Hematoxymethyltransferase CsOMT80 mutant and application thereof in synthesis of chalcone compound

Abstract

The invention discloses a hematoxyloxymethyltransferase CsOMT80 mutant and an application of the hematoxyloxymethyltransferase CsOMT80 mutant in synthesis of chalcone compounds. Through structural biological modeling and functional site analysis, on the basis of hematoxylon multi-omics data and identification, an oxygen methyltransferase coding gene CsOMT80 closely related to methoxychalcone synthesis is obtained, structural prediction and molecular docking are performed on an amino acid sequence of the oxygen methyltransferase coding gene CsOMT80, and key sites influencing substrate binding and catalytic efficiency are screened out. On the basis, amino acid replacement is carried out on a target site by adopting a site-directed mutagenesis strategy, and a plurality of mutants are constructed and obtained. Functional verification finds that the mutant CsOMT80G112R shows remarkably enhanced catalytic efficiency when a catalytic reaction is carried out by taking isoliquiritigenin as a substrate, and the enzymatic activity of the mutant CsOMT80G112R is 1278 times that of a wild type and is superior to that of reported O-methyltransferase (ChOMT) from alfalfa.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant biotechnology, and relates to a Caesalpinia sappan L. O-methyltransferase CsOMT80 mutant and application thereof in synthesis of chalcone compounds. BACKGROUND

[0002] Caesalpinia sappan L. is a leguminous plant widely distributed in tropical and subtropical regions, and has been used as a medicine for more than 1800 years in China. The core efficacy of Caesalpinia sappan L. is described in Chinese Pharmacopoeia as “activating blood and resolving stasis, relieving swelling and relieving pain”, and modern pharmacological studies have shown that Caesalpinia sappan L. is rich in various bioactive flavonoid secondary metabolites, especially characterized by homoisoflavonoids. Homoisoflavonoids not only exhibit good efficacy in the fields of neuroinflammatory inhibition and ischemic stroke protection, but are also widely used in food, dyes, cosmetics and other industries.

[0003] Homoisoflavonoids belong to a special subclass of flavonoids, and their parent skeleton is C6-C3-C1-C6 structure. Compared with ordinary isoflavones (C6-C3-C6), homoisoflavonoids have an additional carbon atom between the B ring and the C ring, which endows them with higher biological activity and structural diversity. According to the structure, homoisoflavonoids can be further divided into four categories: Brazilin-type, Sappanins-type, Caesalpin-type and Protosappanin-type. At present, nearly 30 homoisoflavonoid derivatives have been isolated from Caesalpinia sappan L., which is one of the most abundant species in leguminous plants. Among them, representative compounds such as Brazilin and Sappanone A have significant neuroprotective and anti-inflammatory effects, and are potential targets for modern natural drug development.

[0004] Although the pharmacological research of high isoflavones is relatively in-depth, the biosynthetic pathway thereof has not been fully elucidated. It has been shown in research that chalcone compounds, especially 2', 4', 4-trihydroxychalcone, are important precursors for the biosynthesis of high isoflavones. The key enzyme catalyzing the O-methylation reaction of such chalcones to generate 4, 4'-dihydroxy-2'-methoxychalcone is the core enzyme for synthesizing the skeleton of high isoflavones. However, the O-methyltransferase catalyzing this reaction in Caesalpinia sappan has not been systematically identified, and the enzyme activity, substrate specificity and transfer efficiency thereof still have technical gaps, which seriously limit the directional synthesis and medicinal development of high isoflavones. In traditional research, although similar glycyrrhizin 2'-O-methyltransferase (ChOMT) can catalyze 2', 4', 4-trihydroxychalcone to generate 2'-methoxy product in Escherichia coli, the catalytic efficiency thereof is limited and difficult to meet the requirements of industrial preparation. Therefore, developing new O-methyltransferase variants with high activity, especially the key enzymes from Caesalpinia sappan, is a key bottleneck and research focus for realizing the efficient biosynthesis of high isoflavones.

