Glycosyltransferase mutant and use thereof

Abstract

A glycosyltransferase mutant and the use thereof. The glycosyltransferase mutant comprises: (a) a protein having an amino acid sequence represented by SEQ ID NO: 1; or (b) a protein having a glycosyltransferase function and undergone amino acid mutation at at least one of the following sites R56, G126, V216, V223, V306 or S386 in the amino acid sequence of (a); or (c) a protein having a glycosyltransferase function and having homology of 80% or above with the amino acid sequence defined in any one of (a) and (b). The present invention solves the problem of low activity of glycosyltransferases in the prior art, has an excellent property high activity, and is suitable for the technical field of biology.

Description

A glycosyltransferase mutant and application thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202410638089.8, filed on May 22, 2024, the disclosure of which is incorporated herein in its entirety by reference as part of the present application. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, in particular, to a glycosyltransferase mutant and application thereof. BACKGROUND

[0004] Salidroside (2-(4-Hydroxyphenyl)ethyl-betta-D-glucopyranoside) has a molecular formula of C 14 H 20 O7, and a structural formula of Salidroside is a phenylethanoid glycoside compound derived from plants, which has functions of anti-fatigue, anti-hypoxia, anti-aging, anti-cancer, anti-inflammatory, protection of cardiovascular and central nervous system, etc., and is the main active ingredient of many medicinal plants such as Rhodiola, Ligustri Lucidi Fructus, Aconitum tanguticum and the like. In recent years, salidroside has been widely used in the pharmaceutical, food and health product industries, and there has been no report on the side effects or drug interactions of salidroside.

[0005] Currently, salidroside is mainly obtained by extraction from the plant Rhodiola, but it is limited by the special high-altitude growth environment of Rhodiola, the low content of salidroside in the body, and the scarcity of plant resources due to overexploitation. Chemical synthesis method requires complex hydroxyl protection and deprotection steps and expensive catalysts, and has poor regioselectivity. Therefore, developing a mild and efficient biological method to synthesize salidroside is conducive to reducing production costs and expanding market value.

[0006] Glycosyltransferase (Glycosyltransferase) has an enzyme classification number of EC (2.4.X.X), which catalyzes the transfer of a sugar group from an activated donor molecule to a specific aglycone, and can change the hydrophilicity, stability and subcellular localization of natural products, thereby further improving the chemical properties and biological activities. It is a key catalytic enzyme for the biosynthesis of glycoside compounds. Although a variety of glycosyltransferases that can catalyze the synthesis of salidroside have been identified, there is still room for improvement in their specificity and catalytic activity. Therefore, it is essential to use protein engineering to obtain efficient and specific glycosyltransferases through directed evolution, which is crucial for improving the yield of salidroside and reducing the subsequent separation cost.

[0007] SUMMARY

[0008] The main object of the present application is to provide a glycosyltransferase mutant and application thereof, so as to solve the problem of low catalytic activity of glycosyltransferase in synthesizing salidroside in the prior art.

[0009] In order to achieve the above object, according to a first aspect of the present application, a glycosyltransferase mutant is provided, comprising: (a) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having an amino acid sequence in (a) which is mutated at at least one of the following positions: R56, G126, V216, V223, V306, S386, I15, Y17, A19, N24, P25, N28, K31, T45, N48, S55, A83, T84, Q85, I87, P88, C91, S93, T94, T95, S124, A127, S129, T148, C151, G152, G155, T157, Y159, F202, R204, I211, N214, R218, E219, E221, A222, L258, M261, M262, H270, S274, S275, S276, E280, L282, S303, A305, V307, D333, M355, S358, E364, S378, N381, T383, C395, F399, Q403, N405, C406, R407, S409 or N421, and has a glycosyltransferase function; or (c) a protein having 80% or more homology with the amino acid sequence defined in any one of (a) and (b) and having a glycosyltransferase function.

[0010] Further, the amino acid mutations of (b) above are each independently selected from the group consisting of R56F or R56L or R56W or R56C or R56A, G126F or G126W or G126Y, V216F or V216W or V216Y or V216H, V223A or V223L or V223R, V306D or V306E or V306K or V306R or V306H or V306T, S386A or S386G or S386Q or S386R, I15F or I15L or I15V, Y17F or Y17A or Y17L or Y17R or Y17W, A19M, N24A or N24L or N24V, P25A or P25L or P25V, N28L or N28M or N28W, K31L or K31M, T45F or T45Y, N48F or N48M or N48Y, S55A or S55H or S55W, A83F or A83H or A83Y, T84M, Q85F or Q85L, I87L or I87W or I87Y, P88I or P88K or P88T or P88V, C91F or C91H or C91K or C91Y, S93A or S93F or S93S or S93V, T94L or T94V, T95F or T95Q or T95Y, S124A or S124C or S124V, A127E or A127F or A127M or A127W or A127Y, S129E or S129F or S129K or S129R or S129W or S129Y, T148E or T148I or T148R or T148V or T148Y, C151A or C151K or C151R, G152A, G155A or G155H or G155R, T157L or T157M, Y159F, F202I, F202Q or F202W, R204F or R204K or R204W, I211K or I211P, N214L or N214M or N214W, R218F or R218H or R218W or R218Y, E219L or E219Q or E219V, E221L or E221R or E221W, A222L or A222M or A222Y, L258F, M261L or M262I, M262L or M262V, H270V, S274C or S274I or S274V, S275F or S275W or S275Y, S276I or S276L, E280L or E280P or E280W, L282Y, S303E or S303L or S303T or S303D, A305P, V307P, D333F or D333W or D333Y, M355F or M355L or M355W, S358H or S358L or S358R, E364L, S378C or S378L or S378M, N381L or N381Q or N381W,T383I or T383L or T383V, C395F or C395H or C395M, F399D or F399G or F399Y, Q403F or Q403S or Q403V or Q403W, N405I or N405L or N405V, C406A or C406M or C406T, R407F or R407W or R407Y, S409I or S409L or S409M or S409T or N421H or N421M or N421W or N421Y; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid; preferably, the above-mentioned protein having 85% or more, preferably 90% or more, more preferably 95% or more, further preferably 99% or more homology with the above-mentioned amino acid sequence defined in (a) or (b) and having a glycosyltransferase function in (c) above.

