CN116622664A - Method for generating C-glycoside through biocatalysis - Google Patents

Method for generating C-glycoside through biocatalysis Download PDF

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CN116622664A
CN116622664A CN202310761371.0A CN202310761371A CN116622664A CN 116622664 A CN116622664 A CN 116622664A CN 202310761371 A CN202310761371 A CN 202310761371A CN 116622664 A CN116622664 A CN 116622664A
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mutant
qdp
genetically engineered
engineered bacterium
glycosyltransferase
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王健博
李敏
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Hunan Normal University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01047—N-Acylsphingosine galactosyltransferase (2.4.1.47)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Abstract

The invention discloses a method for generating C-glycoside by biocatalysis, belonging to the technical field of bioengineering. The glycosyltransferase is engineered through protein engineering to obtain mutants with higher yield and regioselectivity, the obtained mutant QDP is used as a biocatalyst, BP-2, 4-dihydroxypropiophenone, 4-fluoro-2 ',4' -dihydroxybenzophenone or 4' -hydroxyphenylheptanone are used as acceptor substrates, UDP-glucose is used as donor substrates, and the corresponding C-glucoside modification products are synthesized in a catalytic manner, so that the method has application value in the aspect of the synthesis of glucoside compounds.

Description

Method for generating C-glycoside through biocatalysis
Technical Field
The invention relates to a method for generating C-glycoside by biological catalysis, belonging to the technical field of biological engineering.
Background
Glycosides are a class of compounds that are widely found in nature, with diverse structures and wide range of physiological activities. The biological activity of the compounds is determined by glycosyl groups in the structure to a great extent, so that a method capable of catalyzing glycosylation of corresponding aglycone with high efficiency is developed, and the method is a focus of attention of organic chemists and pharmaceutical chemists. The glycosylation reaction in natural product biosynthesis is catalyzed by glycosyltransferases (GTs; EC 2.4.) which catalyze the transfer of sugar molecules from an activated glycosyl donor to a substrate acceptor, and directly construct the glycosylation product in a simple manner in one step, avoiding the lengthy protection-deprotection steps in chemical methods, so that the enzymes are widely accepted by scientists as powerful glycosylation tools.
Most glycoside natural products are O-glycosylation modification products, and besides glycoside compounds also comprise C-, N-, S-glycosylation products. From a chemical point of view, the C-glycoside imparts a longer in vivo half-life to the corresponding glycosylation product due to its remarkable resistance to spontaneous, acid and enzyme-catalyzed hydrolysis. However, the presence of relatively few GTs in nature that have the activity of catalyzing the formation of C-glycosidic bonds, and the range of substrates identified is relatively narrow, and the catalytic efficiency is mostly relatively low, which limits their practical application.
Disclosure of Invention
The invention aims to improve productivity and chemoselectivity by modifying glycosyltransferase MicGT derived from Indian mango (Mangiferandica) through protein engineering means. The invention provides a method for synthesizing different C-glucosides by biocatalysis by utilizing glycosyltransferase mutant QDP (E152Q/V190D/S122P). The method has the advantages of mild condition, environmental friendliness, catalytic synthesis of glycosylated products with different chemical selectivities and the like.
The first object of the invention is to provide a glycosyltransferase MiCGT mutant QDP, which is characterized in that 152 th mutant glutamic acid of an amino acid sequence shown in SEQ ID NO.1 is glutamine, 190 th valine is aspartic acid, and 122 th serine is proline.
In one embodiment of the invention, the nucleotide sequence encoding the glycosyltransferase is shown in SEQ ID NO. 2.
A second object of the present invention is to provide a gene encoding the above glycosyltransferase MiCGT mutant QDP.
It is a third object of the present invention to provide an expression vector containing the gene.
In one embodiment of the invention, the expression vector includes, but is not limited to, pET series vectors, pRSF series vectors, or pCDF series vectors.
The fourth object of the present invention is to provide a genetically engineered bacterium expressing the mutant.
In one embodiment of the invention, the host cell of the genetically engineered bacterium includes, but is not limited to, E.coli.
In one embodiment of the present invention, the construction of the genetically engineered bacterium specifically includes the following steps: and (3) connecting a gene encoding the mutant with a vector by taking pET28a as the vector, and recombining and expressing the glycosyltransferase mutant in E.coli BL21 (DE 3).
