further increase the efficiency of vanillin synthesis by microorganisms to meet the increasing market demand.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the invention aims to provide a construction method of a recombinant strain of saccharomyces cerevisiae for producing vanillin. The recombinant strain can improve the purity and yield of vanillin.
To this end, in one aspect of the invention, the invention proposes a method for constructing a recombinant strain of Saccharomyces cerevisiae for the production of vanillin, comprising:
Knocking out ADH6 gene, ADH7 gene, SFA1 gene, GRE2 gene, HFD1 gene, GRE3 gene, GCY1 gene, YPR1 gene, YDL124W gene, ARI1 gene, AAD3 gene and YDR541C gene of Saccharomyces cerevisiae to obtain engineering strain;
The engineering strain is heterologously expressed with 3-dehydroshikimate dehydratase ASBF or AROZ, 2-oxygen-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE to obtain the Saccharomyces cerevisiae recombinant strain.
According to the construction method of the saccharomyces cerevisiae recombinant strain for producing vanillin, disclosed by the embodiment of the invention, the strain capable of avoiding the conversion of vanillin into vanillyl alcohol and vanillic acid by endogenous oxidoreductase is obtained by knocking out saccharomyces cerevisiae oxidoreductase ADH6, ADH7, SFA1, GRE2, HFD1, GRE3, GCY1, YPR1, YDL124W, ARI, AAD3 and YDR541C, the strain capable of heterologously expressing 3-dehydroshikimate dehydratase ASBF or AROZ, 2-oxo-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE is integrated, the recombinant strain is obtained, and after the recombinant strain is cultured for 120 hours, the content of extracellular vanillin (purity > 99%) is 85.39 +/-0.94 mg/L, so that the problem that the saccharomyces cerevisiae cannot produce high-purity vanillin is solved, and the method has a wide industrial application prospect.
In addition, the construction method of the recombinant strain of saccharomyces cerevisiae for producing vanillin provided by the embodiment of the invention can also have the following additional technical characteristics:
Optionally, the method comprises the following steps:
(1) PCR amplification to obtain gRNA expression fragment of gene editing technology: the gRNA expression fragments were ligated to expression vectors p426-SNR52-GGA, respectively, ADH6, ADH7, SFA1, GRE2, HFD1, GRE3, GCY1, YPR1, YDL124W, ARI, AAD3 and YDR541C to obtain recombinant plasmids p426-gRNA (ADH 6), recombinant plasmids p426-gRNA (ADH 7), recombinant plasmids p426-gRNA (SFA 1), recombinant plasmids p426-gRNA (GRE 2), recombinant plasmids p426-gRNA (HFD 1), recombinant plasmids p426-gRNA (GRE 3), recombinant plasmids p426-gRNA (GCY 1), recombinant plasmids p426-gRNA (YPR 1), recombinant plasmids p426-gRNA (YDL 124W), recombinant plasmids p426-gRNA (ARI 1), recombinant plasmids p426-gRNA (AAD 3) and recombinant plasmids p426-gRNA (YDR C);
(2) Sequentially transferring the recombinant plasmids p426-gRNA (ADH 6), the recombinant plasmids p426-gRNA (ADH 7), the recombinant plasmids p426-gRNA (SFA 1), the recombinant plasmids p426-gRNA (GRE 2), the recombinant plasmids p426-gRNA (HFD 1), the recombinant plasmids p426-gRNA (GRE 3), the recombinant plasmids p426-gRNA (GCY 1), the recombinant plasmids p426-gRNA (YPR 1), the recombinant plasmids p426-gRNA (YDL 124W), the recombinant plasmids p426-gRNA (ARI 1), the recombinant plasmids p426-gRNA (AAD 3) and the recombinant plasmids p426-gRNA (YDR 541C) and corresponding gene editing integration fragments thereof into Saccharomyces cerevisiae competent cells to obtain recombinant strains JS-RARE3;
(3) Recombinant plasmids pRS423-ASBF/HsOMT and pRS425-CAR/PPTASE were introduced into recombinant strain JS-RARE3 to obtain a recombinant strain JS-A4 of Saccharomyces cerevisiae; or introducing recombinant plasmids pRS423-AROZ/HsOMT and pRS425-CAR/PPTASE into the recombinant strain JS-RARE3 to obtain a Saccharomyces cerevisiae recombinant strain JS-A5.
Further, the method further comprises the steps of electrically transforming the recombinant plasmid P426-gRNA (ROX 1) and the gene editing integration fragment DeltaROX 1P GAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1 into Saccharomyces cerevisiae JS-RARE3 competent cells which are transformed into Cas9, obtaining positive monoclonal through PCR identification, and removing the gRNA expression vector from the positive monoclonal through 5-fluoroorotic acid screening to obtain the recombinant strain JS-B1.
Further, the method further comprises the steps of electrically transforming the recombinant plasmid P426-gRNA (BTS 1) and the gene editing integration fragment delta BTS 1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1 into Saccharomyces cerevisiae JS-B1 competent cells which are transformed into Cas9, obtaining positive monoclonal through PCR identification, removing the gRNA expression vector from the positive monoclonal through 5-fluoroorotic acid screening, and removing the Cas9 expression vector through substitution culture to obtain the recombinant strain JS-B2.
Optionally, the recombinant plasmid pRS423-ASBF/HsOMT uses Bacillus cereus genome as a template, uses ASBF _P1_fwd with a nucleotide sequence shown as SEQ ID NO. 72 and ASBF _P1_rev with a nucleotide sequence shown as SEQ ID NO. 73 as primers, obtains ASBF genes through PCR amplification, and recycles a target strip through a DNA purification kit to obtain a gel recycling product; the method comprises the steps of taking an artificially synthesized gene HsOMT with a nucleotide sequence shown as SEQ ID NO. 82 as a template, taking HsOMT _P2_fwd with a nucleotide sequence shown as SEQ ID NO. 74 and HsOMT _P2_rev with a nucleotide sequence shown as SEQ ID NO. 75 as primers, carrying out PCR amplification to obtain HsOMT genes, and recovering a target strip through a DNA purification kit to obtain a gel recovery product; after pRS423A-GGA1 plasmid is digested for 4h by BsaI, large fragment and small fragment are recovered by glue, then ASBF gene fragment and HsOMT gene fragment are mixed, the obtained product is transferred into competent cells of E.coli DH5 alpha by using T4 ligase and BsaI enzyme, positive clone colony is obtained by verifying primers with nucleotide sequences shown as SEQ ID NO:72 and SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, and expression plasmid pRS423-ASBF/HsOMT is extracted.
Optionally, the recombinant plasmid pRS425-CAR/PPTASE uses a Sagnac bacillus Segniliparus rugosus genome as a template, uses CAR_P1_fwd with a nucleotide sequence shown as SEQ ID NO. 78 and CAR_P1_rev with a nucleotide sequence shown as SEQ ID NO. 79 as primers, obtains a CAR gene through PCR amplification, and recycles a target strip through a DNA purification kit to obtain a gel recycling product; taking Ai Ahua nocardia Nocardia iowensis genome as a template, taking PPTASE _P2_fwd with a nucleotide sequence shown as SEQ ID NO. 80 and PPTASE _P2_rev with a nucleotide sequence shown as SEQ ID NO. 81 as primers, performing PCR amplification to obtain PPTASE genes, and recovering a target strip through a DNA purification kit to obtain a gel recovery product; after pRS425A-GGA1 plasmid is digested for 4h by BsaI, large fragment and small fragment are recovered by glue, then the large fragment and the small fragment are mixed with CAR gene fragment and PPTASE gene fragment, the mixture is connected by using T4 ligase and BsaI enzyme, the connected product is transferred into competent cells of escherichia coli DH5 alpha, positive clone colony is obtained by verifying with nucleotide sequences shown as SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, and expression plasmid pRS425-CAR/PPTASE is extracted.
