CN115975833A

CN115975833A - Saccharomyces cerevisiae recombinant strain for producing vanillin and construction method thereof

Info

Publication number: CN115975833A
Application number: CN202211313001.2A
Authority: CN
Inventors: 袁吉锋; 莫棋文
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2022-10-25
Filing date: 2022-10-25
Publication date: 2023-04-18

Abstract

The invention relates to a saccharomyces cerevisiae recombinant strain for producing vanillin and a construction method thereof, the construction method comprises the steps of knocking out an ADH6 gene, an ADH7 gene, an SFA1 gene, a GRE2 gene, an HFD1 gene, a GRE3 gene, a GCY1 gene, a YPR1 gene, an YDL124W gene, an ARI1 gene, an AAD3 gene and an YDR541C gene of saccharomyces cerevisiae to obtain an engineering strain, and the engineering strain heterologously expresses 3-dehydroshikimic acid dehydratase ASBF or AROZ, 2-oxygen-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase ASE to obtain the saccharomyces cerevisiae recombinant strain. The recombinant strain can improve the purity and yield of vanillin, solves the problem that high-purity vanillin can not be produced by saccharomyces cerevisiae, and has wide industrial application prospect.

Description

Saccharomyces cerevisiae recombinant strain for producing vanillin and construction method thereof

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 is also known as vanillin, is one of the most widely used seasonings in the world, has the name of spice king, and is widely applied to the fields of food, medicines, cosmetics, agriculture and the like. Vanillin also has antibacterial, antioxidant, antimutagenic, hypolipidemic, anti-stinging and anti-inflammatory activity. In addition, vanillin is also an important raw material for preparing L-dopa, dopamine, aldehyde amine and other medicines. As a plant secondary metabolite, natural vanillin is extracted from the seed bulb of orchid. Vanillin is consumed worldwide in excess of 16,000 tons per year, making it one of the most widely used flavor additives in foods and beverages. However, as vanilla grows slowly, the concentration of vanillin in its mature seed pods is very low, and only a small fraction (about 0.25%) of the overall vanillin yield is from vanilla. Thus, the market demand for such flavors is mainly met by chemical synthesis of lignin and/or fossil hydrocarbons, which is also considered to be an environmentally unfriendly and unsustainable process. In addition, the vanillin can also be produced by a biocatalysis method and can be sold as a 'natural extracted product', so that the vanillin price is 250 times higher than that of a 'chemically synthesized product'.

The increasing demand has prompted a number of biotechnologies for the synthesis of natural vanillin. Phytochemicals such as ferulic acid are the main substrates used in the production process of natural vanillin. Although in the last decade different microorganisms have been isolated and studied, which are capable of converting ferulic acid to vanillin, the high price of ferulic acid limits its application.

The biosynthesis of vanillin from simple carbon sources (e.g. glucose) is attractive because of its availability and cost. In 2009 Hansen et al introduced 3-dehydroshikimate dehydratase from copromomyces, bacterial aromatic acid reductase (CAR) from Nocardia genus and O-methyltransferase (OMT) from Homo sapiens source into Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, using glucose as an initial substrate, and knocked out vanillin degrading genes at the same time, yielding 0.065g/L and 0.045g/L vanillin, respectively. However, most of vanillin synthesized in the fermentation liquor is converted into vanillyl alcohol, and the downstream separation cost is high, so that the purity and yield of vanillin are limited. Therefore, the efficiency of synthesizing vanillin by using microorganisms needs to be further improved to meet the increasing market demand.

Disclosure of Invention

The present invention is directed to solving, at least in part, one of the technical problems in the related art. Therefore, the invention aims to provide a construction method of a saccharomyces cerevisiae recombinant strain for producing vanillin. The recombinant strain can improve the purity and yield of vanillin.

To this end, in one aspect of the present invention, the present invention provides a method for constructing a recombinant strain of saccharomyces cerevisiae for producing 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;

and (3) heterologously expressing 3-dehydroshikimate dehydratase ASBF or AROZ, 2-oxygen-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE by using the engineering strain 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, saccharomyces cerevisiae oxidoreductase ADH6, ADH7, SFA1, GRE2, HFD1, GRE3, GCY1, YPR1, YDL124W, ARI, AAD3 and YDR541C are knocked out to obtain a strain capable of preventing vanillin from being converted into vanillyl alcohol and vanillic acid by an endogenous oxidoreductase, and then the strain is integrated to heterologously express 3-dehydroshikimate dehydratase ASBF or AROZ, 2-oxo-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase PPTASE to obtain a recombinant strain, and after the recombinant strain is cultured for 120 hours, the extracellular vanillin (purity is more than 99%) content is 85.39 +/-0.94 mg/L, so that the problem that high-purity vanillin cannot be produced by saccharomyces cerevisiae 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 above 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: ADH6, ADH7, SFA1, GRE2, HFD1, GRE3, GCY1, YPR1, YDL124W, ARI, AAD3 and YDR541C, wherein the gRNA expression fragments are respectively connected with an expression vector p426-SNR52-GGA to obtain a recombinant plasmid p426-gRNA (ADH 6), a recombinant plasmid p426-gRNA (ADH 7), a recombinant plasmid p426-gRNA (SFA 1), a recombinant plasmid p426-gRNA (GRE 2), a recombinant plasmid p426-gRNA (HFD 1), a recombinant plasmid p426-gRNA (GRE 3), a recombinant plasmid p426-gRNA (GCY 1), a recombinant plasmid p426-gRNA (GCR 1), a recombinant plasmid p426-gRNA (YDL 124W), a recombinant plasmid p426-gRNA (ARI 1), a recombinant plasmid p426-gRNA (AAD 3) and a recombinant plasmid p-gRNA (YDR 541C);

(2) Sequentially transferring the recombinant plasmid p426-gRNA (ADH 6), the recombinant plasmid p426-gRNA (ADH 7), the recombinant plasmid p426-gRNA (SFA 1), the recombinant plasmid p426-gRNA (GRE 2), the recombinant plasmid p426-gRNA (HFD 1), the recombinant plasmid p426-gRNA (GRE 3), the recombinant plasmid p426-gRNA (GCY 1), the recombinant plasmid p426-gRNA (YPR 1), the recombinant plasmid p426-gRNA (YDL 124W), the recombinant plasmid p426-gRNA (ARI 1), the recombinant plasmid p426-gRNA (AAD 3) and the recombinant plasmid p426-gRNA (YDR 541C) and corresponding gene editing integration fragments thereof into saccharomyces cerevisiae competent cells to obtain a recombinant strain JS-RARE3;

(3) Introducing a recombinant plasmid pRS423-ASBF/HsOMT and a recombinant plasmid pRS425-CAR/PPTASE into the recombinant strain JS-RARE3 so as to obtain a saccharomyces cerevisiae recombinant strain JS-A4; or introducing a recombinant plasmid pRS423-AROZ/HsOMT plasmid and pRS425-CAR/PPTASE plasmid into the recombinant strain JS-RARE3 to obtain the saccharomyces cerevisiae recombinant strain JS-A5.

