A mutant of the 415th position of the sugar transferase mutant and its application in the production of salidroside

Abstract

This invention proposes an acid-loving glycosyltransferase mutant with a mutation at position 415 and its application in the production of rhodioloside, belonging to the field of bioengineering technology. It solves the problem of low synthesis efficiency of rhodioloside under acidic conditions. It comprises an acid-loving glycosyltransferase mutant obtained by mutating any one of the amino acids shown in SEQ ID NO.2 at position 415. It is mainly used for high-yield production of rhodioloside under acidic conditions.

Description

[0001] This application is a divisional application of application number 202510901721.8, application date July 1, 2025, entitled "A sour glycosyltransferase mutant and its application in the production of rhodioloside". Technical Field

[0002] This invention belongs to the field of bioengineering technology, and in particular relates to a mutant of acid-saccharide transferase with a mutation at position 415 and its application in the production of rhodioloside. Background Technology

[0003] Salidroside (CAS 10338-51-9) is a natural glycoside compound, chemically named 2-(4-hydroxyphenyl)ethyl-β-D-glucoside (molecular formula C64-). 14 H 20 O7 (molecular weight 300.3). Its physicochemical properties include a sweet taste and high water solubility (extremely soluble in water and methanol, sparingly soluble in ether). Rhodioloside is the core active ingredient of plants in the genus Rhodiola of the Rosaceae family, possessing multiple pharmacological activities such as anti-hypoxia, anti-fatigue, antioxidant, neuroprotective, and anti-tumor effects. With its expanding applications in functional foods, cosmetics, and cardiovascular drugs, global market demand is increasing by more than 15% annually.

[0004] The extraction of rhodioloside from natural plants faces serious bottlenecks. Wild rhodiola resources are scarce, with a growth cycle of 5-7 years, and the content is only 0.1-1% (dry weight), which is insufficient to meet market demand. Although chemical synthesis can achieve large-scale production, it faces problems such as the use of toxic reagents (such as halogenated hydrocarbons and heavy metal catalysts), serious pollution, and violation of green manufacturing principles. However, biosynthesis is considered an alternative due to its mild conditions, fewer byproducts, and environmental friendliness. Summary of the Invention

[0005] In view of this, the present invention aims to provide an acid-loving glycosyltransferase mutant and its application in the production of rhodioloside, in order to solve the problem of low synthesis efficiency of rhodioloside under acidic conditions in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an acid-loving glycosyltransferase mutant, which is obtained by mutating any one of the following positions: 87, 210, 293, 356, 399, 400, and 415, starting from the amino acid sequence shown in SEQ ID NO. 2.

[0007] This invention provides a glycosyltransferase mutant, wherein the glycosyltransferase mutant is any one of the following: (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace isoleucine in position 87 with phenylalanine; (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace valine at position 210 with threonine; (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the asparagine in position 293 with tryptophan; (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the glutamic acid at position 356 with valine; (5) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the cysteine ​​in position 399 with alanine; (6) Using the amino acid sequence shown in SEQ ID NO.2 as the starting sequence, replace the arginine at position 400 with valine; (7) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the aspartic acid in position 415 with phenylalanine.

[0008] The present invention provides a gene encoding the above-mentioned glycosyltransferase mutant.

[0009] The present invention provides a recombinant vector containing the above-mentioned genes.

[0010] Further specifying, the launch carriers are the pRSFDuet series and the pETDuet series.

[0011] This invention provides a recombinant microbial cell containing the above-mentioned genes.

[0012] Further specifying, it includes genes containing phenylpyruvate decarboxylase. ARO10 Ethanol dehydrogenase gene ADH6 phosphoglucosuricase gene pgm and UDP-glucose pyrophosphorylase gene gaul Overexpression plasmids and genes containing mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase. aroG fbr Cyclohexadiene dehydrogenase gene tyrC The genetically engineered bacteria were obtained by transferring the overexpression plasmid of the mutant glycosyltransferase gene into Escherichia coli BL21(DE3) competent cells.

[0013] This invention provides the application of the above-mentioned glycosyltransferase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cell in the production of rhodioloside.

[0014] The present invention provides a method for producing rhodioloside, the steps of which are as follows: fermenting the genetically engineered bacteria described in claim 7 at 30°C for 72 hours, and adjusting the pH to 4.0-6.0.

[0015] Further specified, the fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract, with 10 g / L CaCO3 used to maintain the pH of the fermentation medium. Add 0.1% by volume of trace elements to the culture medium. The trace element composition is: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O and 15 g / L ZnSO4·7H2O. Adjust the pH to 4.0-6.0.