Optionally, the recombinant plasmid pRS423-AROZ/HsOMT uses P423-H7-ArCatA, taGDC1, paAroZ vector as template, AROZ _P1_fwd with nucleotide sequence shown as SEQ ID NO. 76 and AROZ _P1_rev with nucleotide sequence shown as SEQ ID NO. 77 as primers, AROZ gene is obtained by PCR amplification, target band is recovered by DNA purification kit, and gel recovery product is obtained; after pRS423A-GGA1 plasmid is digested for 4h by BsaI, large fragment and small fragment are recovered by glue, AROZ gene fragment and HsOMT gene fragment are mixed, T4 ligase and BsaI enzyme are used for connecting, the connected product is transferred into E.coli DH5 alpha competent cells, and positive clone colony is obtained by verifying with primers with nucleotide sequences as SEQ ID NO. 74 and SEQ ID NO. 75, SEQ ID NO. 76 and SEQ ID NO. 77, and expression plasmid pRS423-AROZ/HsOMT is extracted.
Optionally, the recombinant plasmid p426-gRNA (ROX 1) takes a p426-SNR52-gRNA vector as a template, takes F_gRNA.ROX1 with a nucleotide sequence shown as SEQ ID NO. 14 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 as primers, obtains gRNA (ROX 1) fragments through PCR amplification, and obtains a gel recovery product through recovery of a DNA purification kit; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.
Alternatively, the gene editing integration fragment DeltaROX 1P GAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1 is obtained by PCR amplification by taking pRS423-AROZ/HsOMT plasmid as a template, taking F-ROX1-Int with a nucleotide sequence shown as SEQ ID NO. 42 and R-ROX1-Int with a nucleotide sequence shown as SEQ ID NO. 43 as primers.
Further, the recombinant plasmid p426-gRNA (BTS 1) takes a p426-SNR52-gRNA vector as a template, takes F_gRNA.BTS1 with a nucleotide sequence shown as SEQ ID NO. 13 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 as primers, obtains a gRNA (BTS 1) fragment through PCR amplification, and the target strip is recovered through a DNA purification kit to obtain a gel recovery product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.
Furthermore, the gene editing integration fragment delta BTS 1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1 is obtained by taking pRS423-CAR/PPTASE plasmid as a template, taking F-BTS1-Int with a nucleotide sequence shown as SEQ ID NO. 40 and R-BTS1-Int with a nucleotide sequence shown as SEQ ID NO. 41 as primers, and carrying out PCR amplification to obtain the knockout integration fragment delta BTS 1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1.
In a second aspect of the invention, the invention provides recombinant strains of Saccharomyces cerevisiae for producing vanillin constructed by the construction method described above.
According to the recombinant saccharomyces cerevisiae provided by the embodiment of the invention, the recombinant strain can improve the purity and the yield of vanillin.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a pathway for the catalytic synthesis of vanillin by four enzymes of heterologous expression ASBF or AROZ, hsOMT, CAR, PPTASE in Saccharomyces cerevisiae;
FIG. 2 is a graph showing consumption analysis of the Saccharomyces cerevisiae engineering bacteria JS-CR, JS-RARE1, JS-RARE2, JS-RARE3 vanillin according to an embodiment of the invention;
FIG. 3 is a diagram of fermentation yields of Saccharomyces cerevisiae engineering bacteria JS-A1, JS-A2, JS-A3, JS-A4, JS-A5, according to an embodiment of the present invention;
fig. 4 is a diagram of fermentation yield of saccharomyces cerevisiae engineering bacteria JS-B2 according to an embodiment of the invention.
Detailed Description
The technical scheme of the invention is described below through specific examples. It is to be understood that the mention of one or more method steps of the present invention does not exclude the presence of other method steps before and after the combination step or that other method steps may be interposed between these explicitly mentioned steps; it should also be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.
In order to better understand the above technical solution, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market; the related experiments are conventional experiments unless otherwise specified.
The sources of the materials are as follows: the engineering strain JS-CR is Saccharomyces cerevisiae BY4741 derived (Fan,C.,Engineering Saccharomyces cerevisiae-based biosensors for copper detection,Microbial Biotechnology.2022,00,1–7.Available from:https://doi.org/10.1111/1751-7915.14105) and DH5 alpha is commercially available, and DH5 alpha is used for carrier construction. Saccharomyces cerevisiae expression vectors p426-SNR52-gRNA, p426-SNR52-GGA, p414-TEF2-Cas9, pRS423-GGA1, pRS425-GGA1 are commercially available. Phusion high fidelity DNA polymerase, T4 ligase, restriction enzymes were purchased from Xiaomenlong Biotechnology development Co. Plasmid extraction kits, DNA purification kits, gel recovery kits, and yeast genomic DNA extraction kits were purchased from Shanghai bioengineering limited.
The LB medium consists of: 10g/L peptone, 5g/L yeast powder, 10g/L NaCl, and sterilizing at 115 deg.C under 0.1MPa for 30min.
YPD medium consisted of: 10g/L yeast powder, 20g/L peptone, 20g/L glucose, and double distilled water to 1L, and sterilizing at 115 deg.C under 0.1mpa for 30min.
YNBD-TRP medium composition is: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotroph medium supplement (without tryptophan), 20g/L D-glucose, double distilled water to 1L, and sterilizing at 115 deg.C under 0.1MPa for 20min.
YNBD-TRP-URA medium consists of: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotroph medium supplement (without tryptophan and uracil), 20g/L D-glucose, double distilled water was added to 1L, and sterilized at 115℃under 0.1MPa for 20min.
The YNBD-HIS-LEU culture medium comprises the following components: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotroph medium supplement (without histidine and leucine), 20g/L D-glucose, double distilled water to 1L, and sterilizing at 115 deg.C under 0.1MPa for 20min.
YNBD complete medium composition is: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotroph medium supplement, 20g/L D-glucose, double distilled water to 1L, and sterilizing at 115 deg.C under 0.1mpa for 20min.
100X 5-fluoroorotic acid: 100mg of 5-fluoroorotic acid was dissolved in 1mL using DMSO.
Detection of vanillin/vanillyl alcohol content by high performance liquid chromatography:
Sucking 400 mu L of fermented liquid, adding 400 mu L of 100% ethanol, centrifuging at 14000rpm for 5min, and filtering to obtain liquid phase bottle; detection analysis was performed using Shimadzu high performance liquid chromatograph using a photodiode array detector (operating wavelength 275 nm); the chromatographic conditions are as follows: 0 minutes, 95% solvent a+5% solvent B;8 minutes, 20% solvent A+80% solvent B;10 minutes, 80% solvent a+20% solvent B;11 minutes, 95% solvent A+5% solvent B. The contents of vanillin and vanillyl alcohol were determined using a Shimadzu C18 column (4.6X250 mm,5 μm) with a flow rate of 1mL/min, the mobile phase comprising solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (0.1% acetonitrile trifluoroacetic acid) at 35℃and a sample injection of 10. Mu.L.