Further, recombinant plasmid P426-gRNA (ROX 1) and gene editing integration fragment delta ROX 1P are included _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 And (3) transferring the electric transformation into a Saccharomyces cerevisiae JS-RARE3 competent cell which is transferred into Cas9, obtaining a positive monoclonal through PCR identification, and removing a gRNA expression vector from the positive monoclonal through 5-fluoroorotic acid screening to obtain a recombinant strain JS-B1.

Further, the recombinant plasmid P426-gRNA (BTS 1) and the gene editing integration fragment delta BTS 1P are also included _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 And (3) transferring the electric transformation into a Saccharomyces cerevisiae JS-B1 competent cell which is transferred into Cas9, obtaining a positive monoclonal through PCR identification, removing a gRNA expression vector from the positive monoclonal through 5-fluoroorotic acid screening, and removing the Cas9 expression vector through subculture to obtain a recombinant strain JS-B2.

Optionally, the recombinant plasmid pRS423-ASBF/HsOMT is obtained 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 an ASBF gene, and recovering a target band by using a DNA purification kit to obtain a gel recovery product; using an artificially synthesized gene HsOMT with a nucleotide sequence shown as SEQ ID NO. 82 as a template, using 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, obtaining an HsOMT gene through PCR amplification, and recycling a target strip through a DNA purification kit to obtain a gel recycling product; after the pRS423A-GGA1 plasmid is digested for 4h by BsaI, a large fragment and a small fragment are recovered by glue, then an ASBF gene fragment and an HsOMT gene fragment are mixed, T4 ligase and BsaI enzyme are used for connection, the connected product is transferred into a competent cell of Escherichia coli DH5 alpha, primers with nucleotide sequences shown as SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75 are used for verifying to obtain a positive clone bacterial colony, and the expression plasmid pRS423-ASBF/HsOMT is extracted.

Optionally, the recombinant plasmid pRS425-CAR/PPTASE is a CAR gene obtained by PCR amplification by using a Segniliparus rugosus genome as a template, a CAR _ P1_ fwd with a nucleotide sequence shown in SEQ ID NO:78 and a CAR _ P1_ rev with a nucleotide sequence shown in SEQ ID NO:79 as primers, and a gel recovery product is obtained by recovering a target band through a DNA purification kit; using Ai Ahua Nocardia iowensis genome as template, PPTASE _ P2_ fwd with nucleotide sequence as shown in SEQ ID NO. 80 and PPTASE _ P2_ rev with nucleotide sequence as shown in SEQ ID NO. 81 as primer, obtaining PPTASE gene by PCR amplification, recovering target band by DNA purification kit to obtain gel recovery product; after the pRS425A-GGA1 plasmid is digested for 4h by BsaI, large fragments and small fragments are recovered by glue, then mixed with CAR gene fragments and PPTAE gene fragments, T4 ligase and BsaI enzyme are used for connection, the connected products are transferred into Escherichia coli DH5 alpha competent cells, positive clone colonies are obtained by verification of nucleotide sequences such as SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, and the expression plasmid pRS 425-CAR/PPTAE is extracted.

Optionally, the recombinant plasmid pRS423-AROZ/HsOMT is prepared by taking P423-H7-ArCatA, taGDC1 and PaAroZ vectors as templates, 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 through PCR amplification to obtain an AROZ gene, and recovering a target band through a DNA purification kit to obtain a gel recovery product; after the pRS423A-GGA1 plasmid is digested for 4h by BsaI, large fragments and small fragments are recovered by glue, an AROZ gene fragment and an HsOMT gene fragment are mixed, T4 ligase and BsaI enzyme are used for connection, the connected product is transferred into a competent cell of escherichia coli DH5 alpha, primers with nucleotide sequences shown as SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76 and SEQ ID NO 77 are used for verifying to obtain a positive clone bacterial colony, and the expression plasmid pRS423-AROZ/HsOMT is extracted.

Optionally, the recombinant plasmid p426-gRNA (ROX 1) is a gRNA (gRNA) p426-SNR52-gRNA vector serving as a template, F _ gRNA, ROX1 and R _ SUP4, wherein the F _ gRNA and the ROX1 have nucleotide sequences shown in SEQ ID NO:14 and SEQ ID NO:15 respectively, are used as primers, 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; the gel recovered product was ligated with the expression vector p426-SNR 52-GGA.

Alternatively, the gene editing integration fragment Δ ROX 1P _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 Using pRS423-AROZ/HsOMT plasmid as template, F-ROX1-Int with nucleotide sequence shown in SEQ ID NO. 42 and R-ROX1-Int with nucleotide sequence shown in SEQ ID NO. 43 as primers, and performing PCR amplification to obtain a knock-out integration fragment delta ROX 1P _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 。

Further, the recombinant plasmid p426-gRNA (BTS 1) is prepared by taking a p426-SNR52-gRNA vector as a template, taking 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, carrying out PCR amplification to obtain a gRNA (BTS 1) fragment, and recovering a target band 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.

Further, the gene editing integration fragment Δ BTS 1P _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 Using pRS423-CAR/PPTASE plasmid as template, F-BTS1-Int with nucleotide sequence shown as SEQ ID NO. 40 and R-BTS1-Int with nucleotide sequence shown as SEQ ID NO. 41 as primers, and performing PCR amplification to obtain integration fragment Δ BTS 1P _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 。

In the second aspect of the invention, the invention provides a vanillin-producing saccharomyces cerevisiae recombinant strain constructed by the construction method.

According to the recombinant saccharomyces cerevisiae disclosed by the embodiment of the invention, the purity and the yield of vanillin can be improved by the recombinant strain.

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 shows the path of the catalytic synthesis of vanillin by the heterologous expression of ASBF or AROZ, hsOMT, CAR, PPTASE in Saccharomyces cerevisiae;

FIG. 2 is a chart of analyzing vanillin consumption of engineering bacteria JS-CR, JS-RARE1, JS-RARE2 and JS-RARE3 of Saccharomyces cerevisiae according to the embodiment of the present invention;

FIG. 3 is a graph of fermentation yields of engineered saccharomyces cerevisiae bacteria JS-A1, JS-A2, JS-A3, JS-A4, JS-A5 according to an embodiment of the present invention;

FIG. 4 is a graph of fermentation yield of engineered saccharomyces cerevisiae JS-B2 according to the embodiment of the invention.

Detailed Description

The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the method steps is only a convenient tool for identifying each method step, and is not intended to limit the order of the method steps or the scope of the invention, and changes or modifications in the relative relationship thereof may be regarded as the scope of the invention without substantial change in the technical content.

In order to better understand the above technical solutions, 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 all routine experimental methods if not specifically stated.

Sources of materials used: the engineered strain JS-CR is derived from Saccharomyces cerevisiae BY4741 (Fan, C., engineering Saccharomyces cerevisiae-based biosensions for coater detection, microbiological Biotechnology.2022,00,1-7.Available from https:// doi.org/10.1111/1751-7915.14105) and DH5 α are commercially available, DH5 α is used for vector construction. Saccharomyces cerevisiae expression vectors p426-SNR52-gRNA, p426-SNR52-GGA, p414-TEF2-Cas9, pRS423-GGA1 and pRS425-GGA1 are commercially available. Phusion high fidelity DNA polymerase, T4 ligase, restriction enzyme were purchased from Xiamen Lulong Biotech development Inc. Plasmid extraction kit, DNA purification kit, gel recovery kit and yeast genome DNA extraction kit were purchased from Shanghai bioengineering, inc.

