Glycosyl transferase mutant and application thereof in preparation of salidroside

Abstract

The invention discloses a glycosyl transferase mutant and application thereof in preparation of salidroside, and belongs to the technical field of bioengineering. The mutant is obtained by mutating UDP (User Datagram Protocol) glycosyl transferase AtUGT85A1 from arabidopsis thaliana, and the mutant comprises at least one mutation of C128G, F204A and F217V. The invention also discloses a gene for coding the mutant, a recombinant expression vector containing the gene and a genetically engineered bacterium. According to the present invention, the glycosyl transferase mutant with significantly improved catalytic activity is obtained through the structure-oriented rational design, the glycosyl transferase mutant is integrated into the de novo synthesis path of Escherichia coli, and after the constructed gene engineering bacterium is fermented for 72 h, the salidroside titer can achieve 9.1 g / L, and is improved by 200% compared with the wild type; the method provided by the invention gets rid of dependence on exogenous addition of expensive glycosyl donors, has the advantages of simple process, low cost, environmental friendliness and the like, and provides a new strategy for green industrial production of salidroside.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a glycosyltransferase mutant and its application in the preparation of rhodioloside. Background Technology

[0002] Rhodioloside is the main active ingredient in the natural plant Rhodiola rosea, possessing various pharmacological effects such as anti-fatigue, anti-aging, and anti-hypoxia. It has been widely used in the food, health product, cosmetic, and pharmaceutical industries. Currently, the production of rhodioloside mainly relies on plant extraction and chemical synthesis.

[0003] Plant extraction directly obtains rhodioloside from Rhodiola rosea plants. However, due to the harsh growing environment and long growth cycle of Rhodiola rosea, and the low natural content of rhodioloside in plant tissues, the resource utilization rate of this method is extremely low, making it difficult to meet the needs of large-scale production. While chemical synthesis can solve the resource shortage problem, the formation of glycosidic bonds in rhodioloside requires precise selective protection and deprotection of multiple hydroxyl groups in the glucose substrate, resulting in lengthy synthetic routes and numerous steps. Furthermore, the process often involves highly toxic reagents such as heavy metal catalysts and organotin compounds, which does not meet the requirements of green chemistry development.

[0004] Compared with the two methods mentioned above, biosynthesis offers advantages such as mild reaction conditions, simple processes, low cost, high product yield, and environmental friendliness, demonstrating greater development potential. In the biosynthesis research of rhodioloside, although glycosyltransferases using tyrosol as a substrate have been widely identified and heterologously expressed, the expression levels of plant-derived glycosyltransferases in host bacteria are generally low, and existing catalytic systems are highly dependent on expensive glycosyl donors (such as uridine diphosphate glucose, UDPG). These factors severely restrict the efficient and low-cost biosynthesis of rhodioloside. Therefore, developing glycosyltransferase mutants with high catalytic efficiency and low dependence on glycosyl donors is of great significance for realizing the green industrial production of rhodioloside. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a glycosyltransferase mutant and its application in the preparation of rhodioloside.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: To achieve the above objectives, the present invention first provides a glycosyltransferase mutant, which is obtained by mutating the UDP glycosyltransferase AtUGT85A1 derived from Arabidopsis thaliana. The amino acid sequence of AtUGT85A1 is shown below: MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSC IVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVH AMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSITVLSVKQLVEFAWGLAGSGKEFLWVIRPDLVAGEEAMVPPDFLMETKDRSMLASWCPQE KVLSHPAIGGFLTHCGWNSILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD* The codon-optimized nucleotide sequence of its encoding gene is shown below: Specifically, the mutant is at least one of the following mutations: cysteine ​​at position 128 is mutated to glycine (C128G), phenylalanine at position 204 is mutated to alanine (F204A), and phenylalanine at position 217 is mutated to valine (F217V).

[0007] Furthermore, the mutant is a combined mutant, preferably a C128G / F217V double mutant.

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

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

[0010] The present invention also provides a genetically engineered bacterium containing the above-mentioned gene or the above-mentioned recombinant expression vector. The starting strain of the genetically engineered bacterium is preferably *Escherichia coli*.