[0011] Further, the mutation of the above-mentioned glycosyltransferase mutant includes any one of I15F, I15L, I15V, Y17F, Y17A, Y17L, Y17R, Y17W, A19M, N24A, N24L, N24V, P25A, P25L, P25V, N28L, N28M, N28W, K31L, K31M, T45F, T45Y, N48F, N48M, N48Y, S55A, S55H, S55W, R56F, R56L, R56W, R56C, R56A, A83F, A83H, A83Y, T84M, Q85F, Q85L, I87L, I87W, I87Y, P88I, P88K, P88T, P88V, C91F, C91H, C91K, C91Y, S93A, S93F, S93S, S93V, T94L, T94V, T95F, T95Q, T95Y, S124A, S124C, S124V, G126F, G126W, G126Y, A127E, A127F, A127M, A127W, A127Y, S129E, S129F, S129K, S129R, S129W, S129Y, T148E, T148I, T148R, T148V, T148Y, C151A, C151K, C151R, G152A, G155A, G155H, G155R, T157L, T157M, Y159F, F202I, F202Q, F202W, R204F, R204K, R204W, I211K, I211P, N214L, N214M, N214W, V216F, V216W, V216Y, V216H, R218F, R218H, R218W, R218Y, E219L, E219Q, E219V, E221L, E221R, E221W, A222L, A222M, A222Y, V223A, V223L, V223R, L258F, M261L, M262I, M262L, M262V, H270V, S274C, S274I, S274V, S275F, S275W, S275Y, S276I, S276L, E280L, E280P, E280W, L282Y, S303E, S303L, S303T, S303D, A305P, V306D, V306E, V306K, V306R, V306H, V306T, V307P, D333F, D333W, D333Y, M355F, M355L, M355W, S358H, S358L, S358R, E364L, S378C, S378L, S378M, N381L, N381Q, N381W, T383I, T383L, T383V, S386A,S386G, S386Q, S386R, C395F, C395H, C395M, F399D, F399G, F399Y, Q403F, Q403S, Q403V, Q403W, N405I, N405L, N405V, C406A, C406M, C406T, R407F, R407W, R407Y, S409I, S409L, S409M, S409T, N421H, N421M, N421W, N421Y, R56C+G126F, R56C+V216W, R56C+V216H, R56A+V216W, R56A+V216H, R56F+V216W, R56F+V216H, R56C+V223A, R56C+V223L, R56C+V223R, R56A+V223A, R56A+V223L, R56A+V223R, R56F+V223A, R56F+V223L, R56F+V223R, R56C+V306K, R56C+V306H, R56C+V306T, R56A+V306K, R56A+V306H, R56A+V306T, R56F+V306K, R56F+V306H, R56F+V306T, R56C+S386G, R56C+S386R, R56A+S386G, R56A+S386R, R56F+S386G, R56F+S386R, G126F+V216W, G126F+V216H, G126F+V223A, G126F+V223L, G126F+V223R, G126F+V306K, G126F+V306H, G126F+V306T, G126F+S386G, G126F+S386R, V216W+V223A, V216W+V223L, V216W+V223R, V216H+V223A, V216H+V223L, V216H+V223R, V216W+V306K, V216W+V306H, V216W+V306T, V216H+V306K, V216H+V306H, V216H+V306T, V216W+S386G, V216W+S386R, V216H+S386G, V216H+S386R, V223A+V306K, V223A+V306H, V223A+V306T, V223L+V306K, V223L+V306H, V223L+V306T, V223R+V306K, V223R+V306H, V223R+V306T, V223A+S386G, V223A+S386R, V223L+S386G, V223L+S386R, V223R+S386G,V223R+S386R, V306K+S386G, V306K+S386R, V306H+S386G, V306H+S386R, V306T+S386G, V306T+S386R, R56F+G126F+V216W, R56F+G126F+V223L, R56F+G126F+S386G, R56F+G126F+V306H, R56F+V216W+V223L, R56F+V216W+S386G, R56F+V216W+V306H, R56F+V223L+S386G, R56F+V223L+V306H, R56F+S386G+V306H, G126F+V216W+V223L, G126F+V216W+S386G, G126F+V216W+V306H, G126F+V223L+S386G, G126F+V223L+V306H, G126F+S386G+V306H, V216W+V223L+S386G, V216W+V223L+V306H, V216W+S386G+V306H, V223L+S386G+V306H, V223L+S386R+V306H, V223A+S386G+V306H, V223A+S386R+V306H, R56F+G126F+V223A, R56F+G126F+S386R, R56F+G126F+V306T, R56F+V216W+V223A, R56F+V216W+S386R, R56F+V216W+V306T, R56F+V223L+S386R, R56F+V223L+V306T, R56F+S386G+V306T, G126F+V216W+V223A, G126F+V216W+S386R, G126F+V216W+V306T, G126F+V223L+S386R, G126F+V223L+V306T, G126F+S386G+V306T, V216W+V223L+S386R, V216W+V223L+V306T, V216W+S386G+V306T, V223L+S386G+V306T, V223L+S386R+V306T, V223A+S386G+V306T, V223A+S386R+V306T, R56F+G126F+V306K, R56F+V216W+V306K, R56F+V223A+S386G, R56F+V223L+V306K, R56F+S386G+V306K, G126F+V216W+V306K, G126F+V223A+S386G,G126F+V223L+V306K, G126F+S386G+V306K, V216W+V223A+S386G, V216W+V223L+V306K, V216W+S386G+V306K, V223L+S386G+V306K, V223L+S386R+V306K, V223A+S386G+V306K, V223A+S386R+V306K, R56F+V223A+S386R, R56F+V223A+V306H, R56F+S386R+V306H, G126F+V223A+S386R, G126F+V223A+V306H, G126F+S386R+V306H, V216W+V223A+S386R, V216W+V223A+V306H, V216W+S386R+V306H, R56F+V223A+V306T, R56F+S386R+V306T, G126F+V223A+V306T, G126F+S386R+V306T, V216W+V223A+V306T, V216W+S386R+V306T, R56F+V223A+V306K, R56F+S386R+V306K, G126F+V223A+V306K, G126F+S386R+V306K, V216W+V223A+V306K, or V216W+S386R+V306K.

[0012] To achieve the above object, according to a second aspect of the present application, there is provided a DNA molecule encoding the above-mentioned glycosyltransferase mutant.

[0013] To achieve the above object, according to a third aspect of the present application, there is provided a recombinant plasmid to which the above-mentioned DNA molecule is ligated.

