CN114045273B

CN114045273B - Glycosyl transferase OsUGT91C1 mutant and application thereof

Info

Publication number: CN114045273B
Application number: CN202111350045.8A
Authority: CN
Inventors: 朱晓峰
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-07-22
Anticipated expiration: 2041-11-15
Also published as: WO2023083359A1; CN114045273A

Abstract

The invention discloses a glycosyltransferase OsUGT91C1 mutant and application thereof, wherein an amino acid sequence shown in SEQ ID No.1 is obtained by mutating a 208 th amino acid from Phe to Met on the basis of a glycosyltransferase OsUGT91C1 amino acid sequence; the amino acid sequence shown in SEQ ID No.2 is that the 208 th amino acid is mutated from Phe to Met and the 379 th amino acid is mutated from Phe to Ala on the basis of the amino acid sequence of glycosyltransferase OsUGT91C1, wherein the 1 st to 14 th amino acids of the two mutants have redundancy and can be completely removed or changed without influencing the normal enzyme activity. The mutant provided by the invention improves the capability of catalyzing the same normal reaction, further removes the disadvantage of side reaction of the original OsUGT91C1 enzyme, and can efficiently catalyze and generate a series of stevioside products including Reb E.

Description

Glycosyl transferase OsUGT91C1 mutant and application thereof

Technical Field

The invention belongs to the field of enzymology, and particularly relates to a glycosyltransferase OsUGT91C1 mutant and application thereof.

Background

In recent years, the harm of high calorie sugar is gradually paid attention, but because sweet taste can stimulate the production of reward neurotransmitters such as endogenous opium and dopamine, people have difficulty in quitting the dependence on the sweet taste. Although several kinds of mass-produced artificial sweeteners can meet the requirement of low calorie, the taste, safety and psychological acceptability are still controversial, and the conventional high-calorie sweeteners are difficult to replace effectively. Stevia native to south america has been locally used as a natural source of sweetness for over a hundred years. In 2006, the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United nations in combination with the Special Committee for food additives (JECFA) passed the safety evaluation of the health and harmlessness of stevioside. In 2008 the FDA approved in the united states to allow stevia sugar to be used in food products. In 2011, stevioside obtains a safety certificate of European food safety administration. With the continued increase in consumer demand for low-sugar, low-calorie foods and beverages, stevia has become one of the fastest growing natural sweeteners worldwide.

Stevioside is a collective term for a number of steviol glycosides. All Steviol glycosides have a similar chemical structure pattern, i.e. there are different glucosyl modifications (a in fig. 1) at the C13-hydroxy (also denoted R1 in the figure for simplicity) and/or C19-carboxy (also denoted R2 in the figure for simplicity) core skeleton of the general Steviol (Steviol, CAS 471-80-7). According to the combination condition of glycosyl composition and glycosidic bond of C13-hydroxyl and C19-carboxyl, the Stevioside molecules can be named, but currently, Stevioside products in the market are represented by Stevioside (Stevioside, CAS 57817-89-7 accounts for 57% and is represented by ST) and Stevioside A (Rebaudioside A, CAS 58543-16-1, the content of the Stevioside can reach 32% and is represented by Reb A), and the Stevioside molecules have the disadvantage of poor mouthfeel, so that the market acceptance of the Stevioside is influenced. Research shows that stevioside D (Rebaudioside D, CAS 63279-13-0, represented by Reb D) and M (Rebaudioside M, CAS 1220616-44-3, represented by Reb M) has the sweetness which is 200-fold and 300-fold that of cane sugar, has the mouthfeel which is very close to that of cane sugar which is a sweet standard product, is considered to be the component with the best quality of stevioside, and is the key point of research and development of stevioside manufacturers. Both Peterjoy and Coca Cola were patented with the use of steviol glycosides D and M in beverages.

However, the content of steviol glycosides D and M in the leaves of natural stevia is below 1%, which is difficult to extract directly from naturally planted stevia. Due to the low content, the extraction process from stevia rebaudiana is tedious, the cost is high, and the marketable application of the stevioside D and M with the best mouthfeel is limited. Researches show that the stevia rebaudiana has no catalytic capability of adding No.2 glucosyl in the C19-carboxyl direction of stevioside, and Reb D and Reb M cannot be effectively synthesized, so that the phenomenon of low natural content of the two is caused; meanwhile, the No.2 glucosyl of Reb D and Reb M in the C19-carboxyl direction obviously improves the mouthfeel of the Reb D and the Reb M compared with other stevioside. Therefore, the method for utilizing heterologous enzymatic conversion makes up the defect of insufficient capability of stevia to catalyze and add No.2 glucosyl in the C19-carboxyl direction of stevioside, and is a solution for preparing stevioside D and M in a large scale. A glycosyl transferase OsUGT91C1 (also called EUGT11) from rice can catalyze the addition of glucosyl group No.2 (the glucosyl group No.2 is the glucosyl group which forms beta (1-2) glycosidic bond with the glucosyl group No.1, the glucosyl group No.1 is particularly the glucosyl group which directly forms beta-glycosidic bond with C13-hydroxyl or C19-carboxyl) in the direction of C13-hydroxyl and C19-carboxyl of stevioside (b, C and d in figure 1). The capability of adding 2 # glucosyl in both directions of C13-hydroxyl and C19-carboxyl of stevioside by using the glycosyltransferase OsUGT91C1 makes up the defect of capability of adding 2 # glucosyl in the direction of C19-carboxyl of stevioside by stevia rebaudiana, and is a determining factor for whether stevioside D and M can be produced in a large scale. Through a series of enzymatic reactions including glycosyltransferase OsUGT91C1, a precursor substance is used in vivo (not necessarily stevia rebaudiana Bertoni) or in vitro to perform total synthetic conversion of stevioside, or glucose is added to a natural extract of stevia rebaudiana Bertoni through the enzymatic reaction, particularly to specific positions of Stevioside (ST), stevioside A (Reb A) or other available stevioside molecules with abundant natural content, so as to generate stevioside D and M (Reb D and Reb M).

Since addition of No.2 glucosyl group in C19-carboxyl direction of steviol glycoside is key to generation of Reb D and Reb M, the inventors hoped to further improve the ability of glycosyltransferase OsUGT91C1 to catalyze addition of No.2 glucosyl group (forming β (1-2) glycosidic bond with No.1 glucosyl group) in C19-carboxyl direction of steviol glycoside for more efficient conversion to steviol glycoside D and M. Meanwhile, the inventors found that the glycosyltransferase OsUGT91C1 has poor specificity in catalyzing the enzymatic conversion process of stevioside D, M, and besides the addition of glucose group No.2 (which is a reaction required for the enzymatic conversion to generate stevioside D, M and is called a normal reaction), there are also obvious side reactions, namely, the addition of glucose group No. 6 (or glucose group No. 4) in the direction of C13-hydroxyl or C19-carboxyl, and the side reactions with the glucose group No.1 form a beta (1-6) glycosidic bond (or a beta (1-4) glycosidic bond) (the side reactions mentioned in the patent all refer to the reaction of adding glucose group No. 6 (or glucose group No. 4) (b, C, d in FIG. 1). The side reaction wastes enzyme catalysis resources and substrates, forms obvious byproducts, influences the generation efficiency of the stevioside D and M, also brings impurities, and brings great difficulty for obtaining high-quality stevioside D, M pure products.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to improve the capability of glycosyltransferase OsUGT91C1 for adding No.2 glucosyl in the direction of catalyzing C13-hydroxyl or/and C19-carboxyl of stevioside, and on the other hand, side reaction is eliminated on the premise of improving the catalytic capability.

Based on the three-dimensional space structure of glycosyltransferase OsUGT91C1 independently discovered by the inventor, the original amino acid sequence of glycosyltransferase OsUGT91C1 is rationally optimized, and phenylalanine at position 208 is changed into methionine (Phe208Met) to obtain the novel enzyme A. The example verifies that the new enzyme A can enhance the capability of glycosyltransferase OsUGT91C1 of adding No.2 glucosyl group at C13-hydroxyl, especially at C19-carboxyl.

