Cytochrome P450 enzyme mutant and application thereof in preparation of emamectin benzoate intermediate

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to a cytochrome P450 enzyme mutant and application of the cytochrome P450 enzyme mutant in preparation of an emamectin benzoate intermediate, and the cytochrome P450 enzyme mutant is obtained by performing single-point or multi-point mutation on the 119th site and the 124th site of an amino acid sequence as shown in SEQ ID NO.1. According to the invention, the key point of the cytochrome P450 oxidase from Streptomyces is subjected to rational transformation, and the conversion rate of the mutant obtained by screening is further improved. By using the cytochrome P450 oxidase mutant provided by the invention, the C4 'carbonyl-C5' hydroxyl-abamectin can be directly prepared in the presence of glucose dehydrogenase, glucose and cofactors by taking abamectin as a substrate, and the method is mild in reaction condition and low in catalyst cost, and is an ideal scheme for preparing the C4 'carbonyl-C5' hydroxyl-abamectin.

Description

Technical Field

[0001] The invention belongs to the field of protein engineering, and in particular relates to a cytochrome P450 enzyme mutant and an application thereof in preparing an emamectin benzoate intermediate. Background Art

[0002] Abamectin (AVM for short) is a fermentation product produced by a fungus called Streptomyces avermitilis isolated from the soil. It has insecticidal and anthelmintic properties, and its structure is a series of 16-membered macrolides. Abamectin has become a mainstream agent on the market due to its broad spectrum of pest control on various crops such as rice, cotton, fruit trees, tobacco, and its high efficiency, low toxicity, high selectivity, and environmental friendliness. However, there are many problems with avermectin, such as the increasing resistance of insects to avermectin year by year, and the leaching of less soluble pesticides that are adsorbed on the soil surface in large quantities. Therefore, using avermectin as the parent, finding 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 substance, its biological activity is increased by 1 to 3 orders of magnitude. Since emamectin benzoate is a biogenic pesticide, it is easily degradable, has small residues, and causes minimal pollution to the environment. It is a green and environmentally friendly pesticide with a wider insecticide spectrum, such as cotton bollworm and beet armyworm, and is particularly effective against Lepidoptera pests.

[0004] Emamectin benzoate is made by salifying avermectin with benzoic acid; avermectin is obtained by modification on C4-OH, which requires six steps of protection, oxidation, amination, reduction, deprotection, and salt formation; but there is a more active C5-OH in avermectin, so it needs to be protected and deprotected, which greatly increases the cost of raw materials, 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 average, and the product appearance is not ideal.

[0005] Biocatalysis, also known as biotransformation, refers to the addition of exogenous natural or synthetic organic compounds as substrates to an active biological system or enzyme system, and the cultivation under suitable conditions, so that the substrate interacts with the enzyme in the system, thereby producing structural changes. Its essence is an enzymatic reaction. Biocatalysis has the characteristics of mild reaction conditions, high efficiency and high selectivity (chemical, regional and stereoselectivity). It is considered to be a resource-saving and environmentally friendly technology and an important supplement to chemical synthesis. Seeking cheap, efficient and environmentally friendly biocatalysts to achieve site-specific oxidation of avermectin C4"-O will greatly simplify the synthesis steps of emamectin benzoate and help promote the application of emamectin benzoate. Summary of the invention

[0006] In view of the need for biocatalysts that efficiently and specifically catalyze 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 scheme is as follows: In a first aspect, the present invention provides a cytochrome P450 oxidase mutant, wherein the cytochrome P450 oxidase mutant is obtained by performing single-point or multi-point mutation at positions 119 and 124 of the amino acid sequence shown in SEQ ID NO.1.

[0007] Furthermore, 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.

[0008] In a second aspect, the present invention provides a gene encoding the above-mentioned cytochrome P450 oxidase mutant.

[0009] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned coding gene.

[0010] In a fourth aspect, the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium contains the above-mentioned encoding gene.

[0011] Furthermore, the genetically engineered bacteria uses Escherichia coli BL21 (DE3) as a host bacterium.

[0012] Furthermore, the genetically engineered bacteria can express the spinach electron transfer system.

