Cytochrome P450 enzyme mutant and its application in preparing emamectin benzoate intermediate

Abstract

The present invention belongs to the field of synthetic biology, and specifically relates to a cytochrome P450 enzyme mutant and its application in the preparation of emamectin benzoate intermediates. The cytochrome P450 oxidase mutant is obtained by single-point or multi-point mutations at the 119th and 124th positions of the amino acid sequence shown in SEQ ID NO.1. The present invention rationally modifies the key points of the cytochrome P450 oxidase from Streptomyces tubercidicus , and the conversion rate of the screened mutant is further improved. Using the cytochrome P450 oxidase mutant provided by the present invention, emamectin can be used as a substrate to directly prepare C4"-carbonyl-C5"-hydroxy-emamectin in the presence of glucose dehydrogenase, glucose and cofactors. This method has mild reaction conditions and low catalyst cost, and is an ideal scheme for the preparation of C4"-carbonyl-C5"-hydroxy-emamectin.

Description

Technical Field

[0001] The present invention belongs to the field of protein engineering, and particularly relates to a cytochrome P450 enzyme mutant and its application in the preparation of emamectin benzoate intermediate. Background Art

[0002] Avermectin (abbreviated as AVM) is a fermentation product produced by Streptomyces avermitilis isolated from soil, and has insecticidal and anthelmintic properties. Its structure is a series of 16-membered macrolides. Due to the broad-spectrum, high efficiency, low toxicity, high selectivity and environmental friendliness of avermectin in controlling insect pests on various crops such as rice, cotton, fruit trees and tobacco, it has become the mainstream agent in the market. However, there are also many problems with avermectin. For example, the insect resistance to avermectin increases year by year, and a large amount of pesticides with weak solubility will leach out and be adsorbed on the soil surface. Therefore, taking avermectin as the parent body, searching for derivatives with better activity and higher safety has become a current research hotspot.

[0003] Emamectin benzoate is a derivative of the biological fermentation product avermectin. Compared with the parent body, its biological activity has increased by 1 to 3 orders of magnitude. Since emamectin benzoate belongs to a biogenic pesticide, it is easily degraded, has little residue, and has extremely little pollution to the environment. It is a green and environment-friendly pesticide, and has a wider insecticidal spectrum, such as Helicoverpa armigera and Spodoptera exigua, especially being particularly effective against Lepidoptera pests.

[0004] Emamectin benzoate is prepared by salifying abamectin methylamino with benzoic acid. And abamectin methylamino is obtained by modifying the C4-OH, and a total of six steps of protection, oxidation, amination, reduction, deprotection and salification are required. However, there is a more active C5-OH in avermectin, so its protection and deprotection are required, which greatly increases the raw material cost, and a large amount of by-products will be discharged in the production process. At the same time, the product yield is low, the purity is general, and the product appearance is not ideal.

[0005] Biocatalysis (also known as biotransformation, bioconversion) refers to using exogenous natural or synthetic organic compounds as substrates, adding them to an active biological system or enzyme system, culturing under suitable conditions, so that the substrates interact with the enzymes in the system, resulting in structural changes. Its essence is an enzymatic reaction. Biocatalysis has the characteristics of mild reaction conditions, high efficiency and high selectivity (chemical, regioselective and stereoselective), and is considered a resource-saving and environment-friendly technology, which is an important supplement to chemical synthesis. Seeking cheap, efficient and environmentally friendly biocatalysts to achieve site-specific oxidation of the C4"-O of avermectin will greatly simplify the synthesis steps of emamectin benzoate and contribute to the popularization and application of emamectin benzoate. Summary of the Invention

[0006] In view of the need for a biocatalyst with high efficiency and specificity for catalyzing the C4-OH of avermectin in the prior art, the present invention provides a cytochrome P450 enzyme mutant and its application in the preparation of emamectin benzoate intermediate 4"-carbonyl-5"-hydroxy-avermectin. The specific technical solutions are as follows:

[0007] In a first aspect, the present invention provides a cytochrome P450 oxidase mutant, which is obtained by single-point or multi-point mutation at the 119th and 124th positions of the amino acid sequence shown in SEQ ID NO.1.

[0008] Further, the cytochrome P450 oxidase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 in one of the following mutation forms:

[0009] (1) Leucine at the 119th position is mutated to alanine or phenylalanine;

[0010] (2) Asparagine at the 124th position is mutated to alanine, proline or tryptophan;

[0011] (3) Leucine at the 119th position is mutated to phenylalanine, and asparagine at the 124th position is mutated to proline or tryptophan.

[0012] In a second aspect, the present invention provides a gene encoding the above-mentioned cytochrome P450 oxidase mutant.

[0013] In a third aspect, the present invention provides a recombinant vector, which contains the above-mentioned encoding gene.

[0014] In a fourth aspect, the present invention provides a genetically engineered bacterium, which contains the above-mentioned encoding gene.

