CN114085784A

CN114085784A - Recombinant yeast with high cytochrome P450 expression and application thereof

Info

Publication number: CN114085784A
Application number: CN202111177370.9A
Authority: CN
Inventors: 连佳长; 江丽红; 董昌; 刘腾飞
Original assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Current assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2022-02-25
Anticipated expiration: 2041-10-09
Also published as: CN114085784B

Abstract

The invention discloses a recombinant yeast with high expression of cytochrome P450 and application thereof. A recombinant yeast with high expression of cytochrome P450 is obtained by overexpression of at least one gene of AtGRP7, AtMSBP1 and AtCOL4 derived from Arabidopsis thaliana in yeast. The constructed saccharomyces cerevisiae platform strain construction method for improving cytochrome P450 expression by utilizing the arabidopsis cDNA overexpression library successfully screens the target gene which is derived from arabidopsis and can improve CYP76AD1 expression by utilizing the biosensor for producing betaxanthin and the arabidopsis cDNA overexpression library through the change of strain color and fluorescence. Meanwhile, the target genes are applied to other P450 genes to prove the universality, so that the method can effectively establish a platform yeast strain for the functional expression of various P450 genes.

Description

Recombinant yeast with high cytochrome P450 expression and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to recombinant yeast with high expression of cytochrome P450 and application thereof.

Background

Cytochrome P450 enzymes (P450) are a superfamily of heme-thiolated proteins that are widely found in animals, plants, and microorganisms. Due to various biocatalytic activities like oxidation, epoxidation, hydroxylation and demethylation, P450 plays an important role in the metabolic network and is involved in the biosynthesis of many natural products, such as opioids, artemisinic acid and glycyrrhetinic acid. As a well-studied model organism, Saccharomyces cerevisiae has the advantages of clear genetic background, easy culture, post-translational processing capability and the like. Thus, s.cerevisiae is often the host of choice for functional expression of P450 and biosynthesis of natural products.

Although various P450s have been successfully expressed in Saccharomyces cerevisiae, for example, the invention with publication No. CN106987533A discloses a construction method of an engineered strain of Saccharomyces cerevisiae capable of synthesizing glycyrrhetinic acid. Beta-coumarol synthase gene GgbAS, cytochrome P450 oxidase genes CYP88D6 and CYP72A154 from Phyllanthus emblica and cytochrome P450 oxidoreductase genes CPR1 and CPR2 from Arabidopsis thaliana are respectively constructed to form gene expression cassettes, then the gene expression cassettes are co-transformed in Saccharomyces cerevisiae CEN. PK21C, and Saccharomyces cerevisiae engineering bacteria with a complete glycyrrhetinic acid biosynthesis pathway are assembled by utilizing the homologous recombination capability of the yeast, so that the artificial synthesis of glycyrrhetinic acid in Saccharomyces cerevisiae is realized.

But low expression levels and activity have become the biggest challenge for P450 research and application. Improvements in different levels and activities of P450 expression have been achieved currently by different strategies such as N-terminal truncation, molecular modification of the protein of P450, and co-expression with Cytochrome P450 Reductase (CPR), but these strategies are not applicable to most P450s and lack of versatility (Jiang, l., Huang, l., Cai, J., Xu, z., Lian, J. (2021). Functional expression of eukrastic cytochromes P450s in year.biotechnol.bioeng.118, 1050-1065.). Therefore, it is highly desirable to develop a universally applicable strategy to construct a saccharomyces cerevisiae platform strain to improve P450 expression in yeast.

The plant has a plurality of P450s, can interact with a plurality of substrates, and participates in the synthesis and degradation of alkaloid, terpenoid, flavonoid, fatty acid, phytohormone and signal molecules. Thus, plants have evolved a complex gene regulatory system to control the expression and folding of P450 s. It is speculated that the introduction of key genes involved in plant regulatory networks should contribute to the functional expression of a range of P450s in yeast.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a recombinant yeast with high cytochrome P450 expression and application thereof.

A recombinant yeast with high expression of cytochrome P450 is obtained by overexpression of at least one gene of AtGRP7, AtMSBP1 and AtCOL4 derived from Arabidopsis thaliana in yeast. Preferably, the recombinant yeast is obtained by over-expressing three genes of AtGRP7, AtMSBP1 and AtCOL4 derived from Arabidopsis thaliana in yeast. Preferably, the GenBank numbers of the recombinant yeast AtGRP7, AtMSBP1 and AtCOL4 are NM-127738.5, NM-124603.4 and LR782546.1, respectively. Preferably, the recombinant yeast further comprises a cytochrome P450 gene. More preferably, the recombinant yeast comprises one of the following:

(1) the cytochrome P450 gene is CYP76AD1, CYP76AD5 or CYP76AD6, the recombinant yeast also over-expresses the DOD gene, and the GenBank numbers of the CYP76AD1 and the DOD gene are KU644144.1 and KM502867.1 respectively; the GenBank numbers of the CYP76AD5 and CYP76AD6 genes are KM592961.1 and KM592962.1 respectively;