[0013] Furthermore, the spinach electron transfer system includes ferredoxin and ferredoxin reductase.

[0014] In a fifth aspect, the present invention provides the use of the above-mentioned cytochrome P450 oxidase mutant, or the above-mentioned recombinant vector, or the above-mentioned genetically engineered bacteria in catalyzing the synthesis of avermectin benzoate intermediate 4"carbonyl-5"hydroxy-avermectin from avermectin B1a.

[0015] In a sixth aspect, the present invention provides a method for producing 4"carbonyl-5"hydroxy-avermectin, wherein a reaction system is composed of avermectin B1a as a substrate and the above-mentioned cytochrome P450 oxidase mutant as a catalyst to synthesize 4"carbonyl-5"hydroxy-avermectin.

[0016] Furthermore, the catalyst can be wet bacteria obtained by fermentation culture of the above-mentioned genetically engineered bacteria or a crude enzyme solution obtained by ultrasonically crushing wet bacteria.

[0017] Furthermore, the reaction temperature is 25-30°C.

[0018] Furthermore, the reaction temperature is 30°C.

[0019] Furthermore, the reaction time is 24 to 72 h.

[0020] Furthermore, the reaction time was 72 h.

[0021] Furthermore, in the reaction system, the substrate is dissolved in a Tween 40 / DMSO mixture, and then mixed with a catalyst for reaction; the volume ratio of Tween 40 to DMSO in the Tween 40 / DMSO mixture is 1:1.

[0022] Furthermore, in the reaction system, the cytochrome P450 oxidase mutant is dispersed in a buffer solution.

[0023] Furthermore, the pH of the buffer solution is 7.4-8.4.

[0024] Furthermore, the buffer solution is potassium phosphate buffer, phosphate buffer or Tris-HCl buffer.

[0025] Furthermore, in the reaction system, a coenzyme circulation system is constructed with glucose dehydrogenase (GDH) and NADH as coenzymes and glucose as a cosubstrate.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on Streptomyces tubercidicus The key points of the cytochrome P450 oxidase are rationally modified, and the conversion rate of the mutants obtained by screening 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. The method has mild reaction conditions and low catalyst cost, and is an ideal solution for preparing C4 "carbonyl-C5" hydroxy-avermectin. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the structural formula of avermectin.

[0028] Figure 2 Schematic diagram of the reaction for preparing C4"carbonyl-C5"hydroxy-avermectin from avermectin B1a.

[0029] Figure 3 This is the standard curve of C4 "carbonyl-C5" hydroxy-avermectin.

[0030] Figure 4 This is the HPLC detection spectrum; among them, the retention time is 26.604 min for avermectin B1a, and 30.823 min for C4 "carbonyl-C5" hydroxy-avermectin. DETAILED DESCRIPTION

[0031] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0032] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0034] In the following examples, the structural formula of avermectin is as follows Figure 1 As shown, the present invention uses avermectin B1a.

[0035] In the following examples, the reaction formula for preparing C4"carbonyl-C5"hydroxy-avermectin from avermectin B1a is as follows: Figure 2 shown.

[0036] The culture medium and the required antibiotics, inducers, etc. configured in the embodiment of the present invention are composed of: LB liquid medium: 5 g·L -1 Yeast powder, 10 g·L -1 Peptone, 10 g L -1 Sodium chloride.

[0037] LB solid medium: 5 g·L -1 Yeast powder, 10 g·L -1 Peptone, 10 g L -1 Sodium chloride, 3 g·L -1 Agar powder.

[0038] TB liquid medium: 12 g·L -1 Peptone, 12 g L-1 Yeast powder, 12.5 g·L -1 Potassium hydrogen phosphate, 2.3 g·L -1 Potassium dihydrogen phosphate, 4 mL·L -1 glycerin.

[0039] The above culture medium was dissolved in water and sterilized at a pressure of 0.105 MPa and a temperature of 121°C for 20 min.

[0040] 100 mg / mL ampicillin: Weigh 5 g of ampicillin sodium and dissolve it in 50 mL of sterile water. After it is fully dissolved, filter it with a 0.22 um filter membrane, divide it into EP tubes, and store it at -20℃.