[0005] To solve the above problems, the present application identifies a gene CsOMT80 significantly related to methoxy chalcone synthesis from Caesalpinia sappan based on multi-omics analysis, and constructs and verifies a high-activity mutant CsOMT80G112R of the gene by means of structure prediction, active site analysis and site-directed mutagenesis, etc. The enzyme activity of the mutant is increased by more than 1278 times compared with that of the wild type, and exceeds the activity of ChOMT reported in the prior art, and for the first time realizes the efficient catalysis of glycyrrhizin to 2'-O-methyl glycyrrhizin, which significantly breaks through the key problems of low catalytic efficiency and poor biosynthesis efficiency in the prior art. SUMMARY

[0006] The present application aims to solve the problems of low synthesis efficiency of high isoflavone compounds and insufficient activity of key catalytic enzymes in the prior art, and provides a high-activity mutant of Caesalpinia sappan O-methyltransferase CsOMT80 gene and its application in chalcone compound synthesis, realizes efficient catalysis to generate 2'-O-methyl chalcone compounds, and promotes the directional synthesis and industrialization of high isoflavone natural products. In order to achieve the above-mentioned purpose, the present application provides the following technical solutions including but not limited to:

[0007] In one aspect, the present application provides a mutant of Caesalpinia sappan O-methyltransferase CsOMT80, the amino acid sequence of which is that the 112th position of SEQ ID NO: 1 is mutated from glycine (Gly, G) to arginine (Arg, R), i.e. G112R mutant, the mutant has enhanced O-methyltransferase activity and can catalyze glycyrrhizin to generate 2'-O-methyl glycyrrhizin.

[0008] The present invention also provides a gene encoding hematoxymethyltransferase CsOMT80, the nucleotide sequence of which is SEQ ID NO:2.

[0009] On the other hand, the present invention also provides a recombinant expression plasmid of the above-mentioned mutant, which is capable of expressing the G112R mutant protein in Escherichia coli.

[0010] On the other hand, the present invention also provides a method for preparing the mutant of claim 1, comprising the following steps:

[0011] (1) By predicting the structure of HelixFold3 and analyzing it with PyMOL, the 112th position was determined to be the activity enhancement site;

[0012] (2) The G112R mutation was introduced into the CsOMT80 gene using site-directed mutagenesis.

[0013] (3) Insert the mutated CsOMT80 gene into the expression plasmid and transform it into the E. coli expression system;

[0014] (4) The G112R mutant was obtained by induction expression, protein extraction and Ni-NTA purification.

[0015] According to the present invention, the above-mentioned site-directed mutagenesis adopts Hieff Mut. TM The Site-Directed Mutagenesis Kit uses isopropyl-β-D-thiogalactoside (IPTG) as the inducing agent, with a final concentration of 0.5 mmol / L.

[0016] On the other hand, the present invention also provides the use of the above-mentioned mutant in the biosynthesis of chalcone compounds, the chalcone compounds being 2'-O-methylisoglycyrrhizin, wherein the amino acid sequence of the mutant is SEQ ID NO:1, where position 112 is mutated from glycine (Gly, G) to arginine (Arg, R).

[0017] This invention utilizes structural biology modeling and functional site analysis, based on multi-omics data from *Caesalpinia sappan*, to identify the gene encoding the oxygen methyltransferase CsOMT80, which is closely related to the synthesis of methoxychalcone. The amino acid sequence was then structurally predicted and molecularly docked to screen for key sites affecting substrate binding and catalytic efficiency. Based on this, a site-directed mutagenesis strategy was employed to replace amino acids at the target sites, generating multiple mutants. Functional validation revealed that the mutant CsOMT80G112R exhibited significantly enhanced catalytic efficiency in the reaction catalyzed by isoliquiritigenin, with an enzyme activity 1278 times that of the wild type, and superior to previously reported alfalfa-derived O-methyltransferases (ChOMTs).

