A glycosyltransferase and its mutants and application thereof in synthesis of salidroside

Abstract

The application discloses a glycosyltransferase and a mutant thereof and application thereof in synthesis of rhodiolin, and belongs to the field of bioengineering. The glycosyltransferase ArUGT3 with a Genebank accession number of UYE91529.1 is used to catalyze tyrosol glycosylation, and a mutant thereof is provided on the basis. The mutant comprises at least one amino acid mutation selected from V8I, L22F, A47I, T172E, T248P and V289M, and preferably is a L22F, A47I and T172E triple mutant. The application also protects a gene encoding the mutant, an expression cassette comprising the gene, a recombinant plasmid, a recombinant strain and application thereof. The mutant glycosyltransferase significantly improves catalytic efficiency and strain tolerance to tyrosol, and compared with an existing optimal enzyme UGT85A1-A21G, production performance is improved by 223.6%, growth performance is improved by 150.4%, the problem of high-concentration substrate inhibition is effectively solved, high-efficiency conversion can be realized without strain domestication, and the mutant glycosyltransferase has significant industrial production advantages and application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a glycosyltransferase and its mutants and their application in the synthesis of rhodioloside. Background Technology

[0002] Rhodioloside, hailed as "plateau gold" (a glycosylated product of tyrosol), is a glycoside compound derived from plants in the Rhodiola genus. It possesses activities such as anti-hypoxia, anti-oxidation, anti-aging, and anti-tumor activity, and is now widely used in cosmetics, health products, and pharmaceuticals. However, the yield of plant extracts from Rhodiola is severely limited by factors such as the slow growth rate and low concentration of Rhodiola. Currently, thanks to the rapid development of synthetic biology technology, the biosynthesis of rhodioloside has made significant progress, and some companies have successfully achieved industrial-scale production.

[0003] However, the synthesis of rhodioloside is still limited by three main factors: insufficient glycosyltransferase activity, strain intolerance to the substrate tyrosol, and the supply strategy of the glycosylation donor UDPG. Among these, the efficiency of glycosyltransferases is still insufficient to support the one-time input of high concentrations of the substrate tyrosol, easily leading to impaired activity of the substrate strain and a decrease in conversion capacity. Currently, there are not many reported glycosyltransferases that can convert tyrosol to rhodioloside: Chinese patent application CN201610361309.2 achieved a rhodioloside yield of 0.7 g / L by introducing the glycosyltransferase mutant UGT73B6MK and optimizing the glucose metabolism pathway in *E. coli*. Chinese patent application CN201711479443.3 achieved a rhodioloside yield of 0.227 g / L by introducing AtUGT85A1 (GenBank: At1g22400) from *Arabidopsis thaliana* and modifying the relevant pathway in *Saccharomyces cerevisiae*. CN202210356043.8 describes the introduction of AtUGT85A1 into modified *E. coli*, yielding 9.34 g / L of rhodioloside in a 5 L fermenter containing 2.5 L of M9Y medium. CN202311023305.X describes the introduction of a glycosyltransferase derived from *Rhodiola rosea*, RrUGT33 (GenBank: AUI41147), into high-tyrosine-producing *E. coli*, yielding 0.87 g / L of rhodioloside in a 5 L fermenter. However, in Chinese patent application CN202311560803.8, replacing RrUGT33 with AtUGT85A1 resulted in a yield of 2.1 g / L of rhodioloside, indicating that AtUGT85A1 remains the preferred choice. Chinese patent application CN202410783574.4 discloses an AtUGT85A1-A21G mutant with better glycosylation ability. Combined with metabolic modifications to the *E. coli* chassis and adaptive evolution to tyrosol, it achieves better tyrosol tolerance. The related recombinant strain synthesizes 16.8 g / L of rhodioloside. Chinese patent application CN202411014408.4 screened UGTs from different sources, among which UGT6 (Uniprot ID: A0A7J9DKC8) from *Gossypium trilobum* achieved the highest specific conversion of tyrosol, yielding 4.029 g / L of rhodioloside, with a conversion rate of approximately 37%.

