A glycosyltransferase mutant and a method for catalyzing the synthesis of ginsenoside Rg3

Abstract

The present invention discloses a glycosyltransferase mutant and a method for catalytically synthesizing ginsenoside Rg3. The mutant is a single mutation or a combined mutation based on the amino acid sequence of the original glycosyltransferase as SEQ ID NO: 1, and finally a UGTPg29 mutant strain with a good catalytic effect is screened out to form a coupling system with SuSy to achieve efficient catalytic synthesis of ginsenoside Rg3. The half-life of glycosyltransferase UGTPg29 is extended by site-directed mutagenesis, and the mutant is used to achieve efficient catalytic synthesis of Rg3. The synthesis method has mild conditions, simple operation, short time, high catalytic efficiency, high yield, and good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a glycosyltransferase mutant and a method for catalyzing the synthesis of ginsenoside Rg3. Background Art

[0002] China has a long history of using ginseng as a traditional herbal medicine, among which tetracyclic triterpenoid saponins, known as ginsenosides, are considered to be the key bioactive components with therapeutic value and multiple functions. The content and composition of ginsenosides in ginseng are affected by the years of cultivation, harvest season, and environmental abiotic factors such as light, temperature, humidity, and soil fertility. Among the more than 100 ginsenosides identified, protopanaxadiol (PPD), protopanaxatriol (PPT), cintenolol, and oleanolic acid are the four most common types.

[0003] Ginsenoside Rh2 and Ginsenoside Rg3 belong to the so-called rare ginsenosides due to their relatively low concentrations naturally present in ginseng plants. They are derivatives of PPD, with one or two glucose groups attached to the C3 hydroxyl group of PPD, respectively. They have high pharmacological effects, including anti-inflammatory, anti-cancer, cardiovascular protection, immunomodulatory, neuroprotective, anti-diabetic, anti-fatigue, anti-allergic, anti-aging, and antioxidant effects. Therefore, obtaining a large amount of rare ginsenosides not only has far-reaching research value, but also contains huge economic benefits.

[0004] Since the content of ginsenosides in ginseng is limited by many factors such as genotype, growth conditions, root age and growth cycle, it is extremely difficult to extract and separate rare ginsenosides from cultivated ginseng. The Bs-YjiCm variant derived from Bacillus subtilis can catalyze the C-3' glycosylation reaction of PPD to produce Rh2, while UGTPg29 can transfer the glucose fragment from UDP-Glc to Rh2 to form Rg3, so Rg3 can be obtained by catalyzing PPD with UGTPg29 and Bs-YjiCm variants.

[0005] UGTPg29 is a UDP-glycosyltransferase belonging to the GT1 family. It usually uses UDPG as a glycosyl donor to synthesize glycoside compounds. It can be obtained in large quantities by heterologous expression in Escherichia coli or Saccharomyces cerevisiae, and is used to catalyze Rh2 to obtain Rg3. With the continuous improvement of production methods, UDPG can be synthesized in large quantities by enzymatic methods such as SuSy. When the regeneration rate of UDPG is not a limitation (for example, by increasing the concentration of sucrose), the key to the efficiency of synthesizing Rg3 lies in the stability of the second glycosyltransferase. However, UGTPg29 from ginseng has poor stability, and generally loses serious enzyme activity after 6 hours of catalysis, which limits the efficient application of glycosyltransferases. Summary of the invention

[0006] The purpose of the present invention is to provide a glycosyltransferase mutant and a method for catalyzing the synthesis of Rg3 using the glycosyltransferase mutant; to prepare Rg3 by glycosylation of Rh2 using the UGTPg29 mutant, and to provide a certain reference for industrial production.

[0007] In order to achieve the above purpose, the technical solution adopted in this application is:

[0008] A glycosyltransferase mutant is obtained by mutating any one or more of positions 9, 65, 84, 127, 147, 152, 91, 184, 287, and 342 of the glycosyltransferase UGTPg29 of the amino acid sequence as shown in SEQ ID NO: 1. More preferably, the mutation site is selected from any one or more of positions 91, 184, 287, and 342. More preferably, the mutation site is selected from position 91.

[0009] A glycosyltransferase mutant suitable for Rg3 synthesis, wherein the glycosyltransferase amino acid sequence is the mutant amino acid sequence shown in SEQ ID NO:1, and comprises a mutation of the amino acid residue at at least one site corresponding to S9R, H65W, S84P, R91 M, S127Y, G147L, K152R, D184M, A287V, A342L of SEQ ID NO:1.

[0010] Preferably, the glycosyltransferase mutant suitable for Rg3 synthesis comprises a mutation of the amino acid residue corresponding to one or more of R91M, D184M, A287V and A342L of SEQ ID NO:1.

[0011] A glycosyltransferase mutant R91 M suitable for Rg3 synthesis, wherein the amino acid at position 91 of the glycosyltransferase UGTPg29 sequence shown in SEQ ID NO: 1 is mutated from arginine (R) to methionine (M). The amino acid sequence of the mutant R91 M is shown in SEQ ID NO: 4.

[0012] A glycosyltransferase mutant R91M / A287V suitable for Rg3 synthesis, wherein the 91st amino acid in the glycosyltransferase UGTPg29 sequence shown in SEQ ID NO: 1 is mutated from arginine (R) to methionine (M), and the 287th amino acid is mutated from alanine (A) to valine (V).

[0013] A glycosyltransferase mutant R91 M / A342L suitable for Rg3 synthesis, wherein the amino acid at position 91 of the glycosyltransferase UGTPg29 sequence shown in SEQ ID NO: 1 is mutated from arginine (R) to methionine (M), and the amino acid at position 342 is mutated from alanine (A) to leucine (L).