[0018] The CsOMT80G112R mutant provided by the application can efficiently catalyze the generation of 2'-O-methylisoliquiritigenin from isoliquiritigenin, can be used as a key enzyme tool for synthesizing the high-isoflavone precursor 2'-O-methylisoliquiritigenin, is suitable for application scenarios such as plant metabolic engineering modification, microbial synthesis platform construction, and preparation of high-isoflavone derivatives, and has important scientific research and industrialization values. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the present application and together with the description serve to explain the present application. In the drawings:

[0020] Figure 1 For SDS-PAGE analysis of the CsOMT80G112R protein of the application, the Maker is a double-color pre-stained protein Marker; FT is flow-through liquid; W is washing liquid; E1 and E2 are elution liquids.

[0021] Figure 2 For UPLC analysis of the CsOMT80G112R protein of the application using isoliquiritigenin as a substrate.

[0022] Figure 3 For the secondary mass spectrum of the product 2'-O-methylisoliquiritigenin catalyzed by the CsOMT80G112R protein of the application from isoliquiritigenin.

[0023] Figure 4 For the content diagram of the product 2'-O-methylisoliquiritigenin catalyzed by the CsOMT80G112R protein of the application from isoliquiritigenin. DETAILED DESCRIPTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials are described herein for use in the present application; other methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application.

[0025] Unless otherwise indicated, the materials, reagents, and the like used in the following examples are commercially available.

[0026] To make the objectives, technical solutions, and advantages of the present application clearer, further detailed descriptions will be given to the present application in combination with the accompanying drawings and specific examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0027] 1. Experimental materials and reagents

[0028] 1.1 Experimental materials

[0029] Based on the genome of Caesalpinia sappan and the transcriptome and metabolome data of each sample, the correlation between compounds and genes was calculated using R scripts to screen candidate genes for high isoflavone compounds in C. sappan. With r>0.85 and p<0.05 as the screening condition, combined with the prediction of the biosynthetic pathway, the gene CsOMT80 significantly associated with methoxy chalcone compound synthesis was mined as the orthologous gene of chalcone O-methyltransferase (ChOMT).

[0030] 1.2 Chemical reagents

[0031] The main reagents used in this experiment are shown in Table 1.1 below

[0032] Table 1.1 Reagents used for CsOMT80 gene activity modification

[0033]

[0034] 1.3 Instruments and equipment

[0035] Lanyan constant temperature metal bath; AB104-N electronic analytical balance; PCR instrument; Thermo nanodrop 2000 spectrophotometer; Sigma table top high-speed refrigerated centrifuge; D-37520 high-speed centrifuge; DK-S24 electric heating constant temperature water bath; GENSYS 10S UV-Vis ultraviolet spectrophotometer; ultrasonic cell crusher (Ningbo Xinzhi Biological Technology Co., Ltd.).

[0036] 1.4 Main solution formula

[0037] 1.4.1 LB medium

[0038]

[0039] 121℃ high pressure steam sterilization for 20min, and after cooling, store at 4℃. Liquid LB medium does not add agar.

[0040] 1.4.2 Preparation of antibiotic stock solution

[0041] Kanamycin (50mg·mL -1 )

[0042] Kanamycin (Kan) 500mg

[0043] ddH2O (autoclaved) 10mL

[0044] After dissolving, filter sterilization with 0.22μm microporous filter, aliquot to sterilized 2mL centrifuge tube, and store at -20℃.

[0045] 1.4.3 Preparation of protein inducer

[0046] Isopropyl-β-D-thiogalactopyranoside (1 mol / L)

[0047] Isopropyl-β-D-thiogalactopyranoside (IPTG) 2.38 g

[0048] ddH2O (autoclaved) 50 mL

[0049] After dissolving, filter sterilize with 0.22 μm microfiltration membrane, distribute to sterilized 2 mL centrifuge tube, and store at -20 °C.

[0050] 1.4.4 Buffer and formula required for soluble His-tag protein purification

[0051] (1) Equilibrium buffer

[0052] 50 mM NaH2PO4

[0053] 300 mM NaCl

[0054] 10 mM Imidazole

[0055] Adjust pH to 8.0 with NaOH, filter sterilize with 0.22 μm or 0.45 μm.