[0011] Furthermore, the genetically engineered bacteria were constructed by overexpressing the following genes in an *E. coli* strain BL21(DE3) with the aromatic amino acid transcriptional repressor gene tyrR and the branching acid mutase gene pheA knocked out: 1) The above-mentioned glycosyltransferase mutant genes; 2) 4-Hydroxyphenylacetaldehyde synthase gene aas; 3) Phosphoglucose mutase gene pgm; 4) UDP-glucose pyrophosphorylase gene galU; 5) Mutant 3-deoxy-D-arabinohepulose-7-phosphate synthase gene aroG fbr ; 6) Shikimate kinase II gene aroL; 7) Cyclohexadiene dehydrogenase gene tyrC.

[0012] The present invention further provides the application of the glycosyltransferase mutant or the genetically engineered bacteria in the preparation of rhodioloside.

[0013] Specifically, the application includes: using tyrosol or glucose as a substrate, and utilizing the glycosyltransferase mutant or the genetically engineered bacteria to carry out a biocatalytic reaction to generate rhodioloside.

[0014] More specifically, the application is a method for preparing rhodioloside, comprising the following steps: constructing the above-mentioned genetically engineered bacteria, fermenting and culturing them under suitable conditions, inducing the expression of related genes, and collecting rhodioloside after fermentation.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly enhanced catalytic activity: Through structure-guided rational design, this invention modifies the AtUGT85A1 enzyme, and the mutants screened (especially the C128G / F217V double mutant) show that the glycosylation activity of tyrosol is more than 3 times higher than that of the wild type.

[0016] 2. Significantly improved synthesis efficiency: By integrating the glycosyltransferase mutant of the present invention into the de novo synthesis pathway of Escherichia coli, the constructed genetically engineered bacteria can achieve a rhodioloside titer of 9.1 g / L after 72 hours of shake-flask fermentation, which is 200% higher than that of strains using wild-type enzymes, thus significantly improving the production efficiency of rhodioloside.

[0017] 3. Green and efficient: The biosynthesis method provided by this invention uses inexpensive carbon sources such as glucose as raw materials to directly synthesize rhodioloside de novo through engineered bacteria, eliminating the dependence on the addition of expensive exogenous glycosyl donors UDPG. The process is simple, low-cost, and environmentally friendly, and has good prospects for industrial application. Attached Figure Description

[0018] Figure 1 A comparative diagram of the activities of AtUGT85A1 and its mutants; Figure 2 A diagram illustrating the metabolic pathway of engineered bacteria that synthesize rhodioloside de novo; Figure 3 A comparison of the titers of rhodioloside and tyrosol after 72 h of fermentation by engineered bacteria Sa-0 and Sa-1. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with existing known technologies. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0020] Culture medium: LB liquid medium: 10.0 g tryptone, 5.0 g yeast extract and 10.0 g sodium chloride dissolved in 1 L deionized water and autoclaved at 121°C for 15 min.

[0021] Fermentation broth medium: 2.0 g casein amino acids, 6.8 g disodium hydrogen phosphate, 3.0 g dipotassium hydrogen phosphate, 1.0 g ammonium chloride, 0.5 g sodium chloride, and 15.0 g glucose were dissolved in 1 L of deionized water and autoclaved at 115°C for 30 min. Additionally, a 5 mM magnesium sulfate solution was added and sterilized separately at 121°C for 15 min.

[0022] The amino acid sequence of the UDP glycosyltransferase AtUGT85A1 derived from Arabidopsis thaliana: MGSQIIHNSQKPHVVCVPYPAQGHINPMMRVAKLLHARGFYVTFVNTVYNHNRFLRSRGSNALDGLPSFRFESIADGLPETDMDATQDITALCESTMKNCLAPFRELLQRINAGDNVPPVSC IVSDGCMSFTLDVAEELGVPEVLFWTTSGCAFLAYLHFYLFIEKGLCPLKDESYLTKEYLEDTVIDFIPTMKNVKLKDIPSFIRTTNPDDVMISFALRETERAKRASAIILNTFDDLEHDVVH AMQSILPPVYSVGPLHLLANREIEEGSEIGMMSSNLWKEEMECLDWLDTKTQNSVIYINFGSITVLSVKQLVEFAWGLAGSGKEFLWVIRPDLVAGEEAMVPPDFLMETKDRSMLASWCPQE KVLSHPAIGGFLTHCGWNSILESLSCGVPMVCWPFFADQQMNCKFCCDEWDVGIEIGGDVKREEVEAVVRELMDGEKGKKMREKAVEWQRLAEKATEHKLGSSVMNFETVVSKFLLGQKSQD* Nucleotide sequence (codon optimized) of the UDP glycosyltransferase AtUGT85A1 from Arabidopsis thaliana: Obtaining single-point mutants and combinatorial mutants of glycosyltransferases: AlphaFold was used to simulate the three-dimensional structure of glycosyltransferases, and the substrate binding pocket was determined by molecular docking. Key amino acid residues around the pocket were selected for rational mutation. Sites with high activity were screened out, and mutants with significantly improved catalytic activity were obtained through combined mutations.