[0014] A glycosyltransferase mutant R91M / A287V / A342L suitable for Rg3 synthesis, wherein the amino acid at position 91 of the glycosyltransferase UGTPg29 sequence shown in SEQ ID NO: 1 is mutated from arginine (R) to methionine (M), the amino acid at position 287 is mutated from alanine (A) to valine (V), and the amino acid at position 342 is mutated from alanine (A) to leucine (L).

[0015] A glycosyltransferase mutant R91 M / D184M / A287V / A342L suitable for Rg3 synthesis, wherein the 91st amino acid in the glycosyltransferase UGTPg29 sequence shown in SEQ ID NO:1 is mutated from arginine (R) to methionine (M), the 184th amino acid is mutated from asparagine (D) to methionine (M), the 287th amino acid in the sequence is mutated from alanine (A) to valine (V), and the 342nd amino acid in the sequence is mutated from alanine (A) to leucine (L). The amino acid sequence of the mutant R91M / D184M / A287V / A342L is shown in SEQ ID NO:5.

[0016] An expressed gene encoding any one of the above glycosyltransferase mutants.

[0017] A gene encoding a glycosyltransferase mutant R91M / D184M / A287V / A342L, the nucleotide sequence of which is shown in SEQ ID NO: 9;

[0018] A recombinant plasmid is connected with the above-mentioned glycosyltransferase mutant gene.

[0019] A recombinant plasmid is provided, wherein the glycosyltransferase mutant gene and the sucrose synthase gene SuSy are connected.

[0020] A recombinant cell comprises the above recombinant expression vector or the expression gene of the above glycosyltransferase mutant.

[0021] Application of the above glycosyltransferase mutant in the preparation of Rg3.

[0022] The above-mentioned glycosyltransferase mutants are used in constructing a cascade system with glycosyltransferase YjiCm to prepare Rg3.

[0023] The glycosyltransferase mutant can be used to catalyze Rh2 to obtain Rg3.

[0024] The glycosyltransferase mutant can construct a cascade reaction system with Bs-YjiCm derived from Bacillus subtilis to catalyze PPD to generate Rg3.

[0025] A SuSy-UGTPg29 and SuSy-YjiCm cascade reaction system was used, with SuSy providing a sugar donor for UGTPg29 and Bs-YjiCm to achieve the recycling of UDPG. Bs-YjiCm could catalyze the C-3' glycosylation reaction of PPD to generate Rh2, while UGTPg29 could transfer the glucose fragment from UDP-Glc to Rh2 to form Rg3. Therefore, Rg3 could be obtained by catalyzing PPD with UGTPg29 and Bs-YjiCm.

[0026] The method for synthesizing Rg3 by using Rh2 as a substrate and catalyzing the mutant of glycosyltransferase UGTPg29 comprises the following steps:

[0027] 1) Construction of a recombinant strain containing dual enzyme co-expression: The gene of the mutant of glycosyltransferase UGTPg29 and the gene of sucrose synthase are co-constructed into a plasmid to obtain a recombinant plasmid;

[0028] 2) Transforming the recombinant plasmid into a host bacterium to obtain a recombinant strain containing a dual enzyme co-expression system;

[0029] 3) Activate the recombinant strain, transfer it to LB induction medium, add inducer IPTG to induce culture, centrifuge at low temperature and collect the cells, resuspend the cells in an appropriate amount of buffer and break them, and collect the supernatant by centrifugation as the crude enzyme solution;

[0030] 4) Catalytic synthesis of Rg3: Rh2, sucrose, UDP, DMSO, Tween-80 and crude enzyme solution were added to the catalytic reaction system, reacted for 1-30 hours, inactivated at high temperature, and the supernatant obtained by centrifugation was Rg3.

[0031] In step 3), more specifically, the recombinant bacteria were inoculated into LB liquid medium at 25-40°C and cultured until OD 600 When the pH reaches 0.6-0.8, IPTG is used as an inducer, and the fermentation is induced at 20-37°C and 150-300 r / min for 36 hours. The fermentation broth is centrifuged to collect the bacteria, the bacteria are broken by ultrasonic disruption, and the supernatant is centrifuged to obtain a crude enzyme solution;

[0032] In step 4), ginsenoside Rh2 is dissolved in DMSO, crude enzyme solution, sucrose, UDP, Tween-80 are added, and the mixture is reacted at 20-40°C for 1-30h, the reaction is terminated at high temperature, and the supernatant obtained by centrifugation is ginsenoside Rg3;

[0033] The final concentration of the inducer is 0.02-1 mM, and the induction time is 36 h.

[0034] The concentration of Rh2 is 1-50mM; the concentration of sucrose is 1-500mM; the amount of crude enzyme added is 0.1-1000g / L; Tween-80 is used as a co-solvent to increase the solubility of PPD in the reaction system, and its addition amount is 0.1-10%. DMSO is used as a co-solvent to increase the solubility of PPD in the reaction system, and its addition amount is 0.1-20%.

[0035] The mutant of the glycosyltransferase UGTPg29 and Bs-YjiCm from Bacillus subtilis construct a cascade reaction system to catalyze PPD to generate Rg3, comprising the following steps:

[0036] 1) Construction of a recombinant strain containing dual enzyme co-expression: the gene of the mutant of glycosyltransferase UGTPg29 and the sucrose synthase gene are co-constructed into a plasmid to obtain a recombinant plasmid; the gene of glycosyltransferase BsYjiCm and the sucrose synthase gene are co-constructed into a plasmid to obtain a recombinant plasmid.