The LB medium consisted of: 10g/L peptone, 5g/L yeast powder, 10g/L NaCl, and double distilled water to 1L, sterilizing at 115 deg.C under 0.1Mpa for 30min.

YPD medium composition was: 10g/L yeast powder, 20g/L peptone, 20g/L glucose, and double distilled water to 1L, sterilizing at 0.1Mpa and 115 deg.C for 30min.

YNBD-TRP medium composition is: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotrophic culture medium supplement (without tryptophan), 20g/L D-glucose, double distilled water to 1L,0.1Mpa pressure 115 ℃ sterilization for 20min.

The YNBD-TRP-URA culture medium comprises the following components: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotrophic medium supplement (without tryptophan and uracil), 20g/L D-glucose, double distilled water to 1L,0.1Mpa pressure 115 ℃ sterilization for 20min.

The YNBD-HIS-LEU culture medium comprises the following components: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotrophic medium supplement (without histidine and leucine), 20g/L D-glucose, double distilled water to 1L,0.1Mpa pressure 115 ℃ sterilization for 20min.

The YNBD total culture medium comprises: 6.7g/L yeast nitrogen source base, 1.4g/L yeast auxotrophic culture medium supplement, 20g/L D-glucose, fully filled to 1L by double distilled water, sterilized at 115 ℃ under 0.1Mpa for 20min.

100x 5-Fluorotic acid: 100mg of 5-fluoroorotic acid was dissolved in 1mL using DMSO.

Detecting the content of vanillin/vanillyl alcohol by high performance liquid chromatography:

sucking out 400 μ L of fermented liquid, adding 400 μ L of 100% ethanol, centrifuging at 14000rpm for 5min, and filtering to obtain liquid phase bottle; detecting and analyzing by using a Shimadzu high performance liquid chromatograph, and adopting a photodiode array detector (with the working wavelength of 275 nm); the chromatographic conditions are as follows: 0min, 95% solvent a +5% solvent B;8 minutes, 20% solvent a +80% solvent B;10 minutes, 80% solvent a +20% solvent B;11 min, 95% solvent a +5% solvent B. The content of vanillin and vanillyl alcohol was determined using a Shimadzu C18 column (4.6X 250mm,5 μm) at a flow rate of 1mL/min, a mobile phase comprising solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile), a column temperature of 35 ℃ and a sample volume of 10 μ L.

The construction of the plasmids referred to in the following examples was carried out in e.coli DH5 α, and after the plasmid construction was completed, the plasmids were used as knock-out or expression vectors and transformed into saccharomyces cerevisiae JS-CR for gene knock-out and heterologous expression.

TABLE 1 primers used for PCR amplification

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The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Example 1 construction of a gRNA expression module for CRISPR

(1) Construction of p426-gRNA (ADH 6) plasmid:

a gRNA (ADH 6) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA.AHD6 with a nucleotide sequence shown in SEQ ID NO:1 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:15 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large fragment and the small fragment are recovered by gel, and then mixed with a gRNA (ADH 6) fragment using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulation is carried out for 4 times at 37 ℃ and 10min at 16 ℃; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences such as SEQ ID NO:1 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (ADH 6), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(2) Construction of p426-gRNA (ADH 7) plasmid:

a gRNA (ADH 7) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA. ADH7 with a nucleotide sequence shown in SEQ ID NO:2 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:15 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid was digested for 4h with BsaI, the large and small fragments were recovered from the gel and then mixed with gRNA (ADH 7) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell 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 a positive clone colony, extracting gRNA expression plasmid p426-gRNA (ADH 7), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(3) Construction of p426-gRNA (SFA 1) plasmid:

a gRNA (SFA 1) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA, SFA1 with a nucleotide sequence shown in SEQ ID NO:3 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:15 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid was digested for 4h with BsaI, the large and small fragments were recovered from the gel and then mixed with gRNA (SFA 1) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:3 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (SFA 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(4) Construction of p426-gRNA (GRE 2) plasmid:

a gRNA (GRE 2) fragment is obtained by PCR amplification by taking a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA.GRE2 with a nucleotide sequence shown in SEQ ID NO. 4 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO. 15 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid was digested for 4h with BsaI, the large and small fragments were recovered by gel and then mixed with gRNA (GRE 2) fragments using T4 ligase and BsaI enzyme under the following ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:4 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (GRE 2), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(5) Construction of p426-gRNA (HFD 1) plasmid:

a gRNA (HFD 1) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA. HFD1 with a nucleotide sequence shown in SEQ ID NO:5 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:15 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: cycling at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1min for 30 times; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid was digested for 4h with BsaI, the large and small fragments were recovered from the gel and then mixed with gRNA (HFD 1) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a bacillus coli DH5 alpha competent cell at 20 ℃ for 10min, verifying and obtaining a positive clone colony by using primers with nucleotide sequences shown as SEQ ID NO:5 and SEQ ID NO:15, extracting gRNA expression plasmid p426-gRNA (HFD 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(6) Construction of p426-gRNA (GRE 3) plasmid:

a p426-SNR52-gRNA vector (adddge # 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) segment 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: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid was digested for 4h with BsaI, the large and small fragments were recovered by gel and then mixed with gRNA (GRE 3) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:6 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (GRE 3), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(7) Construction of p426-gRNA (GCY 1) plasmid:

a gRNA (GCY 1) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) 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 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: cycling at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1min for 30 times; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large fragment and the small fragment are recovered by gel, and then mixed with gRNA (GCY 1) fragments, T4 ligase and BsaI enzyme are used, and the ligation conditions are as follows: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:7 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (GCY 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(8) Construction of p426-gRNA (YPR 1) plasmid:

a gRNA (YPR 1) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) 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 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large and small fragments are recovered from the gel and then mixed with gRNA (YPR 1) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell 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 a positive clone colony, extracting gRNA expression plasmid p426-gRNA (YPR 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(9) Construction of p426-gRNA (YDL 124W) plasmid:

a p426-SNR52-gRNA vector (adddge # 43803) is used as a template, F _ gRNA, YDL124W and R _ SUP4 are used as primers (table 1), the nucleotide sequences of the F _ gRNA, YDL124W and the R _ SUP4 are shown in SEQ ID NO:9 and SEQ ID NO:15, gRNA (YDL 124W) fragments are obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, large fragments and small fragments are recovered by glue and then mixed with gRNA (YDL 124W) fragments by using T4 ligase and BsaI enzyme under the connection conditions that: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences such as SEQ ID NO:9 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (YDL 124W), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(10) Construction of p426-gRNA (ARI 1) plasmid:

a p426-SNR52-gRNA vector (adddge # 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) segment 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: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large and small fragments are recovered by gel, and then mixed with gRNA (ARI 1) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell 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 a positive clone colony, extracting gRNA expression plasmid p426-gRNA (ARI 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(11) Construction of p426-gRNA (AAD 3) plasmid:

a gRNA (AAD 3) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) 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 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid was digested for 4h with BsaI, the large and small fragments were recovered from the gel and then mixed with gRNA (AAD 3) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:11 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (AAD 3), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(12) Construction of p426-gRNA (YDR 541C) plasmid:

a p426-SNR52-gRNA carrier (adddge # 43803) is used as a template, F _ gRNA, YDR541C and R _ SUP4 are used as primers (table 1), wherein the nucleotide sequences are shown in SEQ ID NO:12 and SEQ ID NO:15, gRNA (YDR 541C) fragments are obtained through PCR amplification, and a target band is recovered through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI enzyme, large fragments and small fragments are recovered by glue, and then mixed with gRNA (YDR 541C) fragments by using T4 ligase and BsaI enzyme under the connection conditions that: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha for 10min at 20 ℃, verifying by primers with nucleotide sequences such as SEQ ID NO:12 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (YDR 541C), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(13) Construction of p426-gRNA (BTS 1) plasmid:

a gRNA (BTS 1) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) as a template, F _ gRNA.BTS1 with a nucleotide sequence shown in SEQ ID NO:13 and R _ SUP4 with a nucleotide sequence shown in SEQ ID NO:15 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large and small fragments are recovered by gel, and then mixed with gRNA (BTS 1) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulation is carried out for 4 times at 37 ℃ and 10min at 16 ℃; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by primers with nucleotide sequences shown as SEQ ID NO:13 and SEQ ID NO:15 to obtain a positive clone colony, extracting gRNA expression plasmid p426-gRNA (BTS 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

(14) Construction of p426-gRNA (ROX 1) plasmid:

a gRNA (ROX 1) fragment is obtained by PCR amplification by using a p426-SNR52-gRNA vector (adddge # 43803) 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 as primers (Table 1), and a target band is recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: circulating for 30 times at 98 deg.C for 2min,98 deg.C for 10s,56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2min. After the p426-SNR52-GGA plasmid is digested for 4h by BsaI, the large and small fragments are recovered by gel, and then mixed with gRNA (ROX 1) fragments using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃; circulating for 4 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell 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 a positive clone colony, extracting gRNA expression plasmid p426-gRNA (ROX 1), and completely conforming the sequencing result to the designed plasmid DNA sequence.

Example 2 Targeted knock-out fragment preparation

(1) F-ADH6-Del with a nucleotide sequence shown as SEQ ID NO. 16 and R-ADH6-Del with a nucleotide sequence shown as SEQ ID NO. 17 are used as primers (Table 1), an ADH6 knockout integration fragment is obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s, and the cycle is 30 times; 72 ℃ for 1min.

(2) 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 are used as primers (Table 1), ADH7 knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 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), and SFA1 knockout integration fragments are obtained through PCR amplification under the conditions of 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 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), and a GRE2 knockout integration fragment is obtained through PCR amplification under the conditions of 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 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), and HFD1 knockout integration fragments are obtained by PCR amplification under the conditions of 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s and 30 cycles; 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), and a GRE3 knockout integration fragment is obtained through PCR amplification for 30 times under the conditions of 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15 s; 72 ℃ for 1min.

(7) 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 are used as primers (table 1), and GCY1 knockout integration fragments are obtained through PCR amplification under the conditions of 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s and 30 cycles; 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), YPR1 knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 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), YDL124W knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s, and the cycle is 30 times; 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, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 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), and AAD3 knockout integration fragments are obtained through PCR amplification under the conditions of 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s,72 ℃ for 15s, and the cycle is 30 times; 72 ℃ for 1min.

(12) F-YDR541C-Del with a nucleotide sequence shown as SEQ ID NO:38 and R-YDR541C-Del with a nucleotide sequence shown as SEQ ID NO:39 are taken as primers (table 1), YDR541C knockout integration fragments are obtained through PCR amplification, the PCR amplification conditions are 94 ℃ for 2min,94 ℃ for 15s,50 ℃ for 15s and 72 ℃ for 15s, and the cycle is carried out for 30 times; 72 ℃ for 1min.

Example 3 construction of plasmids

(1) The method comprises the steps of taking a Bacillus cereus genome (NZ _ CP 017060.1) 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 (table 1), carrying out PCR amplification to obtain an ASBF gene, and recycling a target band through a DNA purification kit to obtain a glue recycling product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃, 4min at 72 ℃ and 30 times of circulation; 72 ℃ for 2min. An artificially synthesized gene HsOMT with a nucleotide sequence shown as SEQ ID NO. 82 is used 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 are used as primers (table 1), an HsOMT 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 ℃, 4min at 72 ℃ and 30 times of circulation; 72 ℃ for 2min. After pRS423A-GGA1 plasmid is digested for 4h by BsaI, the large fragment and the small fragment are recovered by glue, and then the ASBF gene fragment and the HsOMT gene fragment are mixed by using T4 ligase and BsaI enzyme, and the connection conditions are as follows: 10min at 37 ℃; circulating for 8 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying by using primers with nucleotide sequences shown as SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74 and SEQ ID NO 75 to obtain a positive clone colony, extracting an expression plasmid pRS423-ASBF/HsOMT, and completely conforming the sequencing result to the designed plasmid DNA sequence.

(2) An AROZ gene was obtained by PCR amplification using P423-H7-ArCatA, taGDC1, paAroZ vector (Bruckner Christine, et al, an expanded enzyme vector for the production of cis, cis-multiconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae, FEMS Yeast research.2018, 2:2) as a template, AROZ _ P1_ fwd having a nucleotide sequence shown in SEQ ID NO:76 and AROZ _ P1_ rev having a nucleotide sequence shown in SEQ ID NO:77 as primers (Table 1), and the objective band was recovered by a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃, 4min at 72 ℃ and 30 times of circulation; 72 ℃ for 2min. Subsequently, the AROZ gene fragment and the HsOMT gene fragment obtained in step (1) were mixed, using T4 ligase and BsaI enzyme under the ligation conditions: 10min at 37 ℃;10 min at 37 ℃ and 10min at 16 ℃ and circulating for 8 times; transferring the connected product into a competent cell of Escherichia coli DH5 alpha at 20 ℃ for 10min, verifying 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 to obtain a positive clone colony, extracting an expression plasmid pRS423-AROZ/HsOMT, and completely conforming the sequencing result to the designed plasmid DNA sequence.