[0023] Obtaining the single-point mutant: Amino acids near the substrate-binding pocket were screened as mutation sites. These amino acids were Y19, A21, T86, I89, L92, C128, A153, F204, F217, I308, and A404. Using pET28a-ugt85A1 as a template, the mutant was introduced through the specific primers in Table 1. PCR amplification was performed using DNA polymerase. The PCR product was digested with DpnI digestive enzyme at 37°C for 1 hour. The digested product was transformed into E. coli DH5α, plated on a solid plate containing kanamycin, and incubated overnight at 37°C. Single bacteria were picked and incubated in LB liquid medium containing kanamycin at 37°C for 6 hours. The samples were sent to a sequencing company for sequencing verification. After verification, the recombinant plasmid containing the mutation site was obtained.

[0024] Obtaining combined mutants: Mutants with enhanced activity were screened from the above single-point mutants, namely C128G, F204A, and F217V. Using pET28a-ugt85A1 (C128G) as a template and the specific primers in Table 1, combined mutants C128G / F204A and C128G / F217V were obtained according to the method for obtaining single-point mutants. Using pET28a-ugt85A1 (F204A) as a template and the specific primers in Table 1, combined mutants F204A / F217V were obtained according to the method for obtaining single-point mutants. Table 1 Primers used for the AtUGT85A1 mutant Primer Name Primer Sequence (5'-3') Y19A-F TGCCGGCTCCGGCCCAGGGCCATATTAAT Y19A-R GCCGGAGCCGGCACACACACCACATG A21G-F ATCCGGGCCAGGGCCATATTAATCCG A21G-R CTGGCCCGGATACGGCACACACA T86A-F CAGCCCAGGATATTACCGCACTGTG T86A-R TATCCTGGGCTGCATCCATATCGGTT I89F-F CAGGATTTTACCGCACTGTGTGAAAG I89F-R GCGGTAAAATCCTGGGTTGCATCC L92A-F CCGCAGCCTGTGAAAGTACCATGAAAAATTG L92A-R ACAGGCTGCGGTAATATCCTGGGT C128G-F GGTGGCATGAGTTTTACCCTGGATG C128G-R CATGCCACCATCGCTCACAATGC A153G-F CTGTGGTTTTCTGGCATATCTGCATT A153G-R CAGAAAACCACAGCCACTGGTG F204A-F AGCGCCATTCGCACCACCAATCC F204A-R GCGAATGGCGCTCGGAATATCTTTCAGTT F217V-F AGTGTTGCACTGCGTGAAACC F217V-R GTGCAACACTAATCATCACATCATCCG I308A-F AGCGCAACCGTGCTGAGCGTTA I308A-R CACGGTTGCGCTGCCGAAGTTGATAT A404G-F TTTGGCGATCAGCAGATGAATTGT A404G-R ATCGCCAAAAAACGGCCAACAC Activity assay and screening of glycosyltransferase mutants: The recombinant plasmid containing the mutant was transformed into BL21(DE3) and plated on kanamycin-containing solid plates, then incubated overnight at 37°C. Single bacteria were picked and cultured in 1 mL of LB broth containing kanamycin at 37°C / 220 rpm for 5 h. The culture was then inoculated at a 2% inoculum into 50 mL of fermentation broth containing kanamycin and cultured at 37°C / 220 rpm until OD (Organic Dysplasia) was reached. 600 When the pH value is 0.6-0.8, add IPTG to a final concentration of 1 mM for induction, and add tyrosol to a final concentration of 5 g / L. Ferment and culture at 30℃ / 220 rpm for 48 h.