(2) the cytochrome P450 gene is CYP736A167, the recombinant yeast also overexpresses a santalene synthase SAS gene, an AtCPR2 gene, and an MVA pathway-related gene expression frame: tHMG1-ERG8-ERG13-ERG20-ERG12 and EGR10-MVD1-IDI1-tHMG1, wherein the GenBank number of the CYP736A167 gene is KU169302.1, the sequence of the santalene synthase SAS gene is shown in SEQ ID No.2, the GenBank number of the AtCPR2 gene is KC842188.1, the GenBank numbers of the tHMG1-ERG8-ERG13-ERG20-ERG12 genes are NM _001182434.1, NM _001182727.1, NM _001182489.1, NM _001181600.1 and NM _001182715.1, the GenBank numbers of the EGR10-MVD1-IDI1-tHMG1 genes are NM _001183842.1, NM _001183220.1, NM _001183931.1 and NM _001182434.1, respectively;

(3) the cytochrome P450 gene is F3H, and the recombinant yeast also overexpresses AtCPR1 gene, wherein the GenBank number of the F3H gene is NM-114983.3, and the GenBank number of the AtCPR1 gene is NM-001203894.1;

(4) the cytochrome P450 gene is T16H2, T3O and D4H, the recombinant yeast also overexpresses AtCPR1, 16OMT, T3R, NMT and DAT genes, wherein the GenBank number of the T16H2 gene is JF742645.1, the GenBank number of the T3O gene is KP122967.1, the GenBank number of the D4H gene is U71605.1, the GenBank number of the AtCPR1 gene is NM _001203894.1, the GenBank number of the 16OMT gene is EF444544.1, the GenBank number of the T3R gene is KP122966.1, the GenBank number of the NMT gene is HM584929.1, and the GenBank number of the DAT gene is AF 053307.1.

The invention also provides application of the recombinant yeast in high expression of cytochrome P450. Among them, the cytochrome P450 gene is CYP76AD1, CYP76AD5 or CYP76AD 6. The invention also provides application of the recombinant yeast in preparation of Z-alpha-santalol, wherein the cytochrome P450 gene is CYP736A 167. The invention also provides application of the recombinant yeast in catalyzing naringenin to synthesize flavanonol, wherein the cytochrome P450 gene is F3H. The invention also provides application of the recombinant yeast in catalyzing tabersonine to synthesize vindoline, wherein cytochrome P450 genes are T16H2, T3O and D4H.

The constructed saccharomyces cerevisiae platform strain construction method for improving cytochrome P450 expression by utilizing the arabidopsis cDNA overexpression library successfully screens the target gene which is derived from arabidopsis and can improve CYP76AD1 expression by utilizing the biosensor for producing betaxanthin and the arabidopsis cDNA overexpression library through the change of strain color and fluorescence. Meanwhile, the target genes are applied to other P450 genes to prove the universality, so that the method can effectively establish a platform yeast strain for the functional expression of various P450 genes.

Drawings

FIG. 1 is a schematic (A) and flow chart (B) of the use of Arabidopsis thaliana cDNA overexpression library to screen for target genes that increase cytochrome P450 expression.

FIG. 2 is a plasmid map of pRS 416.

FIG. 3 is a plasmid map of pRS416-TEF1 p.

FIG. 4 is a diagram showing the results of PCR validation of the diversity of Arabidopsis thaliana cDNA overexpression libraries.

FIG. 5 is a graph showing the results of fluorescence intensity detection of clones screened from the plate.

FIG. 6 is a graph showing the results of the test of the effects of AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) on CYP76AD1 expression and betaxanthin yield and the synergistic effect of the combination of these three genes on CYP76AD1 expression.

Fig. 7 is a graph of the results of examining the effects of AtGRP7(A3), AtMSBP1(a6) and AtCOL4(E9) on CYP736a167 expression and santalol synthesis, wherein a: constructing a flow; b: and (5) detecting the conversion rate.

FIG. 8 is a graph showing the results of examining the effects of AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) on F3H expression and flavanonol synthesis, wherein A: constructing a flow; b: and (5) detecting the conversion rate.

FIG. 9 is a graph showing the results of examining the effects of AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) on T16H2, T3O and D4H expression and vindoline synthesis, wherein A: constructing a flow; b: and (5) detecting the conversion rate.

Detailed Description

Vector backbone pRS416 was purchased from Addgene. Coli DH5 α was purchased from Biotech, Inc., of Onghama, Beijing. Saccharomyces cerevisiae BY4741 was purchased from the China center for Industrial culture Collection of microorganisms (CICC). DNA polymerase, restriction enzyme and T4 ligase were purchased from NEB. Coli plasmid extraction kit was purchased from AXYGEN ltd, yeast plasmid extraction kit was purchased from Zymo Research, and PCR product nucleic acid purification kit was purchased from Thermo Scientific.

The Escherichia coli culture medium adopts an LB culture medium, and the formula is as follows: 10g of peptone, 5g of yeast extract, 10g of NaCl and 1L of deionized water. The culture medium of the saccharomyces cerevisiae uses an SCD-URA culture medium, and the formula is as follows: amino-free yeast nitrogen source (Difco, Boom, The Netherlands)1.7g, ammonium sulfate 5g, CMS-URA (MP Biomedicals, Solon, Ohio)0.77g, glucose 20g, deionized water 1L.

