CN115873870B - Gene, vector, microsomal protein of CYP71BE epoxidase in camptotheca acuminata and application thereof - Google Patents

Gene, vector, microsomal protein of CYP71BE epoxidase in camptotheca acuminata and application thereof Download PDF

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CN115873870B
CN115873870B CN202211743330.0A CN202211743330A CN115873870B CN 115873870 B CN115873870 B CN 115873870B CN 202211743330 A CN202211743330 A CN 202211743330A CN 115873870 B CN115873870 B CN 115873870B
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protein
gene
cyp71be
reaction
isoliquiritigenin
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CN115873870A (en
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蒲祥
陈梦涵
张家华
王旻吉
林芯宇
雷明
孙萌萌
杨胜男
王晗光
黄乾明
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Sichuan Agricultural University
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Sichuan Agricultural University
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Abstract

The invention belongs to the technical field of bioengineering, and provides CYP71BE epoxidase genes in camptotheca acuminata and corresponding proteins coded by the genes, and also provides plasmids containing the genes and saccharomyces cerevisiae WAT11 host cells containing the expression plasmids, and CYP71BE microsomal proteins are induced and expressed by the host cells, and are used as epoxidating biocatalysts for the epoxidating of isoliquiritigenin or the epoxidating of isovincoside lactam.

Description

Gene, vector, microsomal protein of CYP71BE epoxidase in camptotheca acuminata and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to a gene, a vector, microsomal proteins and application of CYP71BE epoxidase in camptotheca acuminata.
Background
Epoxides are important intermediates in pharmaceutical synthesis, and currently the main synthesis methods are those prepared by olefin oxidation, such as by oxidation of olefins with an oxidizing agent such as peroxycarboxylic acid (Prilezhaev reaction), potassium peroxymonosulphonate (Schlem asymmetric epoxidation), t-butyl hydroperoxide (Sharpless asymmetric epoxidation), mn (III) salen catalyst (Jacobsen-Katsuki), hydrogen peroxide or carbamide peroxide (Julia-colonna). In addition, epoxides are also commonly present in plants, which are formed mainly by the oxidation of proteins of the CYP71 family. The CYP71 subfamily is belonging to the cytochrome CYP450 oxidase family. CYP71 family genes are generally involved in oxidative modification of indole alkaloids, terpenes, and flavonoid components. Two CYP71D12 (P98183.2) and CYP71D351 (U5HKE8.1) are found, for example, in Vinca rosea to participate in selective hydroxylation modification at C-16 position of vindoline. Two CYP71D521 (MG 873080) and CYP71D347 (MG 873081) were found to be involved in the 6, 7-epoxidation modification of cromolyn from vinca. CYP71D20 (NP 001311564.1) was found to be involved in the hydroxylation modification of aristolene during capsaicin biosynthesis from tobacco. CYP71D13 (Q9XHE7.1) and CYP71D18 (AIS 36970.1) are found from peppermint to be involved in the C-3 and C-6 selective hydroxylations of limonene, respectively. Three CYP71D (MK 209616, MK209619, MK 209625) were found to be involved in hydroxylation modification of thymol and carvacrol from thyme and oregano. Two CYP71D373 and CYP71D375 are found in the root of red-rooted salvia to participate in the hydroxylation and cyclization oxidation step in the biosynthesis process of tanshinone.
Currently, 49 alkaloids are discovered and identified from camptotheca acuminata based on targeted metabonomics analysis of adult camptotheca acuminata flowers, fruits, stems, leaves, and camptotheca acuminata seedlings; according to the rationality of chemical reaction and the route of Camptothecine (CPT) source, it was found that at least fourteen steps such as intramolecular dehydration, 2, 7-epoxidation (CYP 450), epoxidation hydrolysis, 2, 7-oxidative cleavage (CYP 450) are needed after the formation of strictosidinic acid in camptotheca, but the specific participation of 2, 7-epoxidation related to CYP71 family genes and the discovery of functional genes such as CYP71BE in camptotheca are not clear, and further research is needed.
Disclosure of Invention
The invention aims to combine genome, transcriptome and proteome multi-group data to carry out screening and optimization methods of CYP71BE genes in camptotheca acuminata, so as to obtain CYP71BE cyclooxygenase genes in camptotheca acuminata and corresponding proteins coded by the genes, plasmids of the genes and saccharomyces cerevisiae WAT11 host cells containing the expression plasmids are obtained, and simultaneously, host cells are utilized to induce expression of four CYP71BE microsomal proteins of CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE20, and the four microsomal proteins can BE used for selective epoxidation of isoliquiritigenin, and meanwhile, the CYP71BE206 can BE used for 2-7-bit epoxidation of isoliquiritigenin lactam.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides CYP71BE cyclooxygenase genes in camptotheca acuminata, comprising CYP71BE206 gene, CYP71BE207 gene, CYP71BE208 gene or CYP71BE131 gene.
Preferably, the nucleotide sequence of the CYP71BE206 gene is shown in SEQ ID NO. 1; the nucleotide sequence of the CYP71BE207 gene is shown in SEQ ID NO. 2; the nucleotide sequence of the CYP71BE208 gene is shown in SEQ ID NO. 3; the nucleotide sequence of the CYP71BE131 gene is shown in SEQ ID NO. 4.
The invention provides a protein coded by the CYP71BE epoxidase gene, which comprises CYP71BE206 protein, CYP71BE207 protein, CYP71BE208 protein or CYP71BE131 protein.
Preferably, the amino acid sequence of the CYP71BE206 protein is shown in SEQ ID NO. 5; the amino acid sequence of the CYP71BE207 protein is shown in SEQ ID NO. 6; the amino acid sequence of the CYP71BE208 protein is shown in SEQ ID NO. 7; the amino acid sequence of the CYP71BE131 protein is shown in SEQ ID NO. 8.
The invention provides a recombinant expression plasmid containing the CYP71BE epoxidase gene.
Preferably, the original plasmid used is the pYES2/CT plasmid.
Preferably, the CYP71BE epoxidase gene is inserted at the Kpn I site and the Not I site of the original plasmid.
The invention also provides a recombinant strain for expressing the CYP71BE epoxidase gene, which takes WAT11 saccharomyces cerevisiae as a host bacterium and is transformed with the recombinant expression plasmid.
The invention also provides application of the protein coded by the CYP71BE epoxidase gene as an epoxidation biocatalyst.
Preferably, the specific application process of the protein encoded by the CYP71BE epoxidase gene is as follows: the reaction is carried out in a reaction system containing a reaction substrate, NADPH, protein coded by the CYP71BE epoxidase gene and Tris-HCl buffer solution, and then the reaction system is extracted and filtered.
Preferably, the reaction substrate is isoliquiritigenin or isovincoside; the reaction time is 0.5-8h, the reaction temperature is 20-40 ℃, and the reaction pH is 6-8.
Preferably, the protein encoded by the CYP71BE epoxidase gene is used for the selective epoxidation of isoliquiritigenin, and the CYP71BE206 protein is used for the 2-7 epoxidation of isoliquiritigenin lactam.
By adopting the technical scheme, the invention has the following beneficial effects:
1. the invention provides four amino acid sequences of cytochrome CYP71BE enzyme, a nucleotide sequence for encoding the protein, a plasmid containing the gene and a saccharomyces cerevisiae WAT11 host cell containing the expression plasmid;
2. the host cell constructed by the invention can induce and express CYP71BE epoxidase, four CYP71BE microsomal proteins (CYP 71BE206, CYP71BE131, CYP71BE207 and CYP71BE 20) are used for the selective epoxidation of isoliquiritigenin, and one CYP71BE206 can BE used for the 2-7-position epoxidation of the isoliquiritigenin lactam.