[0004] Currently, the efficiency of glycosyltransferases required for rhodioloside production remains insufficient, necessitating strategies including adaptive evolution of strains to improve substrate tolerance. However, adaptive evolution of strains is often subject to uncertainties, such as internal enzyme mutations leading to incorrect conversion or degradation of tyrosol, resulting in substrate loss. Therefore, it is necessary to further develop glycosyltransferases with higher efficiency, enabling strains to rapidly convert high concentrations of substrate, avoiding continuous and irreversible damage to the strain, maintaining strain activity, thereby maintaining high production efficiency and reducing production time and costs. Summary of the Invention

[0005] In view of this, in order to solve the technical problems of insufficient efficiency of glycosyltransferases used in the synthesis of rhodioloside in the prior art and the inability of strains to tolerate high concentrations of the substrate tyrosol, one of the objectives of this invention is to provide a method for producing rhodioloside, which uses glycosyltransferase ArUGT3 to catalyze the glycosylation reaction of tyrosol; the amino acid sequence of said glycosyltransferase ArUGT3 has the Genebank accession number UYE91529.1; and even with the addition of 6 g / L tyrosol, the strain can still maintain good substrate conversion efficiency and growth performance. Compared with the optimal glycosyltransferase UGT85A1-A21G reported in the prior art, the yield of rhodioloside can be increased by more than 30%, which can effectively solve the bottleneck of the prior art.

[0006] The method includes providing a recombinant strain for synthesizing rhodioloside, which is constructed by introducing the encoding genes for the wild-type glycosyltransferase ArUGT3, phosphoglucose mutase pgm, pyrophosphorylase galU, and nucleoside diphosphate kinase ndk into a chassis strain. Preferably, the chassis strain can be Escherichia coli or Saccharomyces cerevisiae, etc.

[0007] The second objective of this invention is to provide a mutant glycosyltransferase, wherein the mutant glycosyltransferase is based on glycosyltransferase ArUGT3 and undergoes at least one amino acid mutation selected from V8I, L22F, A47I, T172E, T248P, and V289M; the mutant glycosyltransferase optimizes the spatial conformation of the enzyme through amino acid substitution at specific sites, thereby significantly improving the glycosylation catalytic efficiency, while enhancing the host strain's tolerance to the substrate tyrosol, reducing the inhibitory effect of high concentrations of substrate on strain growth, and solving the problems of low catalytic efficiency and poor substrate tolerance of wild-type enzymes. Preferably, the mutant glycosyltransferase is a single mutant of V8I, L22F, A47I, T172E, T248P, or V289M; a double mutant of V8I and L22F; a double mutant of V8I and A47I; a double mutant of V8I and T172E; a double mutant of V8I and T248P; a double mutant of V8I and V289M; a double mutant of L22F and A47I; a double mutant of L22F and T172E; a double mutant of L22F and T248P; a double mutant of L22F and V289M; or an A47I single mutant. The following mutants are used: 7I, T172E double mutant, A47I, V289M double mutant, A47I, T248P double mutant, T172E, T248P double mutant, T248P, V289M double mutant, V8I, L22F, A47I triple mutant, V8I, L22F, T172E triple mutant, L22F, A47I, T172E triple mutant, or V8I, L22F, A47I, T172E quadruple mutant. These mutants ensure that the conversion rate of the substrate complexol is not less than 45% and can reach over 90% during the biosynthesis of rhodioloside. More preferably, the mutant glycosyltransferase is a triple mutant of L22F, A47I, and T172E. The synergistic effect of these three mutants can improve the glycosylation production performance by 145.9% compared with the wild type and by 223.6% compared with the currently reported UGT85A1-A21G. Under the condition of high concentration of substrate tyrosol, the growth performance is improved by 101.0% compared with the wild type and by 150.4% compared with UGT85A1-A21G, which is the best performing type among all mutants to date.

[0008] A third objective of this invention is to provide a coding gene for encoding the aforementioned mutant glycosyltransferase. This coding gene can be obtained by modifying the wild-type ArUGT3 gene using site-directed mutagenesis, enabling stable expression in host cells, and the expression product maintains the high catalytic activity and substrate tolerance of the mutant glycosyltransferase. Furthermore, compared to the nucleotide sequence SEQ ID NO.1 of the wild-type glycosyltransferase ArUGT3, the coding gene sequence only exhibits base changes corresponding to the amino acid mutations, without any other redundant base mutations, ensuring the stability and specificity of gene expression. The nucleotides at each mutation site are: V8I corresponding to ATT, L22F corresponding to TTT, A47I corresponding to ATT, T172E corresponding to GAA, T248P corresponding to CCG, and V289M corresponding to ATG. These base mutations precisely achieve the replacement of the corresponding amino acids without affecting the transcription and translation efficiency of the gene.