[0025] After fermentation, 500 μL of fermentation broth was taken, and subjected to three freeze-thaw cycles. Two volumes of HPLC-grade acetonitrile were added, and the supernatant was collected by high-speed centrifugation. The supernatant was filtered through a 0.22 μm organic filter and analyzed by HPLC. The column temperature was 30℃, the column was a GL Sciences InertSustain C18 (5 μm, 4.6 × 250 mm), the injection volume was 20 μL, the detection wavelength was 280 nm, mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The flow rate was 1 mL / min. The procedure was as follows: 0–8 min, 10%–40% B; 8–12 min, 40%–90% B; 12–15 min, 90% B; 15–20 min, 10% B. The results are attached. Figure 1 As shown, the single-point mutants C128G, F204A, and F217V showed increased activity by 1.87, 1.93, and 1.95 times compared to the wild type, respectively. The combined mutants C128G / F204A, C128G / F217V, and F204A / F217V showed increased activity by 1.96, 3.1, and 1.33 times compared to the wild type, respectively. The optimal mutant obtained was C128G / F217V.

[0026] Construction of engineered bacteria for de novo synthesis of rhodioloside: The BL21(DE3) gene was edited using CRISPR / Cas9 to knock out the gene tyrR encoding a transcriptional regulatory protein and the gene pheA encoding prebenzoic acid dehydratase, resulting in the chassis BL21(DE3)-rtyrRrpheA.

[0027] The gene tyrR, which encodes a transcriptional regulatory protein from BL21(DE3), was knocked out using CRISPR / Cas9. Using pEcgRNA as a template, the primers sgRNA-tyrR-F / R in Table 2 were used for amplification. The ccdB toxic gene between the J23119 promoter and the gRNA backbone was replaced with the N20 sequence of tyrR. The PCR product was digested with DpnI digestive enzyme at 37°C for 1 h. The digested product was transformed into E. coli DH5α, plated on solid plates containing spectinomycin, and incubated overnight at 37°C. Single bacteria were picked and incubated in LB liquid medium containing spectinomycin at 37°C for 6 h. The samples were sent to a sequencing company for sequencing verification. After verification, the recombinant plasmid pTargeF-tyrR was obtained. Using the BL21(DE3) genome as a template, the upstream homologous arm fragment of the tyrR gene was amplified using primers tyrR-UP-F / R in Table 2, and the downstream homologous arm of the tyrR gene was amplified using tyrR-DOWN-F / R. The vector fragment was amplified using primers VpTargeT-tyrR-F / R in Table 2 using pTargeF-tyrR as a template. The PCR product using the plasmid as a template was digested with Dpn I digestive enzyme at 37°C for 1 h. The product was washed with a PCR cleaning kit. The three fragments were assembled using the Gibson recombination method at a molar ratio of 1:1:1. The recombinant product was transformed into E. coli DH5α and plated on a solid plate containing spectinomycin. It was incubated overnight at 37°C. Single bacteria were picked and incubated in LB liquid medium containing spectinomycin at 37°C for 6 h. The samples were sent to a sequencing company for sequencing verification. After verification, the recombinant plasmid pTargeT-tyrR was obtained.

[0028] Genome editing: The pEcCas plasmid was introduced into BL21(DE3) competent cells. At OD600 = 0.1, 10 mM L-arabinose was added to induce Cas9 protein expression. At OD600 = 0.4–0.5, the cells were incubated on ice, centrifuged, and the supernatant was discarded. The cell pellet was resuspended in an equal volume of sterile water. After another centrifugation, the cells were resuspended twice in an equal volume of 10% glycerol. Finally, the cells were resuspended in 10% glycerol to obtain BL21(DE3) / pEcCas electroporation competent cells concentrated 100-fold.

[0029] Add 1 μg of pTargeT-tyrR recombinant plasmid to BL21(DE3) / pEcCas electroporation competent cells, mix well, and then add to a pre-cooled 2 mm electroporation cuvette. Electroporate at 2.5 kV. Immediately after electroporation, add 900 μL of LB medium to revive the cells and incubate at 37℃ / 220 rpm for 1 h. Take 50 μL of the bacterial culture and spread it on a double antibiotic plate containing kanamycin and spectinomycin, and incubate overnight at 37℃.