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FIG. 1 is a comparative analysis of five group protein levels of camptotheca acuminata flowers, fruits, stems, leaves and seedlings (A in FIG. 1 is the anabolism of CPT in adult camptotheca acuminata, B in FIG. 1 is SDS-PAGE of five group total proteins, C in FIG. 1 is a thermal map of five group differential proteins, and D in FIG. 1 is a principal component analysis of five group proteins);
FIG. 2 shows a CaCYP71 target protein structure analysis (A in FIG. 2 shows the tertiary structure of CYP71-Cac_g004916 protein, and B in FIG. 2 shows the case of multi-sequence alignment of CaCYP71 target protein and characterized CYP71 in Catharanthus roseus);
FIG. 3 is a phylogenetic tree of the camptotheca CYP71 protein and the characterized CYP71 protein in other plants;
FIG. 4 shows the expression cluster analysis of the target CYP71 gene and genes involved in CPT and FLA biosynthesis (A in FIG. 4 shows the genome-level expression cluster analysis, B in FIG. 4 shows the transcriptome-level expression cluster analysis, and C in FIG. 4 shows the proteomic-level expression cluster analysis);
FIG. 5 shows the result of the verification of the recombinant CYP71BE plasmid (A in FIG. 5 shows the result of the electrophoresis of the recombinant plasmid and the cleavage product: 1 shows pYES2/CT-CYP71BE206,2 shows pYES2/CT-CYP71BE206 cleavage product, 3 shows pYES2/CT-CYP71BE131,4 shows pYES2/CT-CYP71BE131 cleavage product, 5 shows pYES2/CT-CYP71BE207,6 shows pYES2/CT-CYP71BE207 cleavage product, 7 shows pYES2/CT-CYP71BE208,8 shows pYES2/CT-CYP71BE208 cleavage product, and B in FIG. 5 shows the result of the electrophoresis of the PCR verification product plasmid: 1 shows pYES2/CT-CYP71BE206 plasmid PCR product, 2 shows pYES2/CT-CYP71BE131 PCR product, 3 shows pYES 2/CT-71 BE207 plasmid PCR product, 4 shows pYES 2/CT-71 BE208 plasmid PCR product;
FIG. 6 shows the results of PCR verification of colonies of WAT11 recombinant Saccharomyces cerevisiae strain (M represents DNA Marker,1 represents negative control, 2 represents CYP71BE206 recombinant strain, 3 represents CYP71BE131 recombinant strain, 4 represents CYP71BE207 recombinant strain, 5 represents CYP71BE208 recombinant strain);
FIG. 7 shows the WAT11 yeast cells before and after disruption (A in FIG. 7 is 10X 20 times before disruption, B in FIG. 7 is 10X 20 times after disruption, C in FIG. 7 is 10X 40 times before disruption, and D in FIG. 7 is 10X 40 times after disruption);
FIG. 8 shows the result of Western blot verification of the target CYP71BE Protein (M represents Protein markers (18 kDa to 100 kDa), 1 represents CYP71BE206,2 represents CYP71BE131,3 represents CYP71BE207,4 represents CYP71BE208,5 represents pYES 2/CT);
FIG. 9 shows the results of LC-MS analysis of the CYP71BE206 enzymatic reaction system (A in FIG. 9 is a graph of EIC and TIC of a reaction system using genistein as a substrate, B in FIG. 9 is a graph of EIC and TIC of a reaction system using isoliquiritigenin as a substrate, C in FIG. 9 is a graph of EIC and TIC of a reaction system using isoliquiritigenin lactam as a substrate);
FIG. 10 shows the results of LC-MS analysis of CYP71BE131 enzymatic reaction system (A in FIG. 10 is a graph of EIC and TIC of a reaction system using genistein as a substrate, B in FIG. 10 is a graph of EIC and TIC of a reaction system using isoliquiritigenin as a substrate, C in FIG. 10 is a graph of EIC and TIC of a reaction system using isoliquiritigenin lactam as a substrate);
FIG. 11 shows the results of LC-MS analysis of the CYP71BE207 enzymatic reaction system (A in FIG. 11 is a graph of EIC and TIC of a reaction system using genistein as a substrate, B in FIG. 11 is a graph of EIC and TIC of a reaction system using isoliquiritigenin as a substrate, C in FIG. 11 is a graph of EIC and TIC of a reaction system using isoliquiritigenin lactam as a substrate);
FIG. 12 is a graph showing the results of LC-MS analysis of the CYP71BE208 enzymatic reaction system (A in FIG. 12 is a graph showing the reaction system EIC and TIC using genistein as a substrate, B in FIG. 12 is a graph showing the reaction system EIC and TIC using isoliquiritigenin as a substrate, and C in FIG. 12 is a graph showing the reaction system EIC and TIC using isoliquiritigenin lactam as a substrate);
FIG. 13 is a secondary mass spectrum and cleavage process of an epoxidation product (A in FIG. 13 is a secondary mass spectrum and cleavage process of a isoliquiritigenin lactam epoxide, B in FIG. 13 is a secondary mass spectrum and cleavage process of an isoliquiritigenin epoxide);
FIG. 14 shows the optimized conditions for CYP71BE206 microsomal protein reaction (A in FIG. 14 is the induction time, B in FIG. 14 is the reaction temperature, C in FIG. 14 is the reaction time, D in FIG. 14 is the reaction pH, E in FIG. 14 is the NADPH concentration, F in FIG. 14 is the substrate concentration, striped columns represent the conditions for substrate isoliquiritigenin, white columns represent the conditions for substrate isoliquiritigenin lactam);
FIG. 15 shows the optimized conditions for CYP71BE131 microsomal protein reaction (A in FIG. 15 is the induction time, B in FIG. 15 is the reaction temperature, C in FIG. 15 is the reaction time, D in FIG. 15 is the reaction pH, E in FIG. 15 is the NADPH concentration, F in FIG. 15 is the substrate concentration, striped columns in the figures represent the reaction conditions for the substrate isoliquiritigenin);
FIG. 16 shows the optimized conditions for CYP71BE207 microsomal protein reaction (A in FIG. 16 is the induction time, B in FIG. 16 is the reaction temperature, C in FIG. 16 is the reaction time, D in FIG. 16 is the reaction pH, E in FIG. 16 is the NADPH concentration, F in FIG. 16 is the substrate concentration, striped columns in the figures represent the reaction conditions for the substrate isoliquiritigenin);
FIG. 17 shows the optimized condition of the reaction conditions of CYP71BE208 microsomal proteins (A in FIG. 17 is the induction time, B in FIG. 17 is the reaction temperature, C in FIG. 17 is the reaction time, D in FIG. 17 is the reaction pH, E in FIG. 17 is the NADPH concentration, F in FIG. 17 is the substrate concentration, striped columns in the figure represent the reaction conditions of the substrate isoliquiritigenin);
FIG. 18 shows the kinetic characteristics of four CYP71BE microsomal proteins (A in FIG. 18 is the steady-state kinetics of CYP71BE206 for isoliquiritigenin, B in FIG. 18 is the steady-state kinetics of CYP71BE206 for isoliquiritigenin, C in FIG. 18 is the steady-state kinetics of CYP71BE131 for isoliquiritigenin, D in FIG. 18 is the steady-state kinetics of CYP71BE207 for isoliquiritigenin, and E in FIG. 18 is the steady-state kinetics of CYP71BE208 for isoliquiritigenin).
Detailed Description
The invention provides CYP71BE cyclooxygenase genes in camptotheca acuminata, comprising CYP71BE206 gene, CYP71BE207 gene, CYP71BE208 gene or CYP71BE131 gene.