[0041] 50 mg / mL kanamycin sulfate: weigh 2.5 g of kanamycin sulfate and dissolve it in 50 mL of sterile water. After it is fully dissolved, filter it with a 0.22 um filter membrane, divide it into EP tubes, and store it at -20℃.

[0042] 120 mg / mL IPTG: Weigh 6 g IPTG and dissolve it in 50 mL sterile water. After it is fully dissolved, filter it with a 0.22 um filter membrane, divide it into EP tubes, and store it at -20℃.

[0043] 84 mg / mL ALA: Weigh 4 g ALA and dissolve it in 50 mL sterile water. After it is fully dissolved, filter it with a 0.22 um filter membrane, dispense it into EP tubes, and store it at -20°C.

[0044] 45 mg / mL FeSO4: Weigh 1.2 g FeSO4 and dissolve it in 50 mL sterile water. After it is fully dissolved, filter it with a 0.22 um filter membrane, divide it into EP tubes, and store it at -20℃.

[0045] The test materials and test instruments used in the embodiments of the present invention are: 1. Test materials E. coli containing plasmid pRSF-Duet containing CYP450 enzyme target gene E. coli BL21 (DE3) strain, plasmid pET-Duet with ferredoxin and ferredoxin reductase target genes E. coli BL21 (DE3) strains were purchased from Beijing Qingke Biological Co., Ltd.; E. coli DH5α and E. coli The BL21 (DE3) competent cells were produced and preserved in our laboratory; SurePAGE™ protein precast gel and protein markers were purchased from GenScript Biotech Co., Ltd.

[0046] Anhydrous dipotassium hydrogen phosphate, anhydrous potassium dihydrogen phosphate, chromatographic grade methanol, and chromatographic grade acetonitrile were purchased from Sinopharm Chemical Reagent Co., Ltd., yeast extract and peptone were purchased from Thermo Fisher Scientific Inc., and Tris base, concentrated hydrochloric acid, sodium dodecyl sulfate, bromophenol blue, glycerol, 2-mercaptoethanol, anhydrous ethanol, glacial acetic acid, Coomassie brilliant blue, phosphoric acid, glycine, and 20% SDS were purchased from Saiguo Biotechnology Co., Ltd.

[0047] Kanamycin was purchased from Shanghai Myrel Biochemical Technology Co., Ltd., ampicillin, NADH, and NADPH were purchased from Shanghai Titan Technology Co., Ltd., 5-aminolevulinic acid (ALA) and ferrous sulfate were purchased from Tianjin Damao Chemical Reagent Factory, isopropylthiogalactoside (IPTG) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and dimethyl sulfoxide and Tween 40 were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0048] Nucleic acid markers and nucleic acid dyes were purchased from Qingke Biotechnology Co., Ltd.

[0049] Preparation of competent cells: Obtain glycerol tubes stored in a -80 ℃ freezer. E. coli BL21(DE3) strain was streaked on an antibiotic-free LB plate and cultured at 37°C for 10 h to obtain a single colony; a single colony on the LB plate was picked and inoculated into a test tube containing 10 mL of LB medium and cultured at 37°C and 180 rpm for 9 h; 2 mL of bacterial solution was taken from the test tube and inoculated into 100 mL of LB medium and cultured at 37°C and 180 rpm until OD600 reached 0.4-0.6; the bacterial solution was precooled on ice, taken into a sterilized centrifuge tube, placed on ice for 10 min, and centrifuged at 4°C and 5000 rpm for 10 min; the supernatant was poured out, and the precipitated cells were resuspended with a 0.1 mol / L CaCl2 aqueous solution precooled at 4°C and placed on ice for 30 min; the supernatant was discarded and the precipitated cells were resuspended with a 0.1 mol / L CaCl2 aqueous solution precooled at 4°C and placed on ice for 30 min; the supernatant was discarded and the precipitated cells were resuspended with a 0.1 mol / L CaCl2 aqueous solution precooled at 4°C and placed on ice for 30 min; the supernatant was discarded and the precipitated cells were resuspended with a 0.1 mol / L CaCl2 aqueous solution precooled at 4°C and containing 15% glycerol Resuspend the precipitated cells with 100 μL of 50% CaCl2 aqueous solution, take 100 μL of the resuspended cells and dispense them into sterile 1.5 mL centrifuge tubes, store them in a -80 ℃ refrigerator, and take them out when needed.