[0016] Compared with the prior art, the beneficial effects of the present invention are: the detection results of the production of rhodioloside and accumulation of tyrosol by different mutants under acidic fermentation culture conditions are as follows: Figure 1 As shown, the strain with the 415th amino acid mutant achieved the highest rhodioloside yield of 10.2 g / L after 72 hours of fermentation, while exhibiting the lowest tyrosol accumulation. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The following diagram illustrates the rhodioloside yield and tyrosol accumulation in shake-flask fermentation of a genetically engineered bacterium overexpressing wild-type glycosyltransferase and seven genetically engineered bacteria overexpressing mutant glycosyltransferases according to Example 1 of the present invention. WT, I87, V210, N293, E356, C399, R400, and D415 represent the overexpression of wild-type glycosyltransferase, mutant glycosyltransferase LrUGT85AF8, and other glycosyltransferases, respectively. I87F LrUGT85AF8 V210T LrUGT85AF8 N293W LrUGT85AF8 E356V LrUGT85AF8 C399A LrUGT85AF8R400V LrUGT85AF8 D415F Genetically engineered bacteria. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other, and the described embodiments are only some embodiments of the present invention, not all embodiments.

[0019] Example 1. Construction of an acid-loving glycosyltransferase mutant A glycosyltransferase mutant for efficient synthesis of rhodioloside is provided, wherein the wild-type glycosyltransferase gene LrUGT85AF8 Derived from robust privet ( Ligustrum robustum Its amino acid sequence is shown in SEQ ID NO: 2; the mutant glycosyltransferase gene LrUGT85AF8 I87F Its amino acid sequence is shown in SEQ ID NO: 4; the mutant glycosyltransferase gene LrUGT85AF8 V210T Its amino acid sequence is shown in SEQ ID NO: 6; the mutant glycosyltransferase gene LrUGT85AF8 N293W Its amino acid sequence is shown in SEQ ID NO: 8; the mutant glycosyltransferase gene LrUGT85AF8 E356V Its amino acid sequence is shown in SEQ ID NO: 10; the mutant glycosyltransferase gene LrUGT85AF8 C399A Its amino acid sequence is shown in SEQ ID NO: 12; the mutant glycosyltransferase gene LrUGT85AF8 R400V Its nucleotide sequence is shown in SEQ ID NO: 14; the mutant glycosyltransferase gene LrUGT85AF8 D415F Its amino acid sequence is shown in SEQ ID NO: 16, wherein the starting engineered bacteria is Escherichia coli BL21(DE3).

[0020] SEQ ID NO: 2 (Amino acid sequence of wild-type glycosyltransferase LrUGT85AF8): MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSISRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNIVQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYINFGSITPLSPEQAVEFAWGLAKSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQE QVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNCRYACAKWDIGVEIEGDVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN*; SEQ ID NO: 3 (Mutant glycosyltransferase LrUGT85AF8) I87F (nucleotide sequence) SEQ ID NO: 4 (Mutant glycosyltransferase LrUGT85AF8) I87F (amino acid sequence) MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSFSRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNIVQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYINFGSITPLSPEQAVEFAWGLAKSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQE QVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNCRYACAKWDIGVEIEGDVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN*; SEQ ID NO: 5 (Mutant glycosyltransferase LrUGT85AF8) V210T (nucleotide sequence) SEQ ID NO: 6 (Mutant glycosyltransferase LrUGT85AF8) V210T (amino acid sequence) MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSISRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNITQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYINFGSITPLSPEQAVEFAWGLAKSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQE QVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNCRYACAKWDIGVEIEGDVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN*; SEQ ID NO: 7 (Mutant glycosyltransferase LrUGT85AF8) N293W (nucleotide sequence) SEQ ID NO: 8 (Mutant glycosyltransferase LrUGT85AF8) N293W (amino acid sequence) MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSISRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNIVQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYIWFGSITPLSPEQAVEFAWGLAKSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQE QVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNCRYACAKWDIGVEIEGDVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN*; SEQ ID NO: 9 (Mutant glycosyltransferase LrUGT85AF8) E356V (nucleotide sequence) SEQ ID NO: 10 (Mutant glycosyltransferase LrUGT85AF8) E356V (amino acid sequence) MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSISRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNIVQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYINFGSITPLSPEQAVEFAWGLAKSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQV QVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNCRYACAKWDIGVEIEGDVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN*; SEQ ID NO: 11 (Mutant glycosyltransferase LrUGT85AF8) C399A (nucleotide sequence) SEQ ID NO: 12 (Mutant glycosyltransferase LrUGT85AF8) C399A (amino acid sequence) MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSISRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNIVQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYINFGSITPLSPEQAVEFAWGLAKSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQE QVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNARYACAKWDIGVEIEGDVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN*; SEQ ID NO: 13 (Mutant glycosyltransferase LrUGT85AF8) R400V (nucleotide sequence) SEQ ID NO: 14 (Mutant glycosyltransferase LrUGT85AF8) R400V (amino acid sequence) MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSISRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNIVQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYINFGSITPLSPEQAVEFAWGLAKSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQE QVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNCVYACAKWDIGVEIEGDVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN*; SEQ ID NO: 15 (Mutant glycosyltransferase LrUGT85AF8) D415F (nucleotide sequence) SEQ ID NO: 16 (Mutant glycosyltransferase LrUGT85AF8) D415F (amino acid sequence) MKSHAVVIPYPAQGHIAPVLKLAKLLHYKGFFITFVNTEFNHNRLVRARGADAVKGLEDFQFKTIPDGLPPSDDDATQDIPLLSQSISRNCLAPFLDLIKNLNESSDCPNVSCIVSDL VMSFTLDAADQLNIPEALFYTASACGFMGYTHYAELVARGYVPLKDESCLTNGYLETEIDWIPGMKGIRLKDIPTFIRTTDPNAIMLKYNIVQHGNASRAKAIIFNTFDELEEEVLEAI RDRYDQVYTIGPLQLLEKEIYITKKLKSIGSNLWKEDMDCLNWLDQREENSVLYINFGSITPLSPEQAVEFAWGLAKSSNHHFLWIIRPDLMNGKGSILPEGFLEETQGRGLMVGWCPQ EQVLAHPAIGGFLTHCGWNSTIETISEGVPMVCWPFFAEQQTNCRYACAKWDIGVEIEGFVNRGKVEKMVRVMMEGEEGKEMRKKALEWKEKAHLAAKPGGSSYHDFEKLIHDTLLDN* .