In one embodiment of the invention, the components of the fermentation medium are: TB medium, inducer is 4-6g/L alpha-lactose monohydrate, preferably the temperature is 18-25 ℃, the rotating speed is 160-200 rpm, and the expression time is 18-20h.
In one embodiment of the invention, about 0.05% glucose is added at the time of inoculation.
In one embodiment of the invention, the protein purification of the genetically engineered bacterium specifically comprises the following steps: collecting the expressed thalli by a precooling centrifuge, washing with a buffer solution twice, adding a small amount of lysozyme, quick freezing with liquid nitrogen, thawing with ice water bath, ultrasonic crushing, centrifuging at high speed, and adopting His-nickel column affinity purification and desalting by a desalting column.
It is a fifth object of the present invention to provide a method for synthesizing various C-glycosides using the glycosyltransferase MiCGT mutant QDP of the present invention.
In one embodiment of the present invention, the method is performed in a range of 40-60mM NaH 2 PO 4 -Na 2 HPO 4 In the buffer system, enzyme amount of 40-100 μg/100 μl or corresponding whole cells (OD 600nm =40-60), pH=7.0-8.0, acceptor substrate concentration 0.1-5mM, UDP-glucose concentration 0.1-10mM or 2% glucose at 30-40 ℃, reactionThe rotation speed is 800-1200rpm, and the reaction time is 2-6h.
The sixth object of the present invention is to provide the use of the mutant, or the gene, or the vector, or the genetically engineered bacterium, or the method for producing the mutant, or the method for synthesizing C-glycoside in the fields of food, pharmaceutical, etc.
The beneficial effects are that:
the invention uses glycosyltransferase MiCGT from Indian mango (Mangiferandica) to produce mangiferin, and carries out protein engineering modification, and the obtained mutant QDP (E152Q/V190D/S122P) is used as a biocatalyst to catalyze BP-2, 4-dihydroxypropiophenone, phloretin or 2-phenyl-2 ',4',6' -trihydroxyacetophenone to generate a corresponding C-glucoside modified product.
(1) Compared with the wild enzyme MiCGT, the mutant QDP (E152Q/V190D/S122P) can obtain the C-glucoside modified product with high chemical selectivity, the conversion rate is improved to over 99 percent from nearly 3 percent, TON is improved to 10375 from 136, the specific enzyme activity is improved to 939.4U/mg from 135.8U/mg, and gram-scale preparation (1.2 g) of the C-glucoside modified product with high chemical selectivity is realized in a 1.5L reaction system.
(2) 2, 4-dihydroxypropiophenone is used as a substrate, a C-glucoside modified product with high chemical selectivity can be obtained, and the conversion rate is improved from <3% to 54%; the phloretin is used as a substrate, a C-glucoside modified product with high chemical selectivity can be obtained, and the conversion rate is improved from 17% to more than 99%; the 2-phenyl-2 ',4',6' -trihydroxyacetophenone is used as a substrate, so that a C-glucoside modified product with high chemical selectivity can be obtained, and the conversion rate is improved from 11% to over 99%.
Drawings
Fig. 1: HPLC chromatogram (A) and bar graph (B) of the reaction mixture of BP-2 with mutant QDP and wild-type of glycosyltransferase MiCGT.
Fig. 2: primary mass spectrum and secondary mass spectrum of products 1a and 1b generated by the reaction of BP-2 and mutant QDP; a) 1a MS; b) 1a MS 2 The method comprises the steps of carrying out a first treatment on the surface of the C) 1 b; d) 1b MS 2 。
Fig. 3: HPLC chromatograms (A) and bar charts (B) of the reaction mixtures of 2, 4-dihydroxypropiophenone with mutant QDP and wild-type of glycosyltransferase MiCGT.
Fig. 4: primary mass spectrum and secondary mass spectrum of products 2a and 2b generated by the reaction of 2, 4-dihydroxypropiophenone and mutant QDP; a) 2a MS; b) 2a MS 2 The method comprises the steps of carrying out a first treatment on the surface of the C) 2b MS; d) 2b MS 2 。
Fig. 5: HPLC chromatogram (A) and bar graph (B) of the reaction mixture of phloretin and mutant QDP and wild type of glycosyltransferase MiCGT.