[0015] Further, the genetically engineered bacterium uses Escherichia coli BL21 (DE3) as the host bacterium.

[0016] Even further, the genetically engineered bacterium can express the spinach electron transfer system.

[0017] Even further, the spinach electron transfer system includes ferredoxin and ferredoxin reductase.

[0018] In a fifth aspect, the present invention provides the application of the above-mentioned cytochrome P450 oxidase mutant, or the above-mentioned recombinant vector, or the above-mentioned genetically engineered bacterium in catalyzing the synthesis of emamectin benzoate intermediate 4"-carbonyl-5"-hydroxy-avermectin from avermectin B1a.

[0019] Sixth aspect, the present invention provides a method for producing 4"-carbonyl-5"-hydroxy-avermectin. Using avermectin B1a as a substrate and the above cytochrome P450 oxidase mutant as a catalyst to form a reaction system for synthesizing 4"-carbonyl-5"-hydroxy-avermectin.

[0020] Furthermore, the catalyst can be wet cells obtained by fermenting and culturing the above genetic engineering bacteria or a crude enzyme solution obtained by ultrasonically disrupting the wet cells.

[0021] Furthermore, the reaction temperature is 25 - 30 °C.

[0022] Even further, the reaction temperature is 30 °C.

[0023] Furthermore, the reaction time is 24 - 72 h.

[0024] Even further, the reaction time is 72 h.

[0025] Furthermore, in the reaction system, the substrate is dissolved in a Tween 40 / DMSO mixture and then mixed with the catalyst for reaction; the volume ratio of Tween 40 to DMSO in the Tween 40 / DMSO mixture is 1:1.

[0026] Furthermore, in the reaction system, the cytochrome P450 oxidase mutant is dispersed in a buffer solution.

[0027] Even further, the pH of the buffer solution is 7.4 - 8.4.

[0028] Even further, the buffer solution is potassium phosphate buffer, phosphate buffer or Tris-HCl buffer.

[0029] Furthermore, in the reaction system, a coenzyme recycling system is constructed with glucose dehydrogenase (GDH), NADH as coenzymes and glucose as a cosubstrate.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention rationally modifies the key points of cytochrome P450 oxidase from Streptomycestubercidicus and the conversion rate of the screened mutant is further improved. Using the cytochrome P450 oxidase mutant provided by the present invention, C4"-carbonyl-C5"-hydroxy-avermectin can be directly prepared with avermectin as a substrate in the presence of glucose dehydrogenase, glucose and cofactors. This method has mild reaction conditions and low catalyst cost, and is an ideal scheme for preparing C4"-carbonyl-C5"-hydroxy-avermectin. Description of the Drawings

[0032] Figure 1 It is a schematic structural diagram of avermectin.

[0033] Figure 2 It is a schematic reaction formula diagram for preparing C4"-carbonyl-C5"-hydroxy-avermectin from avermectin B1a.

[0034] Figure 3 It is a standard curve graph of C4"-carbonyl-C5"-hydroxy-avermectin.

[0035] Figure 4 It is an HPLC detection spectrum; among them, the retention time of 26.604 min is for avermectin B1a, and 30.823 min is for C4"-carbonyl-C5"-hydroxy-avermectin. Detailed implementation manners

[0036] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and only represents a part of the embodiments of the present invention, rather than all of the embodiments.

[0037] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the scope of protection of the present invention.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels. The experimental methods without specifying detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

[0039] In the following embodiments, the structural formula of avermectin is as Figure 1 shown, and the present invention uses avermectin B1a.

[0040] In the following embodiments, the reaction formula for preparing C4"-carbonyl-C5"-hydroxy-avermectin from avermectin B1a is as Figure 2 shown.

[0041] The culture medium, required antibiotics, inducers, etc. configured in the embodiments of the present invention are composed of:

[0042] LB liquid medium: 5 g·L -1 yeast extract, 10 g·L -1 peptone, 10 g·L -1 sodium chloride.

[0043] LB solid medium: 5 g·L -1Yeast powder, 10 g·L -1 Peptone, 10 g·L -1 Sodium chloride, 3 g·L -1 Agar powder.

[0044] TB liquid medium: 12 g·L -1 Peptone, 12 g·L -1 Yeast powder, 12.5 g·L -1 Dipotassium hydrogen phosphate, 2.3 g·L -1 Potassium dihydrogen phosphate, 4 mL·L -1 Glycerol.

[0045] The above media were dissolved in water and sterilized at 121 °C under a pressure of 0.105 MPa for 20 min.

[0046] 100 mg / mL Ampicillin: Weigh 5 g of sodium ampicillin and dissolve it in 50 mL of sterile water. After complete dissolution, filter it through a 0.22-μm filter membrane, dispense it into ep tubes, and store it at -20 °C.