FIG. 1 is a schematic diagram (FIG. 1A) and flow chart (FIG. 1B) of the present invention for creating a Saccharomyces cerevisiae platform strain capable of enhanced cytochrome P450 expression by whole genome-scale library screening. The primer sequences used in the following examples or application examples are shown in Table 1. The gene sequences used in the present invention are shown in Table 2, wherein N represents A, T, G or C.

TABLE 1 primer Table used in the present invention

TABLE 2 Gene sequences used in the invention

Example 1: saccharomyces cerevisiae platform strain construction method for improving cytochrome P450 expression by utilizing arabidopsis cDNA overexpression library

1. The betaxanthin-producing biosensor is prepared through the following steps:

a. starting from saccharomyces cerevisiae BY4741, one of betaxanthin synthesis pathway genes CYP76AD1/CYP76AD5/CYP76AD6 and DOD are integrated into a genome together, 3 betaxanthin-producing yeast strains are constructed, and an expression CYP76AD1 strain is selected as a biosensor for subsequent experiments. The GenBank numbers of the CYP76AD1 gene sequence and the DOD gene sequence are KU644144.1 and KM502867.1 respectively, and the GenBank numbers of the CYP76AD5 gene sequence and the CYP76AD6 gene sequence are KM592961.1 and KM592962.1 respectively. Betaxanthin is yellow and fluorescent (excitation wavelength is 498, emission wavelength is 533), and high-throughput screening can be performed through enhancement of color and fluorescence.

The PCR reaction system is as follows: the 50. mu.L reaction system contained 25. mu.L of Q5 DNA polymerase mix, 2.5. mu.L of each of the forward primer and the reverse primer, 1. mu.L of the template, and ddH₂O19. mu.L. Wherein the primers for amplifying CYP76AD1/CYP76AD5/CYP76AD6 fragments are CYP76-F and CYP 76-R; the primers used for amplification of the DOD fragment are DOD-F and DOD-R. The PCR cycle program of CYP76AD1 fragment, CYP76AD5 fragment, CYP76AD6 fragment and DOD fragment amplification is pre-denaturation at 98 ℃ for 2 min; denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 10s, extension at 72 deg.C for 1min for 30s, and circulation for 31 times; the extension was carried out at 72 ℃ for 3min and stored at 4 ℃. And (3) purifying and recovering the fragment obtained BY PCR BY using a PCR product kit, and integrating the CYP76AD1/CYP76AD5/CYP76AD6 fragment (one of three) and the DOD fragment onto a BY4741 genome BY using a CRISPR technology to construct 3 betaxanthin-producing yeast strains.

2. An arabidopsis cDNA overexpression library was prepared as follows:

(a.) starting with pRS416 plasmid (FIG. 2), the plasmid was double digested with KpnI and SacI; (b.) PCR amplification of a TEF1p fragment containing the TEF1 promoter, 40bp homology arms upstream and downstream of the Arabidopsis cDNA containing the ADH1 terminator, and EcoRI and BamHI cleavage sites using primers TEF1p-F and TEF1 p-R; (c.) construction of helper plasmid pRS416-TEF1p (FIG. 3) by inserting the TEF1p fragment into pRS416 plasmid using enzymatic ligation; (d.) EcoRI and BamHI double digestion of plasmid pRS416-TEF1p, PCR amplification of Arabidopsis cDNA library fragments containing regions homologous to plasmid pRS416-TEF1p using primers cDNA-F and cDNA-R; (e.) the digested pRS416-TEF1p plasmid skeleton and Arabidopsis cDNA fragment are transferred into a betaxanthin-producing strain, and an Arabidopsis cDNA overexpression library is constructed by utilizing the yeast homologous recombination capability. The GeneBank number of the TEF1 promoter sequence is KY704476.1, and the ADH1 terminator sequence is shown in SEQ ID No. 1.

The PCR reaction system is the same as that in step 1. Wherein primers for TEF1p fragment amplification containing homology arms and enzyme cutting sites are TEF1p-F and TEF1 p-R; the primers used for cDNA fragment amplification are cDNA-F and cDNA-R. The TEF1p fragment amplification PCR cycle program is the same as that in step 1, and only the extension time in the cycle step is changed to 3 min. The PCR cycle program for cDNA fragment amplification is the same as that in step 1, the annealing temperature in the cycle step is changed to 56 ℃, and the extension time is changed to 4 min. The restriction enzyme plasmid skeleton and the fragment obtained by PCR are purified and recovered by a PCR product kit. Wherein 50ng of pRS416 enzyme-cleaved skeleton and TEF1p fragment, 1 mu L T4 buffer and 0.5 mu L T4 ligase were added to the enzyme-linked reaction system, and ddH was added thereto₂O make the final reaction system 10. mu.L, 25 ℃ for 60 min. Thermally shocking and transforming escherichia coli DH5 alpha competent cells, uniformly coating on an LB solid culture medium of 100 mu g/mL ampicillin, and performing inverted culture at 37 ℃ for 16 h; and selecting positive clones, and extracting plasmids, namely pRS416-TEF1p plasmids. 500 mu g of pRS416-TEF1p enzyme cutting skeleton and 500 mu g of Arabidopsis thaliana cDNA overexpression fragment are added into a Saccharomyces cerevisiae transformation system, and the Arabidopsis thaliana cDNA overexpression library is expressed in Saccharomyces cerevisiae by utilizing homologous recombination.