Fig. 6: primary mass spectrum and secondary mass spectrum of products 3a and 3b generated by the reaction of phloretin and mutant QDP; a) 3a MS; b) 3a MS 2 The method comprises the steps of carrying out a first treatment on the surface of the C) 3b MS; d) 3b MS 2 。
Fig. 7: HPLC chromatograms (A) and bar charts (B) of the reaction mixtures of 2-phenyl-2 ',4',6' -trihydroxyacetophenone with mutant QDP and wild-type glycosyltransferase MiCGT.
Fig. 8: primary mass spectrum and secondary mass spectrum of products 4a and 4b generated by the reaction of 2-phenyl-2 ',4',6' -trihydroxyacetophenone and mutant QDP; a) 4a MS; b) MS of 4a 2 The method comprises the steps of carrying out a first treatment on the surface of the C) 4b MS; d) 4b MS 2 。
Detailed Description
The chemicals used in the present invention are commercially available.
The medium formulation used in the examples is as follows:
solid medium formulation (1L): 5g of yeast extract, 10g of peptone, 10g of sodium chloride and 15g of agar, and sterilizing by deionized water and high-pressure steam.
LB medium formulation (1L): 5g of yeast extract powder, 10g of peptone and 10g of sodium chloride, and sterilizing by deionized water and high-pressure steam.
TB Medium formulation (1L): 24g of yeast extract, 12g of peptone and 4mL of glycerol. Adding 900mL deionized water, sterilizing with high pressure steam after dissolution, and adding 100mL 0.17MKH after the same sterilization 2 PO 4 /0.72MK 2 HPO 4 Is a solution of (a) and (b). ( And (3) injection: sodium chloride was purchased from Alatine and the remainder from Biotechnology Co., ltd )
The product analysis method comprises the following steps: high performance liquid chromatography and mass spectrometry. The corresponding liquid phase detection method is a 5-mu mC18 column, the PDA detector, the mobile phase is: phase A (H) 2 O contained 0.1% formic acid) and phase B (methanol contained 0.1% formic acid). The gradient elution procedure was: 10-50% B8min, 50-85% B2min,85% B3min, 80-10% B7min.
Conversion number (TurnoverNumber, TON) definition: indicating the number of catalytic reactions per unit time, per unit active site, or the number of target products formed or the number of reactants consumed at a given temperature, pressure, reactant ratio and degree of reaction.
Conversion number (TurnoverNumber, TON) calculation: at 50mM NaH 2 PO 4 -Na 2 HPO 4 To (pH 8.0), 3/4/5mM of the substrate and purified MiCGT mutants QDP were added at different concentrations, respectively, at 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 3. Mu.M, and reacted at 40℃for 24 hours in a total volume of 100. Mu.L. The reaction was terminated by adding 300. Mu.L of glacial methanol, and detected by liquid chromatography. TON is calculated as the amount of substrate converted/amount of enzyme.
Enzymatic activity assay of glycosyltransferase: the activity of the glycosyltransferase was determined in 200. Mu.L of reaction buffer, which included 6mM DP-glucose, 50mM NaH 2 PO 4 -Na 2 HPO 4 (pH 8.0) and about 1. Mu.g of the mutant purified enzyme. Incubation was carried out at 50℃for 5 minutes, after which the reaction was stopped by adding the same volume of methanol and analyzed by HPLC. One unit of enzyme activity is defined as the amount of enzyme that consumes 1. Mu. Mol of acceptor substrate or generates 1. Mu. Mol of product for 1 min.
Protein content detection: protein absorption at 280nm was measured using a Nano-300 micro-spectrophotometer.
Calculating the specific enzyme activity: specific activity=1u· (mg protein) -1 =1 μmol· (min mg protein) -1 。
Calculation of conversion: conversion = amount of converted feedstock/total amount of feedstock 100%.
Example 1: glycosyltransferase MiCGT protein engineering
(1) Design and preparation of mutants:
the gene with the sequence shown as SEQ ID NO.2 is taken as a template, primers are designed, and mutation is carried out on the position points S122, E152 and V190, so that the coding gene of the QDP (E152Q/V190D/S122P) mutant is obtained.
TABLE 1QDP mutant primers
(2) Construction of recombinant plasmids:
using commercial VazymeThe coding gene of the mutant prepared in the step (1) is connected with a pET28a vector by a MultiSOneStepclaning kit to obtain a recombinant vector pET28a-MiCGT mutant Specific steps are shown in the specification. The wild type gene shown in SEQ ID NO.2 is connected with a pET28a vector by the same method to obtain a recombinant vector pET28a-MiCGT.