The construction of plasmids involved in the following examples was performed in E.coli DH 5. Alpha. And the plasmids were transformed into Saccharomyces cerevisiae JS-CR as knockout or expression vectors for gene knockout and heterologous expression after completion of the plasmid construction.
TABLE 1 PCR primers for amplification
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not limiting in any way.
EXAMPLE 1 construction of CRISPR gRNA expression Module
(1) Construction of the p426-gRNA (ADH 6) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.AHD6 with a nucleotide sequence shown as SEQ ID NO. 1 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), gRNA (ADH 6) fragments are obtained through PCR amplification, and target bands are recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 10s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (ADH 6) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (ADH 6), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(2) Construction of the p426-gRNA (ADH 7) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.ADH7 with a nucleotide sequence shown as SEQ ID NO. 2 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (ADH 7) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 10s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (ADH 7) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 2 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (ADH 7), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(3) Construction of the p426-gRNA (SFA 1) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.SFA1 with a nucleotide sequence shown as SEQ ID NO. 3 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (SFA 1) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 10s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (SFA 1) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 3 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (SFA 1), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(4) Construction of the p426-gRNA (GRE 2) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.GRE2 with a nucleotide sequence shown as SEQ ID NO. 4 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (GRE 2) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃,30 s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (GRE 2) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (GRE 2), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(5) Construction of the p426-gRNA (HFD 1) plasmid:
the p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.HFD1 with a nucleotide sequence shown as SEQ ID NO. 5 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (HFD 1) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃,30 s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (HFD 1) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 5 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (HFD 1), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(6) Construction of the p426-gRNA (GRE 3) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.GRE3 with a nucleotide sequence shown as SEQ ID NO. 6 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (GRE 3) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃,30 s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (GRE 3) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 6 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (GRE 3), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(7) Construction of the p426-gRNA (GCY 1) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.GCY1 with a nucleotide sequence shown as SEQ ID NO.7 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (GCY 1) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃,30 s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (GCY 1) fragment, using T4 ligase and BsaI enzyme, under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (GCY 1), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(8) Construction of the p426-gRNA (YPR 1) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.YPR1 with a nucleotide sequence shown as SEQ ID NO. 8 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (YPR 1) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃,30 s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (YPR 1) fragment, using T4 ligase and BsaI enzyme, under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 8 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (YPR 1), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(9) Construction of the p426-gRNA (YDL 124W) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.YDL124W with a nucleotide sequence shown as SEQ ID NO. 9 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (YDL 124W) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (YDL 124W) fragment, using T4 ligase and BsaI enzyme, under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (YDL 124W), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(10) Construction of the p426-gRNA (ARI 1) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.ARI1 with a nucleotide sequence shown as SEQ ID NO. 10 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (ARI 1) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (ARI 1) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 10 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (ARI 1), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(11) Construction of the p426-gRNA (AAD 3) plasmid:
the p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.AAD3 with a nucleotide sequence shown as SEQ ID NO. 11 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), gRNA (AAD 3) fragments are obtained through PCR amplification, and the target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (AAD 3) fragment, using T4 ligase and BsaI enzyme, under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 11 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (AAD 3), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(12) Construction of the p426-gRNA (YDR 541C) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.YDR541C with a nucleotide sequence shown as SEQ ID NO. 12 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), gRNA (YDR 541C) fragments are obtained through PCR amplification, and target bands are recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (YDR 541C) fragment, using T4 ligase and BsaI enzyme, under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 12 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (YDR 541C), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(13) Construction of the p426-gRNA (BTS 1) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.BTS1 with a nucleotide sequence shown as SEQ ID NO. 13 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), gRNA (BTS 1) fragments are obtained through PCR amplification, and target bands are recovered through a DNA purification kit, so that a gel recovery product is obtained. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (BTS 1) fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 13 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (BTS 1), and completely conforming the sequencing result with the designed plasmid DNA sequence.
(14) Construction of the p426-gRNA (ROX 1) plasmid:
The p426-SNR52-gRNA vector (addgene # 43803) is used as a template, F_gRNA.ROX1 with a nucleotide sequence shown as SEQ ID NO. 14 and R_SUP4 with a nucleotide sequence shown as SEQ ID NO. 15 are used as primers (table 1), a gRNA (ROX 1) fragment is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,10 s at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃, and 30 times of circulation; 72 ℃ for 2min. After 4h cleavage of the p426-SNR52-GGA plasmid by BsaI, the large and small fragments were recovered in the gel and subsequently mixed with the gRNA (ROX 1) fragment, using T4 ligase and BsaI enzyme, under the following ligation conditions: 37 ℃ for 10min; cycling for 4 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO. 14 and SEQ ID NO. 15 to obtain positive clone colonies, extracting gRNA expression plasmid p426-gRNA (ROX 1), and completely conforming the sequencing result with the designed plasmid DNA sequence.
Example 2 preparation of Targeted knockout fragments
(1) F-ADH6-Del with nucleotide sequence shown as SEQ ID NO. 16 and R-ADH6-Del with nucleotide sequence shown as SEQ ID NO. 17 are used as primers (table 1), the integrated fragment of ADH6 knockout is obtained through PCR amplification, the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s,72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(2) Taking F-ADH7-Del with a nucleotide sequence shown as SEQ ID NO. 18 and R-ADH-Del with a nucleotide sequence shown as SEQ ID NO. 19 as primers (table 1), and performing PCR amplification to obtain an ADH7 knockout integration fragment, wherein the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(3) F-SFA1-Del with a nucleotide sequence shown as SEQ ID NO. 20 and R-SFA1-Del with a nucleotide sequence shown as SEQ ID NO. 21 are used as primers (table 1), the SFA1 knockout integration fragment is obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(4) F-GRE2-Del with a nucleotide sequence shown as SEQ ID NO. 22 and R-GRE2-Del with a nucleotide sequence shown as SEQ ID NO. 23 are used as primers (table 1), GRE2 knockout integration fragments are obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(5) F-HFD1-Del with a nucleotide sequence shown as SEQ ID NO. 24 and R-HFD1-Del with a nucleotide sequence shown as SEQ ID NO. 25 are used as primers (table 1), the HFD1 knockout integration fragment is obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(6) F-GRE3-Del with a nucleotide sequence shown as SEQ ID NO. 26 and R-GRE3-Del with a nucleotide sequence shown as SEQ ID NO. 27 are used as primers (table 1), GRE3 knockout integration fragments are obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(7) Taking F-GCY1-Del with a nucleotide sequence shown as SEQ ID NO. 28 and R-GCY1-Del with a nucleotide sequence shown as SEQ ID NO. 29 as primers (table 1), and performing PCR amplification to obtain a GCY1 knockout integration fragment, wherein the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(8) F-YPR1-Del with a nucleotide sequence shown as SEQ ID NO. 30 and R-YPR1-Del with a nucleotide sequence shown as SEQ ID NO. 31 are used as primers (table 1), the YPR1 knockout integration fragment is obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(9) F-YDL124W-Del with a nucleotide sequence shown as SEQ ID NO. 32 and R-YDL124W-Del with a nucleotide sequence shown as SEQ ID NO. 33 are used as primers (table 1), the YDL124W knockout integration fragment is obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s,72 ℃ 15s, and 30 times of circulation; 72 ℃ for 1min.