[0050] 2. Test instruments Table 1 Test instruments

[0051] In the following examples, the method for determining the conversion activity of avermectin B1a catalyzed by cytochrome P450 oxidase is as follows: The determination system is as follows: the total reaction system is 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mM NAD(P)H, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40: DMSO = 1:1), and 200 uL supernatant obtained by wet bacterial cell crushing and centrifugation. The reaction is carried out at 30°C and 800 rpm for 72 h, and the reaction solution is extracted with ethyl acetate. The extract is blown dry in a fume hood and then re-dissolved with pure acetonitrile and the volume is adjusted to 1 mL. It is then filtered with a 0.22 μm microporous nylon membrane to obtain the test sample solution, and the content is calculated according to the standard curve.

[0052] In the following examples, the determination method of the 4"carbonyl-5"hydroxy-avermectin standard curve is: Prepare 4"carbonyl-5"hydroxy-avermectin standard solution: weigh 0.87 g and dissolve it in 60 mL 50% acetonitrile (acetonitrile: water = 1:1), transfer it to a volumetric flask after dissolving it in a clean beaker, rinse the beaker three times with 20 mL 50% acetonitrile and transfer it to the volumetric flask with a glass rod, then dilute to 100 mL, cover with a lid and shake to obtain a 10 mM 4"carbonyl-5"hydroxy-avermectin 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-avermectin sample solutions for preparing a standard curve. The standard curve of 4"carbonyl-5"hydroxy-avermectin is as follows Figure 3 shown.

[0053] In the following examples, the method for detecting the content of 4"carbonyl-5"hydroxy-avermectin is as follows: HPLC was used for detection. The detection conditions were: Thermo Fisher U3000 liquid chromatograph; C18 column (4.6 mm×250mm); column temperature: 30℃; 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 sample solution at the time corresponding to the peak of 4"carbonyl-5"hydroxy-avermectin in the standard sample solution was inserted into the 4"carbonyl-5"hydroxy-avermectin standard curve to calculate the concentration of catalytically produced 4"carbonyl-5"hydroxy-avermectin. The HPLC detection spectrum of avermectin B1a and C4"carbonyl-C5"hydroxy-avermectin is shown in the figure. Figure 4 As shown, the one at retention time 26.604 min is avermectin B1a, and the one at retention time 30.823 min is C4 "carbonyl-C5" hydroxy-avermectin.

[0054] In the following examples, the conversion rate is calculated as follows: (initial molar concentration of substrate - residual molar concentration of substrate) / initial molar concentration of substrate.

[0055] In the following examples, the yield is calculated as follows: molar concentration of generated product / initial molar concentration of substrate.

[0056] In the following examples, the electron transfer system of spinach refers to a system for transferring electrons in the plant body of spinach. It plays an important role in the physiological processes of spinach such as photosynthesis and respiration. Specifically, electrons can be transferred from one molecule to another through this system, thereby achieving energy conversion and material metabolism.

[0057] 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.

