A glycosyltransferase pgUGT-M8 mutant and its application in the synthesis of rebaudioside D
By modifying the site-specific amino acid sequence of pgUGT-M8, a mutant with high catalytic activity was obtained, which solved the problem of low catalytic activity and achieved efficient conversion of Reb A to Reb D, meeting the needs of industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2025-11-17
- Publication Date
- 2026-07-31
AI Technical Summary
The existing glycosyltransferase pgUGT-M8 has low catalytic activity, resulting in insufficient efficiency in the synthesis of rebaudioside A from rebaudioside D, which makes it difficult to meet market demand.
By performing site-directed mutagenesis on pgUGT-M8, especially modifying the amino acid sequences at M87, I146, N178, L182, and K207, a series of highly catalytically active mutants were obtained, including combinations such as M87H and N178H/W/Y, which improved the efficiency of catalytic synthesis of Reb D from Reb A.
The mutant exhibits significantly enhanced catalytic activity, more than 30 times higher than the original enzyme, with a 30-fold increase in kcat/Km, providing a production solution with great industrial potential.
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Figure CN121737072B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering and relates to a pgUGT mutant glycosyltransferase that efficiently synthesizes Reb A into Rebdi glycoside D. Background Technology
[0002] Excessive intake of calorie compounds can lead to health problems including obesity, sweet tooth addiction, diabetes, high blood pressure, and cardiovascular disease. This has resulted in a growing demand for sweeteners that are high in sweetness, low in calories, and do not cause tooth decay. Stevia is a third-generation sugar substitute, and its leaves contain steviol glycosides (SGs), which can be used to purify natural, plant-based, zero-calorie sweeteners. Stevia glycosides (ST) and rebaudioside A (Reb A) are the main sweet compounds in steviol glycosides, and they are 200-300 times sweeter than sucrose, but they are characterized by a pronounced bitter aftertaste. Theoretically, the glycosides on steviol glycosides have two glycosylated hydroxyl groups, one of which is C. 19 The carboxyl group is linked, and the other is C. 13 The hydroxyl group is linked. Studies have shown that the degree of glycosylation of glycosides significantly affects the sweetness and mouthfeel of SGs. Rebaudioside D (Reb D) is linked to C 13 and C 19 All exhibit a high glycosylation pattern, making them significantly sweeter than sucrose (200-350 times sweeter) and less bitter than ST and Reb A. The cost of SGs is related to the complexity of their extraction process. This makes Rebaudioside D, which accounts for less than 0.4-0.5% of the dry stevia leaf weight, both expensive and difficult to purify.
[0003] Currently, through continuous exploration and research by scientists, the enzymatic synthesis of Reb D using Reb A as a substrate is considered a feasible technical route to increase Reb D production. However, the glycosyltransferase pgUGT-M8, responsible for catalyzing the synthesis of Reb D from Reb A, suffers from low enzyme activity and low heterologous expression levels. Therefore, the large-scale production of Reb D catalyzed by pgUGT to meet market demand has not yet been achieved. Thus, protein engineering to further improve the activity of pgUGT in catalyzing Reb A to achieve high-speed Reb D synthesis is of great significance. Summary of the Invention
[0004] To address the aforementioned problems, this invention, through rational design of the regulation of interactions between adjacent residues in the substrate pocket ring structure and the regulation of protein-substrate residue-molecule interactions, successfully obtained a highly efficient mutant catalyzing rebaudioside A. This provides a series of promising pgUGT-M8 mutants for Reb D biosynthesis (modification sites such as...). Figure 1 (As shown).
[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: Based on the thermostable modified strain Mut8 (M8) of the UDP-glucosyltransferase mutant pgUGT (UGT94B1), this invention obtains high catalytic activity mutant sites M87, I146, N178, L182 and K207 through rational design, and superimposes sense mutations to obtain a series of high catalytic mutants.
[0006] A glycosyltransferase pgUGT-M8 mutant, wherein the mutant is based on the amino acid sequence shown in SEQ ID NO. 1 and undergoes at least one of the following mutations: M87H, I146 F / Y / H / W / P / M / C, Y164W / H / F / M / C, N178 H / W / Y, L182V, K207R.
[0007] Preferably, the mutation is a combination of M87H with any one or more other mutations; or a combination of N178 H / W / Y with any one or more other mutations.
[0008] Preferably, the mutation is at least a combination of M87H and N178 H / W / Y.