Previous studies have found that side reactions can be completely eliminated by changing the phenylalanine at position 379 of the glycosyltransferase OsUGT91C1 to alanine (Phe379 Ala). The inventor combines the characteristics of enhanced catalytic ability of the novel enzyme A (Phe208Met) and combines amino acid changes of two sites of Phe208Met and Phe379Ala to successfully construct a novel enzyme B (Phe208Met/Phe379Ala) which can eliminate side reactions and enhance the target catalytic ability (adding No.2 glucosyl). The new enzyme B (Phe208Met/Phe379Ala) is verified to eliminate the side reaction of adding the No. 6 glucosyl (forming beta (1-6) glycosidic bond), have about 3-7 times of enhancement effect on the normal reaction of adding the No.2 glucosyl, and can participate in the enzymatic conversion of stevioside more efficiently and more specifically.

Further research shows that on the basis of the mutation, the deletion or change of 1-14 amino acids (MDSGYSSSYAAAAG) at the front end of the glycosyltransferase OsUGT91C1 does not influence the enzyme property after mutation. The 14 amino acids at the front end are redundant and can be completely removed or changed.

The technical scheme of the invention is as follows: the glycosyltransferase OsUGT91C1 mutant has an amino acid sequence of any one of the following a, b, C and d:

a. SEQ ID No. 1;

b. SEQ ID No. 2;

c. the 1 st to 14 th amino acids are arbitrarily deleted or changed on the basis of the amino acid sequence shown in SEQ ID No. 1;

d. the 1 st to 14 th amino acids are arbitrarily deleted or changed on the basis of the amino acid sequence shown in SEQ ID No. 2.

The amino acid sequence shown in SEQ ID No.1 is obtained by mutating 208 th amino acid from Phe to Met on the basis of a glycosyltransferase OsUGT91C1 amino acid sequence; the amino acid sequence shown in SEQ ID No.2 is obtained by mutating the 208 th amino acid from Phe to Met and the 379 th amino acid from Phe to Ala on the basis of the glycosyltransferase OsUGT91C1 amino acid sequence.

An expression gene for coding the glycosyltransferase OsUGT91C1 mutant.

An expression vector containing the above-described expression gene.

Recombinant bacteria or cells carrying the expression vector.

The glycosyltransferase OsUGT91C1 mutant is applied to the enzymatic synthesis or conversion process of stevioside D or M and other stevioside molecules.

Further, the enzymatic synthesis or conversion process of the glycosyltransferase OsUGT91C1 mutant in stevioside D or M and other stevioside molecules refers to adding glucose group No.2 in the direction of C13-hydroxyl or/and C19-carboxyl of stevioside, wherein the glucose group No.2 is a glucose group which forms a beta (1-2) glycosidic bond with the glucose group No.1, and the glucose group No.1 is a glucose group which directly forms a beta-glycosidic bond with the C13-hydroxyl or C19-carboxyl.

The substrates that catalyze the normal reaction of adding No.2 glucosyl group in the C13-hydroxyl or C-19 carboxyl direction are not exclusive and include, but are not limited to, Steviol glycoside substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monogluside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding No.2 glucosyl group in the C13-hydroxyl or C-19 carboxyl direction can occur as long as No.1 glucosyl group is present in the C13-hydroxyl or C-19 carboxyl direction, but No. 3 glucosyl group is absent.

Besides being capable of expressing, purifying and preparing the natural OsUGT91C1 or the two novel enzymes of the invention in Escherichia coli, the enzyme can also be expressed and prepared in other expression systems such as other biological systems, non-biological systems, cells, non-cells (Cell-free) and the like; the novel enzyme with the side reaction eliminated is introduced into an organism, an abiotic body, a cell and a non-cell (cell-free) through a transgenic or other gene introduction means to carry out the enzymatic conversion of stevioside, and is used for the enzymatic conversion of adding No.2 glucosyl to the stevioside in an in vitro system such as enzyme and enzyme immobilization, and the enzymatic conversion process including but not limited to the enzymatic conversion process of preparing stevioside D and M achieves the purpose of eliminating the side reaction.

The side reaction of the patent is defined as the side reaction that glucose group No. 6 (or glucose group No. 4) is added in the two directions of C13-hydroxyl and C19-carboxyl of steviol or stevioside to form beta (1-6) glycosidic bond (or beta (1-4) glycosidic bond) with glucose group No. 1; the definition of the normal reaction of the patent is that glucose group No.2 is added in the directions of C13-hydroxyl and C19-carboxyl of steviol or stevioside, and forms beta (1-2) glycosidic bond with glucose group No. 1.

A liquid chromatography-mass spectrometry combination is utilized to verify that the novel enzyme A (Phe208Met) enhances the capability of catalytically adding the No.2 glucosyl, particularly adding the No.2 glucosyl at a C19-carboxyl position. The novel enzyme B (Phe208Met/Phe379Ala) has no side reaction while enhancing the ability to add glucose group 2. The Michaelis dynamics parameters of two new enzyme catalytic reactions are measured by methods such as liquid chromatography-mass spectrometry, a fluorescence conversion method and the like, and the new enzyme B (Phe208Met/Phe379Ala) is verified to eliminate the side reaction of adding No. 6 glucosyl (forming beta (1-6) glycosidic bond), have about 3-7 times of enhancement effect on the normal reaction of adding No.2 glucosyl, and can participate in the enzymatic conversion of stevioside more efficiently and more specifically. The new enzyme B catalyzes enzymatic conversion, and the obtained normal product added with No.2 glucosyl group does not contain byproducts and can be used for synthesizing stevioside D and M.

Compared with the prior art, the invention has the following beneficial effects:

the invention solves the enzymatic defect of rice-derived glycosyltransferase OsUGT91C1 in stevioside conversion, and simultaneously remarkably improves the catalytic capability of the target catalytic reaction. The new enzyme B (Phe208Met/Phe379Ala) can more efficiently and specifically complete the normal enzymatic reaction of adding No.2 glucosyl to stevioside, and is used for converting to obtain high-quality and high-purity stevioside D and M serving as health sweeteners.

Drawings

FIG. 1 shows a part of steviol glycoside structure, simplified representation of structure and schematic representation of normal reaction and side reaction in OsUGT91C1 enzymatic conversion process to stevioside D and M

a part of the structure of steviol glycosides, including steviol, steviol glycosides D and M and simplified forms of structures. Steviol is represented by the complete chemical structure, and steviol glycosides D and M are represented by the complete chemical structure and the simplified diagram, both in parentheses are the sweetness multiples compared to sucrose. In the simplified diagram, the C13-hydroxy group (simplified in R1) and the C19-carboxy group (simplified in R2) are shown. The glucosyl group is represented by a hexagon, the 1-hydroxyl group of each glucose is marked by a black dot, the number in the middle of each glucose represents a number of glucosyl groups, and also represents the type of glycosidic bond formed by the glucosyl group and the glucosyl group number 1, such as 2 represents a beta (1-2) glycosidic bond, 3 represents a beta (1-3) glycosidic bond, 4 represents a beta (1-4) glycosidic bond, and 6 represents a beta (1-6) glycosidic bond.

b OsUGT91C1 is a schematic diagram of catalyzing and adding 2 # glucosyl normal reaction and 6 # glucosyl (or 4 # glucosyl) side reaction at two ends of C13-hydroxyl (simplified by R1) or (and) C19-carboxyl (simplified by R2) of a stevioside substrate.

C, the OsUGT91C1 is a schematic diagram of a normal reaction for catalyzing addition of No.2 glucosyl group and a side reaction for adding No. 6 glucosyl group (or No. 4 glucosyl group) in a stevioside substrate C13-hydroxyl (simplified and represented by R1).

A schematic diagram of d OsUGT91C1 in a normal reaction of catalyzing and adding No.2 glucosyl on a stevioside substrate C19-carboxyl (simplified and shown by R2) and a side reaction of adding No. 6 glucosyl (or No. 4 glucosyl).

FIG. 2 SDS-PAGE analysis of novel enzyme A (Phe208Met), OsUGT91C1 in its native state, and novel enzyme B (Phe208Met/Phe379Ala)

a SDS-PAGE analysis of the novel enzyme A (Phe208 Met). M, Marker; 1, post-induction sample; 2, thallus lysate; 3, supernatant of the thallus lysate; 4, precipitating thallus lysate; 5, Ni column flowing through the sample; 6-12, Ni column eluting protein sample.

B SDS-PAGE analysis of the native state OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379 Ala). M, Marker; WT, native state OsUGT91C 1; 2, flowing a Q column through the sample; 3-7, eluting the protein sample by the Q column. The right arrows of the two figures indicate the position of the band of the protein of interest.