(10) F-ARI1-Del with a nucleotide sequence shown as SEQ ID NO. 34 and R-ARI1-Del with a nucleotide sequence shown as SEQ ID NO. 35 are used as primers (table 1), ARI1 knockout integration fragments are obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(11) F-AAD3-Del with a nucleotide sequence shown as SEQ ID NO. 36 and R-AAD3-Del with a nucleotide sequence shown as SEQ ID NO. 37 are used as primers (table 1), an AAD3 knockout integration fragment is obtained through PCR amplification, and the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s and 72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
(12) F-YDR541C-Del with nucleotide sequence shown as SEQ ID NO. 38 and R-YDR541C-Del with nucleotide sequence shown as SEQ ID NO. 39 are used as primers (table 1), the YDR541C knockout integration fragment is obtained through PCR amplification, the PCR amplification conditions are 94 ℃ 2min,94 ℃ 15s,50 ℃ 15s,72 ℃ 15s, and the cycle is 30 times; 72 ℃ for 1min.
Example 3 construction of plasmids
(1) The target band is recovered by a DNA purification kit by taking the Bacillus cereus genome (NZ_CP 017060.1) as a template and taking ASBF _P1_fwd with a nucleotide sequence shown as SEQ ID NO. 72 and ASBF _P1_rev with a nucleotide sequence shown as SEQ ID NO. 73 as primers (table 1), and obtaining ASBF genes through PCR amplification, thus obtaining a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃ and 4min at 72 ℃, and circulating for 30 times; 72 ℃ for 2min. The HsOMT gene is obtained by PCR amplification by using an artificially synthesized gene HsOMT with a nucleotide sequence shown as SEQ ID NO. 82 as a template, hsOMT _P2_fwd with a nucleotide sequence shown as SEQ ID NO. 74 and HsOMT _P2_rev with a nucleotide sequence shown as SEQ ID NO. 75 as primers (Table 1), and a target band is recovered by using a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃ and 4min at 72 ℃, and circulating for 30 times; 72 ℃ for 2min. After cleavage of pRS423A-GGA1 plasmid by BsaI for 4h, the large and small fragments were recovered by gel followed by mixing ASBF gene fragment and HsOMT gene fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 8 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying with primers with nucleotide sequences shown as SEQ ID NO. 72 and SEQ ID NO. 73, SEQ ID NO. 74 and SEQ ID NO. 75 to obtain positive clone colonies, extracting expression plasmid pRS423-ASBF/HsOMT, and completely conforming the sequencing result with the designed plasmid DNA sequence.
(2) The target band is recovered by a DNA purification kit by taking a P423-H7-ArCatA, taGDC1, paAroZ vector (Brückner Christine,et al.,An expanded enzyme toolbox for production of cis,cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae,FEMS Yeast Research.2018,2:2.) as a template, taking AROZ _P1_fwd with a nucleotide sequence shown as SEQ ID NO. 76 and AROZ _P1_rev with a nucleotide sequence shown as SEQ ID NO. 77 as primers (table 1), and obtaining AROZ genes through PCR amplification, thus obtaining a gel recovery product. PCR amplification conditions: 2min at 98 ℃,15 s at 98 ℃, 2min at 56 ℃ and 4min at 72 ℃, and circulating for 30 times; 72 ℃ for 2min. Subsequently AROZ gene fragment and HsOMT gene fragment obtained in step (1) were mixed and T4 ligase and BsaI enzyme were used under the following ligation conditions: 37 ℃ for 10min; cycling for 8 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of escherichia coli DH5 alpha at 20 ℃ for 10min, verifying and obtaining positive clone colonies by using primers with nucleotide sequences shown as SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76 and SEQ ID NO. 77, extracting expression plasmid pRS423-AROZ/HsOMT, and completely conforming the sequencing result with the designed plasmid DNA sequence.
(3) The genome of Sagnac bacillus Segniliparus rugosus (NZ_KI 391953.1) is used as a template, CAR_P1_fwd with a nucleotide sequence shown as SEQ ID NO. 78 and CAR_P1_rev with a nucleotide sequence shown as SEQ ID NO. 79 are used as primers (table 1), a CAR gene is obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃ and 4min at 72 ℃, and circulating for 30 times; 72 ℃ for 2min. The PPTASE gene is obtained by PCR amplification with Ai Ahua Nocardia Nocardia iowensis genome (NZ_CP 078145.1) as a template, PPTASE _P2_fwd with a nucleotide sequence shown as SEQ ID NO. 80 and PPTASE _P2_rev with a nucleotide sequence shown as SEQ ID NO. 81 as primers (Table 1), and the target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃ and 4min at 72 ℃, and circulating for 30 times; 72 ℃ for 2min. After cleavage of pRS425A-GGA1 plasmid by BsaI for 4h, the large and small fragments were recovered in the gel and subsequently mixed with the CAR gene fragment and PPTASE gene fragment using T4 ligase and BsaI enzyme under the following ligation conditions: 37 ℃ for 10min; cycling for 8 times at 37 ℃ for 10min and 16 ℃ for 10min; transferring the connected product into competent cells of escherichia coli DH5 alpha at 20 ℃ for 10min, verifying and obtaining positive clone colonies by using nucleotide sequences shown as SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80 and SEQ ID NO. 81, extracting expression plasmid pRS425-CAR/PPTASE, and completely conforming the sequencing result to the designed plasmid DNA sequence.
Example 4 preparation of targeted integration expression fragments
(1) The pRS423-AROZ/HsOMT plasmid in step (2) of example 3 was used as a template, F-ROX1-Int having a nucleotide sequence shown as SEQ ID NO. 42 and R-ROX1-Int having a nucleotide sequence shown as SEQ ID NO. 43 were used as primers (Table 1), the knockout integrated fragment DeltaROX 1P GAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1 was obtained by PCR amplification, and the target band was recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,15 s at 98 ℃, 2min at 56 ℃ and 4min at 72 ℃, and circulating for 30 times; 72 ℃ for 2min.
(2) The pRS423-CAR/PPTASE plasmid in step (3) of example 3 was used as a template, F-BTS1-Int having a nucleotide sequence shown as SEQ ID NO. 40 and R-BTS1-Int having a nucleotide sequence shown as SEQ ID NO. 41 were used as primers (Table 1), the knockout integrated fragment DeltaBTS 1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1 was obtained by PCR amplification, and the target band was recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃,15 s at 98 ℃, 2min at 56 ℃ and 4min at 72 ℃, and circulating for 30 times; 72 ℃ for 2min.
Example 5: construction of engineering strain JS-RARE1
(1) Competent cells prepared by Saccharomyces cerevisiae JS-CR are transformed into p414-TEF2-Cas9 plasmid, and cultured on YNBD-TRP plate at 30 ℃ for 2-4 days, and the obtained single colony is named as Saccharomyces cerevisiae Y1. Then Y1 is prepared into competent cells, the plasmid p426-gRNA (ADH 6) obtained in the step (1) of example 1 and the ADH6 knockout integration fragment obtained in the step (1) of example 2 are transformed into Y1 competent cells, the competent cells are cultured on YNBD-TRP-URA plates at 30 ℃ for 2 to 4 days, the single colonies which grow out are respectively streaked on YNBD-TRP-URA solid plates, and PCR verification is carried out by using primers SEQ ID NO:44 and SEQ ID NO:45, and the correct Saccharomyces cerevisiae strain JS-CR delta ADH6 is named as recombinant strain JS-M1.