[0047] 50 mg / mL Kanamycin sulfate: Weigh 2.5 g of kanamycin sulfate and dissolve it in 50 mL of sterile water. After complete dissolution, filter it through a 0.22-μm filter membrane, dispense it into ep tubes, and store it at -20 °C.

[0048] 120 mg / mL IPTG: Weigh 6 g of IPTG and dissolve it in 50 mL of sterile water. After complete dissolution, filter it through a 0.22-μm filter membrane, dispense it into ep tubes, and store it at -20 °C.

[0049] 84 mg / mL ALA: Weigh 4 g of ALA and dissolve it in 50 mL of sterile water. After complete dissolution, filter it through a 0.22-μm filter membrane, dispense it into ep tubes, and store it at -20 °C.

[0050] 45 mg / mL FeSO4: Weigh 1.2 g of FeSO4 and dissolve it in 50 mL of sterile water. After complete dissolution, filter it through a 0.22-μm filter membrane, dispense it into ep tubes, and store it at -20 °C.

[0051] The test materials and test instruments used in the examples of the present invention:

[0052] 1. Test materials

[0053] E. coli with plasmid pRSF-Duet carrying the CYP450 enzyme target gene E.coli BL21(DE3) strain, with plasmid pET-Duet carrying the ferredoxin and ferredoxin reductase target genes E.coliThe BL21(DE3) strain was purchased from Beijing Tsingke Biotechnology Co., Ltd.; E.coli DH5α and E.coli The BL21(DE3) competent cells were prepared and stored in our laboratory; The SurePAGE™ precast protein gel and protein marker were purchased from GenScript Biotech Corporation in China.

[0054] Dipotassium hydrogen phosphate anhydrous, potassium dihydrogen phosphate anhydrous, methanol for chromatography, acetonitrile for chromatography were purchased from Sinopharm Chemical Reagent Co., Ltd.; Yeast extract, peptone were purchased from Thermo Fisher Scientific Inc.; Tris base, concentrated hydrochloric acid, sodium dodecyl sulfate, bromophenol blue, glycerol, 2-mercaptoethanol, absolute ethanol, glacial acetic acid, Coomassie brilliant blue, phosphoric acid, glycine, 20% SDS were purchased from Saiguo Biotechnology Co., Ltd.

[0055] Kanamycin was purchased from Shanghai Merck Chemical Technology Co., Ltd.; Ampicillin, NADH, NADPH were purchased from Shanghai Titan Scientific Co., Ltd.; 5-aminolevulinic acid (ALA), ferrous sulfate were purchased from Tianjin Damao Chemical Reagent Factory; Isopropyl β-D-thiogalactopyranoside (IPTG) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Dimethyl sulfoxide, Tween 40 were purchased from Shanghai Macklin Biochemical Co., Ltd.

[0056] Nucleic acid Marker, nucleic acid dye were purchased from Tsingke Biotechnology Co., Ltd.

[0057] Preparation of competent cells: Obtain the glycerol stock of E.coli the BL21(DE3) strain from the -80 °C refrigerator, streak it on an antibiotic-free LB plate, and culture it at 37 °C for 10 h to obtain single colonies; Pick a single colony from the LB plate and inoculate it into a test tube containing 10 mL of LB medium, culture it at 37 °C and 180 rpm for 9 h; Take 2 mL of the bacterial solution from the test tube and inoculate it into 100 mL of LB medium, culture it at 37 °C and 180 rpm until the OD600 reaches 0.4 - 0.6; Pre-cool the bacterial solution on ice, transfer the bacterial solution to a sterilized centrifuge tube, place it on ice for 10 min, centrifuge at 4 °C and 5000 rpm for 10 min; Pour out the supernatant, resuspend the precipitated cells with 0.1 mol / L CaCl2 aqueous solution pre-cooled at 4 °C, and place it on ice for 30 min; Centrifuge at 4 °C and 5000 rpm for 10 min, discard the supernatant, resuspend the precipitated cells with 0.1 mol / L CaCl2 aqueous solution containing 15% glycerol pre-cooled at 4 °C, take 100 μL of the resuspended cells and aliquot them into sterilized 1.5 mL centrifuge tubes, store them in the -80 °C refrigerator, and take them out when needed.

[0058] 2. Test instruments

[0059] Table 1 Test instruments

[0060]

[0061] In the following examples, the method for determining the conversion activity of cytochrome P450 oxidase to catalyze abamectin B1a is as follows:

[0062] The assay system is as follows: The total reaction system is 1 mL, including 450 μL of potassium phosphate buffer, 200 μL of 10 mM NAD(P)H, 50 μL of 1 M glucose, 2 g / L GDH, 50 μL of 20 mM abamectin B1a dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), and 200 uL of the supernatant obtained by centrifuging the disrupted wet cells. The reaction is carried out at 30 °C and 800 rpm for 72 h. The reaction solution is extracted with ethyl acetate, and the extract is dried in a fume hood and then redissolved with pure acetonitrile and made up to 1 mL; then it is filtered through a 0.22 μm microporous nylon membrane to obtain the test sample solution, and the content is calculated according to the standard curve.