[0030] Using JunP-tyrR-F / R primers from Table 2, colony PCR verification was performed. Positive clones were inoculated into 1 mL of LB liquid medium containing kanamycin and 10 mM rhamnose and incubated at 30 °C for 6 h to eliminate the pTargeT-tyrR plasmid. The relatively turbid bacterial culture was diluted 10,000 times and plated onto kanamycin monoclonal antibody plates, incubated overnight at 37 °C. Single colonies from the plates were then spotted sequentially onto spectinomycin monoclonal antibody and kanamycin monoclonal antibody plates using sterile toothpicks. Single colonies that did not grow on the spectinomycin plate but grew on the kanamycin plate were the knockout bacteria with pTargeT-tyrR plasmid elimination.

[0031] Bacteria capable of growing on kanamycin agar plates but not on spectinomycin agar plates were picked and cultured in 1 mL of antibiotic-free LB broth containing 5 g / L glucose at 37 °C for 6 h to eliminate the pEcCas plasmid. The culture was then diluted 10,000 times and plated onto agar plates containing 5 g / L glucose and 10 g / L sucrose. Single colonies from these plates were then spotted sequentially onto kanamycin-antibiotic and antibiotic-free agar plates. Single colonies that could not grow on kanamycin agar plates but could grow on antibiotic-free agar plates were identified as the pEcCas plasmid-eliminated knockout bacteria. Select bacteria that can grow on antibiotic-free plates but not on kanamycin plates and culture them in 1 mL of antibiotic-free LB medium for 6 h. Add an equal volume of sterilized 50% glycerol to make glycerol bacteria and store them in a -80℃ freezer.

[0032] The gene pheA, representing the BL21(DE3)-rtyrR prephenylacetic acid dehydratase, was knocked out using CRISPR / Cas9. The pheA gene knockout was performed in the same manner as above, using primers listed in Table 2: The recombinant plasmid pRSFDuet-aas-ugt85A1-pgm-galU, which overexpressed the 4-hydroxyphenylacetaldehyde synthase gene aas from parsley, the glycosyltransferase gene ugt85A1 from Arabidopsis thaliana, the phosphogluconomutase gene pgm and the UDP-glucose pyrophosphorylase gene galU from Escherichia coli, and the mutant 3-deoxy-D-arabinohepenoyl-7-phosphate (DAHP) synthase gene aroGfbr and shikimate kinase 2 aroL from Escherichia coli, the cyclohexadiene dehydrogenase gene tyrC from *Monosporium molybdenum*, and the ethanol dehydrogenase gene adh from *Yersinia lipolytica*, was developed in chassis BL21(DE3)-rtyrRrpheA. fbr -aroL-tyrC-adh.