In the invention, the nucleotide sequence of the CYP71BE206 gene is shown in SEQ ID NO: 1:
ATGGAGCTCCAATTTCCCTCCTCCCCTGTGCTCTTTTCCTTCCTCCTCTTCATGTTAATGGCAGTCAAAGTCGTTAAGAGATCCAAAGCCAAGAACCCAACTTCAAAACTGCCCCCAGGGCCATGGAAACTCCCTCTCATAGGAAACATGCACCAGCTTGTTGGCTCACTACCTCATCACATTCTAAGAGACCTGGCCATGAAATATGGACCACTTATGCACCTAAAACTAGGCCAACTTTCCACTGTCATTGTTTCTTCTCCCGAAATTGCCGAAGAGGTTATGAAAACCCATGACATAATCTTTTCTCAAAGGCCATATCTTCTTGCTTCTAGAATCTTATCATATGATTCTACAAATATTGCCTTCTCTCCTTATGGTGACTACTGGAGACAACTAAGGAAAATTTGCACAATTGAGCTTTTAAGCTCAAAGCGGGTCCAAAGTTTCAGATCCATTAGGGAAGAAGAGGTTTTGAGTCTCATTAGATCAATTTCTTCGAATGCAAAATCTCCAATCAATTTTAGCAAGAAAATATTTGACATGACTTACGGTATAACTGGGAGAGCTGCTTTTGGTGAAAAAAATGAAGATCAAGAAGCATTCATATCATTAATGGATGAAGCTTTGCCACTTTTGGGTGGTTTTAGTATTGTTGATATGTACCCTTCAATTAAAGTGCTTTCAGTGATCACAGGAATGAGGCCCAGGCTTGAGAAGATTCATAAAAAAATTGATCAGATACTTCAGAATATCATCAACCAGCACAAAAAAGGAGAGAAAACAATAAAACGTGAAGTGGAGGATTTAGTTGATGTTCTCTTAAAGTTTCAGAAGCATGGAAACCTAGAATTTCCCTTAACTGACAAAAACATCAAAGCCGTCATCTTGGATATTTTCAGTGGTGGGAGTGATACATCATCAACAGCAGTAGAGTGGGCAATGTCAGAAATGCTGAGAAACCCAAGAGTGATGAAAAGGGCACAAGCTGAGGTAAGAAAGGTATTCGGCAAAAATGGAAATGTCAATGAAGCTGGTCTTCATCACCTAAAATACTTAAATTCTGTAATCAAAGAAACCTTGAGGCTACATCCTCCTGCTCCTCTGTTAGTTCCAAGAGAATGCAGTGAGCAATGTGAAATCAATGGATACCAAATACCTGCCAAGACAAGAGTCGTTGTCAATGCATGGGCTATTGGTCGGGATCCCAGATACTGGACTGAAGCAGAGAGATTTCAACCAGAGAGATTTCTTGATAGTTCATTGGATTTCAAGGGGGTAGATTTTGAATACATCCCATTTGGTGCTGGAAGAAGGATATGTCCAGGCATATTGTTTGCTCTACCTAATATTGGGCTTCCACTAGCACAGTTGCTGTATCATTTTGATTGGAAACTCCCCATTGGATTGAAGGAAGGACAACTTGAAATGAATGAAGCCTTTGGCTTGACAGTAAGAAGAAAACAAAATTTGTACTTGATTCCTGTACCTTATTAA。
in the invention, the nucleotide sequence of the CYP71BE207 gene is shown in SEQ ID NO. 2:
ATGGAGCTTCAATATCCTTCCATCTCTGTCCTCTTTGCCTTCCTCCTCTTCATGTTAATGGTTGTGAAAGTTGTTAAGAGATCCAAAGGCAAGAACTCAACTGCTAAATTGCCCCCAGGGCCATGGCAACTACCTCTCATAGGAAACATGCACCAGCTCATGGGCTCACAACTACCGCATCATATCCTGAGAGATTTGGCCGAGAAATATGGACCACTCATGCACCTCAAACTAGGAGAACTTTCCACTATCATTGTTTCTTCACCAGAAATTGCCAAAGAGGTTTTGAAAACCCATGACATCATTTTTTCTGAAAGGCCATATCTTCTTGCTTCCACGATCATGTCATATGATTGTATAGATATTGTGTTCTCTCCTTATGGTGACTACTGGAGGCAGTTAAGGAAAATTTGTATAACTGAGCTTTTAAGTTCGAAGCGCGTCCAAACATTCAGATCCATTAGGGAAGAAGAAGTCTCGAATCTAATTAGCTCAATCTCTTCAAATATTGCAAAATCACCATTAATCAATCTCAGCAATAAAATTTTCTCCCTGATACATGGTATAACATCAAGAGCTGCTTTTGGTGAAAAAAGTAAAGATCAGGAAGAGTTCATATCAATTATGGTGGATCTCCTAAGACTTGCGTCTGGTTTCAGCATTGCTGATATGTACCCTTCGGTTAAAATACTTTCAGTGATCTCTGGAGTAAGGACAAGGCTTGAGGAGATGCATAAAAGAAGTGATCAGATACTTGAGAATATCCTCAATCAACATAAAGAGGGAAGGAAAACGAAACAAGCGAATGAGGATTTAGTTGATGTTCTTCTAAAGTTTCAGGAGGATGGTAATCTTGAAAATCAATTAACTGACAATAGCATTAAAGCCGTCATCTTGGACGTTTTCAGTGCTGGAAGTGAATCAACTACAACTATAGAGTGGGCAATGTCAGAAATGTTTAAAAACCCAACAGTAATGGCAAAGGCACAAGCTGAAGTAAGAGAGGTATTTGACAAAAGGGGAGATGTTGATGAAATGGGCCTTAATCAATTAAATTACTTAAAATTAGTAATCAAAGAAACCTTGAGGCTACATCCCCCTGCTCCTCTATTGGTCCCGAGAGAATGTAGTGCGCAATGTGAAATCAATGGATACCAAATACCTGCCAAGACAAGGGTCATTGTTAATGGATGGGCAATTGGCAGAGACCCCAGATATTGGACTGAAGCAGAGAGATTTCAACCAGAGAGATTTATCGATAGTCCAATTGATTTCAAGGGGACACATTTTGAATTTATTCCATTTGGTGCTGGAAGAAGGATTTGTCCAGGAATATTGTTTGCACTTCCTCTTATTGAGTTGCCCCTTGCGCAATTGCTGTACCATTTCAATTGGAAACTCCCCAATGGATTAAAGCAAGAAGAACTAGATATGACTGAGGCTGTAGGCATTACCAGCACTGAGAAGAGGGCAGCAGTGCAGCTGGAATGGATCGGAGGCTTGATGATCGGGAAACAGCCTTTGGGAGTCGGAACGGAGATGGTGCGCTGCTGTTACAAAGATGGGAAATAG。
in the invention, the nucleotide sequence of the CYP71BE208 gene is shown as SEQ ID NO: 3:
ATGGAGCTCCAATTTCCTTCCATCTCAGTCCTCTTTGCCTTCCTCCTCTTCATCTTAATGGTTATGAAAGTTGTTAAGAGATCCAAAGGCAAAAACACAACCGCAAAATTGCCCCCAGGGCCATGGCAACTACCTCTCATAGGTAACATGCACCAGCTGATGGGCTCACAACTACCCCATCATACCCTGAGAGATTTGGCCAAGAAATATGGAGCACTCATGCACCTCAAACTAGGAGAACTTTCCACTATCATTGTTTCTTCACCAGAAATTGCCAAAGAGGTCATGAAAACCCATGACATCATTTTTTCTCAAAGACCGTATCTTCTTGCTTTCAGGATCATTTCATATGATTCTATAAATATTGTGTTCTCTCCTTATGGTGATTACTGGAGGCAGTTAAGGAAAATTTGTATAACTGAGCTTTTAAGTTCGAAGCGCGTCCAAACATTCAGATCCATTAGGGAAGAAGAGGTTTCGAATCTGATTAGCTCAATCTCTTCAAATATTGCAAAATCACCATTAATCAATCTCAGCAAGAAAATTTCCTCCCTGACATATGGCATAACTTCTAGAGCCGCTTTTGGTGAAAAAAGTAAAGATCAGGAAGAATTCATATCAATGATCGAAGAAACCACAGGACTTGCAGCTGGTTTCAGCATTGCTGATATGTACCCTTCGGTTAAAATGCTTTCAGTGATCACTGGAATTAGGCCAAGGCTTGAGGAGCTGCATAAAAGAATTGATCAGATACTTGAGAATATCCTCAGTCAACATAAAGAGGGAAAGAAAATAAAACAAGTAGATGAGGATTTAGTTGATGTTCTTCTAAAGTTTCAGAAGAATGGTGATCTTGAATTTCCATTAACTGACAGAAGTATCAAAGCAGTCATCATGGATGTTTTCAGTGCTGGTAGTGATACATCAACTAGAGTTGTAGAATGGGCAATGTCAGAAATGCTTAAAAACCCAAGAGTAATGGAAAAGGCACAAGCTGAAGTAAGAGATGTATTTGATAAAAAGGGAAATGTTGATGAAATGGGCCTTAATCAATTAAATTACTTAAAATTAGTAATCAAAGAAACCTTGAGGCTACATCCTCCTGCTCCTCTGTTGGTCCCAAGAGAATGTAGTGAGCAATGTGAAATCAATGGATACCAAATACCTGCAAAGACAAGGGTCATTGTTAATGTATGGGCAATTGGCAGAGATCCCAGATATTGGAGTGAAGCAGAGAGATTTCAACCAGAAAGATTTATCGATAGTCCAATTGATTTCAAGGGGACACATTTCGAATTCATCCCATTTGGTGCTGGAAGAAGGATTTGTCCGGGAATATTGTTTGCCCTTCCTAATATTGAGTTGCCTCTTGCGCAATTTCTGTACCATTTCAATTGGAAGCTCCCCAAGGGATTGAAACTTGAAGAACTAGATATGACTGAGTCTTTTGGTTTTACTGTACGTAGAAAAAATGATCTATACTTGATTCCTCATCCTTATGTTCCTTCTATTTGA。