[0009] The fourth objective of this invention is to provide an expression cassette comprising the coding gene of the aforementioned glycosyltransferase ArUGT3 mutant, which can be used to drive the stable expression of the coding gene in host cells.

[0010] A fifth objective of this invention is to provide a recombinant plasmid comprising the coding gene of the aforementioned glycosyltransferase ArUGT3 mutant. Preferably, the vector for the recombinant plasmid is pZE12.

[0011] The sixth objective of this invention is to provide a recombinant strain comprising expressing the coding gene of the above-mentioned glycosyltransferase ArUGT3 mutant, or containing the above-mentioned recombinant plasmid.

[0012] The seventh objective of this invention is to provide a recombinant strain for synthesizing rhodioloside, wherein the recombinant strain comprises genes encoding the aforementioned glycosyltransferase ArUGT3 mutant, phosphoglucose mutase pgm, pyrophosphorylase galU, and nucleoside diphosphate kinase ndk, introduced into a substrate strain. Preferably, the substrate strain can be *Escherichia coli*, *Saccharomyces cerevisiae*, etc.

[0013] The eighth objective of this invention is to provide a recombinant strain for synthesizing rhodioloside, containing the pZE12-ArUGT3 plasmid and the pLPP-pgm-galU-ndk plasmid with the nucleotide sequence shown in SEQ ID NO.3, wherein the pZE12-ArUGT3 plasmid includes the encoding gene of the above-mentioned glycosyltransferase ArUGT3 mutant.

[0014] The ninth objective of this invention is to provide an application of the above-mentioned mutant of the glycosyltransferase ArUGT3, its encoding gene, the above-mentioned recombinant plasmid, the above-mentioned recombinant strain, or the above-mentioned recombinant strain for synthesizing rhodioloside in the biocatalytic synthesis of rhodioloside or its derivatives.

[0015] In the above-mentioned technical solution provided by the present invention, the wild-type glycosyltransferase ArUGT3 or its mutant can still maintain good substrate transformation efficiency and growth performance under high concentration of tyrosol addition. Compared with the currently reported optimal glycosyltransferase UGT85A1-A21G, the production performance is improved by up to 223.6%, and the growth performance of the recombinant strain is improved by up to 150.4% under high concentration of substrate. At the same time, the technical solution is simple to operate, has mild conditions, and strong stability, which can effectively reduce the production cost of rhodioloside and shorten the production cycle. It has a high efficiency of rhodioloside or its derivative production capacity and broad industrial application prospects, and is suitable for large-scale promotion and application. Attached Figure Description

[0016] Figure 1 Metabolic pathway diagram of Escherichia coli converting tyrosol to rhodioloside; Figure 2 Image of pZE12 plasmid; Figure 3 The pZE12-ArUGT3 plasmid map; Figure 4 The image shows the pLPP-pgm-galU-ndk plasmid.

[0017] The nucleotide sequence used in this invention is as follows: SEQ ID NO.1 is the nucleotide sequence of the wild-type glycosyltransferase ArUGT3; SEQ ID NO.2 is the nucleotide sequence of glycosyltransferase UGT85A1-A21G; SEQ ID NO.3 is the nucleotide sequence of plasmid pZE12; SEQ ID NO.4 is the nucleotide sequence of plasmid pLPP-pgm-galU-ndk. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0019] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available. The molecular biology procedures involved, including PCR polymerase chain reaction, homologous recombination ligation, chemical transformation, and electroporation, are all routine and universally applicable procedures. The DNA polymerase used was PrimeSTAR Max from TaKaRa, the homologous recombination enzyme was the ClonExpress II One Step Cloning Kit-C115 from Novizumi Biotechnology Co., Ltd., and the plasmid miniprep kit and gel extraction kit were purchased from OMEGA. All related operating methods and procedures were performed in accordance with the corresponding instruction manuals. Example 1: Screening for highly efficient wild-type glycosyltransferase ArUGT3 and its application