[0021] Example 2: Construction of genetically engineered bacteria containing different glycosyltransferase mutants Overexpression originates from Escherichia coli (E. coli) Escherichia coli ) phosphoglucose mutase gene pgm and UDP-glucose pyrophosphorylase gene galU, Derived from brewer's yeast ( Saccharomyces cerevisiae ) phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm-galU-ARO10-ADH6 and overexpression of a mutant 3-deoxy-D-arabinohepulose-7-phosphate (DAHP) synthase gene derived from Escherichia coli. aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC and derived from Arabidopsis thaliana ( Arabidopsis thaliana mutant glycosyltransferase gene AtUGT85A1 A21G Recombinant plasmid pETDuet- aroGfbr -tyrC-AtUGT85A1 A21G From Zhou Jingwen's team at Jiangnan University (Engineering Escherichia coli for Efficient De Novo Synthesis of Salidroside) ） .

[0022] Overexpression plasmid pETDuet- aroG fbr -tyrC-AtUGT85A1 A21G glycosyltransferase gene AtUGT85A1 A21G Replaced with privet (from thick privet) Ligustrum robustum ) glycosyltransferase LrUGT85AF8 (wild-type, mutant glycosyltransferase LrUGT85AF8) I87F LrUGT85AF8 V210T LrUGT85AF8 N293W LrUGT85AF8 E356V LrUGT85AF8 C399A LrUGT85AF8 R400V LrUGT85AF8 D415F (Gene), to obtain the recombinant plasmid pETDuet- aroG fbr -tyrC-LrUGT85AF8 pETDuet- aroG fbr -tyrC-LrUGT85AF8 I87F pETDuet- aroG fbr -tyrC-LrUGT85AF8 V210T pETDuet- aroG fbr -tyrC-LrUGT85AF8 N293W pETDuet- aroG fbr - tyrC-LrUGT85AF8 E356V pETDuet- aroG fbr -tyrC-LrUGT85AF8 C399A pETDuet- aroG fbr -tyrC- LrUGT85AF8 R400V pETDuet- aroG fbr -tyrC- LrUGT85AF8 D415FThe expression plasmid pRSFDuet- was applied separately. pgm- galU-ARO10-ADH6 Eight plasmid combinations were obtained by combining plasmids with overexpression plasmids containing different mutant glycosyltransferase genes. These plasmids were then introduced into the chassis strain *Escherichia coli* BL21(DE3) to obtain eight engineered strains as shown in Table 1, named HE-1.0, HE-2.0, HE-3.0, HE-4.0, HE-5.0, HE-6.0, HE-7.0, and HE-8.0, respectively.

[0023] Table 1 Genetically engineered bacteria containing different glycosyltransferase mutants

[0024] Example 3: Fermentation experiment of rhodioloside in genetically engineered bacteria containing different glycosyltransferase mutants Eight genetically engineered bacteria were inoculated into LB broth containing antibiotic resistance. The culture system consisted of 10 mL aliquots of the medium in 100 mL Erlenmeyer flasks, supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin. The culture conditions were 37°C and 220 rpm overnight on a constant-temperature shaking incubator to obtain a seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 2%. The culture system consisted of 50 mL aliquots of the medium in 250 mL Erlenmeyer flasks, supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin. The culture conditions were 37°C and 220 rpm on a constant-temperature shaking incubator to obtain an OD (digestive growth rate). 600 When the concentration reaches 0.6-0.8, add 0.1 mM IPTG to induce fermentation. Fermentate at 30℃ and 220 rpm for 72 h to prepare samples.