[0037] 2) Transforming the recombinant plasmid into a host bacterium to obtain a recombinant strain containing a dual enzyme co-expression system;

[0038] 3) Activate the recombinant strain, transfer it to LB induction medium, add inducer IPTG to induce culture, centrifuge at low temperature and collect the cells, resuspend the cells in an appropriate amount of buffer and break them, and collect the supernatant by centrifugation as the crude enzyme solution;

[0039] 4) Catalytic synthesis of Rg3: PPD, sucrose, UDP, DMSO, Tween-80, crude enzyme solution UGTPg29 and crude enzyme solution BsYjiCm were added to the catalytic reaction system, reacted for 1-30 hours, inactivated at high temperature, and centrifuged to obtain the supernatant, which is Rg3. In step 3), more specifically, the recombinant bacteria were inoculated into LB liquid culture medium at 25-40°C and cultured until OD 600 When the pH reaches 0.6-0.8, IPTG is used as an inducer, and the fermentation is induced at 20-37°C and 150-300 r / min for 36 hours. The fermentation broth is centrifuged to collect the bacteria, the bacteria are broken by ultrasonic disruption, and the supernatant is centrifuged to obtain a crude enzyme solution;

[0040] In step 4), protopanaxadiol PPD is dissolved in DMSO, crude enzyme solution UGTPg29 and crude enzyme solution BsYjiCm (the mass ratio of the two is 1:5-20), sucrose, UDP, Tween-80 are added, and the reaction is carried out at 20-40°C for 1-30h, and the reaction is terminated at high temperature. The supernatant obtained by centrifugation is ginsenoside Rg3;

[0041] The final concentration of the inducer is 0.02-1 mM, and the induction time is 36 h.

[0042] The concentration of PPD is 1-100mM; the concentration of sucrose is 1-500mM; the amount of crude enzyme added is 0.1-1000g / L; Tween-80 is used as a co-solvent to increase the solubility of PPD in the reaction system, and its addition amount is 0.1-10%. DMSO is used as a co-solvent to increase the solubility of PPD in the reaction system, and its addition amount is 0.1-20%.

[0043] The method for catalytic synthesis of Rg3 comprises the following steps:

[0044] 1) constructing a recombinant strain containing a dual-enzyme co-expression system: co-expressing a glycosyltransferase UGTPg29 gene sequence or a corresponding mutant gene sequence with a sucrose synthase gene sequence to obtain a recombinant plasmid, transforming the recombinant plasmid into competent Escherichia coli BL21 (DE3) cells to obtain a recombinant strain UGTPg29-At containing a dual-enzyme co-expression system; co-expressing a glycosyltransferase BsYjiCm gene sequence with a sucrose synthase gene sequence to obtain a recombinant plasmid, transforming the recombinant plasmid into competent Escherichia coli BL21 (DE3) cells to obtain a recombinant strain BsYjiCm-At containing a dual-enzyme co-expression system;

[0045] 2) Induced enzyme production of recombinant strains: The recombinant strains UGTPg29-At and BsYjiCm-At were activated and transferred to LB induction medium at 1-3%, cultured at 37°C for about 2 hours, IPTG was added at 0.1mM, the temperature was lowered to 16°C, and the induction culture was continued for 36 hours. The cells were centrifuged at 200rpm and collected at low temperature. The cells were resuspended in an appropriate amount of buffer and broken, and the supernatants collected by centrifugation were crude enzyme solutions UGTPg29-At and BsYjiCm-At respectively;

[0046] 3) Catalytic synthesis of Rg3: PPD, DMSO, Tween-80, sucrose, crude enzyme solution UGTPg29-At and crude enzyme solution BsYjiCm-At were added to the catalytic reaction system, reacted for 30 hours, inactivated the enzyme at high temperature, and the supernatant obtained by centrifugation was Rg3.

[0047] In the catalytic reaction system in step 3), preferably: the ratio of the two glycosyltransferases (i.e., BsYjiCm and UGTPg29) is 1:9; the UGTPg29 enzyme concentration is 810 mU / mL; the initial reaction pH is 7.5; UDP 2 mM; sucrose 300 mM; and the DMSO concentration is 15% (V / V).

[0048] The amino acid sequence of glycosyltransferase UGTPg29 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 6; the amino acid sequence of glycosyltransferase BsYjiCm is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 8; the amino acid sequence of sucrose synthase is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 7.

[0049] Semi-rational design of proteins is a method of designing or modifying proteins by using computational methods and experimental techniques based on bioinformatics methods, protein three-dimensional structure, homologous protein sequence comparison or existing knowledge. This method can effectively change the specific properties of proteins, such as catalytic activity, binding specificity, stability, etc., to meet specific application requirements. The present invention is based on glycosyltransferase UGTPg29, single-point and combined mutations are performed on the predicted residues, and the catalytic ability of glycosyltransferase mutants is investigated by comparing the relative activity and stability of its mutant strains. The glycosyltransferase mutant is coupled with sucrose synthase, and the substrate protopanaxadiol is co-catalyzed with glycosyltransferase Bs-YjiCm, and an appropriate amount of sucrose and UDP are added to catalyze the synthesis of ginsenoside Rg3. The mutant is simple to prepare. Under the same conditions, the mutant maintains enzyme activity within 24 hours, and the wild type does not have enzyme activity outside 6 hours. The catalytic effect of the reaction system based on the mutant strain R91M / D184M / A287V / A342L was better than that of the non-mutated system. The conversion rate of PPD reached 93.55% after 24 hours of reaction, the concentration of Rg3 was 9.72 g / L after 24 hours of reaction, and the yield reached 68.78%.

