CN120485149A

CN120485149A - Enzyme mutant, glycosyltransferase mutant and application thereof in preparation of salidroside

Info

Publication number: CN120485149A
Application number: CN202510660359.XA
Authority: CN
Inventors: 张章; 梁昊雨; 于铁妹; 李加忠; 黄华; 刘建
Original assignee: Shenzhen Readline Biotechnology Co ltd
Current assignee: Shenzhen Readline Biotechnology Co ltd
Priority date: 2025-05-21
Filing date: 2025-05-21
Publication date: 2025-08-15

Abstract

The invention relates to the field of bioengineering, in particular to an enzyme mutant, a glycosyltransferase mutant and application thereof in preparing salidroside. The invention provides an enzyme mutant, which is characterized by comprising the following amino acid site mutation, wherein the 308 th site is mutated from I to Q, the 333 rd site is mutated from V to R, the 363 st site is mutated from W to S and the 386 th site is mutated from S to G on the basis of wild glycosyltransferase, and the amino acid sequence of the wild glycosyltransferase is shown as SEQ ID NO. 1. According to the invention, glycosyltransferase and sucrose synthase with improved thermal stability are obtained through structural design and deep learning transformation, and tyrosol can be glycosylated in a mode of regenerating ADP-glucose to generate salidroside, and the price of ADP is only about 20% of UDP, so that the method has the advantage of cost.

Description

Enzyme mutant, glycosyltransferase mutant and application thereof in preparation of salidroside

Technical Field

The invention relates to the field of bioengineering, in particular to an enzyme mutant, a glycosyltransferase mutant and application thereof in preparing salidroside.

Background

The dried root and rhizome of the perennial herb rhodiola rosea in the plateau can be used as medicines, has the functions of tonifying qi and activating blood, and promoting blood circulation to relieve asthma, and the main active ingredients of the rhodiola rosea are salidroside and tyrosol. The medicinal materials rhodiola rosea, cordyceps sinensis, saffron and snow lotus are together praised as four rare Tibetan medicines, and are used for medicines, health products and skin care products in recent years. The representative plants of Rhodiola include more than 90 kinds of Rhodiola crenulata Rhodiola crenulata, rhodiola angusta Rhodiola kirilowii, rhodiola kurz Rhodiola sachalinensis, rhodiola rosea, etc., and the wild resources are increasingly exhausted due to excessive digging and damage to the growth environment. Although rhodiola angustifolia starts to be cultivated manually in Gannan regions, the long growth period and high cost lead to no large-scale cultivation, and the market demand is not satisfied.

The research of synthesizing the salidroside by fermenting cells by using escherichia coli and saccharomyces cerevisiae is carried out earlier by the university of Tian institute and Tianjin thanks to the gene excavation of synthesizing the salidroside glycosyltransferase in plants, but the conversion of tyrosol to the salidroside is incomplete due to the lower activity of the glycosyltransferase and insufficient supply of intracellular UDP-glucose, so that the industrial application is limited.

Disclosure of Invention

In view of this, the present invention provides mutants of enzymes, mutants of glycosyltransferases and their use in the preparation of salidroside. According to the invention, glycosyltransferase and sucrose synthase with improved thermal stability are obtained through structural design and deep learning transformation, and tyrosol can be glycosylated in a mode of regenerating ADP-glucose to generate salidroside, and the price of ADP is only about 20% of UDP, so that the method has the advantage of cost.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides an enzyme mutant, which is characterized by comprising the following amino acid site mutation, wherein the 308 th site is mutated from I to Q, the 333 rd site is mutated from V to R, the 363 st site is mutated from W to S and the 386 th site is mutated from S to G on the basis of wild glycosyltransferase, and the amino acid sequence of the wild glycosyltransferase is shown as SEQ ID NO. 1.

In some embodiments of the invention, the amino acid sequence of the mutant is shown in SEQ ID NO. 5.

In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule encoding the wild-type glycosyltransferase described above is shown in SEQ ID NO. 2.

The invention also provides a mutant of glycosyltransferase, which comprises the following amino acid site mutation, namely, mutation from A to I at site 21, mutation from M to N at site 83 and mutation from Q to P at site 407.

In some embodiments of the invention, the amino acid sequence of the mutant of the glycosyltransferase is shown in SEQ ID NO. 7.

The invention also provides an enzyme composition comprising the mutant of the enzyme and/or the mutant of the glycosyltransferase, and sucrose synthase.

In some embodiments of the invention, in the above enzyme composition, the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO. 3.

The invention also provides nucleic acid molecules encoding mutants of the above enzymes, mutants of the above glycosyltransferases and/or compositions of the above enzymes.

In some embodiments of the invention, the nucleic acid molecule described above comprises:

The nucleotide sequence of the nucleic acid molecule encoding the mutant of the enzyme is shown as SEQ ID NO. 6, and/or

The nucleotide sequence of the nucleic acid molecule encoding the mutant of glycosyltransferase is shown as SEQ ID NO. 8, and/or

The nucleotide sequence of the nucleic acid molecule for encoding the sucrose synthase is shown as SEQ ID NO. 4.

The invention also provides a recombinant vector comprising the nucleic acid molecule and an acceptable gene element.

The invention also provides a host, transformation and/or transfection of the recombinant vector.