The arabidopsis thaliana overexpression library is expressed in a betaxanthin-producing strain by utilizing yeast homologous recombination, 20 monoclonals are randomly selected to carry out PCR reaction after transformation and plate coating so as to verify the diversity of the library, and the PCR verification result of figure 4 shows that the library expressed in yeast has good diversity.

3. The screening of the arabidopsis target gene for improving the expression of P450(CYP76AD1) comprises the following preparation processes:

(a.) Saccharomyces cerevisiae strains containing betaxanthin-producing biosensors (CYP76AD1) expressing Arabidopsis thaliana cDNA overexpression libraries after transformation were cultured in SCD-URA medium and plated on SCD-URA solid plates; (b.) screening the more yellow colored clones for targets likely to increase P450 expression, followed by fluorescence detection after SCD-URA culture; (c.) extracting the plasmid of the yeast cell with increased fluorescence using a yeast plasmid extraction kit; (d.) the extracted plasmid is transferred back to Escherichia coli DH5 alpha competent cells for plasmid amplification and plasmid extraction; (e.) the ability to increase CYP76AD1 expression was further confirmed by converting the plasmid extracted from E.coli back into a betaxanthin-producing strain. (f.) integrating the target points which can effectively improve the P450 genes into the genome for combined optimization, and verifying the synergistic effect of the genes.

The PCR reaction system is the same as that in step 1. Wherein the primers used for amplification of the AtGRP7(A3) fragment are A3-XII5-F and A3-XII 5-R; primers for amplification of the AtMSBP1(A6) fragment are A6-X4-F and A6-X4-R; the primers used for amplification of the AtCOL4(E9) fragment were E9-XI3-F and E9-XI 3-F. The amplification PCR cycle sequence for the AtGRP7(A3) fragment, AtMSBP1(A6) fragment and AtCOL4(E9) fragment was the same as that in step 1. The fragment obtained by PCR is purified and recovered by a PCR product kit, and the AtGRP7(A3) fragment, AtMSBP1(A6) fragment and AtCOL4(E9) fragment are integrated on the betaxanthin-producing yeast genome by using the CRISPR technology.

76 clones were selected from the SCD-URA plate by using the betaxanthin-producing biosensor and the Arabidopsis thaliana cDNA overexpression library for fluorescent detection, and the results are shown in FIG. 5. After the plasmid in the cell with improved fluorescence is extracted and amplified and then returned to the bacteria for verification, as shown in FIG. 6, 3 targets are successful, and AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) improve the expression of CYP76AD1 and the yield of betaxanthin, and further verify the synergistic effect of 3 genes, wherein the AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) targets respectively improve the yield of betaxanthin by 1.32, 1.86 and 1.10 times. Meanwhile, when AtMSBP1(A6) is respectively combined and expressed with AtGRP7(A3) or AtCOL4(E9) targets, the betaxanthin yield is improved by 2.21 and 2.18 times, but the combined expression of AtGRP7(A3) and AtCOL4(E9) targets does not obviously improve the betaxanthin yield. Indicating that the AtMSBP1(A6) and the other two targets have synergistic effect. When the three targets are expressed simultaneously, the yield of the betaxanthin is improved by 2.36 times at most. GenBank numbers of AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) are NM-127738.5, NM-124603.4 and LR782546.1, respectively.

Application example 1: effects of AtGRP7(A3), AtMSBP1(a6) and AtCOL4(E9) on CYP736a167 expression and santalol synthesis.

A Z-alpha-santalol synthetic strain catalyzed by P450(CYP 736A167 derived from Santalum album) is selected, and the universality of the screened arabidopsis target point is verified.

(a.) selecting a laboratory constructed santalene-producing yeast strain which starts from BY4741 and integrates 4 copies of santalene synthase (SAS) at loci XVI1, IX1, XIII1 and X1 as a starting strain (Dong C, et., al. (2020). A single Cas9-VPR nucleic acid for single amino gene activation, repression, and edition in Saccharomyces cerevisiae ACS Synth. biol.,9:2252 2257), wherein the sequence of the SAS is shown in SEQ ID No. 2; and overexpresses the MVA pathway-associated gene expression cassettes, tmg 1-ERG8-ERG13-ERG20-ERG12 and EGR10-MVD1-IDI 1-tmg 1, to increase the accumulation of the Z- α -santalol precursor santalene, tmg 1, ERG8, ERG13, ERG20, ERG12, EGR10, MVD1 and IDI1, with GenBank numbers NM _001182434.1, NM _001182727.1, NM _001182489.1, NM _001181600.1, NM _001182715.1, NM _001183842.1, NM _001183220.1 and NM _001183931.1, respectively. (b.) integrating CYP736A167 and AtCPR2 genes on a yeast genome to construct a Z-alpha-santalol synthetic strain, wherein the GenBank numbers of the CYP736A167 and AtCPR2 genes are shown as KU169302.1 and KC842188.1 respectively; (c.) integrating target genes on the genome of the Z-alpha-santalol-producing yeast singly or in combination, and measuring the yield of the santalene and the Z-alpha-santalol by using GCMS.