FIG. 3 comparison of the natural state OsUGT91C1 and the novel enzyme A (Phe208Met) catalyzing the normal enzymatic reaction with addition of glucose group No.2 at both ends of the C13-hydroxy (R1) and C19-carboxy (R2) groups of the substrate Rubu, resulting in the formation of a beta (1-2) glycosidic linkage

a, b liquid phase analysis of OsUGT91C1 in natural state, New enzyme A (Phe208Met) reaction with Rubu. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. The products of OsUGT91C1 which show the natural state and are respectively added with the number 2 glucosyl at the C13-hydroxyl (R1) and the C19-carboxyl (R2) ends of the Rubu are increased along with the increase of time and enzyme amount, the new enzyme A (Phe208Met) can also add the number 2 glucosyl at the C13-hydroxyl and the C19-carboxyl of the substrate Rubu, and can catalyze to obtain more products which are added with the number 2 glucosyl at the C19-carboxyl, the target catalytic capability at the C19-carboxyl is enhanced, and finally the product Reb E which is added with the number 2 glucosyl at the C13-hydroxyl and the C19-carboxyl is more than the OsUGT91C1 at the natural state; c, d Natural State OsUGT91C1 and New enzyme A (Phe208Met) catalyzing the C13-hydroxy (R1) and C19-carboxy (R2) ends of substrate Rubu add No.2 glucosyl groups, respectively, generating a reaction scheme of Reb E, and the New enzyme A (Phe208Met) showing the target catalytic ability enhanced in the C19-carboxy direction.

FIG. 4 comparison of the natural states of OsUGT91C1 and New enzyme A (Phe208Met) catalyzing the addition of glucosyl group No.2 at the C19-carboxy (R2) end of substrate Reb A, resulting in the normal enzymatic reaction of beta (1-2) glycosidic bond

a, b liquid phase analysis of the reaction of natural state OsUGT91C1, neoenzyme A (Phe208Met) with Reb A. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. OsUGT91C1 showing natural state adds No.2 glucosyl group at C19-carboxyl (R2) end of Reb A, generated product Reb D increases along with the increase of time and enzyme amount, and new enzyme A (Phe208Met) can obviously consume the substrate Reb A completely and generate target product Reb D at first, and has enhanced target catalytic capability in the direction of C19-carboxyl; c, D natural state OsUGT91C1 and new enzyme A (Phe208Met) catalyze the C19-carboxyl (R2) end of the substrate Reb A to add No.2 glucosyl to generate a reaction scheme of Reb D, and the reaction of the new enzyme A (Phe208Met) is enhanced.

FIG. 5 comparison of the side reactions of OsUGT91C1 in its native state and the addition of glucose group No. 6 (or No. 4) to the C13-hydroxy (R1) end of the substrate STB by the novel enzyme B (Phe208Met/Phe379Ala)

Liquid phase analysis of the reaction of OsUGT91C1(a), neoenzyme B (Phe208Met/Phe379Ala) (B) in the native state with the substrate STB. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.75mg/mL of the enzyme was added for 0 hour as a control, 0.15mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.75mg/mL of the enzyme was reacted for 2 hours and 18 hours. It was shown that OsUGT91C1 side reaction products in the natural state increased with time and the amount of enzyme, while the novel enzyme B (Phe208Met/Phe379Ala) produced no side reaction products. The lower part of the liquid phase diagram of a is a schematic diagram of the OsUGT91C1 catalyzing STB reaction to generate a by-product, and the lower part of the liquid phase diagram of B is a schematic diagram of the new enzyme B catalyzing STB reaction, so that no by-product is generated and no side reaction occurs.

FIG. 6 comparison of the natural state of OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) catalyzing the addition of glucosyl group No.2 to the C13-hydroxy (R1) end of the substrate S13G, the normal enzymatic reaction forming the beta (1-2) glycosidic bond, and the subsequent side reaction of addition of glucosyl group No. 6 (or No. 4)

Liquid phase analysis of the reaction of OsUGT91C1(a), neoenzyme B (Phe208Met/Phe379Ala) (B) in native state with substrate S13G. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.75mg/mL of the enzyme was added for 0 hour as a control, 0.15mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.75mg/mL of the enzyme was reacted for 2 hours and 18 hours. OsUGT91C1 in natural state catalyzes the normal enzymatic reaction of adding No.2 glucosyl to the C13-hydroxyl (R1) end of a substrate S13G to generate STB, then side reaction is carried out on the STB, and No. 6 (or No. 4) glucosyl is added to generate a byproduct; in contrast, the novel enzyme B (Phe208Met/Phe379Ala) catalyzes only the normal reaction of adding glucosyl group No.2 to the C13-hydroxy group (R1) of the substrate S13G to produce STB, and does not catalyze side reactions or by-products; the lower part of the liquid phase diagram is a schematic diagram that natural state OsUGT91C1 catalyzes S13G to firstly catalyze normal reaction to generate STB and then catalyzes side reaction to generate by-product, and the lower part of the liquid phase diagram is a schematic diagram that new enzyme B (Phe208Met/Phe379Ala) catalyzes S13G to generate STB only in normal reaction and does not generate by-product.

FIG. 7 comparison of the natural state of OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) catalyzing the addition of glucosyl group No.2 at the C13-hydroxy (R1) and C19-carboxy (R2) ends of substrate Rubu, resulting in the normal enzymatic reaction of beta (1-2) glycosidic linkages

a, B liquid phase analysis of the reaction of OsUGT91C1 in native state, New enzyme B (Phe208Met/Phe379Ala) and Rubu. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. The products of OsUGT91C1 in a natural state, which add glucosyl group 2 to the C13-hydroxyl (R1) and C19-carboxyl (R2) ends of Rubu respectively, increase along with the increase of time and enzyme amount, while new enzyme B (Phe208Met/Phe379Ala) can catalyze to obtain more products of adding glucosyl group 2 to the C19-carboxyl direction, has enhanced target catalytic capability in the C19-carboxyl direction, and finally obtains a product Reb E which adds glucosyl group 2 to both the C13-hydroxyl and the C19-carboxyl directions and is also more than that of OsUGT91C1 in a natural state; c, d, the natural state OsUGT91C1 and the new enzyme B (Phe208Met/Phe379Ala) catalyze the C13-hydroxyl (R1) and C19-carboxyl (R2) ends of the substrate Rubu to add No.2 glucosyl respectively to generate a reaction scheme of Reb E, and the new enzyme B (Phe208Met/Phe379Ala) shows higher target catalytic capacity.

FIG. 8 comparison of the Normal enzymatic reactions for the production of Reb D with OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) catalyzing the addition of a glucosyl group 2 at the C19-carboxy (R2) end of the substrate Reb A, forming a beta (1-2) glycosidic linkage

a, B liquid phase analysis of the reaction of OsUGT91C1 in its natural state, New enzyme B (Phe208Met/Phe379Ala), and Reb A. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. OsUGT91C1 showing natural state adds No.2 glucosyl group at C19-carboxyl (R2) end of Reb A, generated product Reb D increases along with the increase of time and enzyme amount, while new enzyme B (Phe208Met/Phe379Ala) can obviously take the substrate reaction to be complete, and has enhanced target catalytic capability in the direction of C19-carboxyl; c, D Natural State OsUGT91C1 and New enzyme B (Phe208Met/Phe379Ala) catalyze the addition of a number 2 glucosyl group at the C19-carboxy (R2) end of the substrate Reb A, generating a reaction scheme for Reb D in which this reaction is enhanced for New enzyme B (Phe208Met/Phe379 Ala).

FIG. 9 stevioside substrate Reb E (without side product) obtained by catalysis of neoenzyme B (Phe208Met/Phe379Ala) by addition of glucosyl group No. 3 in C13-hydroxy or (and) C-19 carboxyl direction by glycosyltransferase UGT76G1 to obtain stevioside D and M

a, on the basis of a stevioside substrate Reb E (without generating byproducts) obtained by catalyzing by a new enzyme B (Phe208Met/Phe379Ala), and then converting by using glycosyltransferase UGT76G1 to form a reaction scheme of Reb D and Reb M. The box shows that the two new enzymes do not generate side reaction, and only normally add No.2 glucosyl in the direction of C13-hydroxyl or (and) C-19 carboxyl to form stevioside E (Reb E); then glycosyltransferase UGT76G1 relays to continuously catalyze the process of adding glucosyl group 3 in the direction of C13-hydroxyl or (and) C-19 carboxyl to form stevioside D and M (Reb D and Reb M). B, the steviol glycoside substrate Reb E obtained (without by-product formation) is catalyzed by the novel enzyme B (Phe208Met/Phe379Ala), and the conversion of Reb E to Reb D and Reb M is further catalyzed by the glycosyltransferase UGT76G 1. The reaction conditions in the liquid phase diagram from top to bottom are as follows: 0.03mg/mL glycosyltransferase UGT76G1 was added for 0 hour as a control, 0.03mg/mL glycosyltransferase UGT76G1 was added for 2 hours and 18 hours, and 0.15mg/mL glycosyltransferase UGT76G1 was added for 2 hours and 18 hours.