[0009] Preferably, the mutation is at least a combination of M87H, N178H / W / Y and I146 F / Y / H / W / P / M / C.
[0010] Preferably, the mutation is at least a combination of M87H, N178H / W / Y, I146 F / Y / H / W / P / M / C and Y164W / H / F / M / C.
[0011] Preferably, the mutation is at least a combination of M87H, N178H / W / Y, I146 F / Y / H / W / P / M / C and Y164 W / H / F / M / C with L182V and / or K207R.
[0012] Preferably, the mutation is at least a combination of I146F / Y / H / W / P / M / C and N178 H / W / Y.
[0013] Preferably, the mutation is at least a combination of I146F / Y / H / W / P / M / C, N178 H / W / Y, and Y164 W / H / F / M / C; or the mutation is at least a combination of I146F / Y / H / W / P / M / C, N178H / W / Y, and Y164 W / H / F / M / C with L182V and / or K207R.
[0014] Table 1: Mutation Annotation Table
[0015] The present invention also provides a gene encoding the mutant.
[0016] The application of the mutant or gene in the synthesis of rebaudioside D using rebaudioside A as a substrate.
[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention involves site-directed mutagenesis of the amino acid sequence of the glycosyltransferase pgUGT-M8, resulting in a series of mutants such as Mut8-N178Y / I146F and pgUGT-M87H / I146F / N178Y, which significantly improve the efficiency of catalytic synthesis of Reb A from Reb D. The catalytic activity is greatly enhanced in a 10 mM Rebaudigan A reaction system, with the mutants exhibiting a substantial increase in activity. Examples include M8-M87H / I146F / N178Y, M8-M87H / I146F / Y164W / N178Y / L182V, M8-M87H / I146F / Y164W / N178Y / K207R, M8-M87H / I146M / Y164M / N178Y, and M8M8-M87H / I146C / Y164C / N178Y. Compared to the original UDP-glucosyltransferase mutant M8, the relative activity is increased by up to 30 times. k cat / K m This represents an improvement of over 30 times, providing a series of mutants with significant industrial potential for Reb D production. Attached Figure Description
[0018] Figure 1 The diagram shows the visualized structure of the pgUGT-M8-rebadigan A molecular docking complex. a is the protein-complex structure diagram, and b is the molecular docking conformation diagram of the complex. The modification sites are marked in the diagram.
[0019] Figure 2 The image shows the enzyme kinetic parameters of M8 measured in the concentration range of 0.05 mM to 5 mM.
[0020] Figure 3 The image shows the enzyme kinetic parameters of the mutant strain M8-N178Y, measured at concentrations ranging from 0.05 mM to 5 mM.
[0021] Figure 4 The image shows the enzyme kinetic parameters of the mutant strain M8-I146F, measured at concentrations ranging from 0.05 mM to 5 mM.
[0022] Figure 5The image shows the enzyme kinetic parameters of the mutant strain M8-M87H / I146F / Y164W / N178Y, measured at concentrations ranging from 0.05 mM to 5 mM.
[0023] Figure 6 The image shows the enzyme kinetic parameters of the mutant strain M8-M87H / I146M / Y164M / N178Y, measured at concentrations ranging from 0.05 mM to 5 mM.
[0024] Figure 7 The image shows the enzyme kinetic parameters of the mutant strain M8-M87H / I146C / Y164C / N178Y, measured at concentrations ranging from 0.05 mM to 5 mM.
[0025] Figure 8 The image shows the enzyme kinetic parameters of the mutant strain M8-M87H / I146F / Y164W / N178Y / L182V, measured at concentrations ranging from 0.05 mM to 5 mM.
[0026] Figure 9 The image shows the enzyme kinetic parameters of the mutant strain M8-M87H / I146F / N178Y, measured at concentrations ranging from 0.05 mM to 5 mM.
[0027] Figure 10 The image shows the enzyme kinetic parameters of the mutant strain M8-M87H / I146F / Y164W / N178Y / K207R, measured at concentrations ranging from 0.05 mM to 5 mM.
[0028] Figure 11 A graph showing the change of Reb D production by recombinant strain over time.
[0029] Figure 12 The image shows an SDS-PAGE gel electrophoresis diagram of the purified protein obtained in Example 2; lane 1 is the pgUGT-M8 loading flow-through solution, lane 2 is the pgUGT-M8 purified sample, and lane 3 is the low-concentration pgUGT-M8 eluted sample in gradient elution. Detailed Implementation
[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the methods and equipment used in the present invention are conventional methods and equipment in this technical field.