(2) The JS-M1 in step (1) was streaked on YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL, and intracellular p426-gRNA (AHD 6) plasmids were removed, followed by preparation of competent cells, transformation of the integrated fragment of p426-gRNA (ADH 7) in step (2) of example 1 and AHD7 knockout in step (2) of example 2, and incubation of the grown single colonies on YNBD-TRP-URA plates at 30℃for 2 to 4 days, and PCR verification was performed using primers SEQ ID NO:46 and SEQ ID NO:47, and the correct yeast strain JS-CR. DELTA. ADH 6. ADH7 was designated as recombinant strain JS-M2.
(3) The JS-M2 in step (2) was streaked on YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL, and intracellular p426-gRNA (ADH 7) plasmids were removed, followed by preparation of competent cells, transformation of the SFA1 knockout integration fragments into the p426-gRNA (SFA 1) in step (3) of example 1 and the SFA1 knockout integration fragments in step (3) of example 2, culture at 30℃for 2 to 4 days on YNBD-TRP-URA plates, and the single colonies grown were streaked on YNBD-TRP-URA solid plates, respectively, and PCR verification was performed using primers SEQ ID NO:48 and SEQ ID NO:49, and the correct Saccharomyces cerevisiae strain JS-CR. DELTA. ADH 6. DELTA. ADH 7. SFA1 was designated as recombinant strain JS-M3.
(4) The JS-M3 in step (3) was streaked onto YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL, and the intracellular p426-gRNA (SFA 1) plasmid was removed, and then used to prepare competent cells, which were transformed into the GRE2 knockout integration fragments of the p426-gRNA (GRE 2) in step (4) of example 1 and step (4) of example 2, and the grown single colonies were streaked onto YNBD-TRP-URA solid plates by culturing at 30℃for 2 to 4 days, and PCR was performed using primers SEQ ID NO:50 and SEQ ID NO:51, and the correct Saccharomyces cerevisiae strain JS-CR ΔADH6ΔADH7ΔSFA1ΔGRE2 was designated as recombinant strain JS-M4.
(5) The JS-M4 in step (4) was streaked on YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular p426-gRNA (GRE 2) plasmid, which was then used to prepare competent cells, the HFD1 knockout integration fragment was transformed into the p426-gRNA (HFD 1) of example 1 step (5) and the HFD1 knockout integration fragment of example 2 step (5), the grown single colonies were streaked on YNBD-TRP-URA solid plates at 30℃for 2 to 4 days, PCR was performed with primers SEQ ID NO:52, SEQ ID NO:53, the correct Saccharomyces cerevisiae strain JS-CR ΔADH6ΔADH7ΔSFA1ΔGRE2ΔHFD1 was named as recombinant strain JS-M5, and then streaked on BD-TRP plates containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular p426-gRNA (HFD 1) and named as JS-RARE1 plasmid.
Example 6: construction of engineering strain JS-RARE2
(1) The JS-RARE 1-prepared competent cells in step (5) of example 5 were transformed into the GRE3 knockout integration fragments in step (6) of example 1, p426-gRNA (GRE 3) and step (6) of example 2, cultured on YNBD-TRP-URA plates at 30℃for 2 to 4 days, and the single colonies grown were streaked on YNBD-TRP-URA solid plates, respectively, and PCR was performed using primers SEQ ID NO:54 and SEQ ID NO:55, and the correct Saccharomyces cerevisiae strain JS-CR ΔADH6ΔADH7ΔSFA1ΔGRE2 ΔHFD1ΔGRE3 was designated as recombinant strain JS-M6.
(2) The JS-M6 in step (1) was streaked onto YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular p426-gRNA (GRE 3) plasmid, which was then used to prepare competent cells, the integrated fragment of GCY1 knockout in step (7) of example 1 and p426-gRNA (GCY 1) and step (7) of example 2 was transformed, and the grown single colonies were streaked onto YNBD-TRP-URA solid plates by culturing at 30℃for 2 to 4 days, and PCR was performed using primers SEQ ID NO:56, SEQ ID NO:57, and the correct Saccharomyces cerevisiae strain JS-CR ΔADH6ΔADH7ΔSFA1ΔGRE2ΔHFD1ΔGRE3ΔGCY1, named recombinant strain-M7, respectively.
(3) The JS-M7 in step (2) was streaked on YNBD-TRP plate containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular p426-gRNA (GCY 1) plasmid, which was then used to prepare competent cells, the YPR1 knockout integrated fragment was transformed into the p426-gRNA (YPR 1) of step (8) of example 1 and the YPR1 knockout integrated fragment of step (8) of example 2, and the grown single colonies were streaked on YNBD-TRP-URA solid plate at 30℃for 2 to 4 days, and PCR was performed with primers SEQ ID NO:58, SEQ ID NO:59, and the correct Saccharomyces cerevisiae strain JS-CR ΔADH6ΔADH7ΔSFA1ΔGRE2ΔHFD1ΔGRE3ΔGCY1ΔY1ΔYR1, named as recombinant strain JS-M8, respectively.
(4) The JS-M8 strain in step (3) was streaked on YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL, and then used to prepare competent cells, transformed into the YDL124W knockout integration fragment of p426-gRNA (YDL 124W) in step (9) of example 1 and YDL124W in step (9) of example 2, and cultured on YNBD-TRP-URA plates at 30℃for 2 to 4 days, and the single colonies grown on YNBD-TRP-URA solid plates were streaked on YNBD-TRP-TRA solid plates, respectively, and PCR verified with primers SEQ ID NO:60, SEQ ID NO:61, and the correct yeast strain, ZRAD6ΔADH7ΔSFA1ΔGRE2ΔGRE2ΔGRE3ΔGCYYP1ΔYD1YDL 124W, and then streaked on YDA426-URA solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL, and then the resulting single colony was streaked on YNBR-TRP-124.
Example 7: construction of engineering strain JS-RARE3
(1) Competent cells prepared by JS-RARE2 in step (4) of example 6 were transformed into the ARI1 knockout integration fragments in p426-gRNA (ARI 1) of step (10) of example 1 and step (10) of example 2, and cultured on YNBD-TRP-URA plates at 30℃for 2 to 4 days, and single colonies grown were streaked on YNBD-TRP-URA solid plates, respectively, using primers SEQ ID NO: and (2) performing PCR verification on the strain 62 and SEQ ID NO. 63, and obtaining a correct Saccharomyces cerevisiae strain JS-CR delta ADH6 delta ADH7 delta SFA1 delta GRE2 delta HFD1 delta GRE3 delta GCY1 delta YPR1 delta YDL124W delta ARI1, which is named as recombinant strain JS-M10.
(2) The JS-M10 in step (1) was streaked on YNBD-TRP plate containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular p426-gRNA (ARI 1) plasmid, which was then used to prepare competent cells, the AAD3 knockout integrated fragment transformed into the p426-gRNA (AAD 3) of step (11) of example 1 and the AAD3 knockout integrated fragment of step (11) of example 2, and the grown single colonies were streaked on YNBD-TRP-URA solid plate at 30℃for 2 to 4 days, and PCR verification was performed using primers SEQ ID NO:64, SEQ ID NO:65, and the correct Saccharomyces cerevisiae strain JS-CR ΔADH6ΔADH7ΔSFA1ΔGRE2ΔGRE2ΔHFD1ΔGRE3ΔGCY1ΔY1ΔY1ΔYLΔYLΔYPYD1ΔAYΔARL1 ΔAΔAY1 AAD3, named recombinant strain JS 11, respectively.