[0050] The present invention improves the stability of glycosyltransferase UGTPg29 through semi-rational design, thereby increasing the yield of catalytic synthesis of ginsenoside Rg3.

[0051] Beneficial effects:

[0052] The invention is rationally designed, potential mutation sites are screened, the stability of glycosyltransferase UGTPg29 is improved by site-directed mutagenesis, and the mutant is used to realize efficient catalytic synthesis of Rg3. The synthesis method has mild conditions, simple operation, high catalytic efficiency, high yield, and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1Schematic diagram of the synthesis of Rh2 catalyzed by the coupling system of glycosyltransferase UGTPg29, BsYjiCm and sucrose synthase AtSuSy.

[0054] Figure 2 Liquid phase diagram of the catalytic products of glycosyltransferases UGTPg29 and BsYjiCm. DETAILED DESCRIPTION

[0055] The detection method used in the following examples is as follows:

[0056] HPLC detection method: Agilent Poroshell 5TC-C18 (250x 4.6mm), UV 203nm, UltiMate 3000 was used to determine the concentrations of PPD, Rh2 and Rg3. The flow rate was 1.0mL / min, and the column temperature was 30°C. The mobile phase was water + 0.01% acetic acid (A) and acetonitrile (B), and the gradient program was 32-100% B, 0-12min.

[0057] Example 1 Construction of the dual enzyme expression system pET-UGTPg29-AtSuSy

[0058] The gene fragment encoding UGTPg29 was codon-optimized and cloned into the NdeⅠ and XhoⅠ restriction sites of pETDuet-1 (Novagen), and the resulting plasmid was named pET-UGTPg29. The codon-optimized gene of sucrose synthase AtSuSy was synthesized and cloned into the NcoⅠ and EcoRⅠ restriction sites of pET-UGTPg29 to generate the plasmid pET-UGTPg29-AtSuSy. The optimized nucleotide sequences of UGTPg29 and AtSuSy are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0059] The recombinant plasmid pET-UGTPg29-AtSuSy was added to the competent cells of E. coli BL21 (DE3) (TransGen Biotech), and the recombinant plasmid and competent cells were mixed by flicking, and then placed on ice for 30 minutes, and then placed in a 42°C water bath for heat shock for 90 seconds. After a 2-minute ice bath, 400 μL of non-resistant LB liquid culture medium was added, and the mixture was mixed by pipetting and placed in a 37°C shaker for activation for 30 minutes. 70 μL was spread on an LB plate containing ampicillin resistance, and cultured in a 37°C incubator for 12 hours to obtain the recombinant strain UGTPg29-At.

[0060] Example 2 Construction of the dual enzyme expression system pRSF-BsYjiCm-AtSuSy

[0061] The gene fragment encoding BsYjiCm was codon optimized and cloned into the NdeⅠ and XhoⅠ restriction sites of pRSFDuet-1 (Novagen), and the resulting plasmid was named pRSF-BsYjiCm. Sucrose synthase AtSuSy was cloned into the NcoⅠ and EcoRⅠ restriction sites of pRSF-BsYjiCm to generate plasmid pRSF-BsYjiCm-AtSuSy. The optimized nucleotide sequence of BsYjiCm is shown in SEQ ID NO:8.

[0062] The recombinant plasmid pRSF-BsYjiCm-AtSuSy was added to the competent cells of E. coli BL21 (DE3) (TransGen Biotech), and the recombinant plasmid and competent cells were mixed by flicking, and then placed on ice for 30 minutes, and then placed in a 42°C water bath for heat shock for 90 seconds. After a 2-minute ice bath, 400 μL of non-resistant LB liquid culture medium was added, and the mixture was mixed by pipetting and placed in a 37°C shaker for activation for 30 minutes. 70 μL was spread on an LB plate containing kanamycin resistance, and cultured in a 37°C incubator for 12 hours to obtain the recombinant strain BsYjiCm-At.

[0063] Example 3 Liquid Phase Identification of Glycosyltransferase Products

[0064] The recombinant strains BsYjiCm-At and UGTPg29-At were spread on LB solid plates (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar) containing the corresponding resistance, and placed in a 37°C incubator for 12 h. The next day, single colonies were selected from the plates and cultured in LB medium at 37°C and 200 rpm. When the absorbance (OD 600 ) reached 0.6 to 0.8. IPTG was added to the culture medium at a final concentration of 0.1 mM, and the recombinant E. coli BL21 (DE3) strain was incubated at 16°C for 36 h.

[0065] The fermentation broth BsYjiCm-At and UGTPg29-At were collected and centrifuged at 4°C, 8000rpm, 5min, and the supernatant was discarded to obtain bacterial mud, which was washed twice with Tris-HCl buffer. An appropriate amount of Tris-HCl buffer was added, and the mixture was placed in an ice-water mixture. The bacterial cells were ultrasonically disrupted with an ultrasonic disruptor, and the parameters were set to Ф6, 300W, and 15min. The mixture was centrifuged with a refrigerated centrifuge, and the parameters were set to 4°C, 8000rpm, and 20min. The supernatants were taken as crude enzyme solutions BsYjiCm-At and UGTPg29-At, respectively, and stored in a 4°C refrigerator for later use.

[0066] Catalytic reaction of glycosyltransferase YjiCm and UGTPg29: 6 mM PPD was added to the catalytic reaction system (5 mL), and 300 mM sucrose, 2 mM UDP, 2% Tween-80 (v:v), 15% DMSO (v:v), 8 mg / mL crude enzyme solution BsYjiCm-At, 4 mg / mL crude enzyme solution UGTPg29-At and 50 mM Tris-HCl buffer solution (pH 7.5) were added, and incubated at 37°C. An appropriate amount of the reaction solution was taken at intervals for high temperature inactivation, and methanol was added for dilution and then detected by HPLC.