The invention also provides products comprising mutants of the above enzymes, mutants of the above glycosyltransferases, the above enzyme compositions, the above nucleic acid molecules, the above recombinant vectors and/or the above hosts and acceptable adjuvants or adjuvants.

The invention also provides the application of the mutant of the enzyme, the mutant of the glycosyltransferase, the enzyme composition, the nucleic acid molecule, the recombinant vector, the host and/or the product in preparing salidroside.

The invention also provides a preparation method of the salidroside, and the raw materials are transformed into the salidroside by any of the following steps;

(a) Mutants of the above enzymes, or

(B) Mutants of the above glycosyltransferases, or

(C) Or the enzyme composition

(D) Or a nucleic acid molecule as described above, or

(E) Or the recombinant vector

(F) Or the host mentioned above, or

(G) The above products;

the raw materials comprise sucrose, tyrosol and ADP.

In some embodiments of the present invention, in the above preparation method, the temperature at the time of conversion is 30 to 60 ℃.

In some embodiments of the invention, in the above preparation method, when a mutant of the above enzyme is used, the temperature at the time of transformation is 30 ℃.

In some embodiments of the invention, in the above preparation method, when the mutant of the above glycosyltransferase is used, the temperature at the time of transformation is 60 ℃.

In some embodiments of the present invention, in the above preparation method, the enzyme activity of the mutant of the enzyme is 13.3U/mL, the enzyme activity of the mutant of the glycosyltransferase is 80.3U/mL, and the enzyme activity of the sucrose synthase is 20.6-83.8U/mL.

In some embodiments of the present invention, in the above preparation method, the sucrose has a final concentration of 204.5 to 409mM, the tyrosol has a final concentration of 202.7 to 405.3mM, and the ADP has a final concentration of 0.2mM.

The beneficial effects of the invention include:

(1) The modified UDP glycosyltransferase can replace expensive UDP by using cheap ADP as a substrate;

(2) The secondarily modified ADP glycosyltransferase can react at a high temperature of 60 ℃, the reaction time is greatly shortened compared with 30 ℃, and the reaction with a high substrate concentration of more than 400mM can be completed.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the results of shake flask expressed AtUGT A1 and AcSUS proteins electrophoresis;

FIG. 2 shows the alignment of AtUGT A1 predicted structures with SrUGT G1-UDP-RA complex crystal structures, UDP being shown in the club model;

FIG. 3 shows the SrUGT G1 residue (rectangle labeled AtUGT A1 corresponding part residue) interacting with UDP;

FIG. 4 shows the predicted structure of AtUGT A1 for residues Ser307, ile308, val333, trp363, ser386, which may interact with UDP;

FIG. 5 shows a partial sequence alignment of AtUGT A1 residues that potentially interact with UDP;

FIG. 6 shows a global single point mutation Pythia energy heat map of AtUGT A1;

FIG. 7 shows a High Performance Liquid Chromatography (HPLC) profile of a pure salidroside product;

FIG. 8 shows a mass spectrum of the pure salidroside.

Detailed Description

The invention discloses an enzyme mutant, a glycosyltransferase mutant and application thereof in preparing salidroside.

It should be understood that one or more of the expressions ". The expressions" individually include each of the objects recited after the expressions and various combinations of two or more of the recited objects unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood as having the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

It should be understood that the order of steps or order of performing certain actions is not important so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Furthermore, the numerical ranges and parameters setting forth the present invention are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

Amino acid sequence of UDP-glycosyltransferase AtUGT A1 from Arabidopsis Arabidopsis thaliana ：MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSITVLSVKQLVEFAWGLAGSGKEFLWVIRPDLVAGEEAMVPPDFLMETKDRSMLASWCPQEKVLSHPAIGGFLTHCGWNSILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD（SEQ ID NO:1）;