[0025] Example 4: Extraction and liquid chromatography detection of rhodioloside Transfer 1 mL of fermentation broth to a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 5 min. Mix 200 μL of the supernatant with 800 μL of mobile phase A. After diluting the sample 5-fold, determine the rhodioloside yield using a Shimadzu high-performance liquid chromatograph. Quantification was performed using a reversed-phase C18 column. Mobile phase A was water containing 1% trifluoroacetic acid (TFA); mobile phase B was acetonitrile containing 1% TFA. The flow rate was 1 mL / min. -1 The absorbance of the sample was detected at 274 nm. The HPLC program was as follows: 10% B: 0-5 min, 60% B: 5-10 min, 90% B: 10-12 min, 2% B: 12-16 min, 0% B: 16-20 min.

[0026] The results after 72 h of shake-flask fermentation under acidic fermentation conditions are as follows: Figure 1 As shown, the control strain HE-1.0 produced 8.38 g / L of rhodioloside and accumulated 1.02 g / L of tyrosol; glycosyltransferase LrUGT85AF8 I87F The mutant strain HE-2.0 produced 3.86 g / L of rhodioloside and accumulated 5.23 g / L of tyrosol; glycosyltransferase LrUGT85AF8 V210T The mutant strain HE-3.0 produced 6.89 g / L of rhodioloside and accumulated 3.87 g / L of tyrosol; glycosyltransferase LrUGT85AF8 N293W The mutant strain HE-4.0 produced 5.78 g / L of rhodioloside and accumulated 4.01 g / L of tyrosol; glycosyltransferase LrUGT85AF8 E356V The mutant strain HE-5.0 produced 9.71 g / L of rhodioloside and accumulated 1.14 g / L of tyrosol; glycosyltransferase LrUGT85AF8... C399A The mutant strain HE-6.0 produced 6.13 g / L of rhodioloside and accumulated 3.12 g / L of tyrosol; glycosyltransferase LrUGT85AF8 R400V The mutant strain HE-7.0 produced 9.07 g / L of rhodioloside and accumulated 1.02 g / L of tyrosol; glycosyltransferase LrUGT85AF8 D415F The mutant strain HE-8.0 produced 10.20 g / L of rhodioloside and accumulated 0.34 g / L of tyrosol. Among these, the mutant glycosyltransferase LrUGT85AF8 showed higher levels of glycosyltransferase compared to the wild-type glycosyltransferase. E356V Rhodioloside production increased by 16%, mutant glycosyltransferase LrUGT85AF8 D415F The yield of rhodioloside was increased by 22%, and the accumulation of tyrosol was significantly reduced. This invention provides valuable components for constructing high-yielding rhodioloside strains.

[0027] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. An acid-loving glycosyltransferase mutant, characterized in that, The glycosyltransferase mutant is based on the amino acid sequence shown in SEQ ID NO.2, with aspartic acid at position 415 replaced by phenylalanine.

2. The gene encoding the glycosyltransferase mutant of claim 1.

3. A recombinant vector containing the gene described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The launch carriers are the pRSFDuet series and the pETDuet series.

5. A recombinant microbial cell containing the gene of claim 2.

6. The recombinant microbial cell according to claim 5, characterized in that, Gene containing phenylpyruvate decarboxylase ARO10 Ethanol dehydrogenase gene ADH6 phosphoglucosuricase gene pgm and UDP-glucose pyrophosphorylase gene galU Overexpression plasmids and genes containing mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase. aroG fbr Cyclohexadiene dehydrogenase gene tyrC The genetically engineered bacteria were obtained by transferring the overexpression plasmid of the mutant glycosyltransferase gene into Escherichia coli BL21(DE3) competent cells.

7. The use of the glycosyltransferase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of claim 5 or 6 in the production of rhodioloside.

8. A method for producing rhodioloside, characterized in that, The steps of the method are as follows: ferment the genetically engineered bacteria described in claim 7 at 30°C for 72 hours, and adjust the pH to 4.0-6.

0.

9. The method according to claim 8, characterized in that, The fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. The pH of the fermentation medium was maintained using 10 g / L CaCO3. Add 0.1% by volume of trace elements to the culture medium. The trace element composition is: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O and 15 g / L ZnSO4·7H2O. Adjust the pH to 4.0-6.0.

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