[0058] SEQ ID NO.1: MGSSHHHHHHMSELMNSPFAAHVGKHPGEPNVMDPALITDPFTGYGALREQGPVVRGRFMDDSPVWLVTRFEEVRQVLRDQRFVNNPASPSLNYAPEDNPLTRLMEMLG LPEHLRVYLLGSILNYDAPDHTRLRLSDVEMVTLVLTLVLAGHQTTAHLARLPEHAEDGVVDLIQHFAYPLPITVICELVGIPEADRPQWRTWGADLISMDPDRLGASF PAMIEHIHQMVRERREALTDDLLSELIRTHDDDGGRLSDVEMVTMILTLVLAGHETTAHLISNGTAALLTHPDQLRLVKDDPALLPRAVHELMRWCGPVHMTQLRYATA DVDLAGTPIRQGDAVQLILVSANFDPRHYTDPDRLDLTRHPAGHAENHVGFGHGAHYCLGATLAKQEGEVAFGKLLTHYPDISLGIAPEHLERTPLPGNWRLNSLPVRLG SEQ ID NO.2: MGSSHHHHHHMTELTDSPFSEFVGKHPGEPNVMEPALLTDPFAGYGALREQGPVVRGRFVDDTPVWFITRFEEAREVLRDQRFANSPAHSAGGGSADTPIDRLLEIMGLPEHYRAYLSGTILNMDAPDHTRLRRLVSRAFTARKITDLRPRVADIAEDALRRLPEHAVDGVVDLIPHFAYPLPITVICELVGIPEADRPQWREWSTHLVSLRRLVSRAFTARKITDLRPRVEQIADALLTDDLLSELIRVHDDDGSRLSDVEMVTLVLTLVLAGHETTAHLITNGVAALLTHPDQLQLLKSEPALLPRAVHELMRWCGPVHLTQMRYATEDVELAGVRIKKGEAVTPVLVAANHDPRHFADPDRLDLTRQPAGRAENHVGFGHGMHYCLGATLARQEAEVAFGKLLAHYPDVALAVAPEDLQRVPLPGSWRLASLPLRLN SEQ ID NO.3: MGSSHHHHHHMSASPSNTFTEHVGKHPGEPNVMDPALIGDPFAGYGALREQGPVVRGRFMDDSPVWFVTRFEEVREVLRDPRFVNNPAAPSLGRSIDESPTARLLEMMGLPEHFRPYLLGSILTNDAPDHTRLRRLVSRAFTARKITDLRPRVAQITAELLDRLPEHAEDGVVDLIEHFAYPLPITVICELVGIAAEDRPQWRTWGADLVSLQPDRMSRSFPAMIEHIHELIRERRGALTDDLLSELIRTHDDDGSRPELHPETFPEMIDHIHALIRERRTALIGNGTAALLTHPDQLRLLKDDPALLPRAVHELMRWCGPVHMTQLRYATADVDLAGTPIRQGDAVQLILVSANFDPRHYTDPDRLDLTRHPAGHAENHVGFGHGAHYCLGATLAKQEGEVAFEKLFAHYPEVSLAVPPDHLERTPVPGMWGLNSLPVRLG。

[0059] Example 1 Screening of wild-type cytochrome P450 oxidase The sources of cytochrome P450 enzymes (CYP450) and related information of gene sequences were searched through literature search and multiple sequence alignment. 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 three CYP450 enzymes from different sources were screened out. Streptomyces tubercidicus Cytochrome P450 oxidase: Em1, Em18, Em5b. The genes were optimized according to the codon preference of Escherichia coli, and NcoI restriction site, HidIII restriction site, His tag and terminator were introduced into the sequence published in GenBank. The genes were synthesized by Hangzhou Qingke Biotechnology Co., Ltd. to obtain wild-type cytochrome P450 oxidase genes (amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively).

[0060] Example 2 Construction and expression of wild-type cytochrome P450 oxidase recombinant engineering bacteria The three wild-type cytochrome P450 oxidase genes obtained in Example 1 ( Em1 , Em18 , E5b ) was connected with plasmid pRSF-Duet, and the spinach electron transfer system was connected with plasmid pET-Duet to construct a recombinant plasmid. The two recombinant plasmids were then transformed into E. coli BL21(DE3) competent cells, construct wild-type recombinant engineering bacteria, transformation steps are as follows: E. coli BL21(DE3) competent cells on ice, add 1 uL of two recombinant plasmids respectively, place on ice for 30 min, heat shock in a 42 ℃ water bath for 45 s, incubate on ice for 4 min, add LB liquid medium without antibiotics, culture at 37 ℃ for 45-60 min, then centrifuge at 4500 rpm for 5 min, discard 600 μL supernatant. Gently blow and mix the remaining bacteria, absorb and hit on the plate containing the correct resistance, pour 5-10 glass beads on the plate and shake gently until there is no bacterial liquid on the plate, pour the glass beads in pure alcohol, and place the plate in a 37 ℃ incubator and invert for 12-16 h. After colonies grew on the overnight culture plate, a single colony was picked and inoculated into LB liquid medium containing 50 mg / mL ampicillin sodium and 50 mg / mL kanamycin, and cultured at 180rpm and 37℃ for 8-12 h. The colony was then sequenced by Hangzhou Qingke Biotechnology Co., Ltd. The wild-type recombinant engineered bacteria that were successfully sequenced were compared using software. E. coli BL21(DE3)-pRSF-Duet-Em1, E. coli BL21(DE3)-pRSF-Duet-Em18, E. coli BL21(DE3)-pRSF-Duet-Em5b bacteria-preserving glycerol tube.