(3) The JS-M11 strain in step (2) was streaked onto YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL, and then used to prepare competent cells after removal of intracellular p426-gRNA (AAD 3) plasmid, the integrated fragment was transformed into p426-gRNA (YDR 541C) in step (12) of example 1 and YDR541C knockout in step (12) of example 2, and cultured on YNBD-TRP-URA plates at 30℃for 2 to 4 days, and the single colonies grown were streaked onto YNBD-TRP-URA solid plates, respectively, and PCR was verified with primers SEQ ID NO:66, SEQ ID NO:67, and the correct yeast strain-CR ΔADH6ΔADH7ΔSFA1ΔGRE2ΔGRD1ΔGRE3ΔGCY1ΔYPYP1ΔYDAR1WΔARI1ΔD1Δ541 D.DELTA 541C, and the recombinant strain JS-M12 was subsequently streaked onto YNBR solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL and was streaked onto YNBR-TRP solid plates (YRBR 541) for 1.
Example 8: accumulation ability of Vanillin of different modified strains
The recombinant strains JS-RARE1, JS-RARE2, JS-RARE3 and JS-CR of the control strain constructed in the examples 5, 6 and 7 are respectively picked up and respectively subjected to monoclonal inoculation in a 15mL small shaking tube filled with 2mL YNBD full liquid culture medium, and are cultured for 12-16 hours at 30 ℃ and 250rpm to prepare seed liquid, and the prepared seed liquid is inoculated in a 50mL centrifuge tube filled with 5mL YNBD full liquid culture medium according to the inoculum size of 1% (v/v) and is cultured for 16 hours at 30 ℃ and 250 rpm. Centrifuging at 5000rpm for 1min, removing supernatant to collect thallus, washing twice with 5mL phosphate buffer (200 mM, pH=8.0), and removing supernatant to collect thallus; to the collected cells, 5mL of vanillin-depleted solution (containing 5mM vanillin, 20g/L glucose, 200mM phosphate buffer ph=8.0 supplemented to 5 mL) was added at 400uL for 4h, 8h, 24h and 48h, respectively, according to 1: adding 100% ethanol in proportion, mixing with vortex oscillator, centrifuging at 14000rpm for 5min, filtering supernatant with filter membrane in liquid sample bottle, detecting with liquid chromatograph, and converting peak area with vanillin and vanillyl alcohol standard to obtain consumption of recombinant strain.
As a result, as shown in FIG. 2, the control strain JS-CR had depleted vanillin at 48h, while recombinant strains JS-RARE1, JS-RARE2, JS-RARE3 had decreased conversion of vanillin to vanillyl alcohol and the modified strain JS-RARE3 had little conversion of vanillin to vanillyl alcohol with increased oxidoreductase gene knockout.
Example 9: construction of synthetic vanillin Strain JS-A1
Competent cells were prepared from the engineering strain JS-CR, transformed into pRS423-ASBF/HsOMT plasmid and pRS425-CAR/PPTASE plasmid obtained in example 3, and cultured on YNBD-HIS-LEU plates at 30℃for 2-4 days to obtain positive clone JS-A1 s.cerevisiae recombinant strain.
Example 10: construction of synthetic vanillin Strain JS-A2
The engineering strain JS-RARE1 constructed in the step (5) of example 5 was subjected to subculture in YPD solid medium to remove the Cas9 expression vector, and then used to prepare competent cells, transformed into pRS423-ASBF/HsOMT plasmid and pRS425-CAR/PPTASE plasmid obtained in the example 3, and cultured on YNBD-HIS-LEU plate at 30℃for 2 to 4 days to obtain positive clone JS-A2 s.cerevisiae recombinant strain.
Example 11: construction of synthetic vanillin Strain JS-A3
The engineering strain JS-RARE2 constructed in the step (4) of example 6 was subjected to subculture in YPD solid medium to remove the Cas9 expression vector, and then used to prepare competent cells, transformed into pRS423-ASBF/HsOMT plasmid and pRS425-CAR/PPTASE plasmid obtained in the example 3, and cultured on YNBD-HIS-LEU plate at 30℃for 2 to 4 days to obtain positive clone JS-A3 s.cerevisiae recombinant strain.
Example 12: construction of synthetic vanillin Strain JS-A4
The engineering strain JS-RARE3 constructed in the step (3) of example 7 was subjected to subculture in YPD solid medium to remove the Cas9 expression vector, and then used to prepare competent cells, transformed into pRS423-ASBF/HsOMT plasmid and pRS425-CAR/PPTASE plasmid obtained in the step 3, and cultured on YNBD-HIS-LEU plate at 30℃for 2 to 4 days to obtain positive clone JS-A4 s.cerevisiae recombinant strain.
Example 13: construction of synthetic vanillin Strain JS-A5
The engineering strain JS-RARE3 constructed in the step (3) of example 7 was subjected to subculture in YPD solid medium to remove the Cas9 expression vector, and then used to prepare competent cells, transformed into pRS423-AROZ/HsOMT plasmid and pRS425-CAR/PPTASE plasmid obtained in the step 3, and cultured on YNBD-HIS-LEU plate at 30℃for 2 to 4 days to obtain positive clone JS-A5 s.cerevisiae recombinant strain.
Example 14: construction of engineering strain JS-B1
The engineering strain JS-RARE3 constructed in the step (3) of example 7 was used to prepare competent cells, transformed into the knock-out integration fragment DeltaROX 1P GAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1 of the step (14) P426-gRNA (ROX 1) of example 1 and the step (1) of example 4, cultured on YNBD-TRP-URA plates at 30℃for 2 to 4 days, and the grown single colonies were streaked on YNBD-TRP-URA solid plates, respectively, and PCR was performed with primers SEQ ID NO:70 and SEQ ID NO:71 to verify that the correct Saccharomyces cerevisiae strain JS-CRΔADH6ΔADH7ΔSFA1ΔGRE2ΔHFD1ΔGRE3ΔGCY1ΔYPR1ΔYDL124WΔARI1ΔAAD3ΔYDR541CΔROX1 PGAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1, was designated as recombinant strain JS-B1.
Example 15: construction of engineering strain JS-B2
The engineering strain JS-B1 constructed in the step (1) of example 14 was streaked on YNBD-TRP solid plates containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular P426-gRNA (ROX 1) plasmid, which was then used to prepare competent cells, transformed into the knock-out integration fragment ΔBTS1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1 in the step (13) of example 1 and the step (2) of example 4, cultured on YNBD-TRP-URA plates at 30℃for 2 to 4 days, the single colonies grown were streaked on YNBD-TRP-URA solid plates, respectively, and PCR was performed using primers SEQ ID NO:68 and SEQ ID NO:69, the correct Saccharomyces cerevisiae strain JS-CRΔADH6ΔADH7ΔSFA1ΔGRE2ΔHFD1ΔGRE3ΔGCY1ΔYPR1ΔYDL124WΔARI1ΔAAD3ΔYDR541CΔROX1 PGAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1ΔBTS1 PGAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1, was then streaked on YNBD-TRP plates containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular P426-gRNA (BTS 1) plasmid, and the resulting recombinant strain JS-B2 was streaked on YBD solid medium for passaging to remove Cas9 expression vector.