Nucleotide sequence of UDP glycosyltransferase AtUGT A1 derived from arabidopsis Arabidopsis thaliana (codon optimized) ）：ATGGGCAGCCAGATTATTCATAATAGCCAGAAACCGCATGTTGTTTGTGTTCCGTATCCGGCACAGGGTCATATTAATCCGATGATGCGTGTTGCAAAACTGCTGCATGCACGTGGTTTTTATGTTACCTTTGTTAACACCGTGTATAACCACAATCGTTTTCTGCGTAGCCGTGGTAGCAATGCACTGGATGGTCTGCCGAGCTTTCGTTTTGAAAGCATTGCAGATGGCCTGCCGGAAACCGATATGGATGCAACCCAGGATATTACCGCACTGTGTGAAAGCACCATGAAAAATTGTCTGGCACCGTTTCGTGAGCTGCTGCAGCGTATTAATGCCGGTGATAATGTTCCGCCTGTTAGCTGTATTGTTAGTGATGGTTGTATGAGCTTTACCCTGGATGTTGCCGAAGAACTGGGTGTTCCGGAAGTTCTGTTTTGGACCACCAGTGGTTGTGCATTTCTGGCATATCTGCATTTTTACCTGTTTATCGAAAAAGGTCTGTGTCCGCTGAAAGATGAAAGCTATCTGACCAAAGAATACCTGGAAGATACCGTGATTGATTTCATTCCGACGATGAAAAACGTGAAGCTGAAAGACATTCCGAGCTTTATTCGTACCACCAATCCGGATGATGTGATGATTAGCTTTGCACTGCGTGAAACCGAACGTGCAAAACGTGCCAGCGCAATTATTCTGAATACCTTTGATGATCTGGAACACGATGTTGTTCATGCAATGCAGAGCATTCTGCCTCCGGTTTATAGCGTTGGTCCGCTGCATCTGCTGGCAAATCGTGAAATTGAAGAAGGTAGCGAAATTGGTATGATGAGCAGCAATCTGTGGAAAGAAGAAATGGAATGTCTGGATTGGCTGGATACCAAAACACAGAATAGCGTGATCTATATCAACTTTGGTAGCATTACCGTTCTGAGCGTTAAACAGCTGGTTGAATTTGCATGGGGTTTAGCAGGTAGCGGTAAAGAATTTCTGTGGGTTATTCGTCCGGATCTGGTTGCCGGTGAAGAAGCAATGGTTCCTCCGGATTTTCTGATGGAAACCAAAGATCGTAGCATGCTGGCAAGCTGGTGTCCGCAAGAAAAAGTTCTGAGCCATCCGGCAATTGGTGGCTTTCTGACCCATTGTGGTTGGAATAGCATTCTGGAAAGCCTGAGCTGTGGTGTTCCGATGGTTTGTTGGCCGTTTTTCGCAGATCAGCAGATGAATTGTAAATTTTGCTGTGATGAATGGGATGTGGGCATTGAAATTGGTGGTGATGTTAAACGCGAAGAAGTTGAAGCAGTTGTTCGTGAACTGATGGATGGTGAAAAAGGTAAAAAGATGCGCGAAAAAGCAGTTGAATGGCAGCGTCTGGCAGAAAAAGCAACCGAGCATAAACTGGGTAGCAGCGTTATGAATTTTGAAACCGTTGTGAGCAAATTTCTGCTGGGCCAGAAAAGCCAGGATTAA（SEQ ID NO:2）;

Amino acid sequence of sucrose synthase AcSUS from thiobacillus caldus Acidithiobacillus caldus ：MIEALRQQLLDDPRSWYAFLRHLVASQRDSWLYTDLQRACADFREQLPEGYAEGIGPLEDFVAHTQEVIFRDPWMVFAWRPRPGRWIYVRIHREQLALEELSTDAYLQAKEGIVGLGAEGEAVLTVDFRDFRPVSRRLRDESTIGDGLTHLNRRLAGRIFSDLAAGRSQILEFLSLHRLDGQNLMLSNGNTDFDSLRQTVQYLGTLPRETPWAEIREDMRRRGFAPGWGNTAGRVRETMRLLMDLLDSPSPAALESFLDRIPMISRILIVSIHGWFAQDKVLGRPDTGGQVVYILDQARALEREMRNRLRQQGVDVEPRILIATRLIPESDGTTCDQRLEPVVGAENVQILRVPFRYPDGRIHPHWISRFKIWPWLERYAQDLEREVLAELGSRPDLIIGNYSDGNLVATLLSERLGVTQCNIAHALEKSKYLYSDLHWRDHEQDHHFACQFTADLIAMNAADIIVTSTYQEIAGNDREIGQYEGHQDYTLPGLYRVENGIDVFDSKFNIVSPGADPRFYFSYARTEERPSFLEPEIESLLFGREPGADRRGVLEDRQKPLLLSMARMDRIKNLSGLAELYGRSSRLRGLANLVIIGGHVDVGNSRDAEEREEIRRMHEIMDHYQLDGQLRWVGALLDKTVAGELYRVVADGRGVFVQPALFEAFGLTVIEAMSSGLPVFATRFGGPLEIIEDGVSGFHIDPNDHEATAERLADFLEAARERPKYWLEISDAALARVAERYTWERYAERLMTIARIFGFWRFVLDRESQVMERYLQMFRHLQWRPLAHAVPME（SEQ ID NO:3）;