[0056] (2) Washing buffer

[0057] 50 mM NaH2PO4

[0058] 300 mM NaCl

[0059] 20 mM Imidazole

[0060] Adjust pH to 8.0 with NaOH, filter sterilize with 0.22 μm or 0.45 μm.

[0061] (3) Elution buffer

[0062] 50 mM NaH2PO4

[0063] 300 mM NaCl

[0064] 250 mM Imidazole

[0065] Adjust pH to 8.0 with NaOH, filter sterilize with 0.22 μm or 0.45 μm.

[0066] 2 Experimental methods

[0067] 2.1 Site-directed mutagenesis

[0068] 2.1.1 Selection of mutation site

[0069] HelixFold3 (https: / / paddlehelix.baidu.com / app / all / helixfold3 / forecast) was used to predict the binding of CsOMT80 protein to substrate ligands, protein catalytic functional sites, and protein pockets. The 3D model of the substrate ligands was visualized using PyMOL 3.1 (https: / / pymol.org / ) to identify the active amino acid sites for CsOMT80 candidate substrate binding and catalytic activity.

[0070] 2.1.2 Primer Design

[0071] Introducing a single-base site-directed mutation into a plasmid only requires designing a pair of primers to perform reverse PCR amplification of the plasmid.

[0072] Table 2.1 Primer sequences used for the G112R mutation site

[0073]

[0074] 2.1.3 Target plasmid amplification

[0075] 2.1.3.1 Preparation of PCR reaction system

[0076] Use Hieff Mut TM The Site-Directed Mutagenesis Kit was used for site-directed mutagenesis. After each reaction group was de-frozen, it was thoroughly shaken. To increase the specificity of amplification, the reaction system was prepared in an ice-water bath.

[0077] Table 2.2 PCR reaction system

[0078]

[0079] The PCR reaction program was as follows: pre-denaturation: 95℃ for 30s; 34 cycles (denaturation: 95℃ for 15s; annealing: 58℃ for 15s; extension: 72℃ for 1min); final extension: 72℃ for 5min; storage: 4℃.

[0080] 2.1.3.2 The amplified product was digested with DpnI to remove the methylated template plasmid.

[0081] Table 2.3 Reaction System

[0082]

[0083] After gently blowing and mixing, place the above reaction system at a constant temperature of 37°C for 1-2 hours.

[0084] 2.1.4 Recombination reaction

[0085] Table 2.4 Recombination Reaction System

[0086]

[0087] (1) The above system is placed at 50°C for 20 min;

[0088] (2) After the reaction is completed, the reaction tube is immediately placed in an ice water bath for cooling;

[0089] (3) Afterwards, the reaction product can be directly converted; or can be stored at -20°C and thawed for conversion when needed.

[0090] 2.1.5 Transformation of E. coli

[0091] The E. coli used for transformation is TOP10 Chemically Competent Cell from Invitrogen.

[0092] (1) 10 μl of the cooled reaction solution is taken and added to 100 μl of the competent cell, the tube is shaken several times and mixed, and is placed on ice for 30 min;

[0093] (2) 42°C heat shock for 45-90 s, and incubation in an ice water bath for 2 min;

[0094] (3) 700 μl of LB medium is added, and shaking culture is performed at 37°C, 200 rpm-250 rpm, for 1 h;

[0095] (4) Centrifugation is performed at 5000 rpm for 1 min to collect the bacteria, and about 100 μl of supernatant is taken, the bacterial mass is resuspended by gently blowing, and is plated on a plate containing Kan antibiotic. The plate is inverted and incubated at 37°C overnight.

[0096] 2.1.6 Screening of positive clones

[0097] A single colony on the bacterial growth plate is picked and placed in LB liquid containing the corresponding antibiotic, and is placed in a 37°C shaker at 200 rpm, and after 5 h, the turbid bacterial solution is taken for PCR verification, and the reaction system is shown in Table 2.5, and the PCR reaction procedure is as follows: pre-denaturation: 98°C for 2 min; 33 cycles of denaturation: 98°C for 10 s; annealing: 58°C for 15 s; extension: 72°C for 1 min; final extension: 72°C for 3 min; storage: 4°C.