[0063] In the following examples, the method for determining the standard curve of 4''-carbonyl-5''-hydroxy-abamectin is as follows:

[0064] Prepare the 4''-carbonyl-5''-hydroxy-abamectin standard solution: Weigh 0.87 g and dissolve it in 60 mL of 50% acetonitrile (acetonitrile:water = 1:1). After dissolving in a clean beaker, transfer it to a volumetric flask. Rinse the beaker three times with 20 mL of 50% acetonitrile and transfer all to the volumetric flask with a glass rod, and then make up to 100 mL. Cover the lid and shake well to obtain a 10 mM 4''-carbonyl-5''-hydroxy-abamectin standard sample solution; then use the standard solution to prepare 1 mM, 0.8 mM, 0.5 mM, and 0.1 mM 4''-carbonyl-5''-hydroxy-abamectin sample solutions for making the standard curve. The standard curve of 4''-carbonyl-5''-hydroxy-abamectin is as Figure 3 shown.

[0065] In the following examples, the method for detecting the content of 4''-carbonyl-5''-hydroxy-abamectin is as follows:

[0066] Detection was performed using HPLC under the following conditions: Thermo Fisher U3000 liquid chromatograph; C18 chromatographic column (4.6 mm×250 mm); column temperature: 30 °C; mobile phase acetonitrile: water (0 - 30 min, 50% acetonitrile - 100% acetonitrile; 30 - 40 min, 100% acetonitrile - 50% acetonitrile); flow rate: 1.2 mL / min; detection wavelength: 243 nm; injection volume: 5 μL. The peak area of the test sample solution at the retention time corresponding to the peak of 4″-keto-5″-hydroxy-avermectin in the standard sample solution was measured, and then the concentration of 4″-keto-5″-hydroxy-avermectin produced by catalysis was calculated by substituting it into the standard curve of 4″-keto-5″-hydroxy-avermectin. The HPLC chromatograms of avermectin B1a and C4″-keto-C5″-hydroxy-avermectin are as Figure 4 shown, where the retention time of avermectin B1a is 26.604 min and that of C4″-keto-C5″-hydroxy-avermectin is 30.823 min.

[0067] In the following examples, the conversion rate was calculated as: (initial molar concentration of substrate - remaining molar concentration of substrate) / initial molar concentration of substrate.

[0068] In the following examples, the yield was calculated as: molar concentration of product formed / initial molar concentration of substrate.

[0069] In the following examples, the electron transfer system of spinach refers to a set of systems for electron transfer existing in the plant spinach. It plays an important role in physiological processes such as photosynthesis and respiration of spinach. Specifically, through this system, electrons can be transferred from one molecule to another, thereby achieving energy conversion and material metabolism.

[0070] In the following examples, the amino acid sequence of cytochrome P450 oxidase Em1 is shown in SEQ ID NO.1, the amino acid sequence of cytochrome P450 oxidase Em18 is shown in SEQ ID NO.2, and the amino acid sequence of cytochrome P450 oxidase Emv5b is shown in SEQ ID NO.3.

[0071] SEQ ID NO.1:

[0072] MGSSHHHHHHMSELMNSPFAAHVGKHPGEPNVMDPALITDPFTGYGALREQGPVVRGRFMDDSPVWLVTRFEEVRQVLRDQRFVNNPASPSLNYAPEDNPLTRLMEMLGLPEHLRVYLLGSILNYDAPDHTRLRLSDVEMVTLVLTLVLAGHQTTAHLARLPEHAEDGVVDLIQHFAYPLPITVICELVGIPEADRPQWRTWGADLISMDPDRLGASFPAMIEHIHQMVRERREALTDDLLSELIRTHDDDGGRLSDVEMVTMILTLVLAGHETTAHLISNGTAALLTHPDQLRLVKDDPALLPRAVHELMRWCGPVHMTQLRYATADVDLAGTPIRQGDAVQLILVSANFDPRHYTDPDRLDLTRHPAGHAENHVGFGHGAHYCLGATLAKQEGEVAFGKLLTHYPDISLGIAPEHLERTPLPGNWRLNSLPVRLG

[0073] SEQ ID NO.2:

[0074] MGSSHHHHHHMTELTDSPFSEFVGKHPGEPNVMEPALLTDPFAGYGALREQGPVVRGRFVDDTPVWFITRFEEAREVLRDQRFANSPAHSAGGGSADTPIDRLLEIMGLPEHYRAYLSGTILNMDAPDHTRLRRLVSRAFTARKITDLRPRVADIAEDALRRLPEHAVDGVVDLIPHFAYPLPITVICELVGIPEADRPQWREWSTHLVSLRRLVSRAFTARKITDLRPRVEQIADALLTDDLLSELIRVHDDDGSRLSDVEMVTLVLTLVLAGHETTAHLITNGVAALLTHPDQLQLLKSEPALLPRAVHELMRWCGPVHLTQMRYATEDVELAGVRIKKGEAVTPVLVAANHDPRHFADPDRLDLTRQPAGRAENHVGFGHGMHYCLGATLARQEAEVAFGKLLAHYPDVALAVAPEDLQRVPLPGSWRLASLPLRLN

[0075] SEQ ID NO.3:

[0076] MGSSHHHHHHMSASPSNTFTEHVGKHPGEPNVMDPALIGDPFAGYGALREQGPVVRGRFMDDSPVWFVTRFEEVREVLRDPRFVNNPAAPSLGRSIDESPTARLLEMMGLPEHFRPYLLGSILTNDAPDHTRLRRLVSRAFTARKITDLRPRVAQITAELLDRLPEHAEDGVVDLIEHFAYPLPITVICELVGIAAEDRPQWRTWGADLVSLQPDRMSRSFPAMIEHIHELIRERRGALTDDLLSELIRTHDDDGSRPELHPETFPEMIDHIHALIRERRTALIGNGTAALLTHPDQLRLLKDDPALLPRAVHELMRWCGPVHMTQLRYATADVDLAGTPIRQGDAVQLILVSANFDPRHYTDPDRLDLTRHPAGHAENHVGFGHGAHYCLGATLAKQEGEVAFEKLFAHYPEVSLAVPPDHLERTPVPGMWGLNSLPVRLG。

[0077] Example 1 Screening of wild-type cytochrome P450 oxidase

[0078] Through literature retrieval and multiple sequence alignment, the sources of cytochrome P450 enzymes (CYP450) and related information on gene sequences were consulted; the gene sequences or amino acid sequences of cytochrome P450 oxidases from different sources were queried through the gene database (https: / / www.ncbi.nlm.nih.gov / genome / ), and 3 cytochrome P450 oxidases were screened out Streptomycestubercidicus from: Em1, Em18, and Em5b. After optimization according to the codon preference of Escherichia coli and introduction of NcoI restriction site, HidIII restriction site, His tag, and terminator into the sequences published in GenBank, and rational modification of the key points of cytochrome P450 oxidase, they were synthesized by Hangzhou Qingke Biotechnology Co., Ltd. to obtain wild-type cytochrome P450 oxidase genes (the amino acid sequences are shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively).

[0079] Example 2 Construction and expression of recombinant engineering bacteria of wild-type cytochrome P450 oxidase

[0080] The three wild-type cytochrome P450 oxidase genes obtained in Example 1 ( Em1 , Em18 , Em5b ) were ligated with plasmid pRSF-Duet, and the spinach electron transfer system was ligated with plasmid pET-Duet to construct recombinant plasmids. Then the two recombinant plasmids were transformed into E.coli BL21(DE3) competent cells to construct wild-type recombinant engineering bacteria. The transformation steps were as follows: Place the Escherichia coli BL21(DE3) competent cells on ice, add 1 μL of each of the two recombinant plasmids respectively, place on ice for 30 min, heat shock in a 42 °C water bath for 45 s, incubate on ice for 4 min, add LB liquid medium without antibiotics, culture at 37 °C for 45 - 60 min, then centrifuge at 4500 rpm for 5 min, and discard 600 μL of the supernatant. Gently pipette and mix the remaining bacterial cells, aspirate and spread them on a plate with the correct resistance, pour 5 - 10 glass beads onto the plate and gently shake until there is no bacterial liquid on the plate, pour the glass beads into pure alcohol, and place the plate in an inverted position in a 37 °C incubator for 12 - 16 h. After colonies grow on the plate after overnight culture, pick a single colony and inoculate it into LB liquid medium containing 50 mg / mL sodium ampicillin and 50 mg / mL kanamycin, culture at 180 rpm and 37 °C for 8 - 12 h, and send it to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. Use software to compare and identify the successfully sequenced wild-type recombinant engineering bacteria E.coli BL21(DE3)-pRSF-Duet-Em1, E.coli BL21(DE3)-pRSF-Duet-Em18, E.coli BL21(DE3)-pRSF-Duet-Em5b for glycerol preservation tubes.

[0081] Inoculate the successfully constructed engineering bacteria into LB liquid medium, place them in a 37 °C shaker, and shake and culture at 180 rpm for 2 - 3 h. When the cell density OD600 value reaches 0.6, add IPTG and then transfer the shake flask to a 28 °C shaker and continue to culture at 180 rpm for 12 h. After the culture is completed, centrifuge the culture solution at 8000 rpm for 10 min, discard the supernatant, and collect the bacterial cells.