[0014] To achieve the above object, according to a fourth aspect of the present application, there is provided a host cell into which the above-mentioned recombinant plasmid is transformed; further, the host cell is E. coli or yeast.

[0015] To achieve the above object, according to a fifth aspect of the present application, there is provided a method for producing salidroside, the method comprising catalyzing reaction using the above-mentioned glycosyltransferase mutant with 4-hydroxyphenethyl alcohol as a substrate to obtain the above-mentioned salidroside.

[0016] Further, the method comprises inoculating the host cells cultured overnight into a fermentation medium, culturing at 30-37℃, 200-220rpm to OD600 of 0.6-0.8, adding an inducer, and inducing the culture for 12-24h to obtain the rhodiolin; wherein the host cells are transformed with a recombinant plasmid, the recombinant plasmid is linked with a DNA molecule, and the DNA molecule encodes the glycosyltransferase mutant.

[0017] Further, the inducer is IPTG; and the final concentration of the inducer is 0.1-1mM.

[0018] Further, the fermentation medium formula comprises NaCl, tryptone, yeast extract, glucose, and 4-hydroxyphenethyl alcohol (tyrosol).

[0019] Further, the fermentation medium formula comprises 10g / L NaCl, 10g / L tryptone, 5g / L yeast extract, 20-30g / L glucose, and 0.5g / L 4-hydroxyphenethyl alcohol (tyrosol).

[0020] By applying the technical solution of the present application, the glycosyltransferase mutant derived from Osmanthus fragrans shown in SEQ ID NO: 1, i.e., glycosyltransferase OfT8GT1, is used as the female parent to perform site-directed mutation and combination mutation and other protein engineering modifications, and a glycosyltransferase mutant with high enzyme activity is obtained, which can be used for the production of rhodiolin. DETAILED DESCRIPTION

[0021] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0022] As mentioned in the background, rhodiolin is widely used in the pharmaceutical, food and health care industries, and has high economic value. However, the yield of rhodiolin from natural sources is low, and chemical synthesis of rhodiolin has the limitations of complicated steps and poor regioselectivity. Therefore, the mild biological method becomes the preferred method for synthesizing rhodiolin. However, the wild-type glycosyltransferase for biological synthesis has the problem of low enzyme activity. Therefore, in the present application, the inventors attempt to modify the glycosyltransferase by directed evolution, and then improve various properties of the enzyme, so that it can be used in the technical solution for industrial production of rhodiolin. The specific modification includes:

[0023] By homology modeling method, the substrate 4-hydroxyphenethyl alcohol (tyrosol) is docked according to the model structure, the docking results are analyzed, and 69 residues near the active center that may affect the catalytic properties of the protein are selected, including: R56, G126, V216, V223, V306, S386, I15, Y17, A19, N24, P25, N28, K31, T45, N48, S55, A83, T84, Q85, I87, P88, C91, S93, T94, T95, S124, A127, S129, T148, C151, G152, G155, T157, Y159, F202, R204, I211, N214, R218, E219, E221, A222, L258, M261, M262, H270, S274, S275, S276, E280, L282, S303, A305, V307, D333, M355, S358, E364, S378, N381, T383, C395, F399, Q403, N405, C406, R407, S409 or N421, and saturated mutation and site-directed mutation are performed on these residues.

[0024] Among them, the saturated mutation is a method of obtaining a mutant in which the target site amino acid is replaced by other 19 amino acids in a short time by modifying the encoding gene of the target protein. This method is not only a powerful tool for protein directed modification, but also an important means for studying the structure-function relationship of protein. Saturation mutation often obtains more ideal evolution than single-point mutation. For these problems that cannot be solved by site-directed mutation method, saturation mutation method is unique. The mutants obtained by saturation mutation are identified by sequencing, and their activities on different substrates and tolerances at high temperature are tested respectively.

[0025] Site-directed mutation: refers to introducing desired changes (usually changes in a favorable direction) into a target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), including addition, deletion, point mutation, etc. of bases. Site-directed mutation can rapidly and efficiently improve the traits of the target protein expressed by the DNA and is a very useful means in gene research work. The method of introducing site-directed mutation by whole plasmid PCR is simple and effective, and is a means that is currently used more frequently. The principle is that a pair of primers (forward and reverse) containing the mutation site and the template plasmid are annealed and then extended by polymerase. The so-called cycle extension refers to the extension of the primer by polymerase according to the template, and then back to the 5' end of the primer to terminate, and then repeated heating, annealing and extension. This reaction is different from rolling circle amplification, and does not form multiple tandem copies. The extension products of the forward and reverse primers are paired to become a notched open plasmid after annealing. The extension product is cut by Dpn I enzyme, and since the original template plasmid is derived from conventional E. coli and is dam methylated and modified, it is sensitive to Dpn I and is cut into pieces, while the in vitro synthesized plasmid with the mutated sequence is not methylated and is not cut, so it can be successfully transformed in the subsequent transformation, and the mutant plasmid clone can be obtained.

[0026] The mutant screened from the mutant library is sequenced, and a suitable mutant is selected according to the sequencing result, and is subjected to activity test. After multiple rounds of evolution, the inventors obtain a series of glycosyltransferase mutants, and the activity of these mutants on the substrate 4-hydroxyphenethyl alcohol (tyrosol) is obviously improved. These mutants can be used for industrial production, and the catalytic efficiency is greatly improved.

[0027] On the basis of the above-mentioned modification results, the applicant proposes a series of protection schemes of the present application. In a first typical embodiment of the present application, a glycosyltransferase mutant is provided, comprising (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having the amino acid sequence in (a) with amino acid mutation at at least one of the following positions: R56, G126, V216, V223, V306, S386, I15, Y17, A19, N24, P25, N28, K31, T45, N48, S55, A83, T84, Q85, I87, P88, C91, S93, T94, T95, S124, A127, S129, T148, C151, G152, G155, T157, Y159, F202, R204, I211, N214, R218, E219, E221, A222, L258, M261, M262, H270, S274, S275, S276, E280, L282, S303, A305, V307, D333, M355, S358, E364, S378, N381, T383, C395, F399, Q403, N405, C406, R407, S409, or N421, and having glycosyltransferase function; (c) a protein having 80% or more homology with the amino acid sequence defined in any one of (a) and (b) and having glycosyltransferase function.