[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.
[0032] Example 1: Construction of a mutant of the pgUGT-M8 glycosyltransferase gene The resulting series of plasmids were sequenced and identified, and then transformed into E. coli. E. coli BL21(DE3) competent cells were used with 100 μ A series of mutant recombinant strains were obtained by screening on LB agar plates (1 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder) with a concentration of 1 g / mL ampicillin. E. coli BL2l(DE3) .
[0033] Table 2 Changes before and after the mutation site
[0034] Table 3 Primer names and primer sequences
[0035] Example 2: Induction of recombinant strain expression and purification of target protein The series of recombinant pgUGT-Mut8 strains constructed in Example 1 were inoculated into a solution containing 50 μL of... μ The drug was incubated in 100 ml LB liquid medium (1% sodium chloride, 0.5% yeast extract, 1% peptone) at 220 rpm and 37°C until the OD 600 reached 0.6-0.8. Then the temperature was lowered to 16°C, and isopropyl-β-thiogalactoside (IPTG) was added to a final concentration of 0.25 mM. The mixture was then induced to grow for 24 h.
[0036] Centrifuge the induced bacterial culture (6000 rpm, 5 min), discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl) at a ratio of 1 g of bacterial cells per 10 mL of lysis buffer. Disrupt the culture using a high-pressure homogenizer, then centrifuge the disrupted culture (10000 rpm, 30 min), and collect the supernatant to obtain the crude enzyme solution.
[0037] The crude enzyme solution was purified by affinity chromatography using a Ni+ column. After loading, impurities were washed with 10-fold lysis buffer, and the target protein was eluted with elution buffer. The eluted target protein was collected, concentrated and desalted to 8 mg / mL using a concentration tube, and then used for subsequent reactions. The purified protein was detected by 10% SDS-PAGE gel electrophoresis, successfully obtaining the pure enzyme PgUGT-Mut8 with clear target bands and accurate protein size.
[0038] Enzyme activity assay system: 200 μ The L reaction system contains 10 mM Rebaudigan A, 20 mM UDPG, 100 mM pH 7.5 PBS buffer, and 1 μAfter purifying the protein to g, reacting it at 45°C for 3 minutes, then using 200 g of purified protein... μ The reaction was stopped by adding 0.9 M phosphoric acid solution. After 5 minutes of reaction cessation, 200 L of phosphoric acid solution was added. μ Samples for enzyme activity assay were obtained using L 1 M NaOH solution.
[0039] Table 4: Relative activity of mutants and pgUGT-M8
[0040] Table 5: Enzymatic kinetic parameters of mutant and pgUGT-M8 ( k cat / K m )
[0041] Example 3: Fermentation production of Reb D by recombinant strains Production system: 1 ml of reaction system contains 2 mM Rebaudigan A, 10 mM UDPG, 100 mM pH 7.5 PBS buffer, and 0.02 mg / ml of purified protein of mutants: M87H / I146C / Y164C / N178Y. After reacting at 45℃ for 2-30 minutes, 100 ml of the solution is taken. μ Add L of reaction solution to 100 μ The reaction was stopped by adding 0.9 M phosphoric acid solution. After 5 minutes of reaction cessation, 100 L of phosphoric acid solution was added. μ The productivity test sample was obtained from L 1 M NaOH solution. The reaction was essentially completed in 9 minutes, and the yield of Reb D was 96.6% in the reaction system with 2.13 mM Reb A as the substrate. Figure 11 ).