Nucleotide sequence (codon optimized) of sucrose synthase AcSUS from thiobacillus caldus Acidithiobacillus caldus ）：ATGATTGAAGCACTGCGTCAGCAACTGCTGGATGATCCGCGTAGCTGGTATGCATTTCTGCGTCATCTGGTTGCAAGCCAGCGTGATAGCTGGCTGTATACCGATCTGCAGCGTGCATGTGCAGATTTTCGTGAACAGCTGCCGGAAGGTTATGCAGAAGGTATTGGTCCGCTGGAAGATTTTGTTGCACATACCCAAGAAGTGATTTTTCGCGATCCGTGGATGGTTTTTGCATGGCGTCCGCGTCCTGGTCGTTGGATTTATGTTCGTATTCATCGTGAGCAGCTGGCACTGGAAGAACTGAGCACCGATGCATATCTGCAGGCAAAAGAAGGCATTGTTGGTCTGGGTGCCGAAGGTGAAGCAGTTCTGACCGTTGATTTCCGTGATTTTCGTCCGGTTAGCCGTCGTCTGCGTGATGAAAGCACCATTGGTGATGGTCTGACCCATCTGAATCGTCGTCTGGCAGGTCGTATTTTTAGCGATCTGGCAGCCGGTCGTAGCCAGATTCTGGAATTTCTGAGCCTGCATCGTCTGGATGGTCAGAATCTGATGCTGAGCAATGGTAATACCGATTTTGATAGTCTGCGTCAGACCGTTCAGTATCTGGGCACCCTGCCTCGTGAAACCCCGTGGGCAGAAATTCGTGAAGATATGCGTCGTCGTGGTTTTGCACCTGGTTGGGGCAATACCGCAGGTCGTGTGCGTGAAACCATGCGTCTGCTGATGGATCTGCTGGATAGCCCGAGTCCGGCAGCACTGGAAAGTTTTCTGGATCGTATTCCGATGATTAGCCGTATTCTGATTGTTAGCATTCATGGTTGGTTTGCCCAGGATAAAGTTCTGGGTCGTCCGGATACCGGTGGTCAGGTTGTTTATATTCTGGATCAGGCACGTGCACTGGAACGTGAAATGCGTAATCGTCTGCGCCAGCAGGGTGTTGATGTTGAACCGCGTATCCTGATTGCAACCCGTCTGATTCCGGAAAGTGATGGCACCACCTGTGATCAGCGTCTGGAACCGGTTGTTGGTGCAGAAAATGTTCAGATCCTGCGTGTTCCGTTTCGTTATCCGGATGGTCGCATTCATCCGCATTGGATTAGCCGCTTTAAAATCTGGCCGTGGCTGGAACGTTATGCACAGGATCTGGAACGCGAAGTTCTGGCCGAACTGGGTAGCCGTCCGGATCTGATTATTGGTAATTATAGTGATGGTAATCTGGTGGCAACCCTGCTGAGCGAACGTCTGGGTGTTACCCAGTGTAATATTGCACATGCCCTGGAAAAATCCAAATATCTGTATAGTGATCTGCACTGGCGTGATCATGAACAGGATCATCATTTTGCATGTCAGTTTACCGCAGATCTGATTGCTATGAATGCAGCCGATATTATTGTTACCAGCACCTATCAAGAAATCGCAGGTAATGATCGTGAAATCGGTCAGTATGAAGGTCATCAGGATTATACCCTGCCTGGTCTGTATCGTGTTGAAAATGGTATTGATGTGTTCGATAGCAAATTCAACATTGTTTCACCGGGTGCAGATCCGCGTTTTTACTTTAGCTATGCACGTACCGAAGAACGTCCGAGCTTTCTGGAACCAGAAATTGAAAGCCTGCTGTTTGGTCGTGAACCTGGTGCCGATCGTCGCGGTGTTCTGGAAGATCGTCAGAAACCGCTGCTGCTGAGCATGGCACGTATGGATCGCATTAAAAACCTGAGCGGTCTGGCAGAACTGTATGGTCGTAGCAGTCGCCTGCGTGGTCTGGCAAATCTGGTTATTATTGGTGGTCATGTTGATGTGGGTAATAGCCGTGATGCGGAAGAACGTGAAGAAATTCGCCGTATGCATGAAATCATGGATCATTATCAGCTGGATGGCCAGCTGCGTTGGGTTGGTGCACTGCTGGACAAAACCGTTGCCGGTGAACTGTATCGCGTTGTTGCAGATGGTCGTGGTGTTTTTGTTCAGCCTGCACTGTTTGAAGCATTTGGCCTGACCGTTATTGAAGCAATGAGCAGCGGTCTGCCGGTTTTTGCGACCCGTTTTGGTGGTCCTCTGGAAATTATTGAAGATGGTGTTAGCGGCTTTCATATCGATCCGAACGATCATGAAGCAACCGCAGAACGCCTGGCCGATTTTCTGGAAGCAGCACGTGAACGTCCTAAATATTGGCTGGAAATTTCAGATGCAGCCCTGGCACGTGTTGCAGAACGCTATACCTGGGAACGCTATGCAGAACGTCTGATGACCATTGCACGTATTTTTGGTTTTTGGCGTTTTGTGCTGGATCGTGAATCACAGGTTATGGAACGCTACCTGCAGATGTTTCGCCATTTACAGTGGCGTCCTCTGGCACATGCAGTTCCGATGGAATAA（SEQ ID NO:4）;

Amino acid sequence of engineered ADP glycosyltransferase AtUGT A1-I308Q/V333R/W363S/S386G (AGT) ：MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSQTVLSVKQLVEFAWGLAGSGKEFLWRIRPDLVAGEEAMVPPDFLMETKDRSMLASSCPQEKVLSHPAIGGFLTHCGWNGILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD（SEQ ID NO:5）;