Example 16: vanillin and vanillyl alcohol yields of recombinant strains under shake flask fermentation conditions
(1) The recombinant strains JS-A1, JS-A2, JS-A3, JS-A4 and JS-A5 constructed in examples 9, 10, 11, 12 and 13 were respectively picked up and individually inoculated in 15mL small shaking tubes filled with 2mL YNBD-HIS-LEU liquid medium, cultured at 30℃and 250rpm for 12-16 hours to prepare seed solutions, the prepared seed solutions were inoculated in 250mL triangular flasks filled with 20mL YNBD-HIS-LEU liquid medium in an inoculum size of 1% (v/v), and added with 20. Mu.M copper sulfate for induction expression, and cultured at 30℃and 250rpm for 120 hours to prepare fermentation solutions.
(2) The recombinant strain JS-B2 constructed in example 15 was inoculated in a 15mL small shaking tube filled with 2mL YNBD whole liquid medium, cultured at 30℃and 250rpm for 12-16 hours to prepare a seed solution, the prepared seed solution was inoculated in a 250mL triangular flask filled with 20mL YNBD whole liquid medium in an inoculum size of 1% (v/v), and 20. Mu.M copper sulfate was added for induction expression, and cultured at 30℃and 250rpm for 120 hours to prepare a fermentation broth.
(3) The yields of extracellular vanillin, vanillyl alcohol were calculated:
And (3) sucking 400 mu L of fermentation liquor, adding 400 mu L of 100% ethanol, uniformly mixing by a vortex oscillator, centrifuging at 14000rpm for 5min, filtering the supernatant by a filter membrane in a liquid phase sample injection bottle, performing high performance liquid chromatography detection, and obtaining the fermentation yield of the engineering strain by converting the peak area of the product with vanillin and vanillyl alcohol standard.
The results are shown in Table 2 and FIG. 3, wherein the extracellular vanillyl alcohol content of the JS-A1 strain is 29.35 +/-2.00 mg/L; the JS-A2 strain has the extracellular vanillin content of 11.66+/-0.38 mg/L and the vanillyl alcohol content of 28.26+/-1.80 mg/L; the JS-A3 strain has the extracellular vanillin content of 11.82+/-1.44 mg/L and the vanillyl alcohol content of 20.81+/-3.27 mg/L; the JS-A4 strain has the extracellular vanillin content of 21.91 +/-1.08 mg/L; the extracellular vanillin content of the JS-A5 strain is 38.12 +/-1.31 mg/L, which shows that the recombinant strain JS-RARE3 can synthesize high-purity vanillin by using pRS423-ASBF/HsOMT plasmid and pRS425-CAR/PPTASE plasmid for expression. Meanwhile, AROZ is used for replacing ASBF in the recombinant strain JS-A5, so that the yield of vanillin is improved by 73.9%.
As shown in Table 2 and FIG. 4, the JS-B2 strain has an extracellular vanillin content of 85.39 + -0.94 mg/L, indicating that the vanillin synthesis pathway is integrated into the genome, and the vanillin yield is improved by 124% compared with the plasmid expression.
Table 2: production of vanillin and vanillyl alcohol by extracellular secretion of different recombinant Saccharomyces cerevisiae
| Strain | Extracellular vanillin (mg/L) | Extracellular vanillyl alcohol (mg/L) |
| JS-A1 | Not measured | 29.35±2.00 |
| JS-A2 | 11.66±0.38 | 28.26±1.80 |
| JS-A3 | 11.82±1.44 | 20.81±3.27 |
| JS-A4 | 21.91±1.08 | Not measured |
| JS-A5 | 38.12±1.31 | Not measured |
| JS-B2 | 85.39±0.94 | Not measured |
In summary, according to the Saccharomyces cerevisiae recombinant strain of the embodiment of the present invention, the recombinant strain JS-RARE3 is constructed by knocking out Saccharomyces cerevisiae oxidoreductase ADH6, ADH7, SFA1, GRE2, HFD1, GRE3, GCY1, YPR1, YDL124W, ARI1, AAD3 and YDR541C, so as to avoid the conversion of vanillin into vanillyl alcohol and vanillic acid by endogenous oxidoreductase, 3-dehydroshikimate dehydratase ASBF and 2-oxo-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE are expressed by plasmids in a heterologous manner, after the recombinant strain is cultured for 120 hours, the content of extracellular vanillin (purity > 99%) is 21.91 + -1.08 mg/L, 3-dehydroshikimate dehydratase ASBF is replaced with AROZ with similar functions, and the content of extracellular vanillin (purity > 99%) is 38.12 + -1.31 mg/L in shake flask fermentation of the recombinant strain JS-A5. In order to solve the instability of plasmid expression, 3-dehydroshikimate dehydratase AROZ, 2-oxygen-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE are further integrated into an engineering strain JS-RARE3 to obtain a recombinant strain JS-B2, and after 120 hours of culture, the final yield of extracellular vanillin (purity > 99%) reaches 85.39 +/-0.94 mg/L, so that the problem that Saccharomyces cerevisiae cannot stably produce high-purity vanillin is solved, and the method has wide industrialized application prospect.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (9)
1. A method for constructing a recombinant strain of saccharomyces cerevisiae for producing vanillin, comprising the steps of:
Knocking out ADH6 gene, ADH7 gene, SFA1 gene, GRE2 gene, HFD1 gene, GRE3 gene, GCY1 gene, YPR1 gene, YDL124W gene, ARI1 gene, AAD3 gene and YDR541C gene of Saccharomyces cerevisiae to obtain engineering strain;
Introducing recombinant plasmid pRS423-ASBF/HsOMT or recombinant plasmid pRS423-AROZ/HsOM T plasmid, recombinant plasmid pRS425-CAR/PPTASE to the engineering strain to heterologously express 3-dehydroshikimate dehydratase ASBF or AROZ, 2-oxo-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE to obtain Saccharomyces cerevisiae recombinant strain;
The recombinant plasmid pRS425-CAR/PPTASE is prepared by taking a Bacillus cereus genome as a template, taking ASBF _P1_fwd with a nucleotide sequence shown as SEQ ID NO. 72 and ASBF _P1_rev with a nucleotide sequence shown as SEQ ID NO. 73 as primers, carrying out PCR amplification to obtain ASBF genes, and recovering a target strip through a DNA purification kit to obtain a gel recovery product; the method comprises the steps of taking an artificially synthesized gene HsOM T with a nucleotide sequence shown as SEQ ID NO. 82 as a template, taking HsOMT _P2_fwd with a nucleotide sequence shown as SEQ ID NO. 74 and HsOMT _P2_rev with a nucleotide sequence shown as SEQ ID NO. 75 as primers, carrying out PCR amplification to obtain HsOMT genes, and recovering a target strip through a DNA purification kit to obtain a gel recovery product; after pRS423A-GGA1 plasmid is digested for 4 hours by BsaI, large fragment and small fragment are recovered by glue, then ASBF gene fragment and HsOMT gene fragment are mixed, T4 ligase and BsaI enzyme are used for connecting, the connected product is transferred into competent cells of escherichia coli DH5 alpha, positive clone colony is obtained by verifying primers with nucleotide sequences shown as SEQ ID NO. 72 and SEQ ID NO. 73, SEQ ID NO. 74 and SEQ ID NO. 75, and expression plasmid pRS423-ASBF/HsOMT is extracted;
The recombinant plasmid pRS425-CAR/PPTASE is prepared by taking a genome of Sagnac bacillus Segniliparus rugosus as a template, taking CAR_P1_fwd with a nucleotide sequence shown as SEQ ID NO. 78 and CAR_P1_rev with a nucleotide sequence shown as SEQ ID NO. 79 as primers, amplifying by PCR to obtain a CAR gene, and recovering a target strip by a DNA purification kit to obtain a gel recovery product; taking Ai Ahua nocardia Nocardia iowensis genome as a template, taking PPTASE _P2_fwd with a nucleotide sequence shown as SEQ ID NO. 80 and PPTASE _P2_rev with a nucleotide sequence shown as SEQ ID NO. 81 as primers, performing PCR amplification to obtain PPTASE genes, and recovering a target strip through a DNA purification kit to obtain a gel recovery product; after pRS425A-GGA1 plasmid is digested for 4 hours by BsaI, large fragments and small fragments are recovered by glue, then the large fragments and the small fragments are mixed with CAR gene fragments and PPTASE gene fragments, the mixture is connected by using T4 ligase and BsaI enzyme, the connected product is transferred into competent cells of escherichia coli DH5 alpha, and positive clone colonies are obtained by verification by using nucleotide sequences shown as SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO: 81;
The recombinant plasmid pRS423-AROZ/HsOMT uses P423-H7-ArCatA, taGDC1, paAroZ vector as template, AROZ _P1_fwd with nucleotide sequence shown as SEQ ID NO. 76 and AROZ _P1_rev with nucleotide sequence shown as SEQ ID NO. 77 as primers, AROZ gene is obtained by PCR amplification, target strip is recovered by DNA purification kit, and gel recovery product is obtained; after pRS423A-GGA1 plasmid is digested for 4h by BsaI, large fragment and small fragment are recovered, AROZ gene fragment and HsOMT gene fragment are mixed, T4 ligase and BsaI enzyme are used for connecting, the connected product is transferred into E.coli DH5 alpha competent cells, and the nucleotide sequences are shown as SEQ ID NO. 74 and SEQ ID NO. 75, SEQ ID NO. 76 and SEQ ID NO. 77, and positive clone colony is obtained by verification.