[0028] It should be noted that the homology in the present application refers to the "sequence identity" between two amino acid sequences, i.e. the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, the degree of sequence identity of amino acid sequences is usually measured using sequence analysis software. For example, it can be determined by the BLAST program of the NCBI database. For determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0029] The above-mentioned protein having 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, even 99.9% or more) homology with the mutant shown in SEQ ID NO: 1 and having glycosyltransferase activity, its active site, active pocket, active mechanism, protein structure, etc. are all probably the same as the protein provided in (a) of the corresponding protein.

[0030] Amino acid residues can be represented according to the standard three-letter or one-letter amino acid code known and agreed in the art. In this text, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0031] Conservative amino acid substitutions or replacements are well known in the art, for example, conservative amino acid substitutions are preferably substitutions of one amino acid residue for another within the same group (1)-(5) as follows: (1) smaller aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) residues with polar negative charge and their (uncharged) amides: Asp, Asn, Glu, and Gin; (3) residues with polar positive charge: His, Arg, and Lys; (4) larger aliphatic nonpolar residues: Met, Leu, lie, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala for Gly or Ser; Arg for Lys; Asn for Gin or His; Asp for Glu; Cys for Ser; Gin for Asn; Glu for Asp; Gly for Ala or Pro; His for Asn or Gin; lie for Leu or Val; Leu for lie or Val; Lys for Arg, Gin, or Glu; Met for Leu, Tyr, or lie; Phe for Met, Leu, or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp or Phe; and Val for lie or Leu.

[0032] Conservative amino acid substitutions can also be made according to the rules for amino acid substitution known to those skilled in the art, such as the "blosum62 score matrix" and the like.

[0033] The following examples are mutants of the glycosyltransferase of the amino acid sequence shown in SEQ ID NO: 1, i.e., the glycosyltransferase OfT8GT1 (nucleotide sequence of SEQ ID NO: 2), which were subjected to site-directed mutagenesis to obtain various variants.

[0034] The amino acid sequence shown in SEQ ID NO: 1 is as follows:

[0035] The nucleotide sequence shown in SEQ ID NO: 2 is as follows:

[0036] The amino acid sequence shown in SEQ ID NO: 1 is a mutant of a glycosyltransferase derived from Osmanthus fragrans, i.e., the amino acid sequence of glycosyltransferase OfT8GT1. By homology modeling of the amino acid sequence and using computer simulation to analyze the model structure and molecular docking results of the substrate 4-hydroxyphenethyl alcohol (tyrosol), 69 amino acid residues including R56, G126, V216, V223, V306, S386, I15, Y17, A19, N24, P25, N28, K31, T45, N48, S55, A83, T84, Q85, I87, P88, C91, S93, T94, T95, S124, A127, S129, T148, C151, G152, G155, T157, Y159, F202, R204, I211, N214, R218, E219, E221, A222, L258, M261, M262, H270, S274, S275, S276, E280, L282, S303, A305, V307, D333, M355, S358, E364, S378, N381, T383, C395, F399, Q403, N405, C406, R407, S409, or N421 are found to be located near the active center and to have the potential to affect the catalytic properties of the protein. By mutating the above-mentioned amino acid sites, a protein having glycosyltransferase function or even enhanced glycosyltransferase function can be obtained. For the protein obtained above, changes can be made at non-critical mutation sites and active sites, and a protein having more than 80% homology with the above-mentioned amino acid sequence and having glycosyltransferase function can be obtained.

[0037] In a preferred embodiment, the amino acid mutations of (b) are each independently selected from the following: R56F or R56L or R56W or R56C or R56A, G126F or G126W or G126Y, V216F or V216W or V216Y or V216H, V223A or V223L or V223R, V306D or V306E or V306K or V306R or V306H or V306T, S386A or S386G or S386Q or S386R, I15F or I15L or I15V, Y17F or Y17A or Y17L or Y17R or Y17W, A19M, N24A or N24L or N24V, P25A or P25L or P25V, N28L or N28M or N28W, K31L or K31M, T45F or T45Y, N48F or N48M or N48Y, S55A or S55H or S55W, A83F or A83H or A83Y, T84M, Q85F or Q85L, I87L or I87W or I87Y, P88I or P88K or P88T or P88V, C91F or C91H or C91K or C91Y, S93A or S93F or S93S or S93V, T94L or T94V, T95F or T95Q or T95Y, S124A or S124C or S124V, A127E or A127F or A127M or A127W or A127Y, S129E or S129F or S129K or S129R or S129W or S129Y, T148E or T148I or T148R or T148V or T148Y, C151A or C151K or C151R, G152A, G155A or G155H or G155R, T157L or T157M, Y159F, F202I, F202Q or F202W, R204F or R204K or R204W, I211K or I211P, N214L or N214M or N214W, R218F or R218H or R218W or R218Y, E219L or E219Q or E219V, E221L or E221R or E221W, A222L or A222M or A222Y, L258F, M261L or M262I, M262L or M262V, H270V, S274CS274I or S274V, S275F or S275W or S275Y, S276I or S276L, E280L or E280P or E280W, L282Y, S303E or S303L or S303T or S303D, A305P, V307P, D333F or D333W or D333Y, M355F or M355L or M355W, S358H or S358L or S358R, E364L, S378C or S378L or S378M, N381L or N381Q or N381W, T383I or T383L or T383V, C395F or C395H or C395M, F399D or F399G or F399Y, Q403F or Q403S or Q403V or Q403W, N405I or N405L or N405V, C406A or C406M or C406T, R407F or R407W or R407Y, S409I or S409L or S409M or S409T or N421H or N421M or N421W or N421Y; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid; preferably, the protein having 85% or above, preferably 90% or above, more preferably 95% or above, and further preferably 99% or above homology with the amino acid sequence defined in (a) or (b) and having the glycosyltransferase function in (c) above.

[0038] In the present application, the applicant continues to explore the active site described above, and finds that the corresponding protein activity also has differences when the active site is mutated to different amino acids. A specific mutation can enhance the glycosyltransferase activity. Through experimental exploration, it is found that the protein with enhanced activity can be obtained by performing the specific mutation on the active site described above. The amino acid mutation site of the enzyme protein can be flexibly selected and combined in the mutation described above.