[0098] 1 ml of the positive bacterial solution is taken and sent to Invitrogen Technology Co., Ltd. for sequencing verification.

[0099] Table 2.5 PCR verification reaction system

[0100]

[0101] 2.1.7 Extraction of positive clone plasmid

[0102] The high-purity plasmid DNA mini-extraction kit (Jinsha Biological) was used, and the kit instructions can be found on the Jinsha Biological website (https: / / genesand.com / ).

[0103] 2.2 Prokaryotic expression and purification of CsOMT80G112R

[0104] 2.2.1 Transformation of the target gene

[0105] The CsOMT80G112R plasmid was transformed into the ClearColi BL21(DE3) Competent Cell expression strain.

[0106] (1) The ClearColi BL21(DE3) competent cells were taken out from -80°C and quickly inserted into ice. After 5 min, the bacterial mass was melted, the target plasmid was added, and the EP tube bottom was gently mixed with hand (avoiding gun suction). The mixture was placed in ice for 30 min;

[0107] (2) 42°C water bath heat shock for 45 s, quickly put back on ice and stand for 2 min, shaking will reduce the transformation efficiency;

[0108] (3) Add 700 μl of LB without antibiotics to the centrifuge tube, mix well, and incubate at 37°C, 200 rpm for 60 min;

[0109] (4) Centrifuge at 5000 rpm for 1 min to collect the bacteria, take about 100 μl of supernatant, gently blow and resuspend the bacterial mass, and spread on LB medium containing the corresponding antibiotic. The plate was inverted and incubated at 37°C overnight;

[0110] (5) Pick single colonies and add to 1 mL of 1 mL LB liquid medium containing 50 mg / mL Kan, and incubate at 37°C, 200 rpm until the bacterial solution is turbid. The positive strain was added with 50% glycerol and stored at -80°C.

[0111] 2.2.2 Induced expression of the target protein

[0112] (1) Take 10 μL of CsOMT80G112R bacterial solution and add to 10 mL of LB liquid medium containing Kan, and incubate at 37°C, 200 rpm overnight;

[0113] (2) Take 2 mL of activated bacterial solution and add to 200 mL of LB liquid medium containing Kan, and incubate at 37°C, 200 rpm until the OD600 is about 0.6;

[0114] (3) Add IPTG inducer, and the final concentration of IPTG in the bacterial solution is 0.5 mM;

[0115] (4) 37℃ 200rpm shaking culture for 16h.

[0116] 2.2.3 Extraction of target protein

[0117] HisSep Ni-NTA Agarose Resin (His-tag protein purification resin) was used for protein purification.

[0118] (1) 4℃ 5000rpm centrifugation of the bacteria solution after IPTG induction for 30min, discard the supernatant, collect the bacteria;

[0119] (2) Resuspend with 10ml of equilibration buffer, then add lysozyme to make its working concentration 1mg / mL, and stand on ice for 30min;

[0120] (3) Use ultrasonic cell crusher, 6th amplitude rod, 28% power, crush for 2sec, pause for 5sec, continue for 5min.

[0121] (4) 4℃ 8000rpm centrifugation of the crushed bacteria solution for 20min, transfer the supernatant to the His-tag purification column, and incubate on a 4℃ shaking table for 16h:

[0122] (5) After incubation, filter and collect the filler, and retain the supernatant as the flow-through liquid for electrophoresis identification;

[0123] (6) Wash the filler with 5 times the volume of the filler of the wash buffer. Add 3-5 times the column volume of the elution buffer for elution, and collect the eluate with a gravity column tube to obtain the purified protein, repeat 3 times.