[0061] The successfully constructed engineered bacteria were inoculated into LB liquid medium and cultured at 37°C in a shaker at 180 rpm for 2-3 h. When the bacterial density OD600 value reached 0.6, IPTG was added and the shake flask was transferred to a 28°C shaker at 180 rpm for 12 h. After the culture was completed, the culture solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected.

[0062] Recombinant Engineering Bacteria E. coli BL21(DE3)-pRSF-Duet-Em1, E. coli BL21(DE3)-pRSF-Duet-Em18, E. coli The 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.

[0063] Experimental Example 3 Screening of cytochrome P450 oxidase mutants and construction of engineered bacteria 1. Acquisition of cytochrome P450 oxidase template gene 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.

[0064] 2. Site-directed mutagenesis of cytochrome P450 oxidase genes According to the Genbank Streptomyces tubercidicus 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.

[0065] First round of mutation: The amino acid residues within 6 angstroms around the substrate avermectin were mutated to alanine.

[0066] 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.

[0067] 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, for a total of 30 cycles, and final extension at 72°C for 5 min.

[0068] Take 10 uL of PCR product to 100 uL of E. coli BL21 (DE3) competent cells, place on ice for 30 min, heat shock in a 42°C water bath for 45 s, incubate on ice for 4 min, add 600 uL of LB liquid medium, culture at 37°C for 45-60 min, centrifuge and discard 600 uL of supernatant, then spread on a plate containing 50 mg·L -1 The culture was carried out on LB solid medium plates containing kanamycin at 37°C for 12 h. A single colony was picked and inoculated into a plate containing 50 mg·L -1 The cells were cultured in kanamycin-resistant LB liquid medium at 180 rpm / min and 37°C for 8-12 h, and then sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. The successfully sequenced mutants were compared using software, and the recombinant mutant plasmids were extracted using a kit.

[0069] Then, 1uL of each mutant plasmid and pET-Duet plasmid containing the spinach electron transport system were added to 100uL of E. coli BL21 (DE3) competent cells. -1 Kanamycin and 50 mg·L -1 The remaining steps were the same as the above transformation steps.

[0070] After colonies grew on the overnight plate, a single colony was picked and inoculated into a plate containing 50 mg·L -1 Kanamycin and 50 mg·L -1 The culture medium was incubated at 180 rpm / min and 37°C for 8-12 h, and then the inoculum was transferred to a 2% volume concentration LB medium containing 50 mg·L -1 Kanamycin and 50 mg·L-1 In LB liquid medium with ampicillin sodium resistance, shake flask culture at 37°C and 180 rpm / min until the OD value reaches 0.6-0.8, add a final concentration of 24 mg·L -1 IPTG, 84mg·L -1 ALA, 45 mg·L -1 FeSO4 was added, and the expression was induced at 20℃ and 180 rpm / min for 24 h. The bacterial solution was centrifuged at 8000 rpm for 10 min, and the wet cells were collected. The conversion rates of avermectin B1a catalyzed by mutants M60A, D61A, P87A, S89A, L119A, S121A, N124A, I210A, T269A, V320A, Q324A, and L325A 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. The conversion rates of L119A and N124A were increased by nearly 19.1 and 18.6 times, respectively, compared with the wild type.

[0071] Table 2 Alanine scanning PCR related primers

[0072] The second round of mutation: The L119 and N124 sites that had significantly improved alanine mutations in the first round were subjected to saturation mutation. The resulting mutants L119F, N124P, and N124W had conversion rates of 63.2%, 53.2%, and 50.1%, respectively, compared to the wild type, which were 21.1, 17.7, and 16.7 times higher than the wild type.