[0033] Construction of pRSFDuet-aas-ugt85A1-pgm-galU recombinant plasmid: Using the BL21(DE3) genome as a template, amplification was performed using pgm-F / R and galU-F / R primers from Table 2. Amplification was also performed using pET28a-ugt85A1 as a template and VpET28a-F / R primers from Table 2. Recombinant plasmids pET28a-pgm and pET28a-galU were obtained through two-fragment recombination. Similarly, amplification was performed using pET28a-aas and pET28a-ugt85A1 as templates and ugt85a1-F / R and RBSaas-F / R primers from Table 2. Amplification was also performed using pRSFDuet-1 as a template and Vugt85A1-aas-F / R primers from Table 2. Recombinant plasmid pRSFDuet- was obtained through three-fragment recombination. aas-ugt85A1; using pET28a-pgm and pET28a-galU as templates, amplification was performed using RBSpgm-F / R and RBSgalU-F / R primers in Table 2. Using pRSFDuet-aas-ugt85A1 as template, amplification was performed using Vpgm-galU-F / R primers in Table 2. The recombinant plasmid pRSFDuet-aas-ugt85A1-pgm-galU was obtained through three-fragment recombination. pCDF-tyrC-adh-aroG fbr Construction of -aroL- recombinant plasmid: Using pET28a-ugt85A1 as a template, amplification was performed using primers VpET28a-F / R (Table 2). Using the BL21(DE3) genome as a template, amplification was performed using primers aroG-F / R (Table 2). Recombination of the two fragments yielded the recombinant plasmid pET28a-aroG. Using pET28a-aroG as a template, amplification was performed using primers mutaroG (Table 2). fbr -F / R amplification yielded the recombinant plasmid pET28a-aroG fbr Using pET28a-ugt85A1 as a template, amplification was performed using primers VpET28a-F / R in Table 2. Using the BL21(DE3) genome as a template, amplification was performed using primers aroL-F / R in Table 2. Recombination of the two fragments yielded the recombinant plasmid pET28a-aroL. Using pET28a-ugt85A1 as a template, amplification was performed using primers VpET28a-F / R in Table 2. Using the Yersinia lipolytica genome as a template, amplification was performed using primers adh-F / R in Table 2. Recombination of the two fragments yielded the recombinant plasmid pET28a-adh. Using pCDFDuet-1 as a template, amplification was performed using primers VaroG-aroL-F / R in Table 2; amplification was performed using pET28a-aroG... fbr Use the primers RBSaroG from Table 2 as templates.fbr -F / R amplification; using pET28a-aroL as a template, amplification was performed using primers RBSaroL-F / R in Table 2, and the recombinant plasmid pCDFDuet-aroGfbr-aroL was obtained through three-fragment recombination. Using pCDFDuet-aroG... fbr Using -aroL as a template, amplification was performed using primers VtyrC-adh-F / R in Table 2; using pET28a-tyrC as a template, amplification was performed using primers RBStyrC-F / R in Table 2; using pET28a-adh as a template, amplification was performed using primers RBSadh-F / R in Table 2. The recombinant plasmid pCDFDuet-aroG was obtained through three-fragment recombination. fbr -aroL-tyrC-adh.

[0034] The recombinant plasmid pRSFDuet-aas-ugt85A1-pgm-galU and pCDF-tyrC-adh-aroGfbr-aroL were co-transferred to the substrate BL21(DE3)-rtyrRrpheA to obtain the engineered bacteria that synthesize rhodioloside de novo, as shown in the attached figure. Figure 2 As shown.