[0124] 2.2.4 SDS-PAGE electrophoresis analysis

[0125] (1) Use Omni-Easy TM One-step PAGE gel preparation kit (10%) to prepare electrophoresis gel, refer to the instructions of Yezyme 10% SDS-PAGE gel preparation kit;

[0126] (2) Take 20μL of supernatant, flow-through liquid and purified protein in a PCR tube, respectively add 4μL of 5× protein loading buffer, 95℃ for 10min, take out and stand on ice for 5min;

[0127] (3) Take 20μL of protein sample and load it on a 10% PAGE gel, 140V for 50min;

[0128] (4) Take out the gel, cut off the upper gel, and put the lower gel into the Coomassie brilliant blue fast staining solution, 20rpm shaking table staining for 30min, then decolorize with water overnight;

[0129] (5) The next day, observe the gel decolorization.

[0130] 2.2.5 Concentration and desalination of the target protein

[0131] According to the electrophoresis results, the purified eluate containing CsOMT80G112R was added to the ultrafiltration tube (30 kDa), centrifuged at 8000 rpm, 4°C, for 10 min, and concentrated to less than 1 mL in the ultrafiltration tube, and the lower flow was discarded. 10 mL of PBS buffer was added to the ultrafiltration tube, and centrifuged at 4000 rpm for 30 min, the flow was discarded, and repeated once. The remaining liquid in the ultrafiltration tube was collected as the concentrated protein solution, and stored at -80°C for subsequent experiments.

[0132] 2.3 In vitro enzyme function verification of CsOMT80G112R

[0133] (1) The in vitro enzyme reaction system is as shown in Table 2.6, the substrate is isoflavone (concentration 1 mg / ml), and the methyl donor is S-adenosyl methionine (SAM); the reaction is carried out at 37°C, 200 rpm for 30 min, and 500 μL of cold methanol is added to terminate the reaction after the reaction is completed;

[0134] Table 2.6 In vitro enzyme reaction system

[0135]

[0136] (2) After the reaction is completed, liquid nitrogen is used to quench the reaction;

[0137] (3) Under the condition that the temperature is not higher than 30°C, a 1200 rpm evaporation instrument is used to dry under reduced pressure for 3h, and the reaction liquid is evaporated;

[0138] (4) Resuspend with mass spectrometry grade methanol, ice water bath ultrasonic for 30 min, then vortex for 30 sec;

[0139] (5) 4°C, 12000 rpm centrifugation for 30 min, take 100 μL supernatant and store at -20°C.

[0140] 2.4 Detection of in vitro enzyme activity product

[0141] Qualitative determination of samples was performed by UHPLC-Q-TOF / MS. Chromatographic conditions: samples were separated by Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) C-18 column; column temperature 40℃; flow rate 0.4 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 0.5% formic acid, B: methanol; gradient elution program as follows: 0---0.5 min, 5% B; 0.5---10 min, B from 5% linearly changed to 100%; 10.0---12.0 min, B maintained at 100%; 12.0---12.1 min, B from 100% linearly changed to 5%; 12.1---16 min, B maintained at 5%; the sample was placed in the 4℃ automatic injector during the whole analysis process. To avoid the influence of instrument detection signal fluctuation, random order was used for continuous analysis of samples. Mass spectrometry conditions: AB Triple TOF 6600 mass spectrometer was used for sample primary and secondary spectrum acquisition. ESI source conditions after chromatographic separation as follows: Ion Source Gas1 (Gas1): 60, Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, source temperature: 600℃, Ion Sapary Voltage Floating (ISVF) ±5500V (both positive and negative modes); TOF MS scan m / z range: 60-1000 Da, production scan m / z range: 25-1000 Da, TOF MS scan accumulation time 0.20 s / spectra, product ion scan accumulation time 0.05 s / spectra; secondary mass spectrum was obtained by information dependent acquisition (IDA), and high sensitivity mode was used, Declustering potential (DP): ±60V (both positive and negative modes), Collision Energy: 35±15 eV, IDA settings as follows: Exclude isotopes within 4 Da, Candidate ions to monitor per cycle: 10. Mass spectrometry data was analyzed by MSDIAL (ver. 4.9.221218 Windows x64).