[0067] The liquid phase diagram of the catalytic reaction of YjiCm and UGTPg29 is shown in Figure 2 As shown, the retention times of the catalytic products of YjiCm and UGTPg29 are around 15.4 min and 11.7 min, respectively, which are consistent with the retention times of the Rh2 standard and the Rg3 standard.

[0068] Example 4 Selection of glycosyltransferase mutation sites

[0069] The present invention uses a homology modeling method to simulate the spatial structure of glycosyltransferase UGTPg29 and predict potential thermal stability related residues. These amino acid residues are simulated and mutated to obtain 10 mutants that are beneficial to improving the stability of UGTPg29, so as to continue experimental verification.

[0070] Using the recombinant plasmid pET-UGTPg29-AtSuSy as a template, the following primers and PrimeSTAR DNA polymerase (TaKaRa) were used for PCR amplification. After verification by agarose gel electrophoresis, the template was digested by adding Dpn I restriction enzyme to the PCR reaction product. The digested PCR reaction product was then transformed into E. coli BL21 (DE3) according to the method in Example 1. Finally, a single colony on the plate was picked and inoculated into LB liquid culture medium containing 50 mg / L ampicillin. After culturing at 37°C and 200 rpm for 12 hours, the plasmid was extracted and sent for testing and identification. The following are the mutation primers for UGTPg29:

[0071] The primers for site-directed mutagenesis of S9R are:

[0072] Forward primer: 5'-CGGAAGGATACGTATTGCTCTGTTGCCA-3'

[0073] Reverse primer: 5'-TGGCAACAGAGCAATACGTATCCTTCCG-3'

[0074] The primers for site-directed mutagenesis of H65W are:

[0075] Forward primer: 5'-GGTTGAATTGTGGCTGCCGAGTTCCCCG-3'

[0076] Reverse primer: 5'-CTCGGCAGCCACAATTCAACCAACTTTATGC-3'

[0077] The primers for S84P site-directed mutagenesis are:

[0078] Forward primer: 5'-GCCTGCCGCCGCACCTTATGCTG-3'

[0079] Reverse primer: 5'-CAGCATAAGGTGCGGCGGCAGGC-3'

[0080] The primers for site-directed mutagenesis of R91 M are:

[0081] Forward primer: 5'-CCCCTGATGAATGCATTCGAAACCGCTGGCCCTACG-3'

[0082] Reverse primer: 5'-GAATGCATTCATCAGGGGCAGCATAAGGTGAGACGG-3'

[0083] The primers for S127Y site-directed mutagenesis are:

[0084] Forward primer: 5'-GCCAGAAATCGCGTATTATCACAATATTCCGGC-3'

[0085] Reverse primer: 5'-GCCGGAATATTGTGATAATACGCGATTTCTGGC-3'

[0086] The primers for site-directed mutagenesis of G147L are:

[0087] Forward primer: 5'-CCTCGTCAATCCTGCTGCACGCGTTCAA-3'

[0088] Reverse primer: 5'-TTGAACGCGTGCAGCAGGATTGACGAGGA-3'

[0089] The primers for K152R site-directed mutagenesis are:

[0090] Forward primer: 5'-GCACGCGTTCCGTAACCCGGGTGA-3'

[0091] Reverse primer: 5'-CTCACCCGGGTTACGGAACGCGTG-3'

[0092] The primers for site-directed mutagenesis of D184M are:

[0093] Forward primer: 5'-TTGCACATGTTTATCGCGTGCTTCGAACGTAGCTGT-3'

[0094] Reverse primer: 5'-CGCGATAAACATGTGCAACAACTTCATGTTATCCGC-3'

[0095] The primers for site-directed mutagenesis of A287V are:

[0096] Forward primer: 5'-ATCTGGGTTGTGCGTCTGATCGAAGGCGAGAAGAAG-3'

[0097] Reverse primer: 5'-CAGACGCACAACCCAGATAAAATTAACGGTGCTAAT-3'

[0098] The primers for site-directed mutagenesis of A342L are:

[0099] Forward primer: 5'-AGCATCCTTGAGTCCATGAAGTTTGGTGTTCCGGTT-3'

[0100] Reverse primer: 5'-GGACTCAAGGATGCTGCTCCAACCACAATGGCTAAC-3'

[0101] Example 5 Fermentation induction of mutant strains

[0102] The glycosyltransferase UGTPg29 mutants were spread on LB solid plates (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar) containing 50 μg / L ampicillin and placed in a 37°C incubator for 12 h. The next day, single colonies were selected from the plates and cultured in LB medium at 37°C and 200 rpm. When the absorbance (OD 600 ) reached 0.6 to 0.8. IPTG was added to the culture medium at a final concentration of 0.1 mM, and the glycosyltransferase UGTPg29 mutant strain was incubated at 16°C for 36 h.

[0103] Collect the fermentation liquid and centrifuge it at 4°C, 8000rpm, 5min, discard the supernatant to get the bacterial sludge, and rinse it twice with Tris-HCl buffer. Add an appropriate amount of Tris-HCl buffer, place it in an ice-water mixture, and use an ultrasonic disruptor to ultrasonically disrupt the bacteria, with the parameters set to Ф6, 300W, 15min. Centrifuge it again with a refrigerated centrifuge, with the parameters set to 4°C, 8000rpm, 20min, take the supernatant as the crude enzyme solution, and store it in a 4°C refrigerator for later use.