The PCR reaction was performed as in step 1 of example 1. Wherein the long fragment used for tHMG1-ERG8-ERG13-ERG20-ERG12 is amplified by two fragments, and the primers for amplification are Cyto6-XI3-F, Cyto6-homo-R, Cyto6-homo-F and Cyto6-XI 3-R; the EGR10-MVD1-IDI1-tHMG1 fragment is amplified by two fragments, and primers for amplification are Cyto5-X4-F, Cyto5-homo-R, Cyto5-homo-F and Cyto 5-X4-R; the primers for amplifying the CYP736A167 fragment are CYP736A167-XII5-F and CYP736A 167-R; the primers used for amplification of the AtCPR2 fragment are CPR2-F and CPR2-XII 5-R. Primers used for amplification of the AtGRP7(A3) fragment are A3-XI2-F and A3-XI 2-R; primers for amplification of the AtMSBP1(A6) fragment are A6-XII2-F and A6-XII 2-R; primers used for amplification of AtCOL4(E9) fragment were E9-XII4-F and E9-XII 4-R. the PCR cycle program for amplifying the tHMG1-ERG8-ERG13-ERG20-ERG12 and EGR10-MVD1-IDI1-tHMG1 fragments was the same as that of step 1 in example 1, and the extension time in only the cycle step was changed to 3 min. The PCR cycling program for amplifying the CYP736A167 fragment and the AtCPR2 fragment is the same as that in step 1 of example 1, and the extension time in only the cycling step is changed to 4 min. The amplification PCR cycle sequence for AtGRP7(A3) fragment, AtMSBP1(A6) fragment and AtCOL4(E9) fragment was the same as in step 1 of example 1. And purifying and recovering the fragment obtained by PCR by a PCR product kit. Adding two short fragments of the recovered tHMG1-ERG8-ERG13-ERG20-ERG12 and a gRNA plasmid into a yeast transformation system, and integrating EGR10-MVD1-IDI1-tHMG1 into a genome by using a CRISPR technology and yeast homologous recombination capacity; similarly, EGR10-MVD1-IDI1-tHMG1 and CYP736A167-AtCPR2 are integrated into the corresponding site of the genome to construct the santalol-producing strain. The genes AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) are integrated into the genome of the santalol-producing strain by using CRISPR technology for combinatorial optimization.

As shown in FIG. 7, AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) can improve the expression of CYP736A167, and correspondingly improve the conversion rate of Z-alpha-santalol by 1.89, 1.71 and 1.73 times, and meanwhile, the targets have synergistic effect, and when the three genes are expressed together, the conversion rate of Z-alpha-santalol is improved by 2.97 times.

Application example 2: effects of AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) on F3H expression and dihydroflavonol synthesis.

A Dihydroflavonol (DHK) synthetic strain catalyzed by P450 (F3H, flavanone-3-hydroxylase, derived from Arabidopsis thaliana) was selected to verify the versatility of screening Arabidopsis thaliana targets.

(a.) starting with BY4741, integrating AtCPR1 into the genome, the GenBank number of AtCPR1 gene is NM-001203894.1; (b.) integrating the gene F3H yeast genome to construct DHK-producing yeast strain, wherein the GenBank number of the F3H gene is NM-114983.3; (c.) integrating the target genes into the DHK-producing yeast genome, alone or in combination; (d.) naringenin precursors were added to the SCD medium and naringenin and DHK were detected by LCMS.

The PCR reaction was performed as in step 1 of example 1. Wherein primers used for amplification of the AtCPR1 fragment are AtCPR1-XII5-F and AtCPR1-XII 5-R; primers for amplifying the F3H fragment are F3H-XII4-F and F3H-XII 4-R; primers used for amplification of the AtGRP7(A3) fragment are A3-XI2-F and A3-XI 2-R; primers for amplification of the AtMSBP1(A6) fragment are A6-XII2-F and A6-XII 2-R; the primers used for amplification of the AtCOL4(E9) fragment were E9-XI3-F and E9-XI 3-R. The amplification PCR cycle sequence for the AtCPR1 fragment, F3H fragment, AtGRP7(A3) fragment, AtMSBP1(A6) fragment and AtCOL4(E9) fragment was the same as in step 1 of example 1. Purifying and recovering the fragment obtained BY PCR BY a PCR product kit, integrating an AtCPR1 fragment and an F3H fragment on a BY4741 genome BY using a CRISPR technology, and constructing a DHK-producing strain; similarly, the AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) genes were integrated into the genome of the DHK-producing strain for combinatorial optimization.

As shown in FIG. 8, AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) can increase the expression of F3H and correspondingly increase the conversion rate of naringenin to DHK by 1.41 times, 1.96 times and 1.10 times respectively. Similarly, when the three genes are expressed simultaneously, the conversion rate of synthesizing DHK from naringenin is further improved by 2.43 times.

Application example 3: effects of AtGRP7(A3), AtMSBP1(a6) and AtCOL4(E9) on T16H2, T3O and D4H expression and vindoline synthesis.

A vindoline synthetic strain catalyzed by a plurality of P450(T16H2, T3O and D4H) was selected to verify the versatility of screening arabidopsis target.