[0020] Currently, the glycosyltransferase activity and thermostability of enzymes that can catalyze the formation of rhodioloside from tyrosol are still insufficient, and more efficient glycosyltransferases need to be found. 金线莲 It contains a large amount of glycosides, suggesting that it may contain high glycosyltransferase activity. Therefore, the currently reported superior glycosyltransferase UGT85A1-A21G was used as a probe for sequence alignment on the National Center for Biotechnology Information (NCBI) database. 金线莲 (Genebank: ASM5182053v1) After online phylogenetic analysis of sequences with high similarity, ArUGT3 (Genebank: UYE91529.1) was obtained as the closest relative. ArUGT3 has been annotated as a potential putative glycosyltransferase, but its specific function has not yet been reported.

[0021] The amino acid sequence of wild-type ArUGT3 was aligned to its homology using NCBI (Standard Protein BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi)). After searching all potential databases (including the seventeen databases provided by All non-redundant GenBank CDS, Core nucleotide BLAST database, clustered NR, etc.), the highest homology was found to be from... 广东舌唇兰 The crocetinglucosyltransferase (Genebank: KAK8956073.1) showed 75.68% homology.

[0022] After codon optimization by Suzhou Genewise Biotechnology Co., Ltd. and introduction of the NcoI restriction site into the proprietary vector plasmid pZE12 (SEQ ID NO.3), plasmids pZE12-ArUGT3 containing glycosyltransferase ArUGT3 (nucleotide sequence as shown in SEQ ID NO.1) and pZE12-UGT85A1-A21G containing glycosyltransferase UGT85A1-A21G (nucleotide sequence as shown in SEQ ID NO.2) were obtained.

[0023] Plasmids pZE12-UGT85A1-A21G or pZE12-ArUGT3 were co-transformed into E. coli with plasmid pLPP-pgm-galU-ndk (nucleotide sequence shown in SEQ ID NO. 4) via chemical transformation. 埃希氏菌属 大肠杆菌 Recombinant strains SAL1 and SAL2 were obtained from BW25113. The plasmid pLPP-pgm-galU-ndk contains gene clusters such as pgm, galU, and ndk, which are required for the conversion of glucose to UDPG, thus enhancing the supply of UDPG, the precursor for rhodioloside synthesis.

[0024] Recombinant strains SAL1 or SAL2 were inoculated into test tubes containing 5 mL of LB medium and cultured overnight at 30°C and 200 rpm with shaking. Then, they were transferred at a ratio of 10% (v / v) to 250 mL Erlenmeyer flasks containing 30 mL of fermentation medium and cultured for 4 hours. Then, tyrosol was added exogenously to a final concentration of 6 g / L as a substrate, and fermentation was continued for 44 hours at 30°C and 200 rpm with shaking to obtain the fermentation broth containing rhodioloside.

[0025] The LB medium formula is as follows: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The fermentation medium was formulated with 20 g / L glucose, 6.8 g / L sodium dihydrogen phosphate, 3.0 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 5.0 g / L ammonium sulfate, and 2 g / L yeast extract.

[0026] Take 1 mL of fermentation broth into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 3 minutes, take the supernatant, filter it through a 0.22 μm filter membrane, and perform high performance liquid chromatography (HPLC) to detect the yield of rhodioloside.

[0027] The HPLC detection method was as follows: chromatographic column: Welchrom® C18 (4.6 × 250 mm × 5 µm, Yuexu Technology); column temperature: 30℃; mobile phase A - acetonitrile, mobile phase B - 0.1% TFA aqueous solution; gradient elution conditions: initial ratio A:B = 5:95, ratio A:B = 80:20 for 10~13 min, ratio A:B = 5:95 for 13.1~18 min; flow rate: 1 mL / min; injection volume: 5 µL; detection wavelength: 280 nm.