Nucleotide sequence of engineered ADP glycosyltransferase AtUGT A1-I308Q/V333R/W363S/S386G (AGT) ：ATGGGCAGCCAGATTATTCATAATAGCCAGAAACCGCATGTTGTTTGTGTTCCGTATCCGGCACAGGGTCATATTAATCCGATGATGCGTGTTGCAAAACTGCTGCATGCACGTGGTTTTTATGTTACCTTTGTTAACACCGTGTATAACCACAATCGTTTTCTGCGTAGCCGTGGTAGCAATGCACTGGATGGTCTGCCGAGCTTTCGTTTTGAAAGCATTGCAGATGGCCTGCCGGAAACCGATATGGATGCAACCCAGGATATTACCGCACTGTGTGAAAGCACCATGAAAAATTGTCTGGCACCGTTTCGTGAGCTGCTGCAGCGTATTAATGCCGGTGATAATGTTCCGCCTGTTAGCTGTATTGTTAGTGATGGTTGTATGAGCTTTACCCTGGATGTTGCCGAAGAACTGGGTGTTCCGGAAGTTCTGTTTTGGACCACCAGTGGTTGTGCATTTCTGGCATATCTGCATTTTTACCTGTTTATCGAAAAAGGTCTGTGTCCGCTGAAAGATGAAAGCTATCTGACCAAAGAATACCTGGAAGATACCGTGATTGATTTCATTCCGACGATGAAAAACGTGAAGCTGAAAGACATTCCGAGCTTTATTCGTACCACCAATCCGGATGATGTGATGATTAGCTTTGCACTGCGTGAAACCGAACGTGCAAAACGTGCCAGCGCAATTATTCTGAATACCTTTGATGATCTGGAACACGATGTTGTTCATGCAATGCAGAGCATTCTGCCTCCGGTTTATAGCGTTGGTCCGCTGCATCTGCTGGCAAATCGTGAAATTGAAGAAGGTAGCGAAATTGGTATGATGAGCAGCAATCTGTGGAAAGAAGAAATGGAATGTCTGGATTGGCTGGATACCAAAACACAGAATAGCGTGATCTATATCAACTTTGGTAGCCAAACCGTTCTGAGCGTTAAACAGCTGGTTGAATTTGCATGGGGTTTAGCAGGTAGCGGTAAAGAATTTCTGTGGCGTATTCGTCCGGATCTGGTTGCCGGTGAAGAAGCAATGGTTCCTCCGGATTTTCTGATGGAAACCAAAGATCGTAGCATGCTGGCAAGCTCTTGTCCGCAAGAAAAAGTTCTGAGCCATCCGGCAATTGGTGGCTTTCTGACCCATTGTGGTTGGAATGGCATTCTGGAAAGCCTGAGCTGTGGTGTTCCGATGGTTTGTTGGCCGTTTTTCGCAGATCAGCAGATGAATTGTAAATTTTGCTGTGATGAATGGGATGTGGGCATTGAAATTGGTGGTGATGTTAAACGCGAAGAAGTTGAAGCAGTTGTTCGTGAACTGATGGATGGTGAAAAAGGTAAAAAGATGCGCGAAAAAGCAGTTGAATGGCAGCGTCTGGCAGAAAAAGCAACCGAGCATAAACTGGGTAGCAGCGTTATGAATTTTGAAACCGTTGTGAGCAAATTTCTGCTGGGCCAGAAAAGCCAGGATTAA（SEQ ID NO:6）;

Amino acid sequence of engineered ADP glycosyltransferase AGT-A21I/M83N/Q407P ：MGSQIIHNSQKPHVVCVPYPIQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDNDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSCIVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVHAMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSQTVLSVKQLVEFAWGLAGSGKEFLWRIRPDLVAGEEAMVPPDFLMETKDRSMLASSCPQEKVLSHPAIGGFLTHCGWNGILESLSCGVPMVCWPFFADQPMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD（SEQ ID NO:7）;

Nucleotide sequence of engineered ADP glycosyltransferase AGT-A21I/M83N/Q407P ：ATGGGCAGCCAGATTATTCATAATAGCCAGAAACCGCATGTTGTTTGTGTTCCGTATCCGATACAGGGTCATATTAATCCGATGATGCGTGTTGCAAAACTGCTGCATGCACGTGGTTTTTATGTTACCTTTGTTAACACCGTGTATAACCACAATCGTTTTCTGCGTAGCCGTGGTAGCAATGCACTGGATGGTCTGCCGAGCTTTCGTTTTGAAAGCATTGCAGATGGCCTGCCGGAAACCGATAACGATGCAACCCAGGATATTACCGCACTGTGTGAAAGCACCATGAAAAATTGTCTGGCACCGTTTCGTGAGCTGCTGCAGCGTATTAATGCCGGTGATAATGTTCCGCCTGTTAGCTGTATTGTTAGTGATGGTTGTATGAGCTTTACCCTGGATGTTGCCGAAGAACTGGGTGTTCCGGAAGTTCTGTTTTGGACCACCAGTGGTTGTGCATTTCTGGCATATCTGCATTTTTACCTGTTTATCGAAAAAGGTCTGTGTCCGCTGAAAGATGAAAGCTATCTGACCAAAGAATACCTGGAAGATACCGTGATTGATTTCATTCCGACGATGAAAAACGTGAAGCTGAAAGACATTCCGAGCTTTATTCGTACCACCAATCCGGATGATGTGATGATTAGCTTTGCACTGCGTGAAACCGAACGTGCAAAACGTGCCAGCGCAATTATTCTGAATACCTTTGATGATCTGGAACACGATGTTGTTCATGCAATGCAGAGCATTCTGCCTCCGGTTTATAGCGTTGGTCCGCTGCATCTGCTGGCAAATCGTGAAATTGAAGAAGGTAGCGAAATTGGTATGATGAGCAGCAATCTGTGGAAAGAAGAAATGGAATGTCTGGATTGGCTGGATACCAAAACACAGAATAGCGTGATCTATATCAACTTTGGTAGCCAAACCGTTCTGAGCGTTAAACAGCTGGTTGAATTTGCATGGGGTTTAGCAGGTAGCGGTAAAGAATTTCTGTGGCGTATTCGTCCGGATCTGGTTGCCGGTGAAGAAGCAATGGTTCCTCCGGATTTTCTGATGGAAACCAAAGATCGTAGCATGCTGGCAAGCTCTTGTCCGCAAGAAAAAGTTCTGAGCCATCCGGCAATTGGTGGCTTTCTGACCCATTGTGGTTGGAATGGCATTCTGGAAAGCCTGAGCTGTGGTGTTCCGATGGTTTGTTGGCCGTTTTTCGCAGATCAGCCGATGAATTGTAAATTTTGCTGTGATGAATGGGATGTGGGCATTGAAATTGGTGGTGATGTTAAACGCGAAGAAGTTGAAGCAGTTGTTCGTGAACTGATGGATGGTGAAAAAGGTAAAAAGATGCGCGAAAAAGCAGTTGAATGGCAGCGTCTGGCAGAAAAAGCAACCGAGCATAAACTGGGTAGCAGCGTTATGAATTTTGAAACCGTTGTGAGCAAATTTCTGCTGGGCCAGAAAAGCCAGGATTAA（SEQ ID NO:8）.