(a.) on the basis of step a of application example 2, T16H2 (tabersonine-16-hydroxylase 2), 16OMT (16-hydroxylated tabersonine-O-methyltransferase), T3O (tabersonine-3-oxidase), T3R (tabersonine-3-reductase), NMT (3-hydroxy-16-methyl-2, 3-dihydrotabersonine-N-methyltransferase), D4H (deacetoxyvindoline-4-hydroxylase) and DAT (deacetylvindoline-4-O-acetyltransferase) genes were integrated into the genome, GenBank numbers for T16H2, 16OMT, T3O, T3R, NMT, D4H and DAT genes were JF742645.1, EF444544.1, 685 2, KP122966.1, HM 71605.1 and AF053307.1, respectively, to construct a vindoline-producing strain; (b.) integrating the target genes into the genome of the yeast, or a combination thereof; (c.) the tabersonine precursor was added to the SCD medium, and tabersonine and vindoline were detected by LCMS.

The PCR reaction was performed as in step 1 of example 1. Wherein the primers used for the amplification of the T16H2 fragment are T16H2-XII4-F and T16H 2-R; the primers used for NMT fragment amplification are NMT-F and NMT-XII 4-R; the primers used for the amplification of the 16OMT fragment were 16OMT-XI1-F and 16OMT-XI 1-R; the primers used for the amplification of the T3O fragment are T3O-X2-F and T3O-R; the primers used for the amplification of the T3R fragment are T3R-F and T3R-X2-R; primers for amplifying the D4H fragment are D4H-X4-F and D4H-R; the primers used for amplification of the DAT fragment were DAT-F and DAT-X4-R; primers used for amplification of the AtGRP7(A3) fragment are A3-XI2-F and A3-XI 2-R; primers for amplification of the AtMSBP1(A6) fragment are A6-XII2-F and A6-XII 2-R; the primers used for amplification of the AtCOL4(E9) fragment were E9-XI3-F and E9-XI 3-R. The amplification PCR cycle procedures for T16H2 fragment, T3O fragment, T3R fragment, NMT fragment, D4H fragment, DAT fragment, AtGRP7(A3) fragment, AtMSBP1(A6) fragment and AtCOL4(E9) fragment were the same as in step 1 of example 1. Purifying and recovering the fragments obtained BY PCR BY using a PCR product kit, integrating a T16H2-NMT fragment, a16 OMT fragment, a T3O-T3R fragment and a D4H-DAT fragment to different sites of a BY4741 genome BY using a CRISPR technology, and constructing a vindoline-producing strain; similarly, the AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) genes were integrated into the genome of the Venturi strain for combinatorial optimization.

As shown in FIG. 9, AtGRP7(A3), AtMSBP1(A6) and AtCOL4(E9) can increase the expression of T16H2, T3O and D4H, and correspondingly increase the conversion rate of tabersonine to vindoline by 1.22 times, 1.49 times and 1.34 times respectively. Similarly, when the three genes are expressed simultaneously, the conversion rate of synthesizing the vindoline by the tabersonine is further improved by 1.56 times.

Sequence listing

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atgtctactc aacaagtttc ttctgaaaac atcgttagaa acgctgctaa cttccaccca 60