[0035] Table 2 Primers required for constructing engineered bacteria that synthesize rhodioloside de novo. Primer Name Primer Sequence (5'-3') sgRNA-tyrR-F CTCGATCTACTCGTGCTAAGGTTTTAGAGCTAGAAATAGCAAGTT sgRNA-tyrR-R CTTAGCACGAGTAGATCGAGACTAGTATTATACCTAGGACTGAGCTAG tyrR-UP-F ATTTGGTCCAGCCAGTTTTAGATGC tyrR-UP-R CTGAAACTCTCAAACTCCAGGCAGGAAGGTTTCTGTCAACAATCA tyrR-DOWN-F GTTGACAGAAACCTTCCTGCCTGGAGTTTGAGAGTTTCAGCAGTC tyrR-DOWN-R TTTACCTGTACCTGTGTCACCCGTA VpTargeT-tyrR-F GGTGACACAGGTACAGGTAAAAAGCTTAGATCTATTACCCTGTTATC VpTargeT-tyrR-R TGGCTGGACCAAATCTGCAGGTCGACTCTAGAGA sgRNA-pheA-F AGATTCCGTATTAACTCAGCGTTTAGAGCTAGAAATAGCAAGTT sgRNA-pheA-R GCTGAGTTAATACGGAATCTACTAGTATTATACCTAGGACTGAGCTAG pheA-UP-F GTTGCTGACGCCCAATCAATACAC pheA-UP-R CACATCATCCGGCACCTTTTCAAGTGTTGCCTTTTTGTTATCAATAAAAG pheA-DOWN-F CTTTTTTATTGATAACAAAAAGGCAACACTTGAAAAGGTGCCGGATGATGTG pheA-DOWN-R CTGCATCGTATTAGCGCTGTCG VpTargeT-pheA-F GACAGCGCTAATACGATGCAGAAGCTTAGATCTATTACCCTGTTATC VpTargeT-pheA-R GATTGTGGCGTCAGCAACCTGCAGGTCGACTCTAGAGA pgm-F GTTTAACTTTAAAGGAGATACCATGGCAATCCACAATCGTGC pgm-R GGCTTTGTTAGCAGCCGGATCTTTACGCGTTTTTCAGAACTTCGCT VpET28a-F AGATCCGGCTGCTAACAAAGCCCCGAAA VpET28a-R CATGGTATATCTCCTTCTTAAAGTTAAACAAAATTTTCTAGAGGGA galU-F GTTTAACTTTAAAGGAGATACCATGGCTGCCATTAATACGAAAG galU-R GGCTTTGTTAGCAGCCGGATCTTTACTCTTAATGCCCATCTCTTC VpET28a-F AGATCCGGCTGCTAACAAAGCCCCGAAA VpET28a-R CATGGTATATCTCCTTCTTAAAGTTAAACAAAATTTTCTAGAGGGA ugt85a1-F AATAAGGATATACCATGGGTAGTCAGATTATTCATAACAGTCAG ugt85a1-R TCTCCTTTTAATCCTGGCTTTTCTGACCCAGCAGGAAT RBSass-F GAAAAGCCAGGTAAAAGGAGATATACCATGGGCAGCAGCCA RBSass-R TCGACTTAAGCATTAACTCGAACTTCAACCAG Vugt85A1-AS-F GTTGAAGTTCTGAGTATGCTTAAGTCGAGAAG Vugt85A1-AS-R CTGACTACCCATGGTATCTCCTTATTAGTTAAACAAAATTATTTC RBSpgm-F AGTTATAGTAGAAGGAGATATAACATGGCAATCCACAATCGTGC RBSpgm-R AGCCATGGTATATCTCCTTTTACGCGTTTTTCAGAACTTCGCT RBSonU-F CGCGTAAAAGGAGATATACCATGGCTGCCATTAATACGAAAG RBSgalU-R CGGTGGCAGCAGCCTAGGTTAATTACTCTTAATGCCCATCTCTTC Vpgm-galU-F AAGTAATTAACCTAGGCTGCTGCCACCGC Vpgm-galU-R GTGGATTGCCATATGTATCTCCTTCTTATACTTAACTAATAC andG-F CTTTAAGAGAGATATACCATGAATTATCCAGAACGACTTTACGC andG-R TTTGTTAGCAGCCGGATCTTTACCCGCGACGCGCTTTTA VpET28a-F AGATCCGGCTGCTAAACAAAGCCCGAAA VpET28a-R HELP GGTATATCTCCTTCTTAAGTAAACAAAATTATTTCTAGAGGGGA mutantGfbr-F GAGTTTCTCAATATGATCACCCCACAATATCTCG mutantGfbr-R TGGGGTGATCATATTGAGAAACTCACCTG andL-F GTTTAACTTTAAGAAGGATATACCATGACACAACCTCTTTTTCTG andL-R TGTTAGCAGCCGGATCTTCAACAATTGATCGTCTGTGC VpET28a-F AGATCCGGCTGCTAACAAAGCCCGAAA VpET28a-R CATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGA adh-F CTTTAAGAAGGAGATATACCATGTCTACTACACCCACCACT adh-R TTTGTTAGCAGCCGGATCTTTAGTTCTGGAACGACAGCCATT VpET28a-F AGATCCGGCTGCTAACAAAGCCCGAAA VpET28a-R CATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGA RBSaroGfbr-F CTTTAATAAGGAGATATACCATGAATTATCAGAACGACGATTTACGC RBSaroGfbr-R TGTCATGGTATATCTCCTTTTACCCGCGACGCGCTTTTA RBSaroL-F GCGTCGCGGGTAAAAGGAGATATACCATGACACAACCTC RBSaroL-R TCTGTTCGACTTAAGCATCAACAATTGATCGTCTGTGC VaroGfbr-aroL-F ACGATCAATTGTTGATGCTTAAGTCGAACAGAAAGTAATC VaroGfbr-aroL-R CTGATAATTCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATTTC RBStyrC-F TATAAGAAGGAGATATACATATGGGCAGCAGCCATCATCATCATC RBStyrC-R CATGGTATATCTCCTTTTACGGATGAATATCATGATCGGT RBSadh-F ATTCATCCGTAAAAGGAGATATACCATGTCTACTACACCCACCACT RBSadh-R GCAGCCTAGGTTAATTAGTTCTGGAACGACAGCCATT VtyrC-adh-F AACTAATTAACCTAGGCTGCTGCCACCGC VtyrC-adh-R GCTGCTGCCCATATGTATATCTCCTTCTTATACTTAACTAATATAC Fermentation test of engineered bacteria for de novo synthesis of rhodioloside The engineered bacteria containing wild-type glycosyltransferase that synthesizes rhodioloside de novo is Sa-0, and the engineered bacteria containing mutant glycosyltransferase is Sa-1.