Example 2: expression purification of glycosyltransferase MiCGT mutant QDP
The recombinant vector pET28a-MiCGT constructed in example 1 mutant And pET28a-MiCGT are respectively transformed into competent cells Rosetta-gamiB (DE 3) through chemical transformation, a proper amount of bacterial liquid is taken to be coated on a solid culture medium containing kanamycin and chloramphenicol, and the culture is carried out for 12-15h at 37 ℃. Single colonies containing the transformed recombinant plasmid were picked up separately into LB medium (containing 35. Mu.gmL) -1 Chloramphenicol and 50. Mu.gmL -1 Kanamycin), and incubated overnight at 37 ℃.1% LB cultures were inoculated into TB medium (containing 35. Mu.g mL) -1 Chloramphenicol, 50. Mu.gmL -1 Kanamycin and 0.05% glucose sterilized by film coating), then culturing at 37 ℃ until the growth logarithmic phase, namely OD value is 0.6-0.8, adding 5g/L alpha-lactose monohydrate as inducer, and transferring to 25 ℃ and expressing for 18-20h under the condition of 160 rpm.
The expressed cells were collected with a centrifuge pre-chilled at 4℃and treated with 50mM NaH (pH=8.0) containing 10% glycerol 2 PO 4 -Na 2 HPO 4 Washing with buffer solution twice, adding small amount of lysozyme, and collecting solutionQuick freezing with nitrogen, thawing in ice water bath, ultrasonic crushing, high speed centrifugation, affinity purification with His-nickel column (eluting the protein with 50mM Tris-HCl buffer containing 10% glycerol and 80mM imidazole, eluting the target protein with 50mM Tris-HCl buffer containing 10% glycerol and 500mM imidazole), and desalting with desalting column (desalting buffer is 50mM NaH containing 10% glycerol) 2 PO 4 -Na 2 HPO 4 Buffer solution). The specific activity of the mutant QDP to BP-2 is 939.4U/mg, the specific activity of the wild enzyme to BP-2 is 135.8U/mg, and the specific activity of the QDP is improved by 7 times compared with the specific activity of the wild enzyme to BP-2. The mutant QDP was determined to have a TON of 10375 for BP-2, 136 for the wild-type enzyme and 76-fold higher than the TON of BP-2 for the wild-type enzyme.
Example 3: glycosyltransferase MiCGT and mutant QDP thereof for catalyzing BP-2 to synthesize C-glucoside
In a 100. Mu.L reaction system, UDP-glucose was at a concentration of 4mM, substrate 2,2', 4' -tetrahydroxybenzophenone (BP-2) was at a concentration of 3mM,50mM NaH 2 PO 4 -Na 2 HPO 4 (pH 8.0), DMSO (v/v, 5%), final concentration of biocatalyst pure enzyme mutant QDP was 54. Mu.g, and the corresponding glycosylation product was obtained by 2h reaction at 30℃with wild enzyme as control. The reaction was terminated by adding 3 volumes of glacial methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
As can be seen from FIGS. 1 and 2, the mutant QDP catalyzes BP-2 to obtain a C-glucoside product with a conversion rate of >99%. And the wild enzyme obtains a more mixed glycoside product, and the conversion rate is 3%. The mutant has higher chemical selectivity than the wild enzyme and greatly improves the conversion rate.
1.1g of substrate 2,2', 4' -tetrahydroxybenzophenone (BP-2) 50mM NaH in a 1.5L reaction system 2 PO 4 -Na 2 HPO 4 (pH 8.0), DMSO (v/v, 5%), biocatalyst is whole cell containing mutant QDP, and final catalyst concentration is OD 600 =40, reacted at 30 ℃ for 24h. Subsequently, the sample was centrifuged at 4,000g for 5 minutes and the supernatant was collected. With 20ml ddH 2 The cells were washed and centrifuged 2 times. Filtering supernatant, performing primary purification and concentration by macroporous resin column chromatography, and performing silica gel column chromatographyFurther purification and isolation gave 1.2g of C-glycoside product.