In comparative example 1 and examples 1 to 5 of the present invention, the raw materials and reagents used were all commercially available.

The invention is further illustrated by the following examples:

comparative example 1 UDP Synthesis of salidroside by glycosyltransferase and sucrose synthase

Selecting UDP glycosyltransferase AtUGT A1 from Arabidopsis Arabidopsis thaliana and sucrose synthase AcSUS from thiobacillus caldus Acidithiobacillus caldus, performing codon optimization on host escherichia coli ESCHERICHIA COLI by using a Invitrogen GeneArt online tool GeneOptimazer to obtain a corresponding optimized nucleotide sequence, performing total gene synthesis, and constructing on a pET-22b vector.

The resulting plasmids pET22b-atUGT A1 and pET22b-acSUS transformed BL21 (DE 3) competent cells, respectively, and expanded to 2000mL LB liquid medium (Amp), and induced to express at 30℃under 0.1mM IPTG. The cells were collected by low-temperature centrifugation, and the cells were resuspended in 4mL of a disruption solution (100 mM K ₂HPO₄∙3H₂O, 10mM KH₂PO₄, 200mM NaCl, pH 7.6) and then sonicated to obtain a crude enzyme solution. Protein electrophoresis examined the expression (FIG. 1), atUGT A1 and AcSUS were both predominantly expressed in the supernatant.

35G of sucrose (204.5 mM, volume of reaction solution 500 mL), 14g of tyrosol (202.7 mM), and 0.04g of UDP (0.2 mM) were weighed and dissolved in 400mL of pure water, pH was adjusted to 6.0 and the volume was set to 450mL,30 mL of AtUGT A1 crude enzyme solution (12.5U/mL) and 20mL of AcSUS crude enzyme solution (20.6U/mL) were added after preheating at 30℃to start the reaction.

In the reaction process, 100 mu L of methanol with the concentration of +900 mu L is sampled and diluted, 100 mu L of methanol with the concentration of +900 mu L is sucked from the diluted mixture, and the mixture is subjected to High Performance Liquid Chromatography (HPLC) after 100 times of dilution to detect tyrosol and salidroside. As shown in Table 1, the conversion was 86.85% after 48 hours of reaction, but the time was longer.

TABLE 1

Example 1 glycoside-selective engineering of UDP glycosyltransferases

The predicted structure of AtUGT A1 of AlphaFold was aligned integrally with the stevia-derived glycosyltransferase SrUGT G1-UDP-RA complex (PDB: 6 INI) as a reference model (FIG. 2), and the scaffold RMSD 1.006A demonstrated that the structure was more similar, particularly in regions that bind UDP. With LigPlot residues shown to interact with UDP (FIG. 3), val309 and Trp338 of SrUGT G1 constrain the uracil ring of UDP, and the diphosphate interactions of Ser283 and Ser361 with UDP may be involved in glycosyl transfer. Sequence and structural alignment (FIGS. 4 and 5) shows that AtUGT A1 is the residue Ser307 (Ser 283), ile308 (Thr 284), val333 (Val 309), trp363 (Trp 338), ser386 (Ser 361), in brackets the corresponding SrUGT G1 residue, possibly interacting with UDP.

To accommodate the larger adenine ring, val333 and Trp363 were first selected as the site of the AtUGT A1 modification, while Ser307, ile308 and Ser386 were fine tuned to accommodate the translocation of diphosphate, primers in table 2 were designed to construct mutant plasmids using QuickChange site-directed mutagenesis kit (agilent).

TABLE 2

The resulting plasmids were transformed into BL21 (DE 3) competent cells, and expanded to 200mL LB liquid medium (Amp), and induced to express at 30℃under 0.1mM IPTG. And (3) centrifugally collecting thalli at a low temperature, adding the crushing liquid into the thalli according to the ratio of 1g to 9mL, and carrying out ultrasonic crushing to obtain a corresponding crude enzyme liquid.

1ML of crude enzyme solution was aspirated to a final volume of 10mL of reaction solution (10 mM tyrosol, 5mM ADP-glucose, pH 6.0), reacted at 30℃at 300rpm overnight, the reaction was terminated by adding 5mL of acetonitrile, and the supernatant was centrifuged to detect salidroside by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results are shown in Table 3, V333R is the optimal single point mutation.

TABLE 3 Table 3

In Table 3, ^* defines product concentrations of "-" as less than 1.5 times "+" as 1.5-2.5 times "+ +" as 2.5-3.5 times "+ + +" as greater than 3.5 times.