[0082] Recombinant engineering bacteria E.coli BL21(DE3)-pRSF-Duet-Em1, E.coli BL21(DE3)-pRSF-Duet-Em18, E.coliThe yields of BL21(DE3)-pRSF-Duet-Em5b catalyzing 1 mM substrate were 3%, 1.3%, and 0.8%, respectively. Among them, Em1 had a higher conversion rate, and none of the three enzymes had a clear crystal structure reported, so Em1 with a better conversion rate was selected for subsequent experiments.

[0083] Experimental Example 3 Screening of cytochrome P450 oxidase mutants and construction of engineered bacteria

[0084] 1. Acquisition of cytochrome P450 oxidase template gene

[0085] Will reorganize E.coli BL21(DE3)-pRSF-Duet-Em1 engineered bacteria were streaked and inoculated in a solution containing 50 mg·L -1 The culture was carried out in LB solid medium containing kanamycin at 37 °C for 12 h. A single colony was picked and inoculated into a 50 mg·L -1 In LB liquid medium containing kanamycin, culture at 180 rpm / min and 37°C for 8-12 h. Extract the recombinant engineering bacterial plasmid according to the kit.

[0086] 2. Site-directed mutagenesis of cytochrome P450 oxidase genes

[0087] According to the Genbank Streptomycestubercidicus The wild-type cytochrome P450 oxidase Em1 gene sequence was used to design mutation primers for site-directed mutagenesis. The plasmid extracted in step 1 of this example was used as the original template for site-directed mutagenesis using rapid PCR technology. The primers in Table 2 were used for PCR amplification. After completion, the bands were compared by nucleic acid gel electrophoresis. The correct PCR product was added with 1.5 uL Dpn1 and digested in a shaker at 37°C for more than 2 h.

[0088] First round of mutation: The amino acid residues within 6 angstroms around the substrate avermectin were mutated to alanine.

[0089] PCR reaction system (total reaction system is 50 μL): 2×Phanta max Buffer 25 μL, 10 mM dNTPmixture (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1 μL, Phanta Max Super-FIDelity DNA Polymerase 0.5 μL, 2 μL each of upstream and downstream primers with a concentration of 50 μM (as shown in Table 2), 1 μL of the recombinant vector pRSF-Duet-Em1, and 18.5 μL of ddH2O.

[0090] PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 5 min, a total of 30 cycles, and finally extension at 72°C for 5 min.

[0091] Take 10 μL of the pcr product and add it to 100 μL of Escherichia coli BL21(DE3) competent cells. Place it on ice for 30 min, heat shock it in a 42°C water bath for 45 s, incubate it on ice for 4 min, add 600 μL of LB liquid medium, culture it at 37°C for 45 - 60 min, centrifuge and discard 600 μL of the supernatant, and then spread it on an LB solid medium plate containing 50 mg·L -1 kanamycin, and culture it at 37°C for 12 h. Pick a single colony and inoculate it into an LB liquid medium containing 50 mg·L -1 kanamycin resistance, culture it at 180 rpm / min at 37°C for 8 - 12 h, send it to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing, use software to compare the successfully sequenced mutants, and extract the recombinant mutant plasmid using a kit.

[0092] After that, add 1 μL each of the correctly sequenced mutant plasmid and the pET-Duet plasmid containing the spinach electron transfer system and co-transform them into 100 μL of Escherichia coli BL21(DE3) competent cells. Except for the final plating on an LB solid medium plate containing 50 mg·L -1 kanamycin and 50 mg·L -1 ampicillin sodium resistance, the other steps are the same as the above transformation steps.

[0093] After the overnight plate grows colonies, pick a single colony and inoculate it into an LB liquid medium containing 50 mg·L -1 kanamycin and 50 mg·L -1 ampicillin sodium resistance, culture it at 180 rpm / min at 37°C for 8 - 12 h, and then transfer it to an LB liquid medium containing 50 mg·L -1 kanamycin and 50 mg·L -1 ampicillin sodium resistance at an inoculation amount of 2% by volume. Culture it in a shaking flask at 37°C and 180 rpm / min until the OD value reaches 0.6 - 0.8, and then add IPTG with a final concentration of 24 mg·L -1 , ALA with 84 mg·L -1 , and 45 mg·L -1FeSO4 was used to induce expression at 20 °C and 180 rpm / min for 24 h. The bacterial solution was centrifuged at 8000 rpm for 10 min to collect wet bacterial cells. The conversion rates of mutants M60A, D61A, P87A, S89A, L119A, S121A, N124A, I210A, T269A, V320A, Q324A, and L325A for catalyzing avermectin B1a were 13.2%, 12.1%, 7.4%, 6.5%, 57.4%, 28.3%, 55.8%, 20.6%, 17.8%, 22.3%, 23.5%, and 18.3% respectively. Among them, the conversion rates of L119A and N124A were nearly 19.1 and 18.6 times higher than that of the wild type respectively.