[0039] In a preferred embodiment, the mutation of the glycosyltransferase mutant comprises any one of the following amino acid mutations: I15F, I15L, I15V, Y17F, Y17A, Y17L, Y17R, Y17W, A19M, N24A, N24L, N24V, P25A, P25L, P25V, N28L, N28M, N28W, K31L, K31M, T45F, T45Y, N48F, N48M, N48Y, S55A, S55H, S55W, R56F, R56L, R56W, R56C, R56A, A83F, A83H, A83Y, T84M, Q85F, Q85L, I87L, I87W, I87Y, P88I, P88K, P88T, P88V, C91F, C91H, C91K, C91Y, S93A, S93F, S93S, S93V, T94L, T94V, T95F, T95Q, T95Y, S124A, S124C, S124V, G126F, G126W, G126Y, A127E, A127F, A127M, A127W, A127Y, S129E, S129F, S129K, S129R, S129W, S129Y, T148E, T148I, T148R, T148V, T148Y, C151A, C151K, C151R, G152A, G155A, G155H, G155R, T157L, T157M, Y159F, F202I, F202Q, F202W, R204F, R204K, R204W, I211K, I211P, N214L, N214M, N214W, V216F, V216W, V216Y, V216H, R218F, R218H, R218W, R218Y, E219L, E219Q, E219V, E221L, E221R, E221W, A222L, A222M, A222Y, V223A, V223L, V223R, L258F, M261L, M262I, M262L, M262V, H270V, S274C, S274I, S274V, S275F, S275W, S275Y, S276I, S276L, E280L, E280P, E280W, L282Y, S303E, S303L, S303T, S303D, A305P, V306D, V306E, V306K, V306R, V306H, V306T, V307P, D333F, D333W, D333Y, M355F, M355L, M355W, S358H, S358L, S358R, E364L, S378C, S378L, S378M, N381L, N381Q, N381W, T383I, T383L,T383V, S386A, S386G, S386Q, S386R, C395F, C395H, C395M, F399D, F399G, F399Y, Q403F, Q403S, Q403V, Q403W, N405I, N405L, N405V, C406A, C406M, C406T, R407F, R407W, R407Y, S409I, S409L, S409M, S409T, N421H, N421M, N421W, N421Y, R56C+G126F, R56C+V216W, R56C+V216H, R56A+V216W, R56A+V216H, R56F+V216W, R56F+V216H, R56C+V223A, R56C+V223L, R56C+V223R, R56A+V223A, R56A+V223L, R56A+V223R, R56F+V223A, R56F+V223L, R56F+V223R, R56C+V306K, R56C+V306H, R56C+V306T, R56A+V306K, R56A+V306H, R56A+V306T, R56F+V306K, R56F+V306H, R56F+V306T, R56C+S386G, R56C+S386R, R56A+S386G, R56A+S386R, R56F+S386G, R56F+S386R, G126F+V216W, G126F+V216H, G126F+V223A, G126F+V223L, G126F+V223R, G126F+V306K, G126F+V306H, G126F+V306T, G126F+S386G, G126F+S386R, V216W+V223A, V216W+V223L, V216W+V223R, V216H+V223A, V216H+V223L, V216H+V223R, V216W+V306K, V216W+V306H, V216W+V306T, V216H+V306K, V216H+V306H, V216H+V306T, V216W+S386G, V216W+S386R, V216H+S386G, V216H+S386R, V223A+V306K, V223A+V306H, V223A+V306T, V223L+V306K, V223L+V306H, V223L+V306T, V223R+V306K, V223R+V306H, V223R+V306T, V223A+S386G, V223A+S386R, V223L+S386G,V223L+S386R, V223R+S386G, V223R+S386R, V306K+S386G, V306K+S386R, V306H+S386G, V306H+S386R, V306T+S386G, V306T+S386R, R56F+G126F+V216W, R56F+G126F+V223L, R56F+G126F+S386G, R56F+G126F+V306H, R56F+V216W+V223L, R56F+V216W+S386G, R56F+V216W+V306H, R56F+V223L+S386G, R56F+V223L+V306H, R56F+S386G+V306H, G126F+V216W+V223L, G126F+V216W+S386G, G126F+V216W+V306H, G126F+V223L+S386G, G126F+V223L+V306H, G126F+S386G+V306H, V216W+V223L+S386G, V216W+V223L+V306H, V216W+S386G+V306H, V223L+S386G+V306H, V223L+S386R+V306H, V223A+S386G+V306H, V223A+S386R+V306H, R56F+G126F+V223A, R56F+G126F+S386R, R56F+G126F+V306T, R56F+V216W+V223A, R56F+V216W+S386R, R56F+V216W+V306T, R56F+V223L+S386R, R56F+V223L+V306T, R56F+S386G+V306T, G126F+V216W+V223A, G126F+V216W+S386R, G126F+V216W+V306T, G126F+V223L+S386R, G126F+V223L+V306T, G126F+S386G+V306T, V216W+V223L+S386R, V216W+V223L+V306T, V216W+S386G+V306T, V223L+S386G+V306T, V223L+S386R+V306T, V223A+S386G+V306T, V223A+S386R+V306T, R56F+G126F+V306K, R56F+V216W+V306K, R56F+V223A+S386G, R56F+V223L+V306K, R56F+S386G+V306K,G126F+V216W+V306K, G126F+V223A+S386G, G126F+V223L+V306K, G126F+S386G+V306K, V216W+V223A+S386G, V216W+V223L+V306K, V216W+S386G+V306K, V223L+S386G+V306K, V223L+S386R+V306K, V223A+S386G+V306K, V223A+S386R+V306K, R56F+V223A+S386R, R56F+V223A+V306H, R56F+S386R+V306H, G126F+V223A+S386R, G126F+V223A+V306H, G126F+S386R+V306H, V216W+V223A+S386R, V216W+V223A+V306H, V216W+S386R+V306H, R56F+V223A+V306T, R56F+S386R+V306T, G126F+V223A+V306T, G126F+S386R+V306T, V216W+V223A+V306T, V216W+S386R+V306T, R56F+V223A+V306K, R56F+S386R+V306K, G126F+V223A+V306K, G126F+S386R+V306K, V216W+V223A+V306K, or V216W+S386R+V306K.

[0040] The amino acid mutations described above are all tested in the embodiments of the present application, all have glycosyltransferase activity, and compared to the parent having the amino acid sequence shown in SEQ ID NO: 1, the glycosyltransferase mutants with high enzyme activity can be obtained.

[0041] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the glycosyltransferase mutant described above.