Other mutation sites are superimposed on the AtUGT A1-V333R mutant, and the reaction is constructed, cultured, expressed and broken in the same manner as described above. As a result, atUGT A1-I308Q/V333R/W363S/S386G was the optimal mutation as shown in Table 4.

TABLE 4 Table 4

In Table 3, the product concentration of ^* compared to AtUGT A1-V333R mutant is defined as "+" less than 1.5-fold; "++" 1.5-2-fold; "++" more than 2-fold.

EXAMPLE 2 Synthesis of salidroside by glycosyltransferase mutant and sucrose synthase

AtUGT85A1-I308Q/V333R/W363S/S386G mutant BL21 (DE 3) glycerol bacteria were grown up to 2000mL LB liquid medium (Amp) and induced to express at 30℃under 0.1mM IPTG. The cells were collected by low-temperature centrifugation, and the cells were resuspended in 4mL of a disruption solution (100 mM K ₂HPO₄∙3H₂O, 10mM KH₂PO₄, 200mM NaCl, pH 7.6) and then sonicated to obtain a crude enzyme solution.

70G of sucrose (204.5 mM, volume of reaction solution 1000 mL) and 28G of tyrosol (202.7 mM) were weighed and dissolved in 800mL of pure water, pH was adjusted to 6.0 and the volume was fixed to 900mL, and two portions of the mixture were separated, 0.04G (0.2 mM) of ADP and 0.04G (0.2 mM) of UDP were added to dissolve respectively, and after preheating at 30 ℃, atUGT A1-I308Q/V333R/W363S 386G crude enzyme solution (13.3U/mL) and 30mL of AcSUS crude enzyme solution (20.6U/mL) were added to dissolve respectively, and the reaction was started.

In the reaction process, 100 mu L of methanol with the concentration of +900 mu L is sampled and diluted, 100 mu L of methanol with the concentration of +900 mu L is sucked from the diluted mixture, and the mixture is subjected to High Performance Liquid Chromatography (HPLC) after 100 times of dilution to detect tyrosol and salidroside. As shown in Table 5, the conversion rate 87.37% after 46h of ADP group reaction was equivalent to that of comparative example 1 in which AtUGT A1 enzyme and AcSUS enzyme regenerated UDG-glucose to salidroside, while the conversion rate of UDP group reaction 46h was only 38.01%, indicating that the modified AtUGT A1-I308Q/V333R/W363S/S386G was ADP-glucose selective glycosyltransferase, designated as "AGT".

TABLE 5

Example 3 thermostability modification of ADP glycosyltransferase AGT

The AlphaFold predicted AtUGT A1 structure was uploaded to a protein mutation prediction tool Pythia based on structure self-supervised learning, a global single point mutation energy heat map (fig. 6) was generated, the first 20 single point mutations with the lowest ΔΔg values are shown in table 6, and structural analysis was performed.

TABLE 6

The M83, V310, Q407, L467, M472 and A21, G151 sites affecting Loop flexibility on the surface were selected, and the primers in Table 7 were designed to construct plasmids of mutants using the QuickChange site-directed mutagenesis kit (Agilent) with the nucleotide sequence of AGT (SEQ ID NO. 6) as template.

TABLE 7

In Table 7, ^* defines the product concentration of "-" as less than 1.5 times "+" as 1.5-3 times "+", as 3-6 times "+ + + +", as greater than 6 times.

1ML of crude enzyme solution was aspirated to a final volume of 10mL of reaction solution (10 mM tyrosol, 5mM ADP-glucose, pH 6.0), reacted at 60℃at 300rpm overnight, the reaction was terminated by adding 5mL of acetonitrile, and the supernatant was centrifuged to detect salidroside by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results are shown in Table 7, where Q407P is the optimal single point mutation.

Other mutation sites were added on the basis of AGT-Q407P mutant, and the reaction was constructed, cultured, expressed and broken in the same manner as described above. The results are shown in Table 8, where AGT-A21I/M83N/Q407P is the optimal mutation.

TABLE 8

In Table 8, the product concentration of ^* compared to the AGT-Q407P mutant is defined as "-" being less than 1-fold, "+" 1-2-fold, "++" 2-3-fold and "++" being more than 3-fold.

EXAMPLE 4 ADP Synthesis of salidroside by glycosyltransferase and sucrose synthase

AGT-A21I/M83N/Q407P mutant BL21 (DE 3) glycerinum was grown up to 2000mL LB liquid medium (Amp) and induced to express at 30℃under 0.1mM IPTG. The cells were collected by low-temperature centrifugation, and the cells were resuspended in 4mL of a disruption solution (100 mM K ₂HPO₄∙3H₂O, 10mM KH₂PO₄, 200mM NaCl, pH 7.6) and then sonicated to obtain a crude enzyme solution.

35G (204.5 mM, 500mL of reaction solution volume), 14g (202.7 mM) of tyrosol and 0.04g (0.2 mM) of ADP were weighed and dissolved in 400mL of pure water, the pH was adjusted to 6.0 and the volume was adjusted to 480mL, 10mL of AGT-A21I/M83N/Q407P crude enzyme solution (80.3U/mL) and 10mL of AcSUS crude enzyme solution (83.8U/mL) were added after preheating at 60℃to start the reaction.