[0094] Table 2 Primers related to alanine scanning PCR

[0095]

[0096] Second-round mutation: The sites L119 and N124 with significant improvement in the first-round alanine mutation were subjected to saturation mutation. The conversion rates of the obtained mutants L119F, N124P, and N124W were 63.2%, 53.2%, and 50.1% respectively compared with the wild type, which were 21.1, 17.7, and 16.7 times higher than that of the wild type respectively.

[0097] Third-round mutation: Using the primers shown in Table 3, with the mutant L119F as the template, the following combined mutations were carried out using the same mutation method as the first round: L119F / N124P and L119F / N124W. The conversion rates were calculated to be 65.2% and 67.9% respectively. The conversion rate of the mutant L119F / N124W was 41.5 times higher than that of the wild type.

[0098] Table 3 Primers related to combined mutation PCR

[0099]

[0100] Example 4 Conversion of avermectin B1a by Em1 mutant (L119F / N124W) under pH 7.4 potassium phosphate buffer system

[0101] The mutant L119F / N124W wet cells were prepared by the method in Example 3. 100 mL of potassium phosphate buffer with a pH of 7.4 was prepared. 0.5 g / L of wet cells was dissolved in 5 mL of potassium phosphate buffer, and after sufficient shaking and dissolution, ultrasonic cell disruption was carried out under ice bath conditions. The power of the ultrasonic cell disruptor was set at 100 W, working for 1 s and with an interval of 2 s, and the total disruption time was 3 min. Subsequently, the cell disruption solution was centrifuged at 12,000 rmp and 4 °C for 15 min to remove cell debris, and the supernatant was collected. The total reaction system was 1 mL, and the system included 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), 200 μL of the supernatant obtained by disrupting and centrifuging the wet cells. Then, the reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was extracted with ethyl acetate, the extract was dried in a fume hood, and then redissolved with pure acetonitrile and fixed volume to 1 mL; then it was filtered through a 0.22 μm microporous nylon membrane to obtain the test sample solution, and 10 μL of the test sample solution was injected into a high-performance liquid chromatograph for analysis. The conversion rate of cytochrome P450 mutant L119F / N124W in potassium phosphate buffer was calculated to be 84.8%.

[0102] Example 5 Conversion of Avermectin B1a by Em1 Mutant (L119F) in Phosphate Buffer System at pH 7.4

[0103] The mutant L119F wet cells were prepared by the method in Example 3. 0.5 g / L of wet cells was dissolved in 5 mL of phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, and the system included 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), 200 μL of the supernatant obtained by disrupting and centrifuging the wet cells. Then, the reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the test sample solution was injected into a high-performance liquid chromatograph for analysis. The conversion rate of cytochrome P450 mutant L119F in phosphate buffer was calculated to be 81.8%.

[0104] Example 6 Conversion of Avermectin B1a by Em1 Mutant (N124W) in Potassium Phosphate Buffer System at pH 7.4

[0105] The mutant wet cells were prepared by the method in Example 3. 0.5 g / L of wet cells were dissolved in 5 mL of potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, and the system included 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), and 200 uL of the supernatant obtained by disrupting and centrifuging the wet cells. Then, the reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the sample solution was injected into a high-performance liquid chromatograph for analysis, and the conversion rate of cytochrome P450 mutant N124W in potassium phosphate buffer with a pH of 7.4 was calculated to be 75.2%.

[0106] Example 7 Conversion of avermectin B1a by Em1 mutant (L119F / N124W) in Tris-HCl buffer system at pH 7.4

[0107] The mutant wet cells of L119F / N124W were prepared by the method in Example 3. 0.5 g / L of wet cells were dissolved in 5 mL of Tris-HCl buffer with a pH of 7.4. The total reaction system was 1 mL, and the system included 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), and 200 uL of the supernatant obtained by disrupting and centrifuging the wet cells. Then, the reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the sample solution was injected into a high-performance liquid chromatograph for analysis, and the conversion rate of cytochrome P450 mutant L119F / N124W in Tris-HCl buffer was calculated to be 80.6%.

[0108] Example 8 Conversion of avermectin B1a by Em1 mutant (L119F) in Tris-HCl buffer system at pH 7.4

[0109] The mutant L119F wet cells were prepared by the method in Example 3. 0.5 g / L of wet cells were dissolved in 5 mL of Tris-HCl buffer with a pH of 7.4. The total reaction system was 1 mL, including 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), and 200 μL of the supernatant obtained by disrupting and centrifuging the wet cells. Then, the reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the sample was injected into a high-performance liquid chromatography for analysis. The conversion rate of cytochrome P450 mutant L119F in Tris-HCl buffer at pH 7.4 was calculated to be 61.0%.