FIG. 10 SDS-PAGE analysis of the truncation after removal of amino acids 1-14 of the new enzyme B (Phe208Met/Phe379 Ala);

comparative SDS-PAGE analysis of the neoenzyme B (Phe208Met/Phe379Ala) and of the truncations after removal of amino acids 1-14 of neoenzyme B. M, Marker; 1, new enzyme B; 2, samples through which a truncation of new enzyme B flowed from the Q column; 3-8 samples eluted from the Q column with a truncation of New enzyme B

FIG. 11 shows a comparison of the normal enzymatic reactions of beta (1-2) glycosidic bonds, in which OsUGT91C1, the truncation of neoenzyme B after removal of amino acids 1-14 catalyzes addition of glucosyl group 2 to the C13-hydroxy (R1) and/or C19-carboxy (R2) ends of a substrate Rubu;

OsUGT91C1(a) in a natural state, a truncation (B) after removing 1 st to 14 th amino acids by a novel enzyme B and liquid phase analysis of Rubu reaction are compared. The reaction conditions of the liquid phase diagram from top to bottom are as follows: 0.25mg/mL of the enzyme was added for 0 hour as a control, 0.05mg/mL of the enzyme was reacted for 2 hours and 18 hours, and 0.25mg/mL of the enzyme was reacted for 2 hours and 18 hours. The products of OsUGT91C1 in a natural state, which are obtained by adding No.2 glucosyl groups to the C13-hydroxyl (R1) and C19-carboxyl (R2) ends of Rubu respectively, increase along with the increase of time and enzyme amount, and the new enzyme B truncated body has the same enzymatic catalytic reaction and has better catalytic capability than the natural OsUGT91C1 as the new enzyme B. C, the natural state OsUGT91C1 and the novel enzyme B truncation can perform the normal target reaction of respectively adding No.2 glucosyl to the C13-hydroxyl (R1) and C19-carboxyl (R2) ends of the substrate Rubu under the same catalysis, so as to generate a reaction scheme of Reb E, further verify the redundancy of the amino acids at the 1 st to 14 th positions, and completely remove or change the redundancy.

Detailed Description

The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

Firstly, construction of OsUGT91C1 protein expression vector in natural state

(1) Acquiring an amino acid Sequence (NCBI Reference Sequence: XP _015629141.1) of OsUGT91C1, and optimizing and synthesizing OsUGT91C1 coding codons according to the requirements of a protein expression system. The encoding codons optimized by the inventors based on the expression system of E.coli are as follows. Due to codon degeneracy, codon optimization options can be varied.

In order to prevent the substitution of an unimportant amino acid sequence of OsUGT91C1 and avoid the claims of the patent, the translated protein is considered to belong to OsUGT91C1 as long as the degree of identity between the amino acid sequence of the translated protein and the amino acid sequence of OsUGT91C1 is 50% or more.

Whether in the amino acid sequence or the spatial structure of OsUGT91C1, the aim of improving the normal reaction of adding No.2 glucosyl group into OsUGT91C1 or (and) eliminating the side reaction of OsUGT91C1 is achieved by changing the equivalent sites of the 208 th and 379 th amino acids or other sites (because of the redundancy of the amino acids, the 208 th and 379 th amino acids are not always the 208 th and 379 th numbering by removing one or any amino acids from the 1 st to the 14 th positions), and the invention belongs to the claims of the patent.

OsUGT91C1 (bold amino acids are the modified amino acid sites in this patent, underlined is the artificially added amino acid sequence, wherein LE is for convenient ligation into E.coli expression vector pET21b, and HHHHHHHHHH (6 histidine tag) is for later purification):

nucleotide sequence after codon optimization of OsUGT91C1 (underlined sequence is coding region of the above-mentioned artificially added amino acid sequence):

atgGATAGCGGTTATAGTAGCAGTTATGCCGCAGCCGCCGGCATGCATGTTGTGATTTGCCCGTGGCTGGCCTTTGGTCATCTGCTGCCGTGCTTAGACCTGGCCCAGCGTCTGGCCAGCCGTGGTCACCGTGTTAGCTTTGTGAGCACCCCGCGTAATATCAGCCGTCTGCCGCCGGTTCGTCCGGCATTAGCCCCGCTGGTGGCATTTGTGGCCTTACCGCTGCCGCGTGTTGAGGGTCTGCCTGATGGCGCCGAAAGTACCAACGACGTGCCGCATGACCGCCCGGATATGGTGGAGCTGCATCGTCGCGCCTTTGATGGTCTGGCAGCCCCGTTTAGCGAGTTTCTGGGCACAGCCTGCGCCGATTGGGTGATCGTTGACGTGTTTCATCACTGGGCAGCCGCAGCCGCCCTGGAACATAAAGTTCCGTGCGCAATGATGCTGCTGGGTAGCGCCCACATGATTGCCAGCATTGCCGATCGTCGCCTGGAACGCGCAGAGACCGAAAGCCCGGCAGCAGCAGGTCAAGGTCGTCCTGCCGCAGCCCCGACCTTTGAAGTGGCCCGCATGAAACTGATCCGTACCAAAGGTAGTAGCGGCATGAGCCTGGCCGAACGCTTTAGCCTGACCCTGAGCCGCAGTAGCCTGGTGGTTGGTCGCAGTTGTGTGGAATTCGAGCCGGAAACAGTGCCGCTGCTGAGCACCCTGCGCGGCAAACCGATCACCTTTCTGGGCCTGATGCCGCCGTTACATGAAGGCCGTCGTGAAGATGGTGAAGATGCCACAGTGCGTTGGCTGGATGCACAGCCGGCCAAAAGCGTTGTGTACGTTGCCCTGGGTAGCGAAGTTCCTCTGGGTGTGGAAAAGGTGCACGAACTGGCACTGGGTCTGGAACTGGCCGGTACCCGCTTCCTGTGGGCCTTACGTAAACCTACCGGTGTTAGCGATGCCGATCTGCTGCCGGCAGGTTTTGAGGAACGTACCCGTGGTCGCGGTGTTGTGGCAACACGCTGGGTTCCGCAGATGAGCATTCTGGCCCATGCCGCCGTGGGTGCCTTTCTGACCCATTGTGGCTGGAATAGCACCATCGAAGGCCTGATGTTCGGCCATCCTCTGATCATGCTGCCTATCTTCGGTGATCAGGGTCCGAACGCACGCCTGATTGAAGCAAAGAATGCCGGTCTGCAGGTGGCACGTAACGATGGCGACGGTAGCTTCGATCGTGAAGGCGTTGCCGCCGCAATTCGCGCCGTTGCAGTTGAAGAAGAGAGCAGCAAGGTGTTCCAGGCCAAAGCCAAAAAACTGCAGGAGATCGTGGCCGATATGGCATGCCATGAGCGCTACATCGATGGCTTCATCCAGCAGCTGCGCAGCTATAAAGATctcgagcaccaccaccaccaccac

(2) the OsUGT91C1 coding region obtained by synthesis is amplified by using the primers listed in the table 1, and the 5 'end and the 3' end of the amplified fragment respectively carry Nde I restriction enzyme sites and Xho I restriction enzyme sites.

TABLE 1 primer sequences of pET21b-OsUGT91C1

(3) pET21b expression vector and the OsUGT91C1 coding region amplified fragment of step 2 were digested with Nde I and Xho I, and the coding region of OsUGT91C1 was ligated between Nde I and Xho I cleavage sites of pET21b vector with T4 ligase, to construct pET21b-OsUGT91C1 expression vector capable of expressing glycosyltransferase OsUGT91C1 in E.coli.