(3) The method comprises the steps of taking a Segniliparus segenii rugosus genome (NZ _ KI 391953.1) 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 (Table 1), carrying out PCR amplification to obtain CAR genes, and recycling a target band through a DNA purification kit to obtain a gel recycling product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃, 4min at 72 ℃ and 30 times of circulation; 72 ℃ for 2min. The method comprises the steps of taking Ai Ahua Nocardia iowensis genome (NZ _ CP 078145.1) 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 (table 1), obtaining PPTASE genes through PCR amplification, and recovering a target band through a DNA purification kit to obtain a gel recovery product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃, 4min at 72 ℃ and 30 times of circulation; 72 ℃ for 2min. After cutting pRS425A-GGA1 plasmid by BsaI for 4h, recovering large and small fragments by glue, and then mixing with CAR gene fragment and PPTASE gene fragment using T4 ligase and BsaI enzyme under the conditions: 10min at 37 ℃; circulating for 8 times at 37 deg.C for 10min and 16 deg.C for 10min; transferring the connected product into Escherichia coli DH5 alpha competent cells at 20 ℃ for 10min, verifying by using nucleotide sequences such as SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81 to obtain positive clone colonies, extracting expression plasmid pRS425-CAR/PPTASE, and completely conforming the sequencing result to the designed plasmid DNA sequence.

Example 4 Targeted integration of expression fragment preparation

(1) The pRS423-AROZ/HsOMT plasmid obtained in step (2) of example 3 was used 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 were used as primers (Table 1), and PCR amplification was performed to obtain a knock-out integrated fragment Δ ROX 1P _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 And recovering the target band by a DNA purification kit to obtain a gel recovered product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃, 4min at 72 ℃ and 30 times of circulation; 72 ℃ for 2min.

(2) Using pRS423-CAR/PPTASE plasmid in step (3) of example 3 as a template, F-BTS1-Int 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 (Table 1), and performing PCR amplification to obtain a knockout integration fragment Δ BTS 1P _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 And recovering the target band by a DNA purification kit to obtain a gel recovered product. PCR amplification conditions: 2min at 98 ℃, 15s at 98 ℃, 2min at 56 ℃, 4min at 72 ℃ and 30 times of circulation；72℃2min。

Example 5: construction of engineered Strain JS-RARE1

(1) Preparing competent cells from Saccharomyces cerevisiae JS-CR, transforming into p414-TEF2-Cas9 plasmid, culturing for 2-4 days at 30 ℃ on YNBD-TRP plate, and obtaining a single colony named Saccharomyces cerevisiae Y1. Then preparing competent cells from Y1, transforming the plasmid p426-gRNA (ADH 6) obtained in the step (1) of the example 1 and the ADH6 knockout integration fragment obtained in the step (1) of the example 2 into the Y1 competent cells, culturing the competent cells on an YNBD-TRP-URA plate at 30 ℃ for 2-4 days, respectively scribing the grown single colonies on the YNBD-TRP-URA solid plate, and carrying out PCR verification by using primers SEQ ID NO:44 and SEQ ID NO:45 to obtain the correct saccharomyces cerevisiae strain JS-CR delta ADH6 which is named as a recombinant strain JS-M1.

(2) The JS-M1 in the step (1) is streaked on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (AHD 6) plasmid, then the plasmid is used for preparing competent cells, the plasmid is transformed into p426-gRNA (ADH 7) in the step (2) in the example 1 and an AHD7 knockout integration fragment in the step (2) in the example 2, the integrated fragments are cultured on the YNBD-TRP-URA plate at 30 ℃ for 2-4 days, grown single colonies are streaked on the YNBD-TRP-URA solid plates respectively, PCR verification is carried out by primers of SEQ ID NO:46 and SEQ ID NO:47, and the correct Saccharomyces cerevisiae strain ADH-CR delta ADH6 delta JS 7 is named as a recombinant strain JS-M2.

(3) The JS-M2 in the step (2) is streaked on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (ADH 7) plasmid, then used for preparing competent cells, transformed into the p426-gRNA (SFA 1) in the step (3) in the example 1 and the SFA1 knockout integrated fragment in the step (3) in the example 2, cultured on the YNBD-TRP-URA plate at 30 ℃ for 2-4 days, grown single colonies are streaked on the YNBD-TRP-URA solid plate respectively, and verified by PCR with primers SEQ ID NO:48 and SEQ ID NO:49, and the correct Saccharomyces cerevisiae strain JS-CR delta ADH6 delta ADH7 delta SFA1 is named as JS recombinant strain-M3.

(4) And (3) scribing JS-M3 in the step (3) on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (SFA 1) plasmid, then using the plasmid for preparing competent cells, transforming the plasmid into p426-gRNA (GRE 2) in the step (4) in the example 1 and a GRE2 knockout integration fragment in the step (4) in the example 2, culturing the integration fragments on the YNBD-TRP-URA plate at 30 ℃ for 2-4 days, respectively scribing grown single colonies on the YNBD-TRP-URA solid plate, carrying out PCR verification by using primers SEQ ID NO:50 and SEQ ID NO:51, and naming the correct saccharomyces cerevisiae strain JS-CR delta ADH6 delta ADH7 delta SFA1 delta GRE2 as a recombinant strain JS-M4.

(5) The JS-M4 in step (4) was streaked on YNBD-TRP plate 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, transformed into p426-gRNA (HFD 1) in step (5) in example 1 and HFD1 integration fragment in step (5) in example 2, cultured on YNBD-TRP-URA plate at 30 ℃ for 2 to 4 days, grown single colonies were streaked on YNBD-TRP-URA solid plate, respectively, verified by PCR using primers SEQ ID NO:52 and SEQ ID NO:53, and correct s.cerevisiae strain JS-CR. DELTA. ADH 6. DELTA. SFA 7. SFA 1. DELTA. GRE 2. HFD1, designated as recombinant strain-M5, followed by streaking on TRNBD-TRP plate containing 5-fluoroorotic acid at a concentration of 1mg/mL to remove intracellular p426-gRNA (HFD 1 ) plasmid designated as RARE1.

Example 6: construction of engineered Strain JS-RARE2

(1) The JS-RARE1 in the step (5) of the example 5 is used for preparing competent cells, the integration fragments of the p426-gRNA (GRE 3) in the step (6) of the example 1 and GRE3 in the step (6) of the example 2 are transformed, the integration fragments are cultured on an YNBD-TRP-URA plate for 2 to 4 days at 30 ℃, single colonies are respectively scribed on the YNBD-TRP-URA solid plate, PCR verification is carried out by using primers SEQ ID NO:54 and SEQ ID NO:55, and the correct Saccharomyces cerevisiae strain JS-CR delta ADH6 delta ADH7 delta SFA1 delta GRE2 delta HFD1 delta GRE3 is named as a recombinant strain JS-M6.

(2) The JS-M6 in the step (1) is streaked on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (GRE 3) plasmid, then the plasmid is used for preparing competent cells, p426-gRNA (GCY 1) in the step (7) in the example 1 and GCY1 knockout integration fragments in the step (7) in the example 2 are transformed into the plasmid, the plasmid is cultured on the YNBD-TRP-URA plate for 2 to 4 days at 30 ℃, single colonies which grow out are streaked on the YNBD-TRP-URA solid plate respectively, PCR verification is carried out by primers SEQ ID NO:56 and SEQ ID NO:57, and the correct saccharomyces cerevisiae strain-CR delta ADH6 delta ADH7 delta SFA1 delta GRE2 delta HFD1 delta GRE3 delta GCY1 is named as recombinant strain-JS M7.

(3) The JS-M7 in the step (2) is streaked on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (GCY 1) plasmid, then used for preparing competent cells, the p426-gRNA (YPR 1) in the step (8) in the example 1 and YPR1 knockout integration fragments in the step (8) in the example 2 are transformed into the plasmid, the plasmid is cultured on the YNBD-TRP-URA plate at 30 ℃ for 2-4 days, grown single colonies are streaked on the YNBD-TRP-URA solid plate respectively, PCR verification is carried out by primers SEQ ID NO:58 and SEQ ID NO:59, and the correct Saccharomyces cerevisiae strain-ADH Δ ADH6 Δ JS 7 Δ SFA1 Δ HFA 1 Δ HFD1 Δ GRE3 Δ GCY1 Δ YPR1 is named as strain JS-JS recombinant strain JS-M8.

(4) The JS-M8 in the step (3) is streaked on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (YPR 1) plasmid, then used for preparing competent cells, the p426-gRNA (YDL 124W) in the step (9) in the example 1 and YDL124W in the step (9) in the example 2 are transformed to knock out integrated fragments, the integrated fragments are cultured on the YNBD-TRP-URA plate at 30 ℃ for 2-4 days, grown single colonies are streaked on the YNBD-TRP-URA solid plate respectively, PCR verification is carried out by using primers SEQ ID NO:60 and SEQ ID NO:61, and a correct Saccharomyces cerevisiae strain-CR Delta ADH6 Delta ADH7 Delta SFA1 Delta HFD1 Delta 3 Delta GCY1 Delta YPR1 Delta is named as TREB 2 Delta YDL124, namely the strain RARGRA-M9, and then the JS-TRRP is streaked on the YNBD-gRNA (YPR 1) plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular gRNA 124-gRNA, finally the plasmid YDW and the plasmid is streaked and the plasmid YDL (YDL-TREB-TRPE) is used for preparing competent cells.

Example 7: construction of engineered Strain JS-RARE3

(1) The JS-RARE2 in the step (4) of the example 6 is used for preparing competent cells, the competent cells are transformed into p426-gRNA (ARI 1) in the step (10) of the example 1 and the ARI1 knockout integration fragment in the step (10) of the example 2, the competent cells are cultured on an YNBD-TRP-URA plate for 2 to 4 days at 30 ℃, single colonies which grow out are respectively scribed on the YNBD-TRP-URA solid plate, PCR verification is carried out by using primers SEQ ID NO:62 and SEQ ID NO:63, and 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 is named as a recombinant strain JS-M10.

(2) The JS-M10 in the step (1) is streaked on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (ARI 1) plasmid, then used for preparing competent cells, the p426-gRNA (AAD 3) in the step (11) in the example 1 and the AAD3 knockout integration fragment in the step (11) in the example 2 are transformed into the competent cells, the competent cells are cultured on the YNBD-TRP-URA plate at 30 ℃ for 2 to 4 days, the grown single colonies are streaked on the YNBD-TRP-URA solid plate respectively, PCR verification is carried out by using primers SEQ ID NO:64 and SEQ ID NO:65, and the correct Saccharomyces cerevisiae strain-CR Delta ADH6 Delta ADH 1 Delta SFA1 Delta GRE2 Delta HFD1 Delta GRE3 Delta GCY1 Delta YDR 1 Delta YDL124 Delta ARI1 Delta AAD3 is named as recombinant strain-JS-M11.

(3) The JS-M11 in the step (2) is streaked on an YNBD-TRP plate containing 5-fluoroorotic acid with the concentration of 1mg/mL to remove intracellular p426-gRNA (AAD 3) plasmid, then used for preparing competent cells, the p426-gRNA (YDR 541C) in the step (12) in the example 1 and the YDR541C in the step (12) in the example 2 are transformed to knock out integrated fragments, the YNBD-TRP-URA plate is cultured for 2-4 days at 30 ℃, grown single colonies are streaked on the YNBD-TRP-URA solid plate respectively, PCR verification is carried out by using primers SEQ ID NO:66 and SEQ ID NO:67, and correct saccharomyces cerevisiae strain ADH6 delta ADH7 delta SFA1 delta HFA 2 delta HFD1 delta 3 delta GCYKR 1 delta YPR1 delta YDL is named as TRWB delta ARI1 delta AAD3 delta YDR541C, the recombined strain ADH-M12 is streaked on the YNBD-TRNBD-TRNBR 541P plate containing 5-gRNA with the concentration of 5-fluoroorotic acid, and then the intracellular gRN-gRNA 426-gRNA (YDR 541C) plasmid is streaked on the JS 426-TRBD-TRP-TRBD plate, and the recombinant strain 12 is used for removing intracellular plasmid.

Example 8: accumulation capacity of vanillin of different modified strains

The recombinant strain JS-RARE1, JS-RARE2, JS-RARE3 and JS-CR, which were constructed in example 5, example 6 and example 7, were each picked up and inoculated monoclonally into a 15mL small shake tube containing 2mL YNBD whole liquid medium, and cultured at 30 ℃ and 250rpm for 12-16 h to prepare a seed solution, and the prepared seed solution was inoculated into a 50mL centrifuge tube containing 5mL YNBD whole liquid medium in an inoculum size of 1% (v/v) and cultured at 30 ℃ and 250rpm for 16h. Centrifuging at 5000rpm for 1min by a centrifuge, removing supernatant, collecting thallus, washing twice with 5mL phosphate buffer (200mM, pH = 8.0), removing supernatant, and collecting thallus; to the collected cells, 5mL of vanillin-depleted solution (containing 5mM vanillin, 20g/L glucose, 200mM phosphate buffer PH =8.0 and supplemented to 5 mL) was added, and 400uL of the solution was added at 4h, 8h, 24h and 48h, respectively, in accordance with a 1: adding 100% ethanol at a ratio of 1, mixing with a vortex oscillator, centrifuging at 14000rpm for 5min, filtering the supernatant with a filter membrane, performing liquid chromatograph, and converting with peak areas of vanillin and vanillyl alcohol standard substance to obtain consumption of recombinant strain.

As shown in FIG. 2, the control strain JS-CR depleted vanillin up at 48h, while the recombinant strains JS-RARE1, JS-RARE2, JS-RARE3, with increasing oxidoreductase gene knockout, reduced the amount of vanillin conversion to vanillyl alcohol and the modified strain JS-RARE3 hardly converted vanillin to vanillyl alcohol.

Example 9: construction of synthetic Vanillin Strain JS-A1

Competent cells were prepared from the engineered strain JS-CR, transformed into the pRS423-ASBF/HsOMT plasmid and pRS 425-CAR/PPTAE plasmid obtained in example 3, and cultured on YNBD-HIS-LEU plate at 30 ℃ for 2-4 days to obtain a positive clone JS-A1 Saccharomyces cerevisiae recombinant strain.

Example 10: construction of synthetic Vanillin Strain JS-A2

The engineered strain JS-RARE1 constructed in the step (5) of the example 5 is subjected to subculture in a YPD solid culture medium to remove a Cas9 expression vector, and then is used for preparing competent cells, is transformed into the pRS423-ASBF/HsOMT plasmid and pRS425-CAR/PPTASE plasmid obtained in the example 3, and is cultured on a YNBD-HIS-LEU plate at 30 ℃ for 2-4 days to obtain a positive clone JS-A2 Saccharomyces cerevisiae recombinant strain.

Example 11: construction of synthetic Vanillin Strain JS-A3

The engineered strain JS-RARE2 constructed in the step (4) of the example 6 is subcultured in YPD solid culture medium to remove Cas9 expression vector, and then used for preparing competent cells, and is transformed into pRS423-ASBF/HsOMT plasmid and pRS 425-CAR/PPTAE plasmid obtained in the example 3, and cultured on YNBD-HIS-LEU plate at 30 ℃ for 2-4 days to obtain positive clone JS-A3 Saccharomyces cerevisiae recombinant strain.

Example 12: construction of synthetic Vanillin Strain JS-A4

The engineered strain JS-RARE3 constructed in the step (3) of example 7 is subcultured in YPD solid medium to remove Cas9 expression vector, and then used for preparing competent cells, and is transformed into pRS423-ASBF/HsOMT plasmid and pRS 425-CAR/PPTAE plasmid obtained in the example 3, and cultured on YNBD-HIS-LEU plate at 30 ℃ for 2-4 days to obtain positive clone JS-A4 Saccharomyces cerevisiae recombinant strain.

Example 13: construction of synthetic Vanillin Strain JS-A5

The engineered strain JS-RARE3 constructed in the step (3) of example 7 is used for preparing competent cells after removing Cas9 expression vectors through subculture in YPD solid medium, is transformed into pRS423-AROZ/HsOMT plasmid and pRS 425-CAR/PPASE plasmid obtained in example 3, and is cultured for 2-4 days at 30 ℃ on YNBD-HIS-LEU plate to obtain a positive clone JS-A5 Saccharomyces cerevisiae recombinant strain.

Example 14: construction of engineered Strain JS-B1

The engineered strain JS-RARE3 constructed in step (3) of example 7 was used to prepare competent cells, and the integration fragment Δ ROX 1P knocked out in step (1) of example 1 (14) P426-gRNA (ROX 1) and example 4 (ROX 1) was transformed _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 Culturing on YNBD-TRP-URA plate at 30 ℃ for 2-4 days, respectively scribing the grown single colonies on YNBD-TRP-URA solid plates, and performing PCR verification by using primers SEQ ID NO:70 and SEQ ID NO:71 to obtain the 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 delta AAD3 delta YDR541C delta ROX 1P _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 Designated as recombinant strain JS-B1.

Example 15: construction of engineered Strain JS-B2

The engineered strain JS-B1 constructed in step (1) of example 14 was streaked on YNBD-TRP 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 P426-gRNA (BTS 1) in step (13) of example 1 and a knock-out integration fragment Δ BTS 1P in step (2) of example 4 _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 In Y atCulturing on NBD-TRP-URA plate at 30 ℃ for 2-4 days, respectively scratching the grown single colonies on YNBD-TRP-URA solid plates, and carrying out PCR verification by using primers SEQ ID NO:68 and SEQ ID NO:69 to obtain the 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 delta AAD3 delta YDR541C delta ROX 1P _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 ΔBTS1 P _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 Then, the plasmid of intracellular p426-gRNA (BTS 1) was streaked out on YNBD-TRP plates containing 5-fluoroorotic acid at a concentration of 1mg/mL, and the expression vector of Cas9 was removed by subculture on YPD solid medium, which was designated as recombinant strain JS-B2.

Example 16: yield of vanillin and vanillyl alcohol 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 selected and inoculated into 15mL small shaking tubes containing 2mL YNBD-HIS-LEU liquid culture medium, respectively, and cultured at 30 deg.C and 250rpm for 12-16 h to obtain seed liquid, which was inoculated into 250mL triangular flasks containing 20mL YNBD-HIS-LEU liquid culture medium with 1% (v/v) of inoculum size, and 20. Mu.M copper sulfate was added to induce expression, and cultured at 30 deg.C and 250rpm for 120h to obtain fermentation broth.

(2) The recombinant strain JS-B2 constructed in the example 15 is inoculated in a 15mL small shaking tube containing 2mL YNBD whole liquid culture medium in a single clone mode, and cultured for 12-16 h under the conditions of 30 ℃ and 250rpm to prepare seed liquid, the prepared seed liquid is inoculated in a 250mL triangular flask containing 20mL YNBD whole liquid culture medium according to the inoculation amount of 1% (v/v), 20 mu M copper sulfate is added for induction expression, and the culture is carried out for 120h under the conditions of 30 ℃ and 250rpm to prepare fermentation liquid.

(3) Calculating the yield of extracellular vanillin and vanillyl alcohol:

absorbing 400 mu L of fermentation liquor, adding 400 mu L of 100% ethanol, uniformly mixing with a vortex oscillator, centrifuging for 5min at 14000rpm of a centrifuge, filtering the supernatant with a filter membrane, performing high performance liquid chromatography detection, and converting with the peak areas of standard vanillin and vanillyl alcohol to obtain the fermentation yield of the engineering strain.

As a result, as shown in Table 2 and FIG. 3, the JS-A1 strain has an extracellular vanillyl alcohol content of 29.35. + -. 2.00mg/L; the extracellular vanillin content of the JS-A2 strain is 11.66 +/-0.38 mg/L and the vanillyl alcohol content is 28.26 +/-1.80 mg/L; the extracellular vanillin content of the JS-A3 strain is 11.82 +/-1.44 mg/L and the vanillyl alcohol content is 20.81 +/-3.27 mg/L; the extracellular vanillin content of the JS-A4 strain is 21.91 plus or minus 1.08mg/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 utilizing pRS423-ASBF/HsOMT plasmid and pRS 425-CAR/PPTAE plasmid for expression. Meanwhile, AROZ is used for replacing ASBF in the recombinant strain JS-A5, and the yield of vanillin is improved by 73.9%.

As shown in Table 2 and FIG. 4, the extracellular vanillin content of the JS-B2 strain is 85.39 +/-0.94 mg/L, which indicates that the vanillin synthesis pathway is integrated into the genome, and compared with plasmid expression, the vanillin yield is improved by 124%.

Table 2: yield of extracellular secretion of vanillin and vanillyl alcohol by different recombinant Saccharomyces cerevisiae

Strain of bacillus	Extracellular vanillin (mg/L)	Extracellular Vanillyl alcohol (mg/L)
			JS-A1	Not detected	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 detected
			JS-A5	38.12±1.31	Has not detected
JS-B2	85.39±0.94	Not detected

In conclusion, according to the saccharomyces cerevisiae recombinant strain disclosed by the embodiment of the invention, saccharomyces cerevisiae oxidoreductases ADH6, ADH7, SFA1, GRE2, HFD1, GRE3, GCY1, YPR1, YDL124W, ARI, AAD3 and YDR541C are knocked out, the finally constructed recombinant strain JS-RARE3 can prevent vanillin from being converted into vanillyl alcohol and vanillic acid by endogenous oxidoreductases, 3-dehydroshikimate dehydratase ASBF and 2-oxo-methyltransferase HsOMT, aromatic carboxylic acid reductase CAR and phosphopantothenate transferase ASSE are heterologously expressed by plasmids, after the recombinant strain is cultured for 120 hours, the extracellular vanillin (purity > 99%) content is 21.91 +/-1.08 mg/L, the 3-dehydroshikimate dehydratase ASBF is replaced by PPTASE with similar function, and the extracellular vanillin (purity > 99%) content is 38.12 +/-1.31 mg/L in shake flask fermentation of the recombinant strain-A5. In order to solve the instability of plasmid expression, 3-dehydroshikimate dehydratase AROZ, 2-oxo-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 culturing for 120 hours, the final yield of extracellular vanillin (the purity is more than 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 a wide industrial application prospect.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the terminology used in the description presented above should not be understood as necessarily referring 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. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A construction method of a saccharomyces cerevisiae recombinant strain for producing vanillin is characterized by comprising the following steps:

2. The method of construction as claimed in claim 1, comprising:

3. The method of claim 2, wherein the recombinant plasmid P426-gRNA (ROX 1) and the gene editing integration fragment Δ ROX 1P are introduced _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 And (3) transferring the electric transformation into a Saccharomyces cerevisiae JS-RARE3 competent cell which is transferred into Cas9, obtaining a positive monoclonal through PCR identification, and removing a gRNA expression vector from the positive monoclonal through 5-fluoroorotic acid screening to obtain a recombinant strain JS-B1.

4. The method of claim 2, wherein the recombinant plasmid p426-g is preparedRNA (BTS 1) and Gene editing integration fragment Δ BTS 1P _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 And (3) transferring the electric transformation into a Saccharomyces cerevisiae JS-B1 competent cell which is transferred into Cas9, obtaining a positive monoclonal through PCR identification, removing a gRNA expression vector from the positive monoclonal through 5-fluoroorotic acid screening, and removing the Cas9 expression vector through subculture to obtain a recombinant strain JS-B2.

5. The construction method according to claim 2, wherein the recombinant plasmid pRS423-ASBF/HsOMT is prepared by using a Bacillus cereus genome as a template, using ASBF _ P1_ fwd with a nucleotide sequence shown in SEQ ID NO:72 and ASBF _ P1_ rev with a nucleotide sequence shown in SEQ ID NO:73 as primers, performing PCR amplification to obtain an ASBF gene, and recovering a target band by using a DNA purification kit to obtain a gel recovery product; using artificially synthesized gene HsOMT with a nucleotide sequence shown as SEQ ID NO. 82 as a template, using 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, obtaining HsOMT gene through PCR amplification, and recovering a target band through a DNA purification kit to obtain a gel recovery product; after the pRS423A-GGA1 plasmid is digested for 4h by BsaI, a large fragment and a small fragment are recovered by glue, then an ASBF gene fragment and an HsOMT gene fragment are mixed, T4 ligase and BsaI enzyme are used for connection, the connected product is transferred into a competent cell of Escherichia coli DH5 alpha, primers with nucleotide sequences shown as SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75 are used for verifying to obtain a positive clone bacterial colony, and the expression plasmid pRS423-ASBF/HsOMT is extracted.

6. The construction method of claim 2, wherein the recombinant plasmid pRS425-CAR/PPTASE is prepared by using a genome of Segani Segniliparus rugosus as a template, using CAR _ P1_ fwd with a nucleotide sequence shown in SEQ ID NO:78 and CAR _ P1_ rev with a nucleotide sequence shown in SEQ ID NO:79 as primers, performing PCR amplification to obtain a CAR gene, and recovering a target band by using a DNA purification kit to obtain a gel recovery product; using Ai Ahua Nocardia iowensis genome as template, PPTASE _ P2_ fwd with nucleotide sequence as shown in SEQ ID NO. 80 and PPTASE _ P2_ rev with nucleotide sequence as shown in SEQ ID NO. 81 as primer, obtaining PPTASE gene by PCR amplification, recovering target band by DNA purification kit to obtain gel recovery product; after the pRS425A-GGA1 plasmid is digested for 4h by BsaI, large fragments and small fragments are recovered by glue, then mixed with CAR gene fragments and PPTAE gene fragments, T4 ligase and BsaI enzyme are used for connection, the connected products are transferred into Escherichia coli DH5 alpha competent cells, positive clone colonies are obtained by verification of nucleotide sequences such as SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, and the expression plasmid pRS 425-CAR/PPTAE is extracted.

7. The constructing method of claim 5, wherein the recombinant plasmid pRS423-AROZ/HsOMT is prepared by using P423-H7-ArCatA, taGDC1 and PaAroZ vectors as templates, AROZ _ P1_ fwd with a nucleotide sequence shown in SEQ ID NO. 76 and AROZ _ P1_ rev with a nucleotide sequence shown in SEQ ID NO. 77 as primers, performing PCR amplification to obtain an AROZ gene, and recovering a target band by using a DNA purification kit to obtain a gel recovery product; after the pRS423A-GGA1 plasmid is digested for 4h by BsaI, large fragments and small fragments are recovered by glue, an AROZ gene fragment and an HsOMT gene fragment are mixed, T4 ligase and BsaI enzyme are used for connection, the connected product is transferred into a competent cell of escherichia coli DH5 alpha, primers with nucleotide sequences shown as SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76 and SEQ ID NO 77 are used for verifying to obtain a positive clone bacterial colony, and the expression plasmid pRS423-AROZ/HsOMT is extracted.

8. The construction method according to claim 3, wherein the recombinant plasmid p426-gRNA (ROX 1) is a gRNA (ROX 1) obtained by PCR amplification of a gRNA fragment obtained by 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 a target band is recovered by a DNA purification kit to obtain a gel recovery product; the gel recovered product was ligated with the expression vector p426-SNR 52-GGA.

9. The method of claim 3, wherein the method is performed in the presence of a primerCharacterized in that the gene editing integration fragment delta ROX 1P _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 Using pRS423-AROZ/HsOMT plasmid as template, F-ROX1-Int with nucleotide sequence shown in SEQ ID NO. 42 and R-ROX1-Int with nucleotide sequence shown in SEQ ID NO. 43 as primers, and obtaining the knockout integration fragment delta ROX 1P through PCR amplification _GAL10 -AROZ-T _ADH1 -P _GAL1 -HsOMT-T _CYC1 。

10. 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 a 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.

11. The method of constructing a recombinant plasmid according to claim 4, wherein the gene editing integration fragment Δ BTS 1P _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 Using pRS423-CAR/PPTASE plasmid as template, F-BTS1-Int with nucleotide sequence shown as SEQ ID NO. 40 and R-BTS1-Int with nucleotide sequence shown as SEQ ID NO. 41 as primers, and performing PCR amplification to obtain the integration fragment Δ BTS 1P _GAL10 -CAR-T _ADH1 -P _GAL1 -PPTASE-T _CYC1 。

12. A recombinant strain of saccharomyces cerevisiae producing vanillin, constructed by the construction method of any one of claims 1 to 11.