in the invention, the nucleotide sequence of the CYP71BE131 gene is shown as SEQ ID NO. 4:
ATGGAGCTTCAATTGCCTTCCATCTCTGTCCTCTTTGCTTTCCTCCTCTTCATGTTAATGGTTGTGAAAGTTGTTCAGAGATCCAAAGGCAAGAACTCAACTGCAAAATTGCCCCCAGGGCCATGGCAACTACCTCTTATAGGAAACATGCACCAGCTTATGGGCTCACAACTACCCCATCATATCCTGAGAGATTTGGCCAAAAAATATGGAGCACTCATGCACCTCAAACTAGGAGAACTTTCCACTATCATTGTCTCTTCACCAGAAATTGCTAGAGAGGTTATGAAAACCCATGACATCATTTTTTCTCAAAGGCCATATTTTCTTGCTTCCAGTATCATGTCATACGGTTTTATAGATGTTATATTCTGTCCCTACGGTGACTACTGGAGGCAGTTAAGGAAAATTTGTATAACTGAGCTTTTAAGTTCGAAGCGCGTCCAAACATTCAGATCCATAAGGGAAGAAGAGGTCTCGAATCTGATTAGCTCTATCTCTTCAAATATTGCAAAATCACCATTAATCAATCTCAGCAAGAAAATTTTCTCCTTGACAAATGACATAACTTCAAGAGCTGCTTTTGGTGAAAAAAGTAAAGATCAGGAAGAATTCATATCAATTATGGTGGAAACCGATAAACTTGCGGCTGGTTTCAGCATTGCTGATATGTACCCTTCGGTTAAACTGCTTTCATTGATCACTAGAATAAGGCCAAGGCTTGAGGAGCTGCATAAAAGAACTGATCAGATACTTGAGAATATCATCAATCAACATAAAAAGGGAAACAAAACAAAAGAAGTGAATGAGGATTTAGTTGATGTTCTTCTAAAGTTTCAGAAACATGGTGATCTTGAATTTCCATTAACTGACAAAGGGATCAAAGCTGTCATCTTGGACGTTTTCAGTGCTGGGAGTGAAACATCAACTACAGCTATAGAGTGGGCAATGTCAGAAATGCTTAAAAACCCAAGAGTAATGGAAAAGGCACAAGCTGAAGTAAGAGATGTTTTTAACAAAAAGGGAAATGTTGATGAAATGGGCCTTCATCAATTAAATTATTTAAAATTAGTCATCAAAGAAACCTTGAGGCTACATCCCCCTCTTCCTCTATTGCTCCCAAGAGAATGTAGTGAGCAATGTGAAATCAATGGATACCAAATACCTGCCAAGACAAGGGTCATTGTCAATGGATGGGCAATTGGTAGAGATCCCAGATTTTGGAGTGAAGCAGAGAGATTTCAGCCAGAGAGATTTATCGATAGTCCAATTGATTTCAAGGGAACACATTTCGAATTCATCCCATTTGGTGCTGGAAGAAGGATTTGTCCAGGAATATTATTTGCCCTTCCTAGTATTGAGTTGCCCCTTGCTCAATTGCTGTACCATTTCAATTGGAAGCTCCCCAATGGATTGAAGCAAGAAGAACTAGACATGACCGAGGGTTTAGGCATGGCCGCAAGAAGGAAACATGATCTATATTTGATTCCTGATCCTTATGTTCCTTCAATCGTTGAATAA。
the invention provides a protein coded by the CYP71BE epoxidase gene, which comprises CYP71BE206 protein, CYP71BE207 protein, CYP71BE208 protein or CYP71BE131 protein.
In the invention, the amino acid sequence of the CYP71BE206 protein is shown in SEQ ID NO. 5:
MELQFPSSPVLFSFLLFMLMAVKVVKRSKAKNPTSKLPPGPWKLPLIGNMHQLVGSLPHHILRDLAMKYGPLMHLKLGQLSTVIVSSPEIAEEVMKTHDIIFSQRPYLLASRILSYDSTNIAFSPYGDYWRQLRKICTIELLSSKRVQSFRSIREEEVLSLIRSISSNAKSPINFSKKIFDMTYGITGRAAFGEKNEDQEAFISLMDEALPLLGGFSIVDMYPSIKVLSVITGMRPRLEKIHKKIDQILQNIINQHKKGEKTIKREVEDLVDVLLKFQKHGNLEFPLTDKNIKAVILDIFSGGSDTSSTAVEWAMSEMLRNPRVMKRAQAEVRKVFGKNGNVNEAGLHHLKYLNSVIKETLRLHPPAPLLVPRECSEQCEINGYQIPAKTRVVVNAWAIGRDPRYWTEAERFQPERFLDSSLDFKGVDFEYIPFGAGRRICPGILFALPNIGLPLAQLLYHFDWKLPIGLKEGQLEMNEAFGLTVRRKQNLYLIPVPY。
in the invention, the amino acid sequence of the CYP71BE207 protein is shown in SEQ ID NO. 6:
MELQYPSISVLFAFLLFMLMVVKVVKRSKGKNSTAKLPPGPWQLPLIGNMHQLMGSQLPHHILRDLAEKYGPLMHLKLGELSTIIVSSPEIAKEVLKTHDIIFSERPYLLASTIMSYDCIDIVFSPYGDYWRQLRKICITELLSSKRVQTFRSIREEEVSNLISSISSNIAKSPLINLSNKIFSLIHGITSRAAFGEKSKDQEEFISIMVDLLRLASGFSIADMYPSVKILSVISGVRTRLEEMHKRSDQILENILNQHKEGRKTKQANEDLVDVLLKFQEDGNLENQLTDNSIKAVILDVFSAGSESTTTIEWAMSEMFKNPTVMAKAQAEVREVFDKRGDVDEMGLNQLNYLKLVIKETLRLHPPAPLLVPRECSAQCEINGYQIPAKTRVIVNGWAIGRDPRYWTEAERFQPERFIDSPIDFKGTHFEFIPFGAGRRICPGILFALPLIELPLAQLLYHFNWKLPNGLKQEELDMTEAVGITSTEKRAAVQLEWIGGLMIGKQPLGVGTEMVRCCYKDGK。
in the invention, the amino acid sequence of the CYP71BE208 protein is shown in SEQ ID NO: 7:
MELQFPSISVLFAFLLFILMVMKVVKRSKGKNTTAKLPPGPWQLPLIGNMHQLMGSQLPHHTLRDLAKKYGALMHLKLGELSTIIVSSPEIAKEVMKTHDIIFSQRPYLLAFRIISYDSINIVFSPYGDYWRQLRKICITELLSSKRVQTFRSIREEEVSNLISSISSNIAKSPLINLSKKISSLTYGITSRAAFGEKSKDQEEFISMIEETTGLAAGFSIADMYPSVKMLSVITGIRPRLEELHKRIDQILENILSQHKEGKKIKQVDEDLVDVLLKFQKNGDLEFPLTDRSIKAVIMDVFSAGSDTSTRVVEWAMSEMLKNPRVMEKAQAEVRDVFDKKGNVDEMGLNQLNYLKLVIKETLRLHPPAPLLVPRECSEQCEINGYQIPAKTRVIVNVWAIGRDPRYWSEAERFQPERFIDSPIDFKGTHFEFIPFGAGRRICPGILFALPNIELPLAQFLYHFNWKLPKGLKLEELDMTESFGFTVRRKNDLYLIPHPYVPSI。
in the invention, the amino acid sequence of the CYP71BE131 protein is shown as SEQ ID NO. 8:
MELQLPSISVLFAFLLFMLMVVKVVQRSKGKNSTAKLPPGPWQLPLIGNMHQLMGSQLPHHILRDLAKKYGALMHLKLGELSTIIVSSPEIAREVMKTHDIIFSQRPYFLASSIMSYGFIDVIFCPYGDYWRQLRKICITELLSSKRVQTFRSIREEEVSNLISSISSNIAKSPLINLSKKIFSLTNDITSRAAFGEKSKDQEEFISIMVETDKLAAGFSIADMYPSVKLLSLITRIRPRLEELHKRTDQILENIINQHKKGNKTKEVNEDLVDVLLKFQKHGDLEFPLTDKGIKAVILDVFSAGSETSTTAIEWAMSEMLKNPRVMEKAQAEVRDVFNKKGNVDEMGLHQLNYLKLVIKETLRLHPPLPLLLPRECSEQCEINGYQIPAKTRVIVNGWAIGRDPRFWSEAERFQPERFIDSPIDFKGTHFEFIPFGAGRRICPGILFALPSIELPLAQLLYHFNWKLPNGLKQEELDMTEGLGMAARRKHDLYLIPDPYVPSIVE。
the invention also provides a recombinant expression plasmid containing the CYP71BE epoxidase gene. In the invention, the obtained recombinant expression plasmids are pYES2/CT-CYP71BE206, pYES2/CT-CYP71BE207, pYES2/CT-CYP71BE208 or pYES2/CT-CYP71BE131.