[0142] 3 Experimental results

[0143] 3.1 Expression and purification of CsOMT80G112R protein

[0144] The flow-through and purified protein from prokaryotic expression and purification were subjected to SDS-PAGE electrophoresis, and then stained with Coomassie brilliant blue staining solution and decolorized overnight, and the results are shown in Figure 1 The size of CsOMT80G112R protein is 45 kDa, and the size of His tag on pET-28a vector is 0.84 kDa, so the size of the recombinant protein after fusion of CsOMT80G112R protein and His tag is 45.84 kDa. As can be seen from the figure, there are bands at 45.84 kDa, and the next purification step can be performed to obtain CsOMT80G112R protein with higher purity for subsequent research.

[0145] 3.2 In vitro enzyme activity functional verification of CsOMT80G112R

[0146] The results of the in vitro enzyme activity experiment of CsOMT80G112R are shown in Figure 2 By comparing the in vitro enzyme activity product of CsOMT80G112R with the theoretical product 2'-O-methyl glycyrrhizin standard liquid chromatogram, it is found that the peak time of the product is similar to that of 2'-O-methyl glycyrrhizin. Compared with the control group of empty load, when glycyrrhizin is used as the substrate, the in vitro enzyme activity of CsOMT80G112R obtains the product 2'-O-methyl glycyrrhizin.

[0147] The in vitro catalytic experiment was carried out with the substrate glycyrrhizin. According to the m / z of the substrate glycyrrhizin being 255 [M-H], the mass-to-charge ratio increases by 14 for each substitution of a methyl group, and the LC-MS / MS results show that the in vitro enzyme activity of CsOMT80G112R obtains the product 2'-O-methyl glycyrrhizin with m / z of 269.04 [M-H] - . Figure 3

[0148] Compared with the wild-type CsOMT80, the catalytic activity of the G112R single-point mutant to the substrate is increased by 1278 times Figure 4 under the same reaction conditions, and the activity is not only significantly higher than that of the wild type, but also higher than that of its orthologous gene ChOMT. This result functionally proves that Gly112 is a key "activity gate" site of CsOMT80, thereby establishing G112R as a decisive mutation for the activity enhancement of CsOMT80.

[0149] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the technical principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A mutant of a CsOMT80 of a Broussonetia koenigii, characterized in that, The amino acid sequence of the mutant is that the 112th position of SEQ ID NO: 1 is mutated from glycine (Gly, G) to arginine (Arg, R), namely G112R mutant, which has enhanced oxygen methyltransferase activity and can catalyze the generation of 2'-O-methyl isoliquiritigenin from isoliquiritigenin.

2. The mutant according to claim 1, wherein The nucleotide sequence of the gene encoding the CsOMT80 of the sappan oxygen methyltransferase is SEQ ID NO:

2.

3. A recombinant expression plasmid comprising the mutant according to claim 1 or 2, wherein The plasmid can express the G112R mutant protein in E. coli.

4. A method of preparing the mutant of claim 1, wherein, The method comprises the following steps: (1) determining that the 112th position is an active enhancement site through HelixFold3 structure prediction and PyMOL analysis; (2) introducing the G112R mutation into the CsOMT80 gene by using site-directed mutagenesis technology; (3) inserting the mutated CsOMT80 gene into an expression plasmid and transforming it into an E. coli expression system; (4) obtaining the G112R mutant through induced expression, protein extraction and Ni-NTA purification.

5. The method of claim 4, wherein, The site-directed mutagenesis employs the Hieff Mut TM Site-Directed Mutagenesis Kit, and the inducible expression agent is isopropyl-β-D-thiogalactoside with a final concentration of 0.5 mmol / L.

6. Use of the mutant according to claim 1 in the biosynthesis of chalcone compounds, characterized in that, The chalcone compound is 2'-O-methyl isoliquiritigenin, and the amino acid sequence of the mutant is that the 112th position of SEQ ID NO: 1 is mutated from glycine (Gly, G) to arginine (Arg, R).

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