[0028] The results showed that after 48 hours of fermentation, the recombinant strain SAL1 expressing UGT85A1-A21G had an OD value of [missing information]. 600 The OD2 of the recombinant strain SAL2 expressing wild-type ArUGT3 was 4.68, with a rhodioloside yield of 3.64 g / L and a substrate conversion rate of 27.9%. However, after 48 hours of fermentation, the OD2 of SAL2 showed a lower OD2 value than that of wild-type ArUGT3. 600 The yield of rhodioloside was 4.79 g / L, with a substrate conversion rate of 36.7%, which is 31.6% higher than that of UGT85A1-A21G. This shows that the screened ArUGT3 is a newly discovered glycosyltransferase that can catalyze the production of rhodioloside from tyrosol, and has a higher tyrosol conversion efficiency. Example 2: Glycosyltransferase ArUGT3 mutant and its application

[0029] 2.1 Single point mutation The wild-type glycosyltransferase ArUGT3 was rationally modified. Specifically, the amino acid sequence of ArUGT3 was analyzed and predicted using the online protein stability design tool FireProt (https: / / loschmidt.chemi.muni.cz / fireprotweb / ), and non-conserved sites were selected for mutation. Following the method described in Example 1, the mutant plasmid and plasmid pLPP-pgm-galU-ndk were chemically transformed into *E. coli*. 大肠杆菌 In BW25113, the corresponding single-point mutant recombinant strain was obtained and fermentation test was performed according to the method shown in Example 1. The results are shown in Table 1.

[0030] Table 1. Fermentation results of recombinant expression strains containing different ArUGT3 mutants Y7 H CAT 4.33 33.3 V8 I ATT 6.19 47.5 I11 L CTG 1.2 9.2 L22 F TTT 6.92 53.1 A47 I ATT 7.82 60.1 I50 L CTG 4.17 32.0 A69 S AGC 3.8 29.2 L121 C TGC 4.13 31.7 M161 L CTG 5.14 39.5 T172 E GAA 6.62 50.8 A225 E GAA 2.1 16.1 T248 P CCG 6.41 49.2 G249 L CTG 5.15 39.6 L274 Y TAT 5.07 38.9 V289 M ATG 6.66 51.2 V347 L CTG 5.03 38.6 A354 S AGC 4.88 37.5 M368 L CTG 4.73 36.3 According to Table 1, the rhodioloside production of ArUGT3 single-point mutant recombinant strains showed that V8I, L22F, A47I, T172E, T248P and V289M mutants were better, with A47I showing the best performance, with a rhodioloside production of 7.81 g / L, which was 63.3% higher than that of the original ArUGT3.

[0031] 2.2 Combinatorial Mutation Based on the single-point mutation results in Table 1, multi-point combination mutations were performed on the V8I, L22F, A47I, T172E, T248P and V289M mutants with better results. The corresponding multi-point mutant recombinant expression strains were constructed using a method similar to that in "2.1 Single-point mutation" and fermentation tests were carried out according to the method shown in Example 1. The results are shown in Table 2.

[0032] Table 2. Fermentation results of recombinant strains containing different ArUGT3 mutant combinations V8I-L22F 7.11 54.6 A47I-T248P 7.36 56.5 V8I-A47I 10.33 79.3 A47I-V289M 7.19 55.2 V8I-T172E 8.33 64.0 T172E-T248P 8.11 62.3 V8I-T248P 8.1 62.2 T172E-V289M 4.82 37.0 V8I-V289M 6.33 48.6 T248P-V289M 7.16 55.0 L22F-A47I 10.34 79.4 V8I-L22F-A47I 10.48 80.5 L22F-T172E 9.27 71.2 V8I-L22F-T172E 7.99 61.4 L22F-T248P 7.01 53.8 L22F-A47I-T172E 11.78 90.5 L22F-V289M 6.29 48.3 V8I-L22F-A47I-T172E 10.63 81.6 A47I-T172E 8.77 67.4 Table 2 shows that the triple mutant L22F-A47I-T172E of ArUGT3 exhibited the best performance, achieving a rhodioloside yield of 11.78 g / L and a conversion rate of 90.5%. This represents a 145.9% improvement in production performance compared to ArUGT3 and a 223.6% improvement compared to UGT85A1-A21G. Additionally, the growth OD of its corresponding expression strains... 600 The value was 11.72, which improved the growth performance of the recombinant strain SAL1 expressing the original ArUGT3 by 101.0% and by 150.4% compared with the strain expressing UGT85A1-A21G. This indicates that the efficient glycosyltransferase can enable the strain to rapidly convert the toxic substrate tyrosol, avoiding the continuous toxicity of high-concentration substrates to the strain. It can effectively improve the activity of the strain while improving production efficiency, which is beneficial to the high efficiency and sustainability of industrial production and reduces time costs.