In the present invention, the original plasmid used is preferably a pYES2/CT plasmid.
In the present invention, the CYP71BE epoxidase gene is preferably inserted at the Kpn I site and the Not I site of the original plasmid.
The invention also provides a recombinant strain for expressing the CYP71BE epoxidase gene, wherein the recombinant strain is transformed with the recombinant expression plasmid by preferably taking WAT11 saccharomyces cerevisiae as a host strain.
The invention also provides application of the protein coded by the CYP71BE epoxidase gene as an epoxidation biocatalyst.
In the present invention, the specific application of the protein encoded by the CYP71BE epoxidase gene is carried out in a reaction system containing a reaction substrate, NADPH, the protein encoded by the CYP71BE epoxidase gene and Tris-HCl buffer, the reaction system is preferably 200. Mu.L, wherein the reaction substrate is preferably isoliquiritigenin or isovincoside, the reaction substrate is preferably prepared into standard mother liquor of 50mM respectively with DMSO before use, and the final use concentration of the reaction substrate is preferably 100. Mu.M; the NADPH concentration is preferably 500. Mu.M; the Tris-HCl buffer is preferably 50mM Tris, ph=7.4. In the present invention, the amount of the protein encoded by the CYP71BE epoxidase gene is preferably 5 to 10mg, more preferably 7 to 9mg, still more preferably 8mg; the addition amount of the reaction substrate and NADPH mother solution is preferably 0.1 to 0.7. Mu.L, more preferably 0.3 to 0.5. Mu.L, still more preferably 0.4. Mu.L. In the reaction system of the present invention, the buffer was used to make up to 200. Mu.L.
In the present invention, the reaction time is preferably 0.5 to 8 hours, more preferably 1 to 6 hours, still more preferably 2 to 4 hours; the temperature of the reaction is preferably 20 to 40 ℃, more preferably 25 to 37 ℃, still more preferably 35 ℃.
In the present invention, the reaction is preferably terminated with ethyl acetate after the completion of the reaction, and the amount of ethyl acetate is preferably 150 to 250. Mu.L, more preferably 180 to 230. Mu.L, still more preferably 200. Mu.L.
In the present invention, the extraction centrifugation is performed after the termination of the reaction, and the rotational speed of the centrifugation is preferably 12000 to 18000rpm, more preferably 14000 to 16000rpm, still more preferably 15000rpm; the time of the centrifugation is preferably 2 to 5 minutes, more preferably 2.5 to 4 minutes, still more preferably 3 minutes.
In the present invention, the upper organic phase was transferred and dried with nitrogen, the residue was fixed to 1mL with methanol, and the resulting sample was filtered through a 0.22 μm syringe filter.
In the invention, the protein coded by the CYP71BE epoxidase gene is used for the selective epoxidation of isoliquiritigenin, wherein the CYP71BE206 protein is used for the 2-7-position epoxidation of isoliquiritigenin lactam.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 protein discrimination, functional annotation and group comparison analysis in camptotheca acuminata in the metabolism exuberance and camptotheca acuminata seedlings
First, collecting mature camptotheca acuminata sample and cultivating seedlings
1. Sample collection
The vigorous period of camptothecin anabolism in adult camptotheca (c.acuminata) was determined from laboratory preliminary studies. Fresh flowers, leaves and stems were harvested from 3 adult camptotheca acuminata in the first week of 8 months; mature fruits were collected for the last week of 11 months. All samples were collected at the university of agriculture, sichuan. The fresh samples were sealed and stored at-80 ℃.
2. Seedling cultivation
The aseptic seedlings of camptotheca acuminata are cultivated in the laboratory, mature seeds are collected in a first teaching building (29 DEG 98 '43.79' N,103 DEG 00 '65.74' E) in the Atlantic university of Sichuan agricultural university, dried in the shade at room temperature, the excellent mature seeds are selected to strip the shells, the seeds are washed with a surfactant (3% TritonX-100) for 3min to wash off surface impurities, and the surfactant is removed by repeated washing with sterile water for 3-5 times. Soaking seeds in 75% ethanol, stirring thoroughly for sterilization for 1min, transferring the seeds into 1% NaClO solution, stirring for sterilization for 3min, and washing with sterile water for 3-5 times to remove residual reagents. The sterile filter paper wraps the seed coat to absorb water, and the sterile gun-shaped forceps clamp the seed coat into a culture flask filled with 1/2MS culture medium, and the seed coat is 7-8 grains/flask. Culturing in dark until cotyledons germinate (25 ℃ C., 60% -75% humidity), and then performing illumination culture period (25 ℃ C., 60% -75% humidity, light intensity 7000LX, photoperiod 16 h/d) to two pieces of true She Mengfa (40 d). Seedlings were transferred to MS medium under sterile conditions for 30d with the same light culture period. The samples were removed from the MS medium with forceps, rinsed with sterile water and stored at-80 ℃.
(II) Total protein extraction and proteomic analysis of camptotheca acuminata
Fresh flowers, fruits, leaves, stems and seedlings (100 mg) of camptotheca acuminata were respectively ground into powder with liquid nitrogen, total proteins were prepared using an acetone precipitation method, and protein quantification was performed. The protein profile of each tissue was analyzed by SDS-PAGE. Proteomic analysis was performed on the beijing norelvan source. 100. Mu.g of protein in each sample was digested with 1.5. Mu.L trypsin and protein fragmentation was performed on a Rigol L3000 HPLC system (Evertech, guangzhou, china) and 0.8. Mu.L of iRT reagent (Biognosys, schlieren, switzerland) was analyzed (4. Mu.g) using an Evosep One UHPLC system (Evosep, odense, denmark) of a tandem Q exact HF-X mass spectrometer (Thermo Fisher, MA, united States) and finally a protein mass spectral library was constructed using the original MS/MS dataset.
The protein profile of the different tissues obtained by SDS-PAGE is shown in FIG. 1, and a total of 518466 maps were obtained by proteomic analysis.
(III) identification, quantification and functional analysis of proteins
Protein discrimination was performed on each sample using Proteome Discoverer software (version 2.2,Thermo Fisher) and the search results were imported into Spectronaut software (version 14.0, biognosys) to generate peptide libraries. And selecting qualified peptide and product ions according to a peptide and ion pair selection rule to generate a target list, importing Data and Independently Acquiring (DIA) data, extracting ion pair chromatographic peaks according to the target list, and realizing peptide identification and quantification by matching ions and calculating peak areas. Quantitative results between different tissues were statistically analyzed using the student's t test and varied according to fold (|log) 2 FC|is more than or equal to 2) and P value<0.05 Determining a Differentially Expressed Protein (DEP) for expression clustering and KEGG enrichment analysis. Gene Ortology (GO) and InterPro (IPR) functional analysis was performed using Interascan software (version 5.22-61.0). Protein families and pathways were annotated using Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.
From 101169 matching patterns, 49597 polypeptides with a length ranging from 7 to 25aa were identified, from which finally 10274 proteins (6.1-950.8 kDa) were identified. In the DIA library, 40817 polypeptides were obtained in total, and 8583 proteins were identified, the number of identified proteins in each tissue sample being: flowers (FL 1:6458, FL2:6737, FL3: 6221), fruits (FR 1:6522, FR2:7008, FR3: 6957), leaves (LE 1:4300, LE2:4303, LE3: 4019), stems (ST 1:6666, ST2:6937, ST3: 6715), seedlings (SE 1:6856, SE2:6922, SE3: 6686). The number average of the identified proteins in flowers, fruits, stems and seedlings exceeds 6400, while the number average of the identified proteins in leaves is 4207, which shows that the protein enrichment characteristics of the leaves and other tissue sites are obviously different. COG annotation indicated that 317 protein was involved in secondary metabolite biosynthesis, transport and metabolism. KEGG annotation indicated 86 proteins were assigned to terpenoid and polyketide metabolism, 160 to secondary metabolite biosynthesis, and 319 to amino acid metabolism, respectively. The IPR annotation indicated that 76 proteins belonged to UDP-glucosyltransferase and 109 proteins belonged to cytochrome P450 protein.