(4) And transferring all the connection products into 100 mu LE, coli DH5 alpha competent cells, selecting positive monoclonal colonies on a plate, inoculating the positive monoclonal colonies into 10mL LB culture medium, culturing at 200rpm and 37 ℃ for 12-16 hours, extracting plasmids, sequencing to verify the correctness of the expression plasmids, and completing the construction of the OsUGT91C1 glycosyltransferase expression vector in a natural state.

Secondly, in order to enhance the normal reaction of adding the No.2 glucosyl group, constructing an expression vector of a novel enzyme A (Phe208 Met); in order to improve the target catalytic activity of the enzymatic addition of glucose group 2 and eliminate side reactions, an expression vector of a novel enzyme B (Phe208Met/Phe379Ala) was constructed

1. Construction of expression vector for novel enzyme A (Phe208Met)

(1) Site-directed mutagenesis primers used for designing the novel enzyme A (Phe208Met) and the novel enzyme B (Phe208Met/Phe379Ala) using pET21B-OsUGT91C1 in a natural state as a template are shown in Table 2.

Table 2 mutant primer sequences are as follows: (underlined part indicates the mutation site)

(2) By ddH₂O dissolves the primers in Table 2, and the concentration of the diluted primers is 10. mu.M. PCR amplification is carried out by using a pair of mutation primers of Phe208Met and pET21b-OsUGT91C1 as a template, wherein the system is as follows: dNTP 4. mu.L, 5 XPS buffer 10. mu.L, upstream and downstream primers 2. mu.L each, template 1. mu.L (about 10ng), PCR amplification enzyme Primer star 0.5. mu.L, and the remainder made up to 50. mu.L with ddH 2O. After mixing, PCR amplification is carried out, and the amplification procedure is as follows: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 30s, annealing at 69 deg.C for 30s, extension at 72 deg.C for 8min, amplification for 20 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C.

(3) Taking 10 mu L of the amplification product out for agarose gel to verify the amplification effect of PCR, adding 1 mu L of LDpnI enzyme into the rest 40 mu L of system, incubating for 1-2 hours at 37 ℃, taking 10 mu L of the amplification product, transferring into 100 mu L of LE. coli DH5 alpha competent cells respectively, carrying out ice bath for 30min, carrying out heat shock for 2min at 42 ℃, adding fresh LB 300 mu L after ice bath for 3min, incubating for 1 hour at 200rpm and 37 ℃ by a shaking table, taking 150 mu L of the amplification product, uniformly spreading the 150 mu L of the amplification product on Amp resistant solid LB plates, and standing and culturing overnight at 37 ℃.

(4) And (3) selecting monoclonal colonies on the two plates, inoculating the monoclonal colonies into 10mL LB culture medium, culturing at the temperature of 37 ℃ at 200rpm for 12-16 hours, extracting plasmids, and verifying the correctness of a mutation site Phe208Met by DNA sequencing, wherein the coded amino acid sequence is shown as SEQ ID No. 1.

2. Construction of expression vector for New enzyme B (Phe208Met/Phe379Ala)

(1) Obtaining an expression vector of the new enzyme A (Phe208Met) which is subjected to sequencing verification in the step 4 of the 2.2.2.1 as a template, and constructing an expression vector of the new enzyme B (Phe208Met/Phe379 Ala);

(2) by ddH₂O dissolves the primers in Table 2, and the concentration of the diluted primers is 10. mu.M. A pair of mutant primers of Phe379Ala is used for carrying out PCR amplification by taking an expression vector of a new enzyme A (Phe208Met) as a template, and the system is as follows: dNTP 4. mu.L, 5 XPS buffer 10. mu.L, upstream and downstream primers 2. mu.L each, template 1. mu.L (about 10ng), PCR amplification enzyme Primer star 0.5. mu.L, and ddH for the remainder₂The content of O is filled to 50 mu L. And (3) uniformly mixing, and performing PCR amplification by using an amplification program: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 30s, annealing at 68 deg.C for 30s, extension at 72 deg.C for 8min, amplification for 20 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C.

(3) And taking 10 mu L of the amplification product out to carry out agarose gel to verify the amplification effect of the PCR, adding 1 mu L of LDpnI enzyme into the remaining 40 mu L of the system, incubating for 1-2 hours at 37 ℃, taking 10 mu L of the system, transferring the 10 mu L of the system into 100 mu LE DH5 alpha competent cells respectively, carrying out ice bath for 30min, carrying out heat shock for 2min at 42 ℃, adding 300 mu L of fresh LB after carrying out ice bath for 3min, incubating for 1 hour at 200rpm and 37 ℃ by a shaking table, taking 150 mu L of the system, uniformly spreading the system on Amp resistant solid LB plates, and standing and culturing overnight at 37 ℃.

(4) Single colonies on two plates are picked and inoculated in 10mL LB culture medium, after 12-16 hours of culture at the temperature of 37 ℃ at 200rpm, plasmids are extracted, DNA sequencing verifies the correctness of two mutation sites Phe208Met and Phe379Ala, and the coded amino acid sequence is shown in SEQ ID No. 2.

Thirdly, the induced expression and purification of natural OsUGT91C1 and new enzyme A and B

(1) The expression plasmids corresponding to the three enzymes are transformed into E.coli BL21(DE3) expression strains, on the next day, a single clone is picked and inoculated into 10mL of fresh LB culture medium, after culturing overnight at 200rpm and 37 ℃, glycerol with the final concentration of 8% is added for preserving the strains, and the strains can be preserved at the temperature of minus 80 ℃ for a long time.

(2) The glycerol E.coli BL21(DE3) strain preserved in the previous step was inoculated into 100mL of fresh LB medium containing Amp 50. mu.g/mL and cultured overnight at 200rpm and 37 ℃. The next day, the cells were inoculated into 1L of fresh LB medium containing Amp at an inoculation ratio of 1% and 50. mu.g/mL, cultured at 180rpm and 37 ℃ until OD600 became 1.0, the cells were cooled to 16 ℃, IPTG was added to a final concentration of 0.5mM, and expression was induced at 160rpm and 16 ℃ to 20 ℃ for 18 hours.

(3) After the induction expression is finished, centrifuging at 4000rpm for 15min, discarding the supernatant, and collecting the thalli. After the mycelia were resuspended in a resuspension buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,30mM imidazole), they were disrupted repeatedly at 1000bar pressure for 3 times using a high pressure disrupter. After centrifugation at 13500rpm at 4 ℃ for 60min, the supernatant after centrifugation was applied to an NTA-Ni column, and non-specifically bound hetero-proteins were washed away with the above-mentioned resuspension buffer, the histidine-tagged protein of interest was eluted with an elution buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,250mM imidazole). The eluted protein is further replaced into 20mM HEPES buffer pH 7.2 and 50mM NaCl, and after quick freezing by liquid nitrogen, the protein can be stored at-80 ℃ for a long time. The above procedure allows to purify OsUGT91C1, neoenzyme A, neoenzyme B in their native state and to check the purity by SDS-PAGE (FIG. 2).

Fourth, measurement of enzymatic Activity of novel enzyme A (Phe208Met) and novel enzyme B (Phe208Met/Phe379Ala)

1. Enzymatic product analysis showed that the novel enzyme A (Phe208Met) improved the catalytic ability to add a glucosyl group No.2 (forming a β (1-2) glycosidic bond with glucosyl group No. 1) in the direction of the C13-hydroxyl group, particularly the C19-carboxyl group

(1) Taking the substrate Rubu (Rubusoside, CAS 64849-39-4) of OsUGT91C1 as an example, the OsUGT91C1 in a natural state can add glucose group No.2 (forming beta (1-2) glycosidic bond with glucose group No. 1) in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (normal reaction) (a in figure 3), the new enzyme A (Phe208Met) can also add No.2 glucosyl to both the C13-hydroxyl and the C19-carboxyl of the substrate Rubu, moreover, more products with the addition of the 2 nd glucosyl group in the C19-carboxyl direction can be obtained through catalysis, the target catalytic capability in the C19-carboxyl direction is enhanced, and finally, the product Reb E (Rebaudioside E, CAS 63279-14-1) with the addition of the 2 nd glucosyl group in both the C13-hydroxyl direction and the C19-carboxyl direction is more than the natural OsUGT91C1 (b in the figure 3).