[0033] In summary, this invention provides a glycosyltransferase ArUGT3 and its mutants, and their application in the synthesis of rhodioloside. Even with high concentrations of tyrosol, the ArUGT3 glycosyltransferase and its mutants maintain good substrate transformation efficiency and growth performance, achieving a production performance improvement of up to 223.6% compared to currently reported glycosyltransferases. Simultaneously, the high transformation efficiency protects the expression strain from the continuous toxicity of high substrate concentrations, resulting in a growth performance improvement of up to 150.4% under high substrate feeding conditions, eliminating the need for lengthy and uncertain strain acclimatization to acquire substrate tolerance. Therefore, this invention offers highly efficient rhodioloside production capabilities, has significant industrial application potential, effectively reduces production costs, and is suitable for practical application.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A method for producing rhodioloside, characterized in that, This includes the use of glycosyltransferase ArUGT3 to catalyze the glycosylation reaction of tyrosol; the amino acid sequence accession number of said glycosyltransferase ArUGT3 is UYE91529.

1.

2. A mutant glycosyltransferase, characterized in that, Based on the wild-type glycosyltransferase ArUGT3, it contains at least one amino acid mutation selected from the following: V8I, L22F, A47I, T172E, T248P, V289M; wherein the amino acid sequence accession number of the wild-type glycosyltransferase ArUGT3 is UYE91529.

1.

3. The mutant glycosyltransferase according to claim 2, characterized in that, The following are single mutants: V8I, L22F, A47I, T172E, T248P, V289M, V8I and L22F, V8I and A47I, V8I and T172E, V8I and T248P, V8I and V289M, L22F and A47I, L22F and T172E, L22F and T248P, L2 2F, V289M double mutant, A47I, T172E double mutant, A47I, V289M double mutant, A47I, T248P double mutant, T172E, T248P double mutant, T248P, V289M double mutant, V8I, L22F, A47I triple mutant, V8I, L22F, T172E triple mutant, L22F, A47I, T172E triple mutant, or V8I, L22F, A47I, T172E quadruple mutant.

4. A gene encoding a gene, characterized in that, It encodes the mutant glycosyltransferase as described in claim 2 or 3.

5. The encoding gene according to claim 4, characterized in that, Compared with the wild-type glycosyltransferase ArUGT3 shown in SEQ ID NO.1, it only has base changes corresponding to the amino acid mutations; the corresponding nucleotides of each mutation site are: V8I corresponds to ATT, L22F corresponds to TTT, A47I corresponds to ATT, T172E corresponds to GAA, T248P corresponds to CCG, and V289M corresponds to ATG.

6. An expression cassette or recombinant plasmid, characterized in that, Includes the coding gene as described in claim 4 or 5.

7. A recombinant bacterial strain, characterized in that, This includes expressing the coding gene as described in claim 4 or 5, or containing the expression cassette or recombinant plasmid as described in claim 6.

8. A recombinant strain for synthesizing rhodioloside, characterized in that, This includes introducing the encoding genes for the glycosyltransferase ArUGT3 mutant, phosphoglucose mutase pgm, pyrophosphorylase galU, and nucleoside diphosphate kinase ndk as described in claim 2 or 3 into the chassis strain.

9. A recombinant strain for synthesizing rhodioloside, characterized in that, The plasmid contains pZE12-ArUGT3 and pLPP-pgm-galU-ndk, as shown in SEQ ID NO.3, wherein the pZE12-ArUGT3 plasmid includes the encoding gene as described in claim 4 or 5.

10. The use of the mutant glycosyltransferase of claim 2 or 3, the encoding gene of claim 4 or 5, the expression cassette or recombinant plasmid of claim 6, the recombinant strain of claim 7, and the recombinant strain for synthesizing rhodioloside of claim 8 or 9 in the biocatalytic synthesis of rhodioloside or its derivatives.

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