[0073] The third round of mutation: Using the primers shown in Table 3, the mutant L119F was used as a template, and the same mutation method as the first round was used to perform the following combined mutations: L119F / N124P, L119F / N124W, and 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.

[0074] Table 3 Primers related to combined mutation PCR

[0075] Example 4 Conversion of Em1 mutant (L119F / N124W) to avermectin B1a in pH 7.4 potassium phosphate buffer system The mutant L119F / N124W wet bacteria 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 bacteria was dissolved in 5 mL of potassium phosphate buffer, and the mixture was fully shaken and dissolved. Ultrasonic cell disruption was performed under ice bath conditions. The power of the ultrasonic cell disruptor was set to 100 W, working for 1 s, with an interval of 2 s, and a total disruption time of 3 min. Subsequently, the cell disruption solution was centrifuged at 12,000 rmp and 4 °C for 15 min to remove cell debris and collect the supernatant. The total reaction system was 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mM NADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40: DMSO = 1:1), and 200 uL of supernatant obtained by crushing and centrifuging wet cells. The reaction was then carried out at 30°C and 800 rpm for 72 h. The reaction solution was extracted with ethyl acetate, and the extract was blown dry in a fume hood and then re-dissolved with pure acetonitrile to a constant volume of 1 mL. The extract was then filtered through a 0.22 μm microporous nylon membrane to obtain the test sample solution, and 10 μL was injected for analysis by high performance liquid chromatography. The conversion rate of cytochrome P450 mutant L119F / N124W in potassium phosphate buffer was calculated to be 84.8%.

[0076] Example 5 Conversion of Em1 mutant (L119F) to avermectin B1a in pH 7.4 phosphate buffer system The mutant L119F wet bacteria were prepared by the method in Example 3. 0.5 g / L wet bacteria were dissolved in 5 mL phosphate buffer at pH 7.4. The total reaction system was 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mMNADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40: DMSO = 1: 1), and 200 uL wet bacteria were crushed and centrifuged to obtain the supernatant, and then reacted 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 was injected by high performance liquid chromatography for analysis, and the conversion rate of the cytochrome P450 mutant L119F in phosphate buffer was calculated to be 81.8%.

[0077] Example 6 Conversion of Em1 mutant (N124W) to avermectin B1a in potassium phosphate buffer system at pH 7.4 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 at pH 7.4. The total reaction system was 1 mL, including 450 μL of potassium phosphate buffer, 200 μL of 10mM NADH, 50 μL of 1M glucose, 2 g / L GDH, 50 μL of 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40: DMSO = 1: 1), and 200 uL of wet cells were crushed and centrifuged to obtain the supernatant, and then reacted 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 was injected by high performance liquid chromatography for analysis, and the conversion rate of cytochrome P450 mutant N124W in potassium phosphate buffer at pH 7.4 was calculated to be 75.2%.

[0078] Example 7 Conversion of Em1 mutant (L119F / N124W) to avermectin B1a in pH 7.4 Tris-HCl buffer system The mutant L119F / N124W wet bacteria were prepared by the method in Example 3. 0.5 g / L wet bacteria were dissolved in 5 mL Tris-HCl buffer at pH 7.4. The total reaction system was 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mM NADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40: DMSO = 1: 1), and 200 uL wet bacteria were crushed and centrifuged to obtain the supernatant, and then reacted 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 was injected by high performance liquid chromatography for analysis, and the conversion rate of the cytochrome P450 mutant L119F / N124W in Tris-HCl buffer was calculated to be 80.6%.

[0079] Example 8: Conversion of Em1 mutant (L119F) to avermectin B1a in pH 7.4 Tris-HCl buffer system The mutant L119F wet bacteria were prepared by the method in Example 3. 0.5 g / L wet bacteria were dissolved in 5 mL Tris-HCl buffer at pH 7.4. The total reaction system was 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mM NADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40: DMSO = 1: 1), and 200 uL wet bacteria were crushed and centrifuged to obtain the supernatant, and then reacted 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 was injected by high performance liquid chromatography for analysis, and the conversion rate of the cytochrome P450 mutant L119F in pH7.4 Tris-HCl buffer was calculated to be 61.0%.