In the reaction process, 100 mu L of methanol with the concentration of +900 mu L is sampled and diluted, 100 mu L of methanol with the concentration of +900 mu L is sucked from the diluted mixture, and the mixture is subjected to High Performance Liquid Chromatography (HPLC) after 100 times of dilution to detect tyrosol and salidroside. As shown in Table 9, the conversion rate after 22 hours was 88.72%, and the reaction time was significantly shortened and the enzyme amount was lower than that of the AGT enzyme and AcSUS enzyme reactions in example 2.

TABLE 9

EXAMPLE 5 ADP Synthesis of high-concentration salidroside by glycosyltransferase and sucrose synthase

70G (409.0 mM) of sucrose, 28g (405.3 mM) of tyrosol and 0.04g (0.2 mM) of ADP were weighed and dissolved in 400mL of pure water, the pH was adjusted to 6.0 and the volume was set to 480mL, 15mL of AGT-A21I/M83N/Q407P crude enzyme solution (80.3U/mL) and 10mL of AcSUS crude enzyme solution (83.8U/mL) were added after preheating at 60℃to start the reaction.

In the reaction process, 100 mu L of methanol with the concentration of +900 mu L is sampled and diluted, 100 mu L of methanol with the concentration of +900 mu L is sucked from the diluted mixture, and the mixture is subjected to High Performance Liquid Chromatography (HPLC) after 100 times of dilution to detect tyrosol and salidroside. As shown in Table 10, the conversion rate is 83.84% after 22 hours of reaction, and the highest report is that the salidroside 102g/L is known.

And (3) removing proteins from the reaction solution through a ceramic membrane, purifying a liquid phase prepared by using C18 of 30% methanol in a mobile phase, collecting a target peak, rotary evaporating, and drying at constant temperature in vacuum to obtain a crude product. The crude product is heated and dissolved by 5 times volume of methanol/ethyl formate (1:1, v/v) at 50 ℃, then cooled to 4 ℃ for crystallization, and vacuum dried at constant temperature to obtain a pure product, wherein the purity of the pure product is 99.98 percent (figure 7) detected by High Performance Liquid Chromatography (HPLC), and the pure product is characterized by mass spectrum (figure 8) to be salidroside.

Table 10

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. An enzyme mutant, characterized in that, based on a wild-type glycosyltransferase, it has the following amino acid site mutations: site 308 is mutated from I to Q, site 333 is mutated from V to R, site 363 is mutated from W to S, and site 386 is mutated from S to G, wherein the amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO: 1.

2. The mutant of the enzyme according to claim 1, characterized in that its amino acid sequence is shown in SEQ ID NO: 5.

3. A mutant of a glycosyltransferase, characterized in that, based on the mutant according to claim 1 or 2, it has the following amino acid site mutations: position 21 is mutated from A to I, position 83 is mutated from M to N, and position 407 is mutated from Q to P.

4. The mutant of glycosyltransferase according to claim 3, wherein the amino acid sequence thereof is as shown in SEQ ID NO: 7.

5. An enzyme composition, characterized in that it comprises: the mutant of the enzyme according to claim 1 or 2 and/or the mutant of the glycosyltransferase according to claim 3 or 4, and sucrose synthase.

6. A nucleic acid molecule encoding a mutant of the enzyme according to claim 1 or 2, a mutant of the glycosyltransferase according to claim 3 or 4 and/or an enzyme composition according to claim 5.

7. The nucleic acid molecule according to claim 6, comprising:

The nucleotide sequence of the nucleic acid molecule encoding the mutant of the enzyme is shown in SEQ ID NO: 6; and/or

The nucleotide sequence of the nucleic acid molecule encoding the mutant of the glycosyltransferase is shown in SEQ ID NO: 8; and/or

The nucleotide sequence of the nucleic acid molecule encoding the sucrose synthase is shown in SEQ ID NO: 4.

8. A recombinant vector, characterized in that it comprises: the nucleic acid molecule according to claim 6 or 7 and an acceptable genetic element.

9. A host, characterized in that it is transformed and/or transfected with the recombinant vector according to claim 8.

10. A product, characterized in that it comprises: the mutant of the enzyme according to claim 1 or 2, the mutant of the glycosyltransferase according to claim 3 or 4, the enzyme composition according to claim 5, the nucleic acid molecule according to claim 6 or 7, the recombinant vector according to claim 8 and/or the host according to claim 9, and acceptable adjuvants or excipients.

11. Use of the mutant of the enzyme according to claim 1 or 2, the mutant of the glycosyltransferase according to claim 3 or 4, the enzyme composition according to claim 5, the nucleic acid molecule according to claim 6 or 7, the recombinant vector according to claim 8, the host according to claim 9 and/or the product according to claim 10 in the preparation of salidroside.

12. A method for preparing salidroside, characterized in that the raw materials are converted into salidroside by any of the following methods:

(a) a mutant of the enzyme according to claim 1 or 2; or

(b) a mutant of the glycosyltransferase according to claim 3 or 4; or

(c) an enzyme composition as claimed in claim 5; or

(d) a nucleic acid molecule according to claim 6 or 7; or

(e) the recombinant vector according to claim 8; or

(f) the host according to claim 9; or

(g) The product according to claim 10;

The raw materials include sucrose, tyrosol and ADP.

13. The preparation method according to claim 12, wherein the temperature during the conversion is 30-60°C.