Example 4: glycosyltransferase MiCGT and mutant QDP thereof for catalyzing 2, 4-dihydroxypropiophenone to synthesize C-glucoside
In a 100. Mu.L reaction system, UDP-glucose was at a concentration of 4mM, substrate 2, 4-dihydroxypropiophenone was at a concentration of 3mM,50mM NaH 2 PO 4 -Na 2 HPO 4 (pH 8.0), DMSO (v/v, 5%), final concentration of biocatalyst pure enzyme mutant QDP was 54. Mu.g, and the corresponding glycosylation product was obtained by 2h reaction at 30℃with wild enzyme as control. The reaction was terminated by adding 3 volumes of glacial methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
As can be seen from FIGS. 3 and 4, the mutant QDP catalyzes 2, 4-dihydroxypropiophenone to obtain a C-glucoside product with a conversion rate of 54%. And the wild enzyme obtains more mixed glycoside products, and the conversion rate is less than 3%. The mutant has higher chemical selectivity than the wild enzyme and greatly improves the conversion rate.
Example 5: glycosyltransferase MiCGT and mutant QDP thereof for catalyzing synthesis of C-glucoside by 4-fluoro-2 ',4' -dihydroxybenzophenone
In a 100. Mu.L reaction system, UDP-glucose was at a concentration of 4mM, substrate phloretin (4-fluoro-2 ',4' -dihydroxybenzophenone) was at a concentration of 3mM,50mM NaH 2 PO 4 -Na 2 HPO 4 (pH 8.0), DMSO (v/v, 5%), final concentration of biocatalyst pure enzyme mutant QDP was 54. Mu.g, and the corresponding glycosylation product was obtained by 2h reaction at 30℃with wild enzyme as control. The reaction was terminated by adding 3 volumes of glacial methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
As can be seen from fig. 5 and 6, the mutant QDP catalyzes 4-fluoro-2 ',4' -dihydroxybenzophenone to obtain C-glucoside product with a conversion rate of >99%. Whereas the wild-type enzyme can give a C-glucoside product, the conversion is only 17%. Compared with wild enzyme, the mutant can obtain products with different chemoselectivities, and the conversion rate is greatly improved.
Example 6: glycosyltransferase MiCGT and mutant QDP thereof for catalyzing synthesis of C-glucoside by 4' -hydroxy-phenylheptanone
U in 100. Mu.L of the reaction systemDP-glucose concentration was 4mM, substrate 4' -hydroxyphenylheptanone concentration was 3mM,50mM NaH 2 PO 4 -Na 2 HPO 4 (pH 8.0), DMSO (v/v, 5%), final concentration of biocatalyst pure enzyme mutant QDP was 54. Mu.g, and reaction at 30℃for 2h gave the corresponding glycosylation product. The reaction was terminated by adding 3 volumes of glacial methanol, and analyzed by high performance liquid chromatography and mass spectrometry.
As can be seen from FIGS. 7 and 8, the mutant QDP catalyzes 4' -hydroxyphenylheptanone to obtain a C-glucoside product, and the conversion rate is more than 99%. Whereas the wild-type enzyme can give a C-glucoside product, the conversion is only 11%. Compared with wild enzyme, the mutant can obtain products with different chemoselectivities, and the conversion rate is greatly improved.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (8)

1. A mutant of glycosyltransferase MiCGT, characterized in that glutamic acid at position 152 of the amino acid sequence shown in SEQ ID No.1 is mutated to glutamine, valine at position 190 is mutated to aspartic acid, and serine at position 122 is mutated to proline.
2. A gene encoding the mutant of claim 1.
3. An expression vector comprising the gene of claim 2, wherein the expression vector comprises a pET series vector, a pRSF series vector, or a pCDF series vector.
4. The genetically engineered bacterium expressing the mutant of claim 1, wherein the host cell of the genetically engineered bacterium comprises escherichia coli.
5. A method for producing the mutant according to claim 1, wherein the genetically engineered bacterium according to claim 4 is fermented.
6. The method of claim 5, wherein an inducer is added during the fermentation process, the inducer being 4-alpha-lactose or IPTG.
7. A method for synthesizing C-glycoside, characterized in that UDP-glucose or glucose is used as glycosyl donor, the mutant of claim 1 or the genetically engineered bacterium of claim 4 is used as biocatalyst, and BP-2, 4-dihydroxypropiophenone, phloretin or 2-phenyl-2 ',4',6' -trihydroxyacetophenone is used as acceptor for reaction.
8. The mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3, the genetically engineered bacterium according to claim 4, the method according to claim 5 or 6, or the use of the method according to claim 7 in the fields of food, pharmaceuticals and the like.
CN202310761371.0A 2023-06-27 2023-06-27 Method for generating C-glycoside through biocatalysis Pending CN116622664A (en)

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