CN-122012442-A - Rhamnosyltransferase and its use in the production of rebaudioside N, rebaudioside J

Abstract

The invention belongs to the technical field of biology, relates to a rhamnosyl transferase and application thereof in the production of rebaudioside N and rebaudioside J, and provides the rhamnosyl transferase, a mutant thereof and application thereof, wherein the rhamnosyl transferase has a sequence shown as SEQ ID NO.1, and the rhamnosyl transferase and the mutant thereof have high catalytic efficiency, improve the conversion rate of the rebaudioside N and are more beneficial to realizing large-scale industrial production.

Inventors

• LI WEI
• XU XINYU
• PEI LIANG
• PAN YUE

Assignees

• 四川盈嘉合生科技有限公司

Dates

Publication Date

20260512

Application Date

20260303

Claims (10)

1. 1. The rhamnosyl transferase is characterized in that the amino acid sequence of the rhamnosyl transferase is shown in SEQ ID NO. 1.
2. 2. A nucleic acid molecule having a nucleotide sequence encoding the murine Li Tangtang methyltransferase of claim 1, said nucleotide sequence being set forth in SEQ ID No. 2.
3. 3. The mutant of rhamnosyltransferase of claim 1 wherein the point of mutation is at least one of N49F, N152L.
4. 4. A nucleic acid molecule having a nucleotide sequence encoding the mutant of claim 3.
5. 5. An expression vector comprising any one of the following: (A1) The nucleic acid molecule of claim 2; (A2) The nucleic acid molecule of claim 4; (A3) The nucleic acid molecule of claim 2 and a genetic element expressing one or more of glycosyltransferase UGT76G1, sucrose synthase AtSUS1, UDP-murine Li Tangge enzyme RHM01, bifunctional enzyme NRS; (A4) The nucleic acid molecule of claim 4 and a genetic element expressing one or more of glycosyltransferase UGT76G1, sucrose synthase AtSUS1, UDP-murine Li Tangge enzyme RHM01, bifunctional enzyme NRS.
6. 6. A host cell, excluding cells having totipotency, comprising any one of the following: (B1) The nucleic acid molecule of claim 2; (B2) The nucleic acid molecule of claim 4; (B3) The expression vector of claim 5.
7. 7. Use of the rhamnosyltransferase of claim 1, the mutant of claim 3 or the host cell of claim 6 for the production of steviol glycosides.
8. 8. The use of claim 7, wherein the steviol glycoside is one of rebaudioside N, rebaudioside J.
9. 9. A biosynthesis method of rebaudioside N is characterized in that rebaudioside A, rebaudioside I or rebaudioside J is used as a substrate, and the rebaudioside N is generated under the action of a catalyst; the catalyst comprises at least one of the following: (C1) The rhamnosyltransferase of claim 1; (C2) A mutant according to claim 3; (C3) The host cell of claim 6; (C4) Glycosyltransferases.
10. 10. A method for the biosynthesis of rebaudioside J, wherein rebaudioside a is used as a substrate to produce rebaudioside J using the rhamnosyltransferase of claim 1, the mutant of claim 3 or the host cell of claim 6.

Description

Rhamnosyltransferase and its use in the production of rebaudioside N, rebaudioside J Technical Field The invention belongs to the technical field of biology, and relates to rhamnosyl transferase and application thereof in the production of rebaudioside N and rebaudioside J. Background Steviol glycosides are a series of natural sweeteners extracted from stevia rebaudiana (Stevia rebaudiana), and steviol glycosides of presently known structure include stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside M, rebaudioside N, rebaudioside J, etc. Stevioside has become an important choice for replacing sucrose and synthetic sweeteners in the global food and beverage industry due to its high sweetness and low calorie characteristics and minimal impact on blood glucose and insulin levels. Steviol glycosides, e.g., rebaudioside a, rebaudioside B, etc. are present in the stevia rebaudiana dry leaves in lesser amounts, with other steviol glycosides, such as Reb J and Reb N, being present in significantly lower amounts. The sweetness multiple of the rebaudioside N is 200-300 times that of the sucrose, the sweetness curve is closer to that of the sucrose, no afterbitter taste is generated, and the threshold concentration is as low as 20ppm. However, the traditional extraction method of Reb N has extremely low yield, is difficult to meet the requirement of kilogram-level commercial sample preparation, and cannot support ton-level food addition. The production route of the rebaudioside N mainly comprises a plant extraction method and a biological synthesis method, wherein the plant extraction method takes dry leaves as raw materials, and the dry leaves are subjected to water extraction, flocculation, resin chromatography and repeated recrystallization to obtain the Reb N, the yield is less than 0.02%, the yield is low, the solvent, the resin and the energy consumption are high, the wastewater amount is large, the cost is high, and the mass production is difficult. The biosynthesis method is increasingly paid attention to the characteristics of safety, no pollution and the like, and at present, reb A, reb J or Reb I is usually used as a substrate, UDP-glucose and UDP-rhamnose are used as glycosyl donors, and Reb N is generated through glycosyl transferase catalysis. However, UDP-glucose and UDP-rhamnose are used as glycosyl donors, so that the price is high, the conversion efficiency is low, and the large-scale industrial production is not facilitated. Disclosure of Invention The invention aims to solve the technical problems and provide application of rhamnosyl transferase and a mutant thereof in producing rebaudioside N and rebaudioside J, the rhamnosyl transferase has high catalytic efficiency and good stability, the rhamnosyl transferase or the mutant thereof and glycosyltransferase-linked reaction are utilized to realize high-efficiency catalytic production of the rebaudioside N by an enzymatic method, a reaction system can use sucrose as a substrate to synthesize UDP-glucose, after cascade RHM01+NRS (UDP-rhamnose Li Tangge enzyme+bifunctional enzyme) to synthesize UDP-rhamnose (UDP-Rha), and the coupling of RHM01 and NRS realizes self-circulation of cofactor NADH, so that the cost of glycosyl donors is greatly reduced, and the conversion efficiency is improved. The technical scheme for solving the technical problems is as follows: The invention provides a rhamnosyl transferase, and the amino acid sequence of the rhamnosyl transferase is shown as SEQ ID NO. 1. The invention also provides a nucleic acid molecule having a nucleotide sequence encoding the rhamnosyltransferase described above. Further, the nucleotide sequence is shown as SEQ ID NO. 2. The invention also provides a mutant of the rhamnosyl transferase, and the mutation site of the mutant is at least one of N49F, N L. In some embodiments of the invention, the mutant is obtained by mutating asparagine at position 49 to phenylalanine and/or asparagine at position 152 to leucine on the basis of the rhamnosyl transferase shown in SEQ ID No. 1. In some embodiments of the invention, the rhamnosyl transferase mutant is N49F and the amino acid sequence is shown in SEQ ID NO. 3. In some embodiments of the invention, the rhamnosyl transferase mutant is N152L, and the amino acid sequence is shown in SEQ ID NO. 5. In some embodiments of the invention, the rhamnosyl transferase mutant is N49F/N152L, and the amino acid sequence is shown in SEQ ID NO. 7. The invention also provides a nucleic acid molecule having a nucleotide sequence encoding the rhamnosyltransferase described above. In some embodiments of the invention, the nucleotide sequence is shown as SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO. 8. The invention also provides an expression vector having at least one of: (A1) A nucleic acid molecule having a nucleotide sequence encoding the rhamnosyltransferase described above; (A2) A nucleic acid molecule having a nucleotide sequence encod