2. The method of construction of claim 1, comprising:
(1) PCR amplification to obtain gRNA expression fragment of gene editing technology: the gRNA expression fragments were ligated to expression vectors p426-SNR52-GGA, respectively, ADH6, ADH7, SFA1, GRE2, HFD1, GRE3, GCY1, YPR1, YDL124W, ARI, AAD3 and YDR541C to obtain recombinant plasmids p426-gRNA (ADH 6), recombinant plasmids p426-gRNA (ADH 7), recombinant plasmids p426-gRNA (SFA 1), recombinant plasmids p426-gRNA (GRE 2), recombinant plasmids p426-gRNA (HFD 1), recombinant plasmids p426-gRNA (GRE 3), recombinant plasmids p426-gRNA (GCY 1), recombinant plasmids p426-gRNA (YPR 1), recombinant plasmids p426-gRNA (YDL 124W), recombinant plasmids p426-gRNA (ARI 1), recombinant plasmids p426-gRNA (AAD 3) and recombinant plasmids p426-gRNA (YDR C);
(2) Sequentially transferring the recombinant plasmids p426-gRNA (ADH 6), the recombinant plasmids p426-gRNA (ADH 7), the recombinant plasmids p426-gRNA (SFA 1), the recombinant plasmids p426-gRNA (GRE 2), the recombinant plasmids p426-gRNA (HFD 1), the recombinant plasmids p426-gRNA (GRE 3), the recombinant plasmids p426-gRNA (GCY 1), the recombinant plasmids p426-gRNA (YPR 1), the recombinant plasmids p426-gRNA (YDL 124W), the recombinant plasmids p426-gRNA (ARI 1), the recombinant plasmids p426-gRNA (AAD 3) and the recombinant plasmids p426-gRNA (YDR 541C) and corresponding gene editing integration fragments thereof into Saccharomyces cerevisiae competent cells to obtain recombinant strains JS-RARE3;
(3) Recombinant plasmids pRS423-ASBF/HsOMT and pRS425-CAR/PPTASE were introduced into recombinant strain JS-RARE3 to obtain a recombinant strain JS-A4 of Saccharomyces cerevisiae; or introducing recombinant plasmids pRS423-AROZ/HsOMT and pRS425-CAR/PPTASE into the recombinant strain JS-RARE3 to obtain a Saccharomyces cerevisiae recombinant strain JS-A5.
3. The construction method according to claim 2, wherein the recombinant plasmid P426-gRNA (ROX 1) and the gene editing integration fragment DeltaROX 1P GAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1 are electrically transformed into Saccharomyces cerevisiae JS-RARE3 competent cells which are transferred into Cas9, positive monoclonal is obtained through PCR identification, and the gRNA expression vector is removed from the positive monoclonal through 5-fluoroorotic acid screening, so as to obtain the recombinant strain JS-B1.
4. The construction method of claim 2, wherein the recombinant plasmid P426-gRNA (BTS 1) and the gene editing integration fragment delta BTS 1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1 are electrically transformed into Saccharomyces cerevisiae JS-B1 competent cells which are transferred into Cas9, positive monoclonal is obtained through PCR identification, gRNA expression vectors are removed from the positive monoclonal through 5-fluoroorotic acid screening, and Cas9 expression vectors are removed through transfer culture, so that the recombinant strain JS-B2 is obtained.
5. The construction method according to claim 3, wherein the recombinant plasmid p426-gRNA (ROX 1) is obtained by PCR amplification using a p426-SNR52-gRNA vector as a template, F_gRNA.ROX1 having a nucleotide sequence shown in SEQ ID NO. 14 and R_SUP4 having a nucleotide sequence shown in SEQ ID NO. 15 as primers, and the target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.
6. The construction method according to claim 3, wherein the gene editing integration fragment ΔROX1P GAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1 is a knockout integration fragment ΔROX1P GAL10-AROZ-TADH1-PGAL1-HsOMT-TCYC1 obtained by PCR amplification using pRS423-AROZ/HsOMT plasmid as a template, F-ROX1-Int having a nucleotide sequence shown in SEQ ID NO. 42 and R-ROX1-Int having a nucleotide sequence shown in SEQ ID NO. 43 as primers.
7. The construction method according to claim 4, wherein the recombinant plasmid p426-gRNA (BTS 1) is a gRNA (BTS 1) fragment obtained by PCR amplification using a p426-SNR52-gRNA vector as a template, F_gRNA.BTS1 having a nucleotide sequence shown in SEQ ID NO. 13 and R_SUP4 having a nucleotide sequence shown in SEQ ID NO. 15 as primers, and the target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovery product was ligated with the expression vector p426-SNR 52-GGA.
8. The construction method according to claim 4, wherein the gene editing integration fragment ΔBTS1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1 is a knockout integration fragment ΔBTS1P GAL10-CAR-TADH1-PGAL1-PPTASE-TCYC1 obtained by PCR amplification using pRS423-CAR/PPTASE plasmid as a template, F-BTS1Int having a nucleotide sequence shown in SEQ ID NO. 40 and R-BTS1-Int having a nucleotide sequence shown in SEQ ID NO. 41 as primers.
9. Recombinant strain of saccharomyces cerevisiae producing vanillin, characterized in that it is constructed by the construction method according to any one of claims 1-8.
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