[0110] Example 9 Catalysis of avermectin B1a by Em1 mutant (N124W) in Tris-HCl buffer system at pH 7.4

[0111] The mutant N124W wet cells were prepared by the method in Example 3. 0.5 g / L of wet cells were dissolved in 5 mL of potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, including 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), and 200 μL of the supernatant obtained by disrupting and centrifuging the wet cells. Then, the reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and after obtaining the test sample solution, 10 μL of the sample was injected into a high-performance liquid chromatography for analysis. The conversion rate of cytochrome P450 mutant N124W in potassium phosphate buffer at pH 7.4 was calculated to be 67.4%.

[0112] Example 10 Reaction of bio-oxidizing avermectin B1a with the substrate dissolved in Tween 40 and DMSO

[0113] The mutant L119F / N124W wet cells were prepared by the method in Example 3. 0.5 g / L of wet cells were dissolved in potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, and the system included 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a, and 200 μL of the supernatant obtained by breaking and centrifuging the wet cells. The substrate was dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1). The reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the sample was injected into a high-performance liquid chromatograph for analysis, and the conversion rate was 90.9%.

[0114] Example 11 The substrate was dissolved in propylene glycol, and the reaction of biocatalytic oxidation of avermectin B1a was carried out

[0115] The mutant L119F / N124W wet cells were prepared by the method in Example 3. 0.5 g / L of wet cells were dissolved in potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, and the system included 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a, and 200 μL of the supernatant obtained by breaking and centrifuging the wet cells. The substrate was dissolved in propylene glycol. The reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the sample was injected into a high-performance liquid chromatograph for analysis, and the conversion rate was 58.6%.

[0116] Example 12 The substrate was dissolved in isopropanol, and the reaction of biocatalytic oxidation of avermectin B1a was carried out

[0117] The mutant L119F / N124W wet cells were prepared by the method in Example 3. 0.5 g / L of wet cells were dissolved in potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, including 450 μL of potassium phosphate buffer, 200 μL of 10 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 20 mM avermectin B1a, and 200 μL of the supernatant obtained by breaking and centrifuging the wet cells. The substrate was dissolved in isopropanol. The reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the sample was injected into a high-performance liquid chromatograph for analysis, and the conversion rate was 66.8%.

[0118] Example 13 The substrate was dissolved in Tween 40 and DMSO, and the reaction for biocatalytic oxidation of high-concentration abamectin B1a was carried out.

[0119] The mutant L119F / N124W wet cells were prepared by the method in Example 3. 0.5 g / L of the wet cells were dissolved in potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, which included 450 μL of potassium phosphate buffer, 200 μL of 100 mM NADH, 50 μL of 1 M glucose, 2 g / L of GDH, 50 μL of 200 mM abamectin B1a, and 200 μL of the supernatant obtained by disrupting and centrifuging the wet cells. The substrate was dissolved in Tween 40 and DMSO (Tween 40:DMSO = 1:1), and the reaction was carried out at 30 °C and 800 rpm for 72 h. The reaction solution was treated by the method in Example 4, and then the test sample solution was obtained. 10 μL of the test sample solution was injected into a high-performance liquid chromatograph for analysis. The conversion rate was 88.9%.

Claims

1. A cytochrome P450 oxidase mutant, characterized in that: The cytochrome P450 oxidase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 according to one of the following mutation forms: (1) Leucine at position 119 mutates to alanine or phenylalanine; (2) Asparagine at position 124 mutated to alanine, proline or tryptophan; (3) The leucine at position 119 mutates to phenylalanine, and the asparagine at position 124 mutates to proline or tryptophan.

2. A gene encoding the cytochrome P450 oxidase mutant as claimed in claim 1.

3. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 2.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contains the gene as claimed in claim 2.

5. Use of the cytochrome P450 oxidase mutant according to claim 1, or the recombinant vector according to claim 3, or the genetically engineered bacteria according to claim 4 in catalyzing the synthesis of emamectin benzoate intermediate 4"carbonyl-5"hydroxy-avermectin from avermectin B1a.

6. A method for producing 4"carbonyl-5"hydroxy-avermectin, characterized in that: A reaction system is formed by taking avermectin B1a as a substrate and the cytochrome P450 oxidase mutant described in claim 1 as a catalyst to synthesize 4"carbonyl-5"hydroxy-avermectin.

7. The method according to claim 6, characterized in that The reaction temperature is 25-30°C.

8. The method according to claim 6, characterized in that In the reaction system, the substrate is dissolved in a Tween 40 / DMSO mixed solution, and then mixed with a catalyst for reaction; the volume ratio of Tween 40 to DMSO in the Tween 40 / DMSO mixed solution is 1:

1.

9. The method according to claim 6, characterized in that In the reaction system, the cytochrome P450 oxidase mutant is dispersed in a buffer solution; the pH of the buffer solution is 7.4-8.4; the buffer solution is a potassium phosphate buffer, a phosphate buffer or a Tris-HCl buffer.

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