[0042] SEQUENCE LISTING SEQ ID NO.1: pgUGT-Mut8 MDNQNGRISILLLPFLAHGHISPFFELAKQLAKRNCNVYLCSTPINLSSIKDKDPSASIKLVELHLPSSPDLPPHYHTTNGLPSHLMLPLRNAFETAGPTFSEILKTLKPDLLIYDFNPSWAPEIASSHNIPAVYFLTTAAASSSIGLHAFKNPGEKYPFPDFYDNSNITPEPPSADNMKLLHDFIACFERSCDIILIKSFRELEGKYIDLLSTLSDKTLVPVGPLVQDPMGHNEDPKTEQIINWLDKREESTVVFVCFGSEYFLSNEELEEVAIGLELSTVNFIWAVRLIEGEKKGILPEGFLQRVGDRGLVVEGWAPQARILGHSSIGGFVSHCGWSSIAESMKFGVPVIAMARHLDQPLNGKLAAEVGVGMEVVRDENGKYKREGIAEVIRKVVVEKSGEVIRRKARELSEKMKEKGEQEIDRALEELVQICKKKKDEQ SEQ ID NO.2:pgUGT-Mut8-M87H / I146F / N178Y MDNQNGRISILLLPFLAHGHISPFFELAKQLAKRNCNVYLCSTPINLSSIKDKDPSASIKLVELHLPSSPDLPPHYHTTNGLPSHLHLPLRNAFETAGPTFSEILKTLKPDLLIYDFNPSWAPEIASSHNIPAVYFLTTAAASSSFGLHAFKNPGEKYPFPDFYDNSNITPEPPSADYMKLLHDFIACFERSCDIILIKSFRELEGKYIDLLSTLSDKTLVPVGPLVQDPMGHNEDPKTEQIINWLDKREESTVVFVCFGSEYFLSNEELEEVAIGLELSTVNFIWAVRLIEGEKKGILPEGFLQRVGDRGLVVEGWAPQARILGHSSIGGFVSHCGWSSIAESMKFGVPVIAMARHLDQPLNGKLAAEVGVGMEVVRDENGKYKREGIAEVIRKVVVEKSGEVIRRKARELSEKMKEKGEQEIDRALEELVQICKKKKDEQ SEQ ID NO.3:pgUGT-Mut8-M87H / I146M / Y164M / N178Y MDNQNGRISILLLPFLAHGHISPFFELAKQLAKRNCNVYLCSTPINLSSIKDKDPSASIKLVELHLPSSPDLPPHYHTTNGLPSHLHLPLRNAFETAGPTFSEILKTLKPDLLIYDFNPSWAPEIASSHNIPAVYFLTTAAASSSMGLHAFKNPGEKYPFPDFMDNSNITPEPPSADYMKLLHDFIACFERSCDIILIKSFRELEGKYIDLLSTLSDKTLVPVGPLVQDPMGHNEDPKTEQIINWLDKREESTVVFVCFGSEYFLSNEELEEVAIGLELSTVNFIWAVRLIEGEKKGILPEGFLQRVGDRGLVVEGWAPQARILGHSSIGGFVSHCGWSSIAESMKFGVPVIAMARHLDQPLNGKLAAEVGVGMEVVRDENGKYKREGIAEVIRKVVVEKSGEVIRRKARELSEKMKEKGEQEIDRALEELVQICKKKKDEQ SEQ ID NO.4:pgUGT-Mut8-M87H / I146C / Y164C / N178Y MDNQNGRISILLLPFLAHGHISPFFELAKQLAKRNCNVYLCSTPINLSSIKDKDPSASIKLVELHLPSSPDLPPPHYHTTNGLPSHLHLPLRNAFETAGPTFSEILKTLKPDLLIYDFNPSWAPEIASSHNIPAVYFLTTAAASSSCGLHAFKNPGEKYPFPDFCDNSNITPEPPSADYMKLLHDFIACFERSCDIILIKSFRELEGKYIDLLSTLSDKTLVPVGPLVQDPMGHNEDPKTEQIINWLDKREESTVVFVCFGSEYFLSNEELEEVAIGLELSTVNFIWAVRLIEGEKKKILPEGFLQRVGDRGLVVEGWAPQARILGHSSIGGFVSHCGWSSIAESMKFGVPVIAMARHLDQPLNGKLAAEVGVGMEVVRDENGKYKREGIAEVIRKVVVEKSGEVIRRKARELSEKMKEKGEQEIDRALEELVQICKKKKDEQ
Claims
1. A glycosyltransferase pgUGT-M8 mutant, characterized in that, The mutant is a mutation based on the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation is any one of the following: M87H _N178Y I146F_N178Y M87H_I146F_N178Y M87H_I146H_N178Y M87H_I146W_N178Y M87H_I146Y_N178Y M87H_I146F_Y164W_N178Y M87H_I146F_Y164H_N178Y M87H_I146F_Y164F_N178Y M87H_I146M_Y164M_N178Y M87H_I146C_Y164C_N178Y M87H_I146F_Y164W_N178Y_L182V M87H_I146F_Y164W_N178Y_K207R.
2. The gene encoding the mutant of any one of claims 1.
3. The use of the mutant of claim 1 or the gene of claim 2 in the synthesis of rebaudioside D.