Based on the abundance data set of the identified proteins, the principal component analysis among groups was performed, and the results showed that the differences of the protein enrichment characteristics of the five groups were significant (as shown in fig. 1), and 5426 different proteins DEP (P < 0.05) were determined from the accumulation. The cluster analysis of the inter-group differential protein expression was performed according to the differential protein abundance, and the results are shown in fig. 1. KEGG enrichment analysis results indicate that amino sugar and nucleotide sugar metabolism, cutin, lipid and waxy biosynthesis are enriched in five groups and are metabolized vigorously in flowers; photosynthesis, carbon metabolism and carbon fixation are actively metabolized in the leaves; alkaloid biosynthesis is enriched in all groups and is metabolized vigorously in the stems and leaves; steroid biosynthesis is enriched in flowers and fruits; tryptophan metabolism is enriched in fruits; diterpenes and terpenoid skeleton biosynthesis are enriched in stems. Thus, stems and leaves were identified as tissues in which camptothecin anabolism was vigorous.
EXAMPLE 2 excavation, screening and structural analysis of CYP71 candidate Gene
Based on the sequence conservation domain, the CYP71 candidate sequence is discovered from Dryad Digital Repository genome database, IMPLAD transcriptome data and self-built camptotheca transcriptome database (SCAU-Genometa Resource), 14 CYP71 candidate gene sequences are obtained, wherein 6 CYP718 complete and 6 incomplete CYP71 candidate sequences are obtained.8 CYP71 candidate sequences (Cac_g 004916, cac_g004918, cac_g004919, cac_g004921, cac_g004924, cac_g004925, cac_g024044, cac_g 026785) were obtained using DNAMAN sequence alignment removal redundancy, as shown in Table 1. Conservation analysis was performed with 3 functionally characterized CYP71 (MG 8730080, MG8730081, U5HKE8.1) amino acid sequences, as shown in FIG. 2. CaCYP71 gene coding proteins all comprise a membrane anchoring domain and O 2 Binding and activating domain, E-R-R triplet domain, heme binding domain, highly variable substrate recognition domain, with typical features of plant CYP71 family proteins.
TABLE 1 excavation of camptotheca CYP71 family Gene
EXAMPLE 3 phylogenetic and expression Cluster analysis of CaCYP71 protein
24 CYP71 protein sequences derived from dicots and CYP71 sequences discovered in camptotheca acuminata are selected, and a systematic evolutionary tree of CYP71 with different species classifications is constructed by an adjacent-Joining method, and the result is shown in figure 3. CYP71 proteins of different species are classified into Tubiflorae, contortae, euphorbiales (Euphorbias), rosales (Rosales), labiatae (Lamiales), sapindales (Sapindales), cornales (Cornales). Cac_g004916, cac_g004918, cac_g004919, cac_g004921, cac_g004924, cac_g004925 and Cac_g024044 are aggregated into a single clade adjacent to the two cyclooxygenase CYP71D521 (MG 873080) and CYP71D347 (MG 873081) that have been characterized in Catharanthus roseus; cac_g026785, CYP71D410 (AJD 25163.1) and CYP71D411 (AJD 25164.1) are aggregated into another clade. The above results indicate that the camptotheca CaCYP71 sequence has typical species specificity, with 7 CYP71 proteins adjacent to the periwinkle CYP71 relatives with epoxidation capability.
To narrow the characterization, expression data (FPKM) of the gene sequences known to be involved in the biosynthesis of Camptothecin (CPT) and Flavonoid (FLA) components were tabulated by extracting CaCYP71 of interest from the genome and transcriptome databases of camptotheca acuminataAnd (4) performing clustering analysis, wherein the clustering result is shown in fig. 4. In mature camptotheca acuminata, cac_g004919 and Cac_g004921 are clustered with genes involved in flavone synthesis (C4H-Cac_g 002001, PAL-Cac_g012077, PAL-Cac_g019328, PAL-Cac_g019335, 4 CL-Cac_g007092) and are expressed in enrichment at the lower end of the stem, immature bark, root; cac_g004916 and Cac_g004924 are low expressed in the lower stem, young flowers and fruits and clustered with ASA2-Cac_g018456 and CHS-Cac_g019808 involved in flavone metabolism. While in the warp MeJa, PEG, agNO 3 In the induced camptotheca seedlings, cac_g004919 and Cac_g004921 are clustered with C4H-Cac_g002001 and PAL-Cac_g019328, and the expression level is down-regulated after MeJa and PEG induction treatment. Cac_g004916 aggregates with genes involved in FLA anabolism (CHI-Cac_g 004852 and PAL-Cac_g 019335), CPT anabolism (GOR-Cac_g 027560, LAMT-Cac_g005179, TDC2-Cac_g023139 and STR 2-Cac_g030435), their expression levels are expressed by AgNO 3 Up-regulation after induction treatment. Cac_g004924 aggregates with genes involved in the CPT upstream synthesis pathway (TSB-Cac_g 001032, GPPS-Cac_026508 and IPI1-Cacg 008847), genes involved in FLA anabolism (4 CL-Cac_g 008496), which are up-regulated in expression after MeJa induction treatment.
To further verify the association of the target CYP71 gene with genes involved in CPT and FLA anabolism at the protein level, abundance data of the target protein was extracted from the camptothecin quantitative dataset and expression cluster analysis was performed. Cac_g004916 is aggregated with TDC1-Cac_g018974, 7DLH-Cac_g017137 and enriched in stems, flowers and fruits; cac_g004919 and Cac_g004921 aggregate with IO-Cac_g032639, ASA1_Cac_g008782, and are enriched in stems and seedlings. Cac_g004924 was not detected at the protein level.
In summary, cac_g004916 is highly associated with the expression patterns of genes and proteins involved in CPT downstream biosynthesis, genes and proteins involved in FLA synthesis; cac_g004919 and Cac_g004921 are associated with genes involved in FLA synthesis and protein expression; cac_g004924 is highly associated with genes and proteins involved in the synthesis pathway upstream of CPT, genes and protein expression involved in FLA synthesis. In AgNO passage 3 And the expression level of Cac_g004916 and Cac_g004924 and CPT content in the seedlings of camptotheca acuminata after the MeJa induction treatmentThe amounts are positively correlated; in camptotheca seedlings after PEG and MeJa induction treatment, the expression levels of Cac_g004919 and Cac_g004921 were inversely related to CPT content. Thus, cac_g004916, cac_g004919, cac_g004921 and Cac_g004924 were subsequently selected as target CYP71 genes for functional characterization. They are formally designated CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE208, respectively.
EXAMPLE 4 construction and verification of the recombinant expression vector for CYP71BE of interest
After the gene sequences of CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE208 are subjected to codon optimization, a pYES2/CT-CYP71BE expression vector is constructed. Codon optimization and full sequence synthesis of the target CYP71 gene are completed by Shanghai workers. The Kpn I and Not I sites are used for inserting the target CYP71BE gene into the pYES2/CT plasmid, so that recombinant plasmids pYES2/CT-CYP71BE206, pYES2/CT-CYP71BE131, pYES2/CT-CYP71BE207 and pYES2/CT-CYP71BE208 are constructed.
The recombinant plasmid was amplified in E.coli TOP10 strain, cultured overnight at 37℃and then extracted using plasmid extraction kit I (Omega, georgia, united States), and the obtained plasmid was verified by PCR. PCR program settings: 95 ℃ for 10min; (95 ℃,30s;57 ℃,30s;72 ℃,1 min) 30 cycles; incubate at 12℃for 10 minutes. The PCR primers are shown in Table 2.
The result of plasmid enzyme digestion and PCR product electrophoresis is shown in figure 5, the target plasmid band is bright, the size of enzyme digestion product band is consistent with the length of target CYP71BE gene, the size of PCR product is consistent with the expectation, and 1074 bp, 1197 bp, 1494 bp and 1134bp respectively show that the construction of the target CYP71BE gene recombinant plasmid is successful. The positive plasmid is sequenced in Chengdu Optimu Biotechnology Co., ltd to verify the sequence accuracy, and the sequencing result shows that the target CYP71BE gene has no base mutation, and can BE used for the subsequent protein expression experiment.