The reaction system of the example is as follows: at the temperature of 20-40 ℃, a 200 mu L reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme A (Phe208Met)), at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively, 0.3mM substrate Rubu. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive and the same results can be obtained as long as OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates of OsUGT91C1 and the novel enzyme A (Phe208Met) in the natural state catalyzing the normal reaction of adding glucose group number 2 in the C13-hydroxy or C-19-carboxy direction are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding glucose group number 2 in the C13-hydroxy or C-19-carboxy direction can occur as long as glucose group number 1 exists in the C13-hydroxy or C-19-carboxy direction but no glucose group 3 does not exist.

(2) Taking the substrate Reb A (Rebaudioside A, CAS 58543-16-1) of OsUGT91C1 as an example, the OsUGT91C1 in the natural state can add glucose group No.2 (forming beta (1-2) glycosidic bond with glucose group No. 1) in the C19-carboxyl direction of the substrate Reb A (normal reaction) (a in FIG. 4), while the new enzyme A (Phe208Met) can obviously take the lead of completely consuming the substrate Reb A and generating the target product Reb D (Rebaudioside D, CAS 63279-13-0), has enhanced target catalytic ability in the C19-carboxyl direction, and enhances the ability of adding glucose group No.2 in the C19-carboxyl direction to form the corresponding product (b in FIG. 4).

The reaction system of the example is as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme A (Phe208Met)), 0.3mM substrate Reb A at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butyl alcohol with the same volume, carrying out vortex oscillation, stopping the reaction, extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of extract phase of the upper n-butyl alcohol, carrying out vacuum drying, carrying out heavy suspension with 25% acetonitrile with the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates of OsUGT91C1 and the novel enzyme A in a natural state catalyzing the normal reaction of adding glucosyl group No.2 in the direction of C13-hydroxyl or C-19 carboxyl are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding glucosyl group No.2 in the direction of C13-hydroxyl or C-19 carboxyl can occur as long as glucosyl group No.1 exists in the direction of C13-hydroxyl or C-19 carboxyl, but No. 3 glucosyl group does not exist.

2. Enzymatic product analysis showed that the novel enzyme B (Phe208Met/Phe379Ala) eliminates the side reaction of addition of glucose group No. 6 (or glucose group No. 4), but does not affect the catalytic ability to add glucose group No.2 (forming a beta (1-2) glycosidic bond with glucose group No. 1) in the C13-hydroxy and C19-carboxy directions

(1) Taking the substrate STB of OsUGT91C1 (Steviolbioside, CAS 41093-60-1) as an example, OsUGT91C1 in the native state can catalyze the side reaction of adding glucose group 6 (or glucose group 4) on the substrate STB respectively (a in FIG. 5), and OsUGT91C1 in the native state increases with the time and the amount of enzyme, while the new enzyme B (Phe208Met/Phe379Ala) does not produce side reaction product, and the side reaction of adding glucose group 6 (or glucose group 4) has been eliminated (B in FIG. 5).

Examples of side reactions are as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), at concentrations of 0.15mg/mL (1X) and 0.75mg/mL (5X), respectively, 0.3mM STB. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butyl alcohol with the same volume, carrying out vortex oscillation, stopping the reaction, extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of extract phase of the upper n-butyl alcohol, carrying out vacuum drying, carrying out heavy suspension with 25% acetonitrile with the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive and the same results can be obtained as long as OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrate for catalyzing side reactions by OsUGT91C1 in natural state is not exclusive, and includes but not limited to side reactions on the following steviol glycoside substrates, STB (Steviolbioside, CAS 41093-60-1), Reb E (Rebaudioside E, CAS 63279-14-1) and the like, and the side reaction of adding glucose group 6 (or glucose group 4) in the corresponding C13-hydroxyl or C-19 carboxyl direction can occur as long as glucose groups No.1 and No.2 exist in the C13-hydroxyl or C-19 carboxyl direction, but No. 3 (glucose group 3 refers to a glucose group forming a beta (1-3) glycosidic bond with glucose group No. 1).

(2) Taking a substrate S13G (Steviol-13-O-monogluside, CAS 60129-60-4) of OsUGT91C1 as an example, OsUGT91C1 in a natural state firstly catalyzes a normal enzymatic reaction of adding No.2 glucosyl to a C13-hydroxyl (R1) end of a substrate S13G to generate STB, then carries out a side reaction on the STB, and adds No. 6 (or No. 4) glucosyl to generate a byproduct (a in figure 6); in contrast, the novel enzyme B (Phe208Met/Phe379Ala) catalyzes only the normal reaction of adding glucosyl group No.2 to the C13-hydroxy group (R1) of the substrate S13G to produce STB, and does not catalyze side reactions and by-product formation (B in FIG. 6).

The reaction conditions of the examples are as follows: at the temperature of 20-40 ℃, a 200 mu L reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), at concentrations of 0.15mg/mL (1X) and 0.75mg/mL (5X), respectively, 0.3mM substrate S13G. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates of OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) in the native state catalyzing the normal reaction of adding No.2 glucosyl group in the C13-hydroxy or C-19 carboxy direction are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding No.2 glucosyl group in the C13-hydroxy or C-19 carboxy direction can occur as long as No.1 glucosyl group exists in the C13-hydroxy or C-19 carboxy direction, but No. 3 glucosyl group does not exist; the substrate for catalyzing side reactions by OsUGT91C1 in a natural state is not exclusive, and includes but is not limited to side reactions on the following stevioside substrates, STB (Steviolbioside, CAS 41093-60-1), Reb E (Rebaudioside E, CAS 63279-14-1) and the like, and the side reaction of adding glucose group No. 6 (or glucose group No. 4) to the corresponding C13-hydroxyl or C-19 carboxyl can occur as long as glucose groups No.1 and No.2 exist in the C13-hydroxyl or C-19 carboxyl direction, but No. 3 (glucose group No. 3 refers to a glucose group which forms a beta (1-3) glycosidic bond with glucose group No. 1).

3. Enzymatic product analysis showed that the novel enzyme B (Phe208Met/Phe379Ala) enhances the catalytic ability to add glucose residue No.2 (forming a beta (1-2) glycosidic bond with glucose residue No. 1) in the direction of the C13-hydroxy group, particularly the C19-carboxy group

(1) Taking the substrate Rubu (Rubusoside, CAS 64849-39-4) of OsUGT91C1 as an example, the OsUGT91C1 in the natural state can add glucosyl group 2 (forming beta (1-2) glycosidic bond with glucosyl group 1) in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (normal reaction) (a in figure 7), the novel enzyme B (Phe208Met/Phe379Ala) can also add glucose group No.2 to both the C13-hydroxyl and C19-carboxyl groups of the substrate Rubu, moreover, more products with the addition of the 2 nd glucosyl group in the C19-carboxyl direction can be obtained through catalysis, the target catalytic capability in the C19-carboxyl direction is enhanced, and finally, the product Reb E (Rebaudioside E, CAS 63279-14-1) with the addition of the 2 nd glucosyl group in both the C13-hydroxyl direction and the C19-carboxyl direction is also obviously more than the OsUGT91C1 (b in the figure 7) in the natural state.

The reaction system of the example is as follows: at the temperature of 20-40 ℃, a 200 mu L reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), 0.3mM substrate Rubu, at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

The substrates for the OsUGT91C1 and the novel enzyme B (Phe208Met/Phe379Ala) in their native state catalyzing the normal reaction of adding No.2 glucosyl group in the C13-hydroxy or C-19 carboxy direction are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding No.2 glucosyl group in the C13-hydroxy or C-19 carboxy direction can occur as long as No.1 glucosyl group exists in the C13-hydroxy or C-19 carboxy direction, but No. 3 glucosyl group does not exist.

(2) Taking the substrate Reb A (Rebaudioside A, CAS 58543-16-1) of OsUGT91C1 as an example, the native OsUGT91C1 can add the glucose group No.2 (forming a beta (1-2) glycosidic bond with the glucose group No. 1) in the C19-carboxyl direction of the substrate Reb A (normal reaction) (a in FIG. 8), while the new enzyme B (Phe208Met/Phe379Ala) can obviously take the lead of complete consumption of the substrate Reb A and generation of the target product Reb D (Rebaudioside D, CAS 63279-13-0), has enhanced target catalytic ability in the C19-carboxyl direction, and enhances the ability of adding the glucose group No.2 in the C19-carboxyl direction to form the corresponding product (B in FIG. 8).