[0042] In a third typical embodiment of the present application, a recombinant plasmid is provided, which is connected with the DNA molecule described above. The DNA described above can encode the glycosyltransferase mutant described above, and can be connected to the recombinant plasmid to form a circular DNA. The DNA and the recombinant plasmid can both be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc., to obtain the glycosyltransferase mutant described above.

[0043] In a fourth exemplary embodiment of the present application, a host cell is provided, which is transformed with the recombinant plasmid described above. Using the host cell described above, the replication of the recombinant plasmid in the host cell can be achieved, and the DNA molecule carried by the recombinant plasmid can be transcribed and translated to obtain a large amount of glycosyltransferase mutant. Using the prior art, the host cell can be broken to purify the protein, the crude enzyme can be catalyzed after breaking, or other methods can be used to obtain the glycosyltransferase mutant, and subsequent catalytic synthesis of rhodiolin can be performed. The host cell is preferably E. coli or yeast.

[0044] In a fifth exemplary embodiment of the present application, a preparation method for producing rhodiolin is provided, which comprises catalyzing the reaction of the glycosyltransferase mutant described above with 4-hydroxyphenethyl alcohol (tyrosol) to obtain the rhodiolin described above.

[0045] The preparation method comprises inoculating the host cell cultured overnight into a fermentation medium, culturing at 30-37°C and 200-220 rpm to OD600 of 0.6-0.8, adding an inducer, and inducing the culture for 12-24 hours to obtain the rhodiolin described above. Preferably, the inducer is IPTG. More preferably, the final concentration of the inducer is 0.1-1 mM. Further preferably, the final concentration of the inducer is 0.1 mM. The fermentation medium formula comprises NaCl, tryptone, yeast extract, glucose, and 4-hydroxyphenethyl alcohol (tyrosol). Preferably, 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, 20-30 g / L glucose, and 0.5 g / L 4-hydroxyphenethyl alcohol (tyrosol).

[0046] As understood by those skilled in the art, the catalytic reaction in the present application is not limited to in vivo, but can also be in vitro. Specifically, the substrate 4-hydroxyphenethyl alcohol can be added, and then rhodiolin can be synthesized in vivo and secreted extracellularly, or the enzyme can be purified and then reacted in vitro.

[0047] Based on the preparation method described above, the glycosyltransferase mutant described above can be used to react with the substrate 4-hydroxyphenethyl alcohol (tyrosol) to catalyze the 4-hydroxyphenethyl alcohol (tyrosol) to obtain rhodiolin. The activity of the glycosyltransferase is greatly improved, which can improve the production efficiency of rhodiolin, reduce the cost of industrial production, and be more suitable for industrial production.

[0048] The present application will be further described in detail below in conjunction with specific examples, which should not be construed as limiting the scope of the present application. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Example 1 Rational design and mutant library screening of glycosyltransferase OfT8GT1

[0050] To produce salidroside, we initially screened several glycosyltransferases from literature and patents. Among them, the mutant of glycosyltransferase derived from Osmanthus fragrans, i.e. glycosyltransferase OfT8GT1, had the highest yield. Therefore, we selected this enzyme for mutation. By homology modeling, we used the model structure to perform molecular docking of the substrate 4-hydroxyphenethyl alcohol (tyrosol). According to the results of rational design, we designed site-directed mutation primers. Using site-directed mutation means, we performed single-point mutation at 69 mutation sites, including I15 or Y17 or A19 or N24 or P25 or N28 or K31 or T45 or N48 or S55 or R56 or A83 or T84 or Q85 or I87 or P88 or C91 or S93 or T94 or T95 or S124 or G126 or A127 or S129 or T148 or C151 or G152 or G155 or T157 or Y159 or F202 or R204 or I211 or N214 or V216 or R218 or E219 or E221 or A222 or V223 or L258 or M261 or M262 or H270 or S274 or S275 or S276 or E280 or L282 or S303 or A305 or V306 or V307 or D333 or M355 or S358 or E364 or S378 or N381 or T383 or S386 or C395 or F399 or Q403 or N405 or C406 or R407 or S409 and N421, to obtain 202 glycosyltransferase OfT8GT1 mutants (Table 1).

[0051] Table 1 Glycosyltransferase OfT8GT1 mutant library

[0052] The expression frame of the glycosyltransferase OfT8GT1 mutant library in Table 1 was introduced into E. coli BL21 (DE3). The E. coli engineering strain was activated in 2YT medium, inoculated into a 250 mL shake flask containing 50 mL liquid fermentation medium at a 1% inoculation amount, cultured at 37°C and 220 rpm to an OD600 of 0.6-0.8, and then 0.1 mM IPTG was added. The culture was continued at 30°C and 220 rpm for 12-24 h. The peak area of 4-hydroxyphenethyl alcohol (tyrosol) and the peak area of salidroside were detected. The ratio of the peak area of salidroside to the peak area of 4-hydroxyphenethyl alcohol (tyrosol) was the conversion rate. The mutant catalytic activity was evaluated according to the conversion rate. The degree of improvement of the relative parent conversion rate of the mutant was represented by “+”. The more “+”, the greater the degree of improvement. The specific results are shown in Table 2.

[0053] Table 2 The degree of improvement in conversion rate of the mutant library of glycosyltransferase OfT8GT1 screened by shake flask

[0054] Note: + represents 20-40%, ++ represents 40-60%, +++ represents 60-80%, ++++ represents 80-90%, and +++++ represents more than 90%.

[0055] According to the results of shake flask screening, the yield of rhodioside of the R56C or R56A or R56F or G126F or V216W or V216H or V223A or V223L or V223R or V306K or V306H or V306T or S386G or S386R mutant is significantly higher than that of the parent.

[0056] Example 2 Screening of double combination mutants of glycosyltransferase OfT8GT1

[0057] The better mutation points R56C or R56A or R56F or G126F or V216W or V216H or V223A or V223L or V223R or V306K or V306H or V306T or S386G or S386R screened in Example 1 were combined and introduced into E. coli BL21 (DE3). The synthesis method and detection method were the same as in Example 1. The degree of yield improvement of the mutants compared with the parent is shown in Table 3. Among them, the yield of rhodioside of the R56F+V216W or G126F+V216W or G126F+V223L or G126F+S386G or V223A+V306H or V223A+V306T or V223L+S386R or V306K+S386G or V306T+S386G mutant is significantly higher than that of the parent.