TABLE 2 PCR primer sequences
Example 5 CYP71BE target protein expression and microsomal protein preparation
And selecting WAT11 saccharomyces cerevisiae culture solution in the logarithmic growth phase to prepare competent cells. The sequenced recombinant plasmids pYES2/CT-CYP71BE206, pYES2/CT-CYP71BE131, pYES2/CT-CYP71BE207, pYES2/CT-CYP71BE208 and pYES2/CT empty plasmid were transformed (1.5 kV) into WAT11 Saccharomyces cerevisiae competent strains, respectively, using a Micropulser 165-2100 electrotransport apparatus (Bio-Rad, california, united States), wherein the pYES2/CT empty plasmid served as a negative control. The obtained strain is subjected to shaking culture and recovery for 2 hours in a 1mL sterile YPDA liquid culture medium at 30 ℃ and 180 r/min; centrifuging at 3000r/min at room temperature for 5min, discarding supernatant, re-suspending and washing thallus with 1mL of sterile water, repeating for three times, adding 200 μl of sterile water to re-suspend thallus, coating SC-Ura auxotroph medium, and culturing at 30deg.C for 72h. Selecting a monoclonal antibody, heating the monoclonal antibody in a PCR instrument at 95 ℃ for 10-14 min, breaking cell walls, and then performing colony PCR, wherein the PCR program is as follows: 95 ℃ for 10min; (95 ℃,30s;57 ℃,30s;72 ℃,1 min) 30 cycles; preserving heat at 12 ℃. The PCR primers are shown in Table 2. The result of PCR product electrophoresis is shown in FIG. 6, which shows that 4 CYP71BE sequences of WAT11 recombinant Saccharomyces cerevisiae strains are successfully constructed.
The positive monoclonal is picked by a sterile toothpick and inoculated in 15mL SC-Ura liquid culture medium, and is cultured for 24 hours at 30 ℃ under 180r/min in a shaking way. Inoculating 1mL of the bacterial liquid into a fresh 50mL of SC-Ura liquid culture medium, shaking and culturing at 30 ℃ and 180r/min until the bacterial liquid grows to a flat stage, centrifuging at room temperature for 5min at 3000r/min, discarding the supernatant, and collecting thalli; then 50mL of sterile water is added to resuspend and wash the thalli, the thalli is centrifuged for 5min at room temperature, the supernatant is discarded, the thalli is collected and repeated three times, and the culture medium containing glucose on the surface of the thalli is removed. The washed cells were resuspended in 5mLYPGal liquid medium, transferred to 100mL YPGal liquid medium, induced and cultured at 30℃and 180r/min for 24h, centrifuged at 2500 r/min at 4℃for 5min, and the supernatant was discarded to collect the cells.
The cells (0.5 g cells/mL) were resuspended with pre-chilled TES buffer (50mM Tris,1mM EDTA,0.6M sorbitol,pH 7.4), sterile ceramic beads 0.5mmol/L in diameter were added to submerge the cells, and allowed to stand on ice for 5min. The three-dimensional freeze mill (Jixin, shanghai, china) is used for continuously crushing for 10 times at the oscillating speed of 20m/s for 30s and the cooling time of 1min, a proper amount of supernatant crushing liquid is taken after the mixture is sucked, blown and mixed uniformly, and an electron microscope (Bazhu, shanghai) is used for observing the crushing condition, as shown in figure 7, most cells are completely crushed, and the whole cell structure is not visible by naked eyes, so that the method can be used for extracting microsomal proteins. Diluting the supernatant with TES buffer solution for 2-3 times, centrifuging at 12000r/min at 4deg.C for 12min, collecting crushed supernatant, adding 1.5mol/LNaCl and solid PEG-4000 to a final concentration of 0.15mol/L and 0.1g/mL, precipitating microsomal protein on ice for 40min, centrifuging at 12000r/min at 4deg.C for 20min, discarding supernatant, weighing, re-suspending microsomal protein with TEG buffer solution (50mM Tris,1mM EDTA,20%glycerol) to a concentration of 40mg/mL, sub-packaging to 1 mL/tube, and storing at-80deg.C. Westernblot was examined for expression of the protein of interest as shown in FIG. 8.Westernblot results show that the target recombinant proteins are successfully expressed, the sizes of the bands are consistent with the sizes of the target bands (61770.28 Da, 62698.39Da, 64180.87Da and 62628.32 Da), and the negative control empty vector is free of bands.
Example 6 CYP71BE microsomal protein catalytic substrate screening and product identification
The enzyme activity test substrates of the microsomal proteins CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE208 were respectively prepared from isovinblastine lactam, genistein and isoliquiritigenin in 50mM standard stock solutions using DMSO. In a constant temperature incubator (Bo technology, hangzhou) in 200. Mu.L of Tris-HCl buffer (50 mM Tris, pH 7.4) containing 100. Mu. M, NADPH concentration of the reaction substrate and 8mg of microsomal protein, the reaction temperature was 30℃and the reaction time was 16 hours. After the reaction was completed, the reaction was terminated with 200. Mu.L of ethyl acetate and the product was extracted, centrifuged at 15000rpm for 3min, and the upper organic phase was transferred and dried with nitrogen, and the residue was fixed to 1mL with methanol. The sample was filtered through a 0.22 μm needle filter. Microsomal proteins prepared from strains transformed with pYES2/CT empty plasmid were used as negative controls.
The reaction products were detected using an Agilent G6125B LC-MS system. Chromatographic column SB-C18 column (2.1X105 mm,1.8 μm)) Mobile phase a was 0.1% formic acid and mobile phase B was acetonitrile. Gradient elution conditions: 0-4 min, 5-10% (phase B); 4-6 min, 10-20% (phase B); 6-9 min, 20-50% (phase B); 9-12 min, 50-50% (phase B); 12-13 min, 50-98% (phase B); 13 to 13.5min,98 to 5 percent (phase B); 13.5-15 min, 5-5% (phase B). The column temperature is 35 ℃, the flow rate is 0.20mL/min, and the sample injection amount is 5 mu L. The mass spectrometry conditions were: the ionization mode is API-ES, positive ion mode; capillary current was 10nA; the dry gas is N 2 The method comprises the steps of carrying out a first treatment on the surface of the The gas flow is 12.0L/h, and the gas temperature is 350 ℃; the source temperature was 100 ℃; scanning range is 50-1000Da; the collision energy was 10eV. Quantitative analysis was performed based on substrate consumption. The oxidation products were analyzed by MS2 using a Waters Xex G2-XS QTOF mass spectrometer (Waters, milford, united States), and product differentiation was based on diagnostic ion and feature group cleavage.
The Total Ion (TIC) and Extracted Ion (EIC) flow diagrams of the microsomal protease reaction solutions are shown in FIGS. 9-12, and the results show that genistein (m/z 271.2) is used as a test substrate, and no oxidation product of +16Da is detected in each reaction system; whereas +16Da oxidation products (m/z 273.2) were observed in each reaction system except for the negative control using isoliquiritigenin (m/z 257.2) as a test substrate; using strictosamide (m/z 499.5) as a test substrate, an oxidation product of +16Da was observed in the CYP71BE206 microsomal protein reaction system, whereas no oxidation product was detected in the CYP71BE131, CYP71BE207, CYP71BE208 microsomal protein reaction system.