The reaction system of the example is as follows: at 20-40 ℃, 200 μ L of reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B (Phe208Met/Phe379Ala)), 0.3mM substrate Reb A at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butanol with the same volume, carrying out vortex oscillation, stopping the reaction and extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of the extract phase of the n-butanol at the upper layer, carrying out vacuum drying, carrying out heavy suspension with acetonitrile with 25 percent of the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive, and the same results can be obtained as long as the OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

4. The rates at which the neoenzyme A (Phe208Met) and the neoenzyme B (Phe208Met/Phe379Ala) catalyze the normal reaction of adding a number 2 glucosyl group (forming a β (1-2) glycosidic bond with a number 1 glucosyl group) to a steviol glycoside substrate were determined by the method of fluorescence conversion.

The new enzyme A (Phe208Met) enhances the reaction speed of adding No.2 glucosyl (forming beta (1-2) glycosidic bond with No.1 glucosyl) to stevioside substrates required for catalysis, and is more than 2 times of the activity of the natural OsUGT91C 1; the side reaction of adding No. 6 (or No. 4) glucosyl is eliminated by the novel enzyme B (Phe208Met/Phe379Ala), and simultaneously, the reaction speed of catalyzing the normally required addition of No.2 glucosyl (forming beta (1-2) glycosidic bond with No.1 glucosyl) to the stevioside substrate is enhanced, and is 3-7 times of the activity of the natural OsUGT91C1 (Table 3).

TABLE 3 kinetic parameters of OsUGT91C1, neoenzyme A (Phe208Met), neoenzyme B (Phe208Met/Phe379Ala) in its native state catalyzing the addition of glucosyl group 2 (normally required catalytic reaction) at the C13-hydroxy (R1) and C19-carboxy (R2) ends of the substrates, respectively

(1) The assay was performed directly using a commercial glycosyltransferase activity kit, example UDP-Glo from Promega^TMThe same results can be obtained by using the Glycosyltransferase Assay kit and other methods for detecting the reaction speed of Glycosyltransferase.

UDP-Glo of Promega^TMThe Glycosyltransferase Assay kit is used for detecting the reaction speed of glucosyltransferase taking UDP-Glucose as a glycosyl donor, and the OsUGT91C1 and the two mutant novel enzymes related to the invention are both suitable.

Under the action of glucosyltransferase, the glycosyl donor UDP-Glucose transfers Glucose to a substrate to generate UDP. In this detection method, the amount of UDP or the like produced (1:1) is converted into ATP, which causes luciferase to emit a quantitative fluorescence, and thus the amount of UDP produced by the enzymatic glycosyltransfer reaction can be detected by measuring the amount of fluorescence. When UDP was produced in the concentration range of 0 to 25. mu.M, the fluorescence intensity was linearly related to the molar concentration of UDP. According to the standard curve of the corresponding relation between the UDP concentration and the fluorescence intensity, the generation amount of UDP is calculated and can be converted into the catalytic reaction speed of glycosyltransferase.

(2) A series of solutions of different concentrations of UDP were prepared and the corresponding fluorescence intensities were determined according to the above-mentioned conversion reaction. The natural state OsUGT91C1 and the two novel enzymes related to the invention are used for evaluating the effect of the novel enzyme A (Phe208Met) and the novel enzyme B (Phe208Met/Phe379Ala) on promoting the normal reaction of adding No.2 glucosyl by detecting information such as the speed of various catalytic reactions, enzyme kinetic constants and the like by using different substrates. The catalytic ability of the neoenzyme A (Phe208Met) and the neoenzyme B (Phe208Met/Phe379Ala) to add the No.2 glucosyl group to the C13-hydroxyl group and the C19-carboxyl group is respectively detected by using Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1) as substrates, and compared with the catalytic ability of natural OsUGT91C 1. The substrates of OsUGT91C1 and the novel enzymes A (Phe208Met) and B (Phe208Met/Phe379Ala) in their native state catalyzing the normal reaction of adding glucosyl group 2 in the C13-hydroxyl or C-19 carboxyl direction are not exclusive, and include, but are not limited to, Steviol glycoside substrates such as Rubu (CAS 64849-39-4), S13G (Steviol-13-O-monogluside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding glucosyl group 2 in the C13-hydroxyl or C-19 carboxyl direction can occur as long as glucosyl group 1 exists in the C13-hydroxyl or C-19 carboxyl direction but glucosyl group 3 does not exist.

(3) The enzyme kinetic constants of the natural state OsUGT91C1 and the new enzyme A (Phe208Met) and the new enzyme B (Phe208Met/Phe379Ala) for different substrates are shown in Table 3, and the new enzyme A (Phe208Met) enhances the reaction speed of the addition of the No.2 glucosyl group (forming a beta (1-2) glycosidic bond with the No.1 glucosyl group) for catalyzing the normal reaction, which is more than 2 times of the activity of the natural state OsUGT91C 1; the side reaction of adding No. 6 (or No. 4) glucosyl is eliminated by the novel enzyme B (Phe208Met/Phe379Ala), and simultaneously, the reaction speed of catalyzing the normally required addition of No.2 glucosyl (forming beta (1-2) glycosidic bond with No.1 glucosyl) to the stevioside substrate is enhanced, and is 3-7 times of the activity of the natural OsUGT91C1 (Table 3).

And fifthly, the novel enzyme B (Phe208Met/Phe379Ala) has no side reaction, improves the target catalytic capability, and can catalyze and complete the normal reaction of adding the No.2 glucosyl in the direction of C13-hydroxyl or (and) C-19 carboxyl of the stevioside substrate to generate a series of stevioside products including Rebaudioside E (CAS 63279-14-1). Under the condition, the glycosyltransferase UGT76G1 is utilized to add glucosyl group No. 3 in the direction of C13-hydroxyl or (and) C-19 carboxyl, thus obtaining the stevioside D and M (Rebaudioside D, CAS 63279-13-0 and Rebaudioside M, CAS 1220616-44-3) (figure 9).

Sixthly, the amino acid sequences of the novel enzyme A (Phe208Met) and the novel enzyme B (Phe208Met/Phe379Ala) have redundancy, and any one of the amino acids at positions 1 to 14 may be removed or changed.

Taking the novel enzyme B (Phe208Met/Phe379Ala) as an example, the novel enzyme B is subjected to removal of all amino acids (MDSGYSSSYAAAAG) at positions 1 to 14 to obtain a novel enzyme B truncated body which can still be normally expressed and purified and shows the activity of the normal reaction of adding glucosyl group No.2 in the direction of C13-hydroxyl or C-19 carboxyl of a steviol glycoside substrate.

1. To verify the redundancy of amino acids 1 to 14 (MDSGYSSSYAAAAG), an expression vector was constructed in which all amino acids 1 to 14 (MDSGYSSSYAAAAG) (truncation of neozyme B) were deleted on an expression vector for neozyme B (Phe208Met/Phe379Ala)

(1) Using the expression plasmid for the novel enzyme B (Phe208Met/Phe379Ala) as a template, primers in which all amino acid truncations at positions 1 to 14 were removed were designed as shown in Table 4.

Table 4 mutant primer sequences are as follows:

(2) by ddH₂O dissolve the primers in Table 4, and dilute the primers at 10. mu.M. A pair of primers for removing 1-14-F and removing 1-14-R respectively use an expression plasmid of a new enzyme B (Phe208Met/Phe379Ala) as a template for PCR amplification, and the system is as follows: dNTP 4. mu.L, 5 XPS buffer 10. mu.L, upstream and downstream primers 2. mu.L each, template 1. mu.L (about 10ng), PCR amplification enzyme Primer 0.5. mu.L, and ddH for the remainder₂The content of O is filled to 50 mu L. And (3) uniformly mixing, and performing PCR amplification by using an amplification program: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 30s, annealing at 69 deg.C for 30s, extension at 72 deg.C for 8min, amplification for 20 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C.

(3) And taking 10 mu L of each amplification product out to carry out agarose gel to verify the amplification effect of the PCR, adding 1 mu L of DpnI enzyme into the remaining 40 mu L of system, incubating for 1-2 hours at 37 ℃, taking 10 mu L of the mixture, transferring the 10 mu L of the mixture into 100 mu L of E.coli DH5 alpha competent cells, carrying out ice bath for 30min, carrying out heat shock for 2min at 42 ℃, adding 300 mu L of fresh LB after carrying out ice bath for 3min, carrying out shake cultivation for 1 hour at 37 ℃ at 200rpm, taking 150 mu L of the mixture, uniformly spreading the mixture on Amp-resistant solid LB plates, and carrying out standing cultivation overnight at 37 ℃.