[0080] Example 9 Catalytic activity of avermectin B1a by the Em1 mutant (N124W) in a pH 7.4 Tris-HCl buffer system The mutant N124W wet bacteria were prepared by the method in Example 3. 0.5 g / L wet bacteria were dissolved in 5 mL potassium phosphate buffer at pH 7.4. The total reaction system was 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mMNADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a dissolved in Tween 40 and DMSO (Tween 40: DMSO = 1: 1), and 200 uL wet bacteria were crushed and centrifuged to obtain the supernatant, and then reacted 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 was injected by high performance liquid chromatography for analysis, and the conversion rate of the cytochrome P450 mutant N124W in potassium phosphate buffer at pH 7.4 was calculated to be 67.4%.

[0081] Example 10 The substrate is dissolved in Tween 40 and DMSO to carry out the biological oxidation reaction of avermectin B1a The mutant L119F / N124W wet bacteria were prepared by the method in Example 3. 0.5 g / L wet bacteria were dissolved in potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mMNADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a, and 200 uL of supernatant obtained by crushing and centrifuging the wet bacteria, wherein 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 was injected by high performance liquid chromatography for analysis, and the conversion rate result was 90.9%.

[0082] Example 11 The substrate is dissolved in propylene glycol and the reaction of biooxidation of avermectin B1a is carried out The mutant L119F / N124W wet bacteria were prepared by the method in Example 3. 0.5 g / L wet bacteria were dissolved in a potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, and the system included 450 μL potassium phosphate buffer, 200 μL 10mMNADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a, and 200 uL of supernatant obtained by crushing and centrifugation of wet bacteria, wherein the substrate was dissolved in propylene glycol, 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, and 10 μL was injected by high performance liquid chromatography for analysis, and the conversion rate result was 58.6%.

[0083] Example 12 The substrate is dissolved in isopropanol and the biooxidation reaction of avermectin B1a is carried out The mutant L119F / N124W wet bacteria were prepared by the method in Example 3. 0.5 g / L of wet bacteria was dissolved in a potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, including 450 μL potassium phosphate buffer, 200 μL 10mM NADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 20 mM avermectin B1a, and 200 μL of supernatant obtained by crushing and centrifugation of wet bacteria, wherein the substrate was dissolved in isopropanol, 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, and 10 μL was injected by high performance liquid chromatography for analysis, and the conversion rate result was 66.8%.

[0084] Example 13 The substrate is dissolved in Tween 40 and DMSO to carry out the biooxidation reaction of high concentration avermectin B1a The mutant L119F / N124W wet bacteria were prepared by the method in Example 3. 0.5 g / L wet bacteria were dissolved in a potassium phosphate buffer with a pH of 7.4. The total reaction system was 1 mL, and the system included 450 μL potassium phosphate buffer, 200 μL 100mMNADH, 50 μL 1M glucose, 2 g / L GDH, 50 μL 200 mM avermectin B1a, and 200 μL supernatant obtained by crushing and centrifuging the wet bacteria, wherein 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, and 10 μL was injected by high performance liquid chromatography for analysis, and the conversion rate result was 88.9%.

Claims

1. A cytochrome P450 oxidase mutant, characterized in that: The cytochrome P450 oxidase mutant is obtained by performing single-point or multi-point mutations at positions 119 and 124 of the amino acid sequence shown in SEQ ID NO.

1.

2. The cytochrome P450 oxidase mutant according to claim 1, 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.

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

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

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contains the gene as claimed in claim 3.

6. Use of the cytochrome P450 oxidase mutant according to any one of claims 1 or 2, or the recombinant vector according to claim 4, or the genetically engineered bacteria according to claim 5 in catalyzing the synthesis of emamectin benzoate intermediate 4"carbonyl-5"hydroxy-avermectin from avermectin B1a.

7. 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 according to any one of claims 1 or 2 as a catalyst to synthesize 4"carbonyl-5"hydroxy-avermectin.

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

9. The method according to claim 7, 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.

10. The method according to claim 7, 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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