To identify the structure of the oxidation product, MS/MS analysis was used to obtain the mass spectrum cleavage process. [ M+H ] of oxidation product of Isovirenin lactam (M/z 515.3629)]The +ion molecular formula is determined as C 26 H 30 N 2 O 9 Specific cleavage of-70 Da and-162 Da and one diagnostic ion (m/z 144.0815) were detected in the MS spectra, whereby the oxidation products were rapidly classified as vincosamide class. In the initial cleavage step, 18Da (-H) was detected 2 O) was compared with MS cleavage patterns of VG2 in mature camptotheca and seedlings and the +16Da oxidation product was identified as isovincoside lactam epoxide (strictosamide epoxide), thereby confirming the CYP71BE206 selectionSex-catalyzed C-2 and C-7 epoxidation of strictosamide. [ M+H ] of isoliquiritigenin oxidation products] + Ion formula (m/z 273.2535) was determined to be C 15 H 13 O 5 By cleavage of-42 Da (-C) 2 H 2 O)、-42Da(-C 2 H 2 O) and-16 Da (-O) to yield 3 intermediate ions (m/z 231.1654, 189.0687, 173.0556), indicating the presence of multiple hydroxyl groups on the benzene ring. Cleavage at-16 Da (-O) was detected at the initial cleavage step and a rearranged ion fragment of m/z 257.2472 was generated. We also detected the same diagnostic ions in the isoliquiritigenin secondary mass spectrum 1,3 A + (m/z 137.8835). These results indicate that there is one epoxy group on its unsaturated double bond, two hydroxyl groups on the A ring and one hydroxyl group on the B ring. Thus, the oxidation product thereof was identified as isoliquiritigenin epoxide. The detailed cleavage process for both epoxidation products is shown in figure 13.
Example 7 optimization of reaction conditions and kinetic characterization of CYP71BE microsomal proteins
The enzyme activity test substrates of the microsomal proteins CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE208 were respectively prepared from isoliquiritigenin lactam and isoliquiritigenin in 50mM standard stock solutions by DMSO. The reaction was carried out in a constant temperature incubator (Bo technology, hangzhou) in 200. Mu.L of Tris-HCl buffer (50 mM Tris, pH 7.4) containing the reaction substrate, NADPH and 8mg of microsomal proteins in the total reaction system, the reaction was terminated with 200. Mu.L of ethyl acetate after the completion of the reaction and the product was extracted, centrifuged at 15000rpm for 3min, the upper organic phase was transferred and dried with nitrogen, the residue was fixed to 1mL with methanol, and the sample was filtered with a 0.22 μm needle filter. The optimal reaction conditions of the CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE208 microsomal proteins, including the induction time (2-24 h), the reaction temperature (20-40 ℃), the reaction time (0.5-8 h), the pH (6.0-8.0), the NADPH concentration (0. Mu.M-1000. Mu.M) and the reaction substrate concentration (100-1000. Mu.M) were determined through a single-factor experiment, and the optimization process of the reaction conditions is shown in FIGS. 14-17, respectively. The optimal reaction conditions for the final determination of the CYP71BE206 microsomal protein were: induction time, 4h; the reaction temperature is 35 ℃; pH 7.0; NADPH concentration, 200. Mu.M; reaction substrate concentration, 400. Mu.M. However, the optimal reaction time for the two substrates of isoliquiritigenin lactam and isoliquiritigenin is different, the isoliquiritigenin is 4 hours, and the isoliquiritigenin lactam is 2 hours. In addition, the optimal reaction conditions of CYP71BE131, CYP71BE207 and CYP71BE208 microsomal proteins for the substrate isoliquiritigenin are the same as those of CYP71BE 206. To explore the catalytic performance of the four epoxidases, their catalytic kinetics were evaluated under optimal reaction conditions.
The reaction was performed at 35℃in 200. Mu. LTris-HCl buffer containing a range of substrate concentrations (100-1000. Mu.M), 200. Mu.M NADPH and 8mg microsomal proteins for a reaction time ranging from 10-60min, the reaction rate was calculated from the quantitative results, the rate-substrate curve was constructed from Michaelis-Menten equation, km and Kcat were calculated to evaluate the enzyme properties, the mean.+ -. Standard Error (SE) of the single factor experiment and kinetic results was calculated using Excel software (2019 version), and statistical significance analysis was performed to obtain the catalytic kinetic parameters, see Table 3.
As can be seen from fig. 18: for CYP71BE206 microsomal proteins, its Km for the substrate isoliquiritigenin (140.67. Mu.M) was lower than that for the substrate isoliquiritigenin lactam (301.16. Mu.M), while its Kcat value for isoliquiritigenin lactam was higher than that of isoliquiritigenin, indicating that CYP71BE206 exhibits better affinity for isoliquiritigenin but higher catalytic efficiency for isoliquiritigenin lactam. The catalytic efficiency of CYP71BE131, CYP71BE207 and CYP71BE208 on the substrate isoliquiritigenin is superior to that of CYP71BE206, wherein the catalytic efficiency of CYP71BE207 on isoliquiritigenin is the highest. However, the affinity of CYP71BE131 and CYP71BE208 for isoliquiritigenin is superior to that of CYP71BE206 and CYP71BE207. These results indicate that although CYP71BE206 reacts more rapidly to isovinblastine than isoliquiritigenin, isoliquiritigenin is more preferred as a natural substrate for CYP71BE206 because of its greater affinity. Four microsomal proteins showed similar catalytic ability to isoliquiritigenin, but CYP71BE131 and CYP71BE208 showed stronger substrate affinity to isoliquiritigenin. Therefore, all four CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE208 microsomal proteins can BE used for the preparation of isoliquiritigenin epoxide, wherein the CYP71BE206 microsomal proteins can also BE used for the preparation of isoliquiritigenin lactam epoxide.
TABLE 3 catalytic kinetic parameters of four CYP71BE microsomal proteins
According to the embodiment, screening and optimization of CYP71BE genes in camptotheca acuminata are carried out by combining genome, transcriptome and proteome multiple sets of data, a protein abundance data set is constructed, the CYP71BE genes in camptotheca acuminata are further dug out, the CYP71BE cyclooxygenase gene sequence, the corresponding protein sequence coded by the genes, the recombinant plasmid of the genes and WAT11 saccharomyces cerevisiae host cells containing the recombinant expression plasmid are obtained, four CYP71BE206, CYP71BE131, CYP71BE207 and CYP71BE20 are simultaneously induced and expressed by the host cells, and further verification is carried out, so that the four microsomal proteins can BE used for selective epoxidation of isoliquiritigenin, and meanwhile, the CYP71BE206 can BE also used for 2-7-bit epoxidation of isoliquiritigenin lactam.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

1. The CYP71BE epoxidase gene in camptotheca acuminata is characterized by comprising a CYP71BE206 gene, a CYP71BE207 gene or a CYP71BE208 gene;
the nucleotide sequence of the CYP71BE206 gene is shown in SEQ ID NO. 1;
the nucleotide sequence of the CYP71BE207 gene is shown in SEQ ID NO. 2;
the nucleotide sequence of the CYP71BE208 gene is shown in SEQ ID NO. 3.
2. The protein encoded by the CYP71BE cyclooxygenase gene of claim 1, comprising a CYP71BE206 protein, a CYP71BE207 protein, or a CYP71BE208 protein;
the amino acid sequence of the CYP71BE206 protein is shown in SEQ ID NO. 5;
the amino acid sequence of the CYP71BE207 protein is shown in SEQ ID NO. 6;
the amino acid sequence of the CYP71BE208 protein is shown in SEQ ID NO. 7.
3. A recombinant expression plasmid comprising the CYP71BE cyclooxygenase gene of claim 1.
4. A recombinant expression plasmid according to claim 3, characterized in that the original plasmid used is a pYES2/CT plasmid.
5. The recombinant expression plasmid of claim 4, wherein the CYP71BE cyclooxygenase gene is inserted at the Kpn I site and Not I site of the original plasmid.
6. A recombinant strain expressing the CYP71BE cyclooxygenase gene of claim 1, wherein said recombinant strain is transformed with the recombinant expression plasmid of claim 3 or 4 using WAT11 saccharomyces cerevisiae as host bacteria.
Use of a protein encoded by a CYP71BE cyclooxygenase gene as a biocatalyst for epoxidation, characterized in that the protein encoded by the CYP71BE cyclooxygenase gene comprises the protein of claim 2 or a CYP71BE131 protein; the amino acid sequence of the CYP71BE131 protein is shown in SEQ ID NO. 8;
the CYP71BE epoxidase gene-encoded protein is used for selective epoxidation of isoliquiritigenin, and the CYP71BE206 protein is used for 2-7-bit epoxidation of isoliquiritigenin lactam.
8. The use according to claim 7, wherein the specific application process of the protein encoded by the CYP71BE cyclooxygenase gene is: the reaction is carried out in a reaction system containing a reaction substrate, NADPH, protein coded by the CYP71BE epoxidase gene and Tris-HCl buffer solution, and then the reaction system is extracted and filtered.
9. The use according to claim 8, wherein the reaction substrate is isoliquiritigenin or isoliquiritigenin; the reaction time is 0.5-8h, the reaction temperature is 20-40 ℃, and the reaction pH is 6-8.
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