[0058] Table 3 Screening results of double combination mutants of glycosyltransferase OfT8GT1

[0059] Note: + represents 20-40%, ++ represents 40-60%, +++ represents 60-80%, ++++ represents 80-90%, and +++++ represents more than 90%.

[0060] Example 3 Screening of triple combination mutants of glycosyltransferase OfT8GT1

[0061] The better mutation points R56F or G126F or V216W or V223L or V223A or S386G or S386R or V306H or V306T or V306K screened in example 2 were combined in three sites, and were introduced into E. coli BL21 (DE3). The synthesis method and detection method were the same as in example 1. The yield of the mutants was improved to different degrees compared with the parent, as shown in Table 4. Among them, the R56F+V216W+V223L or R56F+V216W+V306H or G126F+V223L+S386G or V216W+S386G+V306H or R56F+G126F+V306T or G126F+S386G+V306T or V223A+S386R+V306K or G126F+S386R+V306K mutant had a significantly improved yield of salidroside compared with the parent.

[0062] Table 4 Screening of three-combination mutants of glycosyltransferase OfT8GT1

[0063] Note: + represents 20-40%, ++ represents 40-60%, +++ represents 60-80%, ++++ represents 80-90%, and +++++ represents more than 90%.

[0064] In summary, the above-mentioned embodiments of the present application achieve the following technical effects: the above-mentioned glycosyltransferase mutants of the present application have excellent performance with high activity, high economic value, and wide application prospects. Compared with traditional chemical methods, enzyme catalytic synthesis of salidroside is an economical, green and efficient synthesis method. It does not require complex processes and steps, and the operation is more simple and mild, realizing green production. In general, the glycosyltransferase mutants of the present application have high catalytic activity and specific glycosylation site selectivity, and are expected to be applied to the industrialized production of glycoside compounds by microbial fermentation, bringing significant benefits to subsequent downstream applications.

[0065] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification or equivalent replacement or improvement made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A glycosyltransferase mutant, characterized in that, Comprise: (a) a protein having an amino acid mutation at at least one of the following positions of the amino acid sequence shown in SEQ ID NO: 1: R56, G126, V216, V223, V306, S386, I15, Y17, A19, N24, P25, N28, K31, T45, N48, S55, A83, T84, Q85, I87, P88, C91, S93, T94, T95, S124, A127, S129, T148, C151, G152, G155, T157, Y159, F202, R204, I211, N214, R218, E219, E221, A222, L258, M261, M262, H270, S274, S275, S276, E280, L282, S303, A305, V307, D333, M355, S358, E364, S378, N381, T383, C395, F399, Q403, N405, C406, R407, S409, or N421, and has a glycosyltransferase function; or (b) a protein derived from Osmanthus fragrans having more than 99% homology with the amino acid sequence defined in (a) and having a glycosyltransferase function.

2. The glycosyltransferase mutant of claim 1, wherein, The amino acid mutations of (a) are each independently selected from the following: R56F or R56L or R56W or R56C or R56A, G126F or G126W or G126Y, V216F or V216W or V216Y or V216H, V223A or V223L or V223R, V306D or V306E or V306K or V306R or V306H or V306T, S386A or S386G or S386Q or S386R, I15F or I15L or I15V, Y17F or Y17A or Y17L or Y17R or Y17W, A19M, N24A or N24L or N24V, P25A or P25L or P25V, N28L or N28M or N28W, K31L or K31M, T45F or T45Y, N48F or N48M or N48Y, S55A or S55H or S55W, A83F or A83H or A83Y, T84M, Q85F or Q85L, I87L or I87W or I87Y, P88I or P88K or P88T or P88V, C91F or C91H or C91K or C91Y, S93A or S93F or S93S or S93V, T94L or T94V, T95F or T95Q or T95Y, S124A or S124C or S124V, A127E or A127F or A127M or A127W or A127Y, S129E or S129F or S129K or S129R or S129W or S129Y, T148E or T148I or T148R or T148V or T148Y, C151A or C151K or C151R, G152A, G155A or G155H or G155R, T157L or T157M, Y159F, F202I, F202Q or F202W, R204F or R204K or R204W, I211K or I211P, N214L or N214M or N214W, R218F or R218H or R218W or R218Y, E219L or E219Q or E219V, E221L or E221R or E221W, A222L or A222M or A222Y, L258F, M261L or M262I, M262L or M262V, H270V, S274C or S274I or S274V, S275F or S275W or S275Y, S276I or S276L, E280L or E280P or E280W, L282Y, S303E or S303L or S303T or S303D, A305P, V307P, D333F or D333W or D333Y, M355F or M355L or M355W, S358H or S358L or S358R, E364L, S378C or S378L or S378M, N381L or N381Q or N381W, T383I or T383L or T383V,C395F or C395H or C395M, F399D or F399G or F399Y, Q403F or Q403S or Q403V or Q403W, N405I or N405L or N405V, C406A or C406M or C406T, R407F or R407W or R407Y, S409I or S409L or S409M or S409T or N421H or N421M or N421W or N421Y; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid.