[0104] Example 6 Determination of enzyme activity and stability of wild type and single point mutants

[0105] The single-point mutants obtained by screening were compared with the wild type for enzyme activity and stability to evaluate the effect of the single-point mutation.

[0106] Glycosyltransferase UGTPg29 and its mutant enzyme activity assay: In a 2 mL reaction mixture containing 0.05 mg / mL crude protein, 0.5 mM Rh2, 2 mM UDPG, 1% Tween-80 (v:v) and 50 mM Tris-HCl buffer (pH 7.5), incubate at 30°C for 45 min. The sample was inactivated by high temperature, filtered through a 0.24 μm microporous filter, and the concentration of ginsenoside Rg3 was detected by HPLC. UGT activity is measured in units of the amount of enzyme that produces 1 μmol Rg3 per minute.

[0107] Stability determination of UGTPg29 and its variants under enzyme conversion conditions: The crude enzyme concentration was 6 mg / mL in Tris-HCl (pH 8.0) buffer containing 10% DMSO. After incubation at 37°C for a period of time, samples were taken to measure the enzyme activity under the above experimental conditions. All operations were performed in triplicate. The relative enzyme activity of other mutant enzymes was calculated with the activity of the unincubated wild-type UGTPg29 as 100%.

[0108] The enzyme activity results showed that the stability of some single-point mutants was better than that of the wild type, and the specific data are shown in Table 1. Among them, the enzyme activity stability of the S9R, H65W, S127Y, K152R, S84P, and G147L mutants was poor, and the S84P and G147L mutants were basically inactivated; the enzyme activities of the mutants R91M and A342L were slightly lower than those of the wild type, and the enzyme stability was better than that of the wild type; the enzyme activities of the mutants D184M and A287V were severely reduced, and the enzyme stability was better than that of the wild type.

[0109] Table 1 Relative enzyme activities of wild type and single point mutants

[0110]

[0111] Example 7 Wild-type and multi-point mutant stability assay

[0112] The forward single-point mutations were combined to evaluate the mutation effect of the double-point combination mutation. The combined mutation method is as follows:

[0113] Take R91 M / A342L as an example:

[0114] The recombinant plasmid pET-UGTPg29-R91 M-AtSuSy was used as a template, and the combined mutation of A342L was performed using the mutation method in Example 4, and finally sent for testing and identification. The mutation primers of R91M / A342L are as follows:

[0115] The primers for site-directed mutagenesis of R91 M / A342L are:

[0116] Forward primer: 5'-AGCATCCTTGAGTCCATGAAGTTTGGTGTTCCGGTT-3'

[0117] Reverse primer: 5'-GGACTCAAGGATGCTGCTCCAACCACAATGGCTAAC-3'

[0118] The forward single-point mutations were combined to evaluate the mutation effect of multi-point combined mutations. The combined mutation method is as follows:

[0119] Take R91 M / D184M / A287V / A342L as an example:

[0120] Using the recombinant plasmid pET-UGTPg29-R91M-A342L-AtSuSy as a template, the combined mutation of D184M was performed using the mutation method in Example 4, and finally sent for testing and identification. The mutation primers of R91M / D184M / A342L are as follows:

[0121] The primers for site-directed mutagenesis of R91 M / D184M / A342L are:

[0122] Forward primer: 5'-TTGCACATGTTTATCGCGTGCTTCGAACGTAGCTGT-3'

[0123] Reverse primer: 5'-CGCGATAAACATGTGCAACAACTTCATGTTATCCGC-3'

[0124] Using the recombinant plasmid pET-UGTPg29-R91M-D184M-A342L-AtSuSy as a template, the combined mutation of A287V was performed using the mutation method in Example 4, and finally sent for testing and identification. The mutation primers of R91M / D184M / A287V / A342L are as follows:

[0125] The primers for site-directed mutagenesis of R91 M / D184M / A287V / A342L are:

[0126] Forward primer: 5'-ATCTGGGTTGTGCGTCTGATCGAAGGCGAGAAGAAG-3'

[0127] Reverse primer: 5'-CAGACGCACAACCCAGATAAAATTAACGGTGCTAAT-3'

[0128] Glycosyltransferase UGTPg29 and its multi-point mutant enzyme activity assay: In a 2 mL reaction mixture containing 0.05 mg / mL crude protein, 0.5 mM Rh2, 2 mM UDPG, 1% Tween-80 (v:v) and 50 mM Tris-HCl buffer (pH 7.5), incubate at 30°C for 45 min. The sample was inactivated by high temperature, filtered through a 0.24 μm microporous filter, and the concentration of ginsenoside Rg3 was detected by HPLC. UGT activity is measured in units of the amount of enzyme that produces 1 μmol Rg3 per minute.

[0129] Stability determination of UGTPg29 and its multiple mutants under enzyme conversion conditions: 6 mg / mL crude enzyme was placed in Tris-HCl (pH 8.0) buffer containing 10% DMSO. After incubation at 37°C for a period of time, samples were taken to measure the enzyme activity under the above experimental conditions. All operations were performed in triplicate. The relative enzyme activities of other mutant enzymes were calculated with the activity of the unincubated wild-type UGTPg29 as 100%.

[0130] The enzyme activity results show that the enzyme stability of the multi-point mutants is mostly better than that of the wild type. The specific data are shown in Table 2. From the results, it can be seen that the multi-point combination mutation effectively maintains a more stable enzyme activity than the wild type. Among them, the R91M / D184M / A287V / A342L mutant has higher stability.