(4) And (3) selecting the monoclonal colonies on the two plates, inoculating the monoclonal colonies into 10mL LB culture medium, culturing at 200rpm and 37 ℃ for 12-16 hours, extracting plasmids, and verifying the expression plasmid of the new enzyme B truncation by DNA sequencing to successfully remove all amino acids at the 1 st-14 th sites, wherein the amino acid sequences at the 1 st-14 th sites are identical to those of SEQ ID No.2 except that the amino acids at the 1 st-14 th sites are removed.

2. Inducible expression and purification of the truncation after removal of all amino acids from positions 1 to 14 by the novel enzyme B (Phe208Met/Phe379Ala)

(1) Corresponding expression plasmids are transformed into E.coli BL21(DE3) expression strains, a single clone is picked and inoculated into 10mL of fresh LB culture medium the next day, after culturing overnight at the temperature of 37 ℃ at 200rpm, glycerol with the final concentration of 8% is added for seed preservation, and the strains can be preserved at the temperature of 80 ℃ below zero for a long time.

(3) After the induction expression is finished, centrifuging at 4000rpm for 15min, discarding the supernatant, and collecting the thalli. After the cells were resuspended in resuspension buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,30mM imidazole), they were repeatedly disrupted at 1000bar pressure for 3 times using a high pressure disrupter. 13500rpm, at 4 ℃ for 60min, the centrifuged supernatant was applied to an NTA-Ni column, and the non-specifically bound hetero-proteins were washed away with the above-mentioned resuspension buffer, and the target protein with the histidine tag was eluted with an elution buffer (20mM Tris-HCl buffer pH 7.8,0.5M NaCl,250mM imidazole). The eluted protein is further replaced into 20mM HEPES buffer pH 7.2 and 50mM NaCl, and after quick freezing by liquid nitrogen, the protein can be stored at-80 ℃ for a long time. The above procedure resulted in the purification of a truncated form of the novel enzyme B and the purity was checked by SDS-PAGE (FIG. 10). As can be seen from FIG. 10, when the new enzyme B removes the truncation of amino acids 1-14, normal expression and purification can be performed. Since the novel enzyme A and the novel enzyme B have the same amino acids at positions 1 to 14, the example in which the amino acids at positions 1 to 14 are completely removed from the novel enzyme B shows that the amino acids at positions 1 to 14 have redundancy. Similarly, amino acids 1 to 14 of the novel enzyme A are also redundant.

3. The truncation of the novel enzyme B does not affect the normal reaction of adding glucosyl group No.2 in both directions of C13-hydroxyl and C-19 carboxyl of a stevioside substrate, has higher catalytic activity than that of natural trans-glycosyltransferase OsUGT91C1 as the novel enzyme B, and further shows the redundancy of amino acids 1-14 in the novel enzyme A and the novel enzyme B.

(1) Taking a stevioside substrate Rubu (Rubusoside, CAS 64849-39-4) as an example, OsUGT91C1 in a natural state can add glucose group No.2 (forming a beta (1-2) glycosidic bond with glucose group No. 1) in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (normal reaction) (a in FIG. 11), and a truncated body of neozyme B can also add glucose group No.2 in both directions of C13-hydroxyl and C19-carboxyl of the substrate Rubu (B in FIG. 11).

(2) The reaction system of the example is as follows: at the temperature of 20-40 ℃, a 200 mu L reaction system comprises: 1mM UDP-Glucose,20mM Tris-HCl buffer pH 7.2, enzyme samples (native or New enzyme B truncates) at concentrations of 0.05mg/mL (1X) and 0.25mg/mL (5X), 0.3mM substrate Rubu, respectively. Starting the reaction by adding an enzyme sample, sampling 60 mu L at 0, 2 and 18 hours respectively, mixing with n-butyl alcohol with the same volume, carrying out vortex oscillation, stopping the reaction, extracting the corresponding enzymatic reaction product, centrifuging at 17000rpm for 10min at room temperature, standing at room temperature for 1min, taking 50 mu L of extract phase of the upper n-butyl alcohol, carrying out vacuum drying, carrying out heavy suspension with 25% acetonitrile with the same volume, and detecting the enzymatic product by using HPLC. The reaction conditions listed in this example are not exclusive and the same results can be obtained as long as OsUGT91C1 is enzymatically reacted. The detection means is also not unique, as long as each enzymatic product can be distinguished, and the same detection result can be obtained.

(3) The substrates of the OsUGT91C1 and the novel enzyme B truncation in the natural state catalyzing the normal reaction of adding the glucose group 2 in the C13-hydroxyl or C-19 carboxyl direction are not exclusive, and include, but are not limited to, stevia substrates such as Rubu (Rubusoside, CAS 64849-39-4), S13G (Steviol-13-O-monoglucoside, CAS 60129-60-4) and Reb A (Rebaudioside A, CAS 58543-16-1), and the normal reaction of adding the glucose group 2 in the C13-hydroxyl or C-19 carboxyl direction can occur as long as the glucose group 1 exists in the C13-hydroxyl or C-19 carboxyl direction but the glucose group 3 does not exist.

Sequence listing

<110> Sichuan university

<120> glycosyltransferase OsUGT91C1 mutant and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 462

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly Met His

1 5 10 15

Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu

20 25 30

Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser Phe Val

35 40 45

Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ala Leu

50 55 60

Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val Glu Gly

65 70 75 80

Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp Arg Pro

85 90 95

Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala Ala Pro

100 105 110

Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp

115 120 125

Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro

130 135 140

Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala

145 150 155 160

Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala Gly

165 170 175

Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg Met Lys

180 185 190

Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu Arg Met

195 200 205

Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser Cys Val

210 215 220

Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg Gly Lys

225 230 235 240

Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg

245 250 255

Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala

260 265 270

Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val

275 280 285

Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly Thr Arg

290 295 300

Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala Asp Leu

305 310 315 320

Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val Val Ala

325 330 335

Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala Val Gly

340 345 350

Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met

355 360 365

Phe Gly His Pro Leu Ile Met Leu Pro Ile Phe Gly Asp Gln Gly Pro

370 375 380

Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg

385 390 395 400

Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala Ile

405 410 415

Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln Ala Lys

420 425 430

Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His Glu Arg

435 440 445

Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp

450 455 460

<210> 2

<211> 462

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly Met His

1 5 10 15

Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu

20 25 30

Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser Phe Val

35 40 45

Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ala Leu

50 55 60

Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val Glu Gly

65 70 75 80

Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp Arg Pro

85 90 95

Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala Ala Pro

100 105 110

Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp

115 120 125

Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro

130 135 140

Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala

145 150 155 160

Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala Gly

165 170 175

Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg Met Lys

180 185 190

Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu Arg Met

195 200 205

Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser Cys Val

210 215 220

Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg Gly Lys

225 230 235 240

Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg

245 250 255

Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala

260 265 270

Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val

275 280 285

Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly Thr Arg

290 295 300

Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala Asp Leu

305 310 315 320

Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val Val Ala

325 330 335

Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala Val Gly

340 345 350

Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met

355 360 365

Phe Gly His Pro Leu Ile Met Leu Pro Ile Ala Gly Asp Gln Gly Pro

370 375 380

Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg

385 390 395 400

Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala Ile

405 410 415

Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln Ala Lys

420 425 430

Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His Glu Arg

435 440 445

Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp

450 455 460

Claims

1. The glycosyltransferase OsUGT91C1 mutant has an amino acid sequence of any one of the following a, b, C and d:

a. an amino acid sequence shown as SEQ ID No. 1;

b. the amino acid sequence shown as SEQ ID No. 2;

c. the amino acid sequence shown in SEQ ID No.1 of the amino acids at the 1 st to the 14 th positions is deleted;

d. the amino acid sequence shown in SEQ ID No.2 of amino acids 1-14 is deleted.

2. An expressed gene encoding the glycosyltransferase OsUGT91C1 mutant of claim 1.

3. An expression vector containing the expression gene according to claim 2.

4. A recombinant bacterium carrying the expression vector of claim 3.

5. Use of the glycosyltransferase OsUGT91C1 mutant of claim 1 in the enzymatic synthesis or conversion of steviol glycoside D or M.

6. The use according to claim 5, wherein the glycosyltransferase OsUGT91C1 mutant is used for enzymatic synthesis or conversion of stevioside D or M by adding a 2-glucosyl group in the direction of C13-hydroxyl or/and C19-carboxyl of stevioside, wherein the 2-glucosyl group