[0036] The Sa-0 and Sa-1 bacteria grown on the plate were picked and transferred to 1 mL of LB liquid medium containing kanamycin and streptomycin, respectively, and cultured at 37℃ / 220 rpm for 5 h. Then, they were inoculated at a 2% inoculum into 50 mL of fermentation liquid medium containing kanamycin and streptomycin and cultured at 37℃ / 220 rpm until OD (dose retardation). 600 When the concentration of IPTG reaches 0.6-0.8, a final concentration of 1 mM is added for induction, and fermentation is carried out at 30℃ / 220 rpm for 72 h.

[0037] The detection method is the same as above.

[0038] The results are attached. Figure 3 As shown, tyrosol accumulated in the fermentation broth of Sa-0, while no tyrosol accumulated in the fermentation broth of Sa-1; all tyrosol was converted to rhodioloside. Quantitative analysis is shown in the attached figure. Figure 3The wild-type glycosyltransferase engineered bacteria, Sa-0, showed a rhodioloside titer of 2.98 g / L and a tyrosol titer of 1.8 g / L after 72 h. The engineered bacteria containing the mutant glycosyltransferase, Sa-1, showed a rhodioloside titer of 9.1 g / L after 72 h, with no tyrosol accumulation. Sa-1 showed a 200% increase in rhodioloside titer compared to Sa-0.

[0039] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A glycosyltransferase mutant, characterized in that, The mutant was obtained by mutating the UDP glycosyltransferase AtUGT85A1 derived from Arabidopsis thaliana. The amino acid sequence of AtUGT85A1 is shown in Sequence 1. The mutant is at least one of the following mutations: cysteine ​​at position 128 is mutated to glycine, phenylalanine at position 204 is mutated to alanine, and phenylalanine at position 217 is mutated to valine.

2. The glycosyltransferase mutant according to claim 1, characterized in that, The mutant is a C128G / F217V double mutant.

3. A gene encoding a glycosyltransferase mutant as described in claim 1 or 2.

4. A recombinant expression vector comprising the gene of claim 3.

5. A genetically engineered bacterium comprising the gene of claim 3 or the recombinant expression vector of claim 4.

6. The genetically engineered bacterium according to claim 5, characterized in that, The starting strain of the genetically engineered bacteria is Escherichia coli.

7. The genetically engineered bacterium according to claim 6, characterized in that, The genetically engineered bacteria were constructed by overexpressing the following genes in Escherichia coli strain BL21, in which the aromatic amino acid transcriptional repressor gene tyrR and the branching acid mutase gene pheA were knocked out: (1) The gene as described in claim 3; (2) 4-hydroxyphenylacetaldehyde synthase gene aas; (3) Phosphoglucose mutase gene pgm; (4) UDP-glucose pyrophosphorylase gene galU; (5) Mutant 3-deoxy-D-arabinohepulose-7-phosphate synthase gene aroG fbr ; (6) Shikimate kinase II gene aroL; (7) Cyclohexadiene dehydrogenase gene tyrC.

8. The use of the glycosyltransferase mutant of claim 1 or 2 or the genetically engineered bacteria of any one of claims 5-7 in the preparation of rhodioloside.

9. A method for preparing rhodioloside, characterized in that, The process includes the following steps: constructing the genetically engineered bacteria as described in claim 7, fermenting and culturing them under suitable conditions, inducing the expression of related genes, and collecting rhodioloside after fermentation.

10. The method according to claim 9, characterized in that, The fermentation conditions include: fermentation at 30°C and 220 rpm for 72 hours.

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