[0131] Table 2 Relative enzyme activities of wild type and multi-point combination mutants

[0132]

[0133]

[0134] Example 8 Synthesis of Ginsenoside Rg3 Catalyzed by Wild-Type Glycosyltransferase and Multi-site Combination Mutants

[0135] The catalytic effects of wild-type and multiple combined mutations of glycosyltransferase UGTPg29 were evaluated. The catalytic method is as follows:

[0136] Take R91 M / D184M / A287V / A342L as an example:

[0137] The gene fragment encoding the UGTPg29 mutant R91M / D184M / A287V / A342L was codon-optimized and cloned into the NdeⅠ and XhoⅠ restriction sites of pETDuet-1 (Novagen), and the resulting plasmid was named pET-UGTPg29-R91 M-D184M-A287V-A342L. The codon-optimized gene of sucrose synthase AtSuSy was synthesized and cloned into the NcoⅠ and EcoRI restriction sites of pET-UGTPg29-R91M-D184M-A287V-A342L to generate the plasmid pET-UGTPg29-R91M-D184M-A287V-A342L-AtSuSy. The optimized UGTPg29 mutant R91 M / D184M / A287V / A342L and AtSuSy nucleotide sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 7, respectively.

[0138] The recombinant plasmid pET-UGTPg29-R91M-D184M-A287V-A342L-AtSuSy was added to the competent cells of E. coli BL21 (DE3) (TransGen Biotech), and the recombinant plasmid and competent cells were mixed by flicking, and then placed on ice for 30 minutes, and then placed in a 42°C water bath for heat shock for 90 seconds. After a 2-minute ice bath, 400 μL of non-resistant LB liquid culture medium was added, and the mixture was mixed by pipetting and placed in a 37°C shaker for activation for 30 minutes. 70 μL was spread on an LB plate containing ampicillin resistance, and cultured in a 37°C incubator for 12 hours to obtain the recombinant strain UGTPg29-R91M-D184M-A287V-A342L-AtSuSy.

[0139] The recombinant strain UGTPg29-R91M-D184M-A287V-A342L-AtSuSy was spread on a LB solid plate containing the corresponding resistance (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, 15g / L agar) and placed in a 37°C incubator for 12 hours. The next day, a single colony was selected from the plate and cultured in LB medium at 37°C and 200rpm until the absorbance (OD600) reached 0.6-0.8. IPTG was added to the medium at a final concentration of 0.1mM, and the glycosyltransferase was incubated at 16°C for 36 hours.

[0140] Collect the fermentation liquid and centrifuge it at 4°C, 8000rpm, 5min, discard the supernatant to get the bacterial mud, and rinse it twice with Tris-HCl buffer. Add an appropriate amount of Tris-HCl buffer, place it in an ice-water mixture, and use an ultrasonic disruptor to ultrasonically disrupt the bacteria, with the parameters set to Ф6, 300W, 15min. Centrifuge it again with a refrigerated centrifuge, with the parameters set to 4°C, 8000rpm, 20min, and take the supernatant as the crude enzyme solution UGTPg29-R91M-D184M-A287V-A342L-AtSuSy, and store it in a 4°C refrigerator for later use.

[0141] Add 6 mM Rh2 to the catalytic reaction system (5 mL), and add 300 mM sucrose, 2 mM UDP, 2% Tween-80 (v:v), 15% DMSO (v:v), 810 mU / mL UGTPg29-R91M-D184M-A287V-A342L-AtSuSy, and 50 mM Tris-HCl buffer solution (pH 7.5), react at 37°C for 24 hours, inactivate at high temperature, and centrifuge to obtain the supernatant, which is Rg3.

[0142] The catalytic results show that the catalytic ability of the multi-point mutant is better than that of the wild type. The specific data are shown in Table 3. It can be seen from the results that the 91M / D184M / A287V / A342L mutant has higher stability and catalyzes the generation of more Rg3 in a long-term catalytic reaction system.

[0143] Table 3 The yield of Rg3 synthesized by wild type and multi-point combination mutants (g / L)

[0144]

[0145] Example 9 Synthesis of ginsenoside Rg3 catalyzed by glycosyltransferase mutant R91 M / D184M / A287V / A342L and glycosyltransferase BsYjiCm

[0146] The recombinant strains UGTPg29-R91 M-D184M-A287V-A342L-AtSuSy and BsYjiCm-At were spread on LB solid plates (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar) containing the corresponding resistance, and placed in a 37°C incubator for 12 h. The next day, single colonies were selected from the plates and cultured in LB medium at 37°C and 200 rpm. When the absorbance (OD 600 ) reached 0.6 to 0.8. IPTG was added to the culture medium at a final concentration of 0.1 mM, and the glycosyltransferase was incubated at 16°C for 36 h.

[0147] Collect the fermentation liquid and centrifuge it at 4°C, 8000rpm, 5min, discard the supernatant to get the bacterial sludge, and rinse it twice with Tris-HCl buffer. Add an appropriate amount of Tris-HCl buffer, place it in an ice-water mixture, and use an ultrasonic disruptor to ultrasonically disrupt the bacteria, with the parameters set to Ф6, 300W, 15min. Centrifuge it again with a refrigerated centrifuge, with the parameters set to 4°C, 8000rpm, 20min, take the supernatant as the crude enzyme solution, and store it in a 4°C refrigerator for later use.

[0148] Add 6 mM PPD to the catalytic reaction system (5 mL), and add 300 mM sucrose, 2 mM UDP, 2% Tween-80 (v:v), 15% DMSO (v:v), 90 mU / mL BsYjiCm-At, 810 mU / mL UGTPg29-R91 M-D184M-A287V-A342L-AtSuSy, and 50 mM Tris-HCl buffer solution (pH 7.5), react at 37°C for 24 h, inactivate at high temperature, and centrifuge to obtain the supernatant, which is Rg3.