3. The glycosyltransferase mutant of claim 2, wherein The mutations of the glycosyltransferase mutant include any one of the following: I15F, I15L, I15V, Y17F, Y17A, Y17L, Y17R, Y17W, A19M, N24A, N24L, N24V, P25A, P25L, P25V, N28L, N28M, N28W, K31L, K31M, T45F, T45Y, N48F, N48M, N48Y, S55A, S55H, S55W, R56F, R56L, R56W, R56C, R56A, A83F, A83H, A83Y, T84M, Q85F, Q85L, I87L, I87W, I87Y, P88I, P88K, P88T, P88V, C91F, C91H, C91K, C91Y, S93A, S93F, S93S, S93V, T94L, T94V, T95F, T95Q, T95Y, S124A, S124C, S124V, G126F, G126W, G126Y, A127E, A127F, A127M, A127W, A127Y, S129E, S129F, S129K, S129R, S129W, S129Y, T148E, T148I, T148R, T148V, T148Y, C151A, C151K, C151R, G152A, G155A, G155H, G155R, T157L, T157M, Y159F, F202I, F202Q, F202W, R204F, R204K, R204W, I211K, I211P, N214L, N214M, N214W, V216F, V216W, V216Y, V216H, R218F, R218H, R218W, R218Y, E219L, E219Q, E219V, E221L, E221R, E221W, A222L, A222M, A222Y, V223A, V223L, V223R, L258F, M261L, M262I, M262L, M262V, H270V, S274C, S274I, S274V, S275F, S275W, S275Y, S276I, S276L, E280L, E280P, E280W, L282Y, S303E, S303L, S303T, S303D, A305P, V306D, V306E, V306K, V306R, V306H, V306T, V307P, D333F, D333W, D333Y, M355F, M355L, M355W, S358H, S358L, S358R, E364L, S378C, S378L, S378M, N381L, N381Q, N381W, T383I, T383L, T383V, S386A, S386G, S386Q, S386R, C395F,C395H, C395M, F399D, F399G, F399Y, Q403F, Q403S, Q403V, Q403W, N405I, N405L, N405V, C406A, C406M, C406T, R407F, R407W, R407Y, S409I, S409L, S409M, S409T, N421H, N421M, N421W, N421Y, R56C+G126F, R56C+V216W, R56C+V216H, R56A+V216W, R56A+V216H, R56F+V216W, R56F+V216H, R56C+V223A, R56C+V223L, R56C+V223R, R56A+V223A, R56A+V223L, R56A+V223R, R56F+V223A, R56F+V223L, R56F+V223R, R56C+V306K, R56C+V306H, R56C+V306T, R56A+V306K, R56A+V306H, R56A+V306T, R56F+V306K, R56F+V306H, R56F+V306T, R56C+S386G, R56C+S386R, R56A+S386G, R56A+S386R, R56F+S386G, R56F+S386R, G126F+V216W, G126F+V216H, G126F+V223A, G126F+V223L, G126F+V223R, G126F+V306K, G126F+V306H, G126F+V306T, G126F+S386G, G126F+S386R, V216W+V223A, V216W+V223L, V216W+V223R, V216H+V223A, V216H+V223L, V216H+V223R, V216W+V306K, V216W+V306H, V216W+V306T, V216H+V306K, V216H+V306H, V216H+V306T, V216W+S386G, V216W+S386R, V216H+S386G, V216H+S386R, V223A+V306K, V223A+V306H, V223A+V306T, V223L+V306K, V223L+V306H, V223L+V306T, V223R+V306K, V223R+V306H, V223R+V306T, V223A+S386G, V223A+S386R, V223L+S386G, V223L+S386R, V223R+S386G, V223R+S386R, V306K+S386G,V306K+S386R, V306H+S386G, V306H+S386R, V306T+S386G, V306T+S386R, R56F+G126F+V216W, R56F+G126F+V223L, R56F+G126F+S386G, R56F+G126F+V306H, R56F+V216W+V223L, R56F+V216W+S386G, R56F+V216W+V306H, R56F+V223L+S386G, R56F+V223L+V306H, R56F+S386G+V306H, G126F+V216W+V223L, G126F+V216W+S386G, G126F+V216W+V306H, G126F+V223L+S386G, G126F+V223L+V306H, G126F+S386G+V306H, V216W+V223L+S386G, V216W+V223L+V306H, V216W+S386G+V306H, V223L+S386G+V306H, V223L+S386R+V306H, V223A+S386G+V306H, V223A+S386R+V306H, R56F+G126F+V223A, R56F+G126F+S386R, R56F+G126F+V306T, R56F+V216W+V223A, R56F+V216W+S386R, R56F+V216W+V306T, R56F+V223L+S386R, R56F+V223L+V306T, R56F+S386G+V306T, G126F+V216W+V223A, G126F+V216W+S386R, G126F+V216W+V306T, G126F+V223L+S386R, G126F+V223L+V306T, G126F+S386G+V306T, V216W+V223L+S386R, V216W+V223L+V306T, V216W+S386G+V306T, V223L+S386G+V306T, V223L+S386R+V306T, V223A+S386G+V306T, V223A+S386R+V306T, R56F+G126F+V306K, R56F+V216W+V306K, R56F+V223A+S386G, R56F+V223L+V306K, R56F+S386G+V306K, G126F+V216W+V306K, G126F+V223A+S386G, G126F+V223L+V306K, G126F+S386G+V306K, V216W+V223A+S386G, V216W+V223L+V306K,V216W + S386G + V306K, V223L + S386G + V306K, V223L + S386R + V306K, V223A + S386G + V306K, V223A + S386R + V306K, R56F + V223A + S386R, R56F + V223A + V306H, R56F + S386R + V306H, G126F + V223A + S386R, G126F + V223A + V306H, G126F + S386R + V306H, V216W + V223A + S386R, V216W + V223A + V306H, V216W + S386R + V306H, R56F + V223A + V306T, R56F + S386R + V306T, G126F + V223A + V306T, G126F + S386R + V306T, V216W + V223A + V306T, V216W + S386R + V306T, R56F + V223A + V306K, R56F + S386R + V306K, G126F + V223A + V306K, G126F + S386R + V306K, V216W + V223A + V306K, or V216W + S386R + V306K.

4. A DNA molecule, characterized in that, The DNA molecule encodes the glycosyltransferase mutant of any one of claims 1 to 3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid is linked to the DNA molecule of claim 4.

6. A host cell, characterized in that, The host cell is transformed with the recombinant plasmid of claim 5.

7. The host cell of claim 6, wherein, The host cell is E. coli or yeast.

8. A method for producing salidroside, characterized by, The method comprises using the glycosyltransferase mutant of any one of claims 1 to 3 to catalyze a reaction with 4-hydroxyphenethyl alcohol as a substrate to obtain the rhodioloside.

9. The method of claim 8, wherein, The method comprises inoculating the host cell cultured overnight into a fermentation medium, culturing at 30-37°C, 200-220 rpm to OD600 of 0.6-0.8, adding an inducer, and inducing for 12-24 h to obtain the rhodioloside; wherein the host cell is transformed with a recombinant plasmid, the recombinant plasmid is linked to a DNA molecule, and the DNA molecule encodes the glycosyltransferase mutant.

10. The method of claim 9, wherein, The inducer is IPTG; and the final concentration of the inducer is 0.1-1 mM.

11. The method of claim 9, wherein, The fermentation medium formula comprises NaCl, tryptone, yeast extract, glucose, and 4-hydroxyphenethyl alcohol.

12. The method of claim 11, wherein, The fermentation medium formula comprises 10 g / L of the NaCl, 10 g / L of the tryptone, 5 g / L of the yeast extract, 20-30 g / L of the glucose, and 0.5 g / L of the 4-hydroxyphenethyl alcohol.

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