aacatctggg gtaaccactt cttgacttgt ccatctcaaa ctatcgactc ttggactcaa 120

caacaccaca aggaattgaa ggaagaagtt agaaagatga tggtttctga cgctaacaag 180

ccagctcaaa gattgagatt gatcgacact gttcaaagat tgggtgttgc ttaccacttc 240

gaaaaggaaa tcgacgacgc tttggaaaag atcggtcacg acccattcga cgacaaggac 300

gacttgtaca tagttagctt gtgtttcaga ctcttgagac aacacggtat caagatctct 360

tgtgacgttt tcgaaaagtt caaggacgac gacggtaagt tcaaggcttc tttgatgaac 420

gacgttcaag gtatgttgtc tttgtacgaa gctgctcact tggctatcca cggtgaagac 480

atcttggacg aagctatcgt tttcactact actcacttga agtctactgt ttctaactct 540

ccagttaact ctactttcgc tgaacaaatc agacactctt tgagagttcc attgagaaag 600

gctgttccaa gattggaatc tagatacttc ttggacatct actctagaga cgacctccat 660

gacaagacat tgttgaactt cgcgaagttg gacttcaaca tcttgcaagc tatgcaccaa 720

aaggaagctt ctgaaatgac tagatggtgg agagacttcg acttcttgaa gaagttgcca 780

tacatcagag acagagttgt tgaattgtac ttctggatct tggttggtgt ttcttaccaa 840

ccaaagttct ctactggtag aatcttcttg tctaagatca tctgtttgga aactttggtt 900

gacgacactt tcgacgctta cggtactttc gacgaattgg ctatcttcac tgaagctgtt 960

actagatggg acttgggtca cagagacgct ttgccagaat acatgaagtt tatattcaag 1020

actctcatcg acgtttactc tgaagctgaa caagaattgg ctaaggaagg tagatcttac 1080

tctatccact acgctatcag atctttccaa gaattggtta tgaagtactt ctgtgaagct 1140

aagtggttga acaagggtta cgttccatct ttggacgact acaaatctgt gagcttgaga 1200

tctatcggtt tcttgccaat cgctgttgct tctttcgttt tcatgggtga catcgctact 1260

aaggaagttt tcgaatggga aatgaacaac ccaaagatca tcatcgctgc tgaaactatc 1320

ttcagattct tggacgacat cgctggtcac agattcgaac aaaagagaga acactctcca 1380

tctgctatcg aatgttacaa gaaccaacac ggtgtttctg aagaagaagc tgttaaggct 1440

ttgtctttgg aagttgctaa ctcttggaag gacatcaacg aagaattgtt gttgaaccca 1500

atggctatcc cattgccatt gttgcaagtt atcttggact tgtctagatc tgctgacttc 1560

atgtacggta acgctcaaga cagattcact cactctacta tgatgaagga ccaggttgat 1620

ttggtcctga aggacccagt taagttggac gactaa 1656

<210> 3

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttcacgcaag ttaagtccag gaaggtgagc aaatgctcat aagaggatgt ccaatattt 59

<210> 4

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aggcacggaa acggctgcac gggtacgcca gataaggata cagccgttca gggtaatat 59

<210> 5

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgcgtctaac gcttttgcca cttggatttc tattatagga cacccatgaa ccacacggt 59

<210> 6

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aagaaattct tcctgtgctt catcaaaacg cgaaaattcg gcaaatcgct ccccatttc 59

<210> 7

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(5)

<223> n is a, t, c or g.

<400> 7

nnnnnggtac catagcttca aaatgtttct actc 34

<210> 8

<211> 117

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(5)

<223> n is a, t, c or g.

<400> 8

nnnnngagct ccatagggta ggggaatttc gaccgggatc ccgtatccat ggaattcata 60

tgagcgtaat ctggtacgtc gtatgggtat ttgtaattaa aacttagatt agattgc 117

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tacccatacg acgtaccaga ttac 24

<210> 10

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gagctccata gggtagggga atttcg 26

<210> 11

<211> 64

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tttctaactc ttctcacgct gcccctatct gttcttccgc tatatctagg aacccatcag 60

gttg 64

<210> 12

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gattgctatg ctttctttct aatgagcaag aag 33

<210> 13

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccgcggatag cttcaaaatg tttctactc 29

<210> 14

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctagccttat tgttttagtt cagtgacagc gaactgccgt atagcgccga tcaaagtat 59

<210> 15

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cagccacagt tgtagtcacg tgcgcgccat gctgactaat tagtcgtgca atgtatgac 59

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tccttccttt tcggttagag 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aggaaacaat gatggattcc 20

<210> 18

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tggtagttgg agcgcaatta gcgtatcctg taccatacta ccgcggtata taacagttg 59

<210> 19

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ccaatcaaag aagcatcggt tcagatcgag caaactgtag tagtcgtgca atgtatgac 59

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gttgcttttg cggcctaagt 20

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gtatttatag caaacgcaat tg 22

<210> 22

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tgacatccaa actacaaaac cgagattgga catatagcac gatagcgccg atcaaagta 59

<210> 23

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tttctaactc ttctcacgct gcccctatct gttcttccgc gatagcttca aaatgtttc 59

<210> 24

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ctagccttat tgttttagtt cagtgacagc gaactgccgt ccggtagagg tgtggtcaa 59

<210> 25

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cagccacagt tgtagtcacg tgcgcgccat gctgactaat gatagcttca aaatgtttc 59

<210> 26

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tggtagttgg agcgcaatta gcgtatcctg taccatacta ccggtagagg tgtggtcaa 59

<210> 27

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ccaatcaaag aagcatcggt tcagatcgag caaactgtag gatagcttca aaatgtttc 59

<210> 28

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tgacatccaa actacaaaac cgagattgga catatagcac ccggtagagg tgtggtcaa 59

<210> 29

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ttcacgcaag ttaagtccag gaaggtgagc aaatgctcat gatagcttca aaatgtttc 59

<210> 30

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

aggcacggaa acggctgcac gggtacgcca gataaggata ccggtagagg tgtggtcaa 59

<210> 31

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tgcgtctaac gcttttgcca cttggatttc tattatagga atagcttcaa aatgtttct 59

<210> 32

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aagaaattct tcctgtgctt catcaaaacg cgaaaattcg ccggtagagg tgtggtcaa 59

<210> 33

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

actttgtact attccttccc cgtttactca attcttgaag atagcttcaa aatgtttct 59

<210> 34

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

agcgcgggta acatacctcc gtgaggcatc cttttatttg ccggtagagg tgtggtcaa 59

<210> 35

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tttctaactc ttctcacgct gcccctatct gttcttccgc ctatatctag gaacccatc 59

<210> 36

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ctagccttat tgttttagtt cagtgacagc gaactgccgt ccgcggtata aacagttg 58

<210> 37

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

agcgcttata aggttggggc aatactaaaa ctgtgatctt gtgtcgacgc tgcgggtat 59

<210> 38

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ttccgactct gttgtttcct attgtttcta atagggtacg caggaagaat acactatac 59

<210> 39

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

agcgcttata aggttggggc aatactaaaa ctgtgatctt gatagcttca aaatgtttc 59

<210> 40

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

agtattgata atgataaact cttcgagcgt cccaaaacc 39

<210> 41

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ggttttggga cgctcgaaga gtttatcatt atcaatact 39

<210> 42

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ttccgactct gttgtttcct attgtttcta atagggtacg aacgcagaat tttcgagtta 60

<210> 43

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

gcgccggttt tcattttctt ccacggaata ccaagcccat tagtcgtgca atgtatgac 59

<210> 44

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

ctgtacgcag catttagcag agatttgcca atgccaagaa ccggtagagg tgtggtcaa 59

<210> 45

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gccacccata atcggcgctt agtttcggag ttcaatcata gtgtcgacgc tgcgggtat 59

<210> 46

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ctgatgggtt cctagatata aacgcagaat tttcgagtta 40

<210> 47

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

taactcgaaa attctgcgtt tatatctagg aacccatcag 40

<210> 48

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

atatggggtc agtggcgata ttatactata ggagttaaag ccgcggtata taacagttg 59

<210> 49

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

cagccacagt tgtagtcacg tgcgcgccat gctgactaat gatagcttca aaatgtttc 59

<210> 50

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

agtcatacat tgcacgacta atagcgccga tcaaagtatt 40

<210> 51

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

aatactttga tcggcgctat tagtcgtgca atgtatgact 40

<210> 52

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

tggtagttgg agcgcaatta gcgtatcctg taccatacta tgccggtaga ggtgtggtc 59

Claims

1. A recombinant yeast with high cytochrome P450 expression is characterized in that at least one gene of AtGRP7, AtMSBP1 and AtCOL4 derived from Arabidopsis is overexpressed in yeast to obtain the recombinant yeast.

2. The recombinant yeast according to claim 1, wherein the recombinant yeast is obtained by overexpressing three genes AtGRP7, AtMSBP1 and AtCOL4 derived from Arabidopsis thaliana in yeast.

3. The recombinant yeast according to claim 1, wherein the GenBank numbers of AtGRP7, AtMSBP1, and AtCOL4 are NM _127738.5, NM _124603.4, and LR782546.1, respectively.

4. The recombinant yeast according to claim 1, further comprising a cytochrome P450 gene.

5. The recombinant yeast of claim 4, comprising one of:

(1) the cytochrome P450 gene is CYP76AD1, CYP76AD5 or CYP76AD6, the recombinant yeast also overexpresses the DOD gene,

GenBank numbers of CYP76AD1 and DOD genes are KU644144.1 and KM502867.1 respectively;

the GenBank numbers of the CYP76AD5 and CYP76AD6 genes are KM592961.1 and KM592962.1 respectively;

(2) the cytochrome P450 gene is CYP736A167, the recombinant yeast also overexpresses a santalene synthase SAS gene, an AtCPR2 gene, and an MVA pathway-related gene expression frame: tHMG1-ERG8-ERG13-ERG20-ERG12 and EGR10-MVD1-IDI1-tHMG1,

wherein the GenBank number of the CYP736A167 gene is KU169302.1,

the sequence of the santalene synthase SAS gene is shown in SEQ ID No.2,

the GenBank number of the AtCPR2 gene is KC842188.1,

GenBank numbers of tHMG1, ERG8, ERG13, ERG20 and ERG12 in the tHMG1-ERG8-ERG13-ERG20-ERG12 genes are NM _001182434.1, NM _001182727.1, NM _001182489.1, NM _001181600.1 and NM _001182715.1 respectively,

GenBank numbers of EGR10, MVD1, IDI1 and tHMG1 in EGR10-MVD1-IDI1-tHMG1 genes are NM _001183842.1, NM _001183220.1, NM _001183931.1 and NM _001182434.1 respectively;

(3) the cytochrome P450 gene is F3H, the recombinant yeast also overexpresses AtCPR1 gene,

wherein the GenBank number of the F3H gene is NM-114983.3,

GenBank number of AtCPR1 gene is NM-001203894.1;

(4) cytochrome P450 genes are T16H2, T3O and D4H, the recombinant yeast also overexpresses AtCPR1, 16OMT, T3R, NMT and DAT genes,

wherein the GenBank number of the T16H2 gene is JF742645.1,

the GenBank number of the T3O gene is KP122967.1,

the GenBank number of the D4H gene is U71605.1,

GenBank number of AtCPR1 gene is NM-001203894.1,

GenBank number of 16OMT gene is EF444544.1,

the GenBank number of the T3R gene is KP122966.1,

the GenBank number of the NMT gene is HM584929.1,

GenBank number of DAT gene is AF 053307.1.

6. Use of the recombinant yeast according to any one of claims 1 to 4 for highly expressing cytochrome P450.

7. The use of the recombinant yeast of claim 5 for the preparation of betaxanthin, wherein the cytochrome P450 gene is CYP76AD1, CYP76AD5 or CYP76AD 6.

8. The use of the recombinant yeast of claim 5 for the preparation of Z- α -santalol, wherein the cytochrome P450 gene is CYP736A 167.

9. The use of the recombinant yeast of claim 5 for catalyzing the synthesis of flavanonol from naringenin, wherein the cytochrome P450 gene is F3H.

10. The use of the recombinant yeast of claim 5 for catalyzing tabersonine synthesis of vindoline, wherein the cytochrome P450 genes are T16H2, T3O and D4H.