[0149] Finally, after 24 hours of reaction, the conversion rate of PPD was 88.66%, the output of Rg3 was 3.33 g / L, and the yield of Rg3 was 70.67%.

[0150] Example 10 One-pot three-enzyme reaction of Rg3 in fed-batch batch

[0151] The recombinant strains UGTPg29-R91 M-D184M-A287V-A342L-AtSuSy and BsYjiCm-At were spread on LB solid plates containing corresponding resistance (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, 15g / L agar) and placed in a 37°C incubator for 12 hours. The next day, a single colony was selected from the plate and cultured in LB medium at 37°C and 200rpm until the absorbance (OD600) reached 0.6-0.8. IPTG was added to the medium at a final concentration of 0.1mM, and the glycosyltransferase was incubated at 16°C for 36 hours.

[0152] Collect the fermentation liquid and centrifuge it at 4°C, 8000rpm, 5min, discard the supernatant to get the bacterial sludge, and rinse it twice with Tris-HCl buffer. Add an appropriate amount of Tris-HCl buffer, place it in an ice-water mixture, and use an ultrasonic disruptor to ultrasonically disrupt the bacteria, with the parameters set to Ф6, 300W, 15min. Centrifuge it again with a refrigerated centrifuge, with the parameters set to 4°C, 8000rpm, 20min, take the supernatant as the crude enzyme solution, and store it in a 4°C refrigerator for later use.

[0153] 6mM PPD was added to the catalytic reaction system (5mL), and 300mM sucrose, 2mM UDP, 2% Tween-80 (v:v), 15% DMSO (v:v), 90mU / mL BsYjiCm-At, 810mU / mL UGTPg29-R91 M-D184M-A287V-A342L-AtSuSy and 50mM Tris-HCl buffer solution (pH 7.5) were added, and the reaction was carried out at 37°C for 24h. 6mM PPD was added at 3h and 6h of the reaction, and the reaction was inactivated at high temperature. The supernatant obtained by centrifugation was Rg3. Finally, at 24h of the reaction, the conversion rate of PPD was 93.55%, the yield of Rg3 was 9.72g / L, and the yield was 68.78%.

[0154] SEQ ID NO:1UGTPg29 amino acid sequence:

[0155]

[0156] SEQ ID NO:2 Sucrose synthase amino acid sequence:

[0157]

[0158]

[0159] SEQ ID NO:3Bs-YjiCm amino acid sequence:

[0160]

[0161] SEQ ID NO:4R91 M amino acid sequence:

[0162]

[0163] SEQ ID NO:5R91 M / D184M / A287V / A342L amino acid sequence:

[0164]

[0165] SEQ ID NO:6 UGTPg29 nucleotide sequence after codon optimization:

[0166]

[0167]

[0168] SEQ ID NO:7 codon-optimized sucrose synthase nucleotide sequence:

[0169]

[0170]

[0171] SEQ ID NO:8 Optimized BsYjiCm nucleotide sequence:

[0172]

[0173] SEQ ID NO:9R91 M / D184M / A287V / A342L nucleotide sequence:

[0174]

Claims

1. A glycosyltransferase mutant, It is characterized in that The mutant is obtained by mutation of any one of the amino acid residues of R91M, D184M, A342L, R91M / D184M, R91M / A287V, R91M / A342L, R91M / A287V / A342L, and R91M / D184M / A287V / A342L of the glycosyltransferase UGTPg29 whose amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid sequence encoding the glycosyltransferase mutant according to any one of claim 1.

3. A recombinant plasmid, to which the nucleic acid sequence of the glycosyltransferase mutant according to claim 2 is linked.

4. A recombinant bacterium, comprising the nucleic acid sequence of the recombinant plasmid according to claim 3 or the glycosyltransferase mutant according to claim 2.

5. The recombinant bacterium according to claim 4, It is characterized in that The recombinant bacteria is a recombinant Escherichia coli (Escherichia Coli) BL21 (DE3) strain.

6. Use of the glycosyltransferase mutant according to claim 1, the recombinant plasmid according to claim 3, or the recombinant bacteria according to claim 4 or 5 in catalyzing the synthesis of ginsenoside Rg3.

7. The use according to claim 6, It is characterized in that The steps include: 1) Construction of a recombinant strain containing dual enzyme co-expression: The gene of the glycosyltransferase mutant and the sucrose synthase gene are co-constructed into a plasmid to obtain a recombinant plasmid; 2) Transform the recombinant plasmid into the host bacteria to obtain a recombinant strain containing a dual enzyme co-expression system; 3) Activate the recombinant strain, transfer it to LB induction medium, add inducer IPTG to induce culture, centrifuge at low temperature and collect the cells, resuspend the cells in an appropriate amount of buffer and break them, and collect the supernatant by centrifugation as the crude enzyme solution; 4) Catalytic synthesis of Rg3: Add PPD or Rh2, sucrose, UDP, DMSO, Tween-80 and crude enzyme solution to the catalytic reaction system, react for 3-10 hours, inactivate at high temperature, and centrifuge to obtain the supernatant which is Rg3.

8. The use according to claim 7, It is characterized in that The final concentration of the inducer is 0.02-1 g / L, and the induction time is 36h.

9. The use according to claim 7, It is characterized in that The concentration of PPD is 1-100 mM; the concentration of Rh2 is 1-100 mM; the concentration of sucrose is 1-500 mM; and the amount of crude enzyme added is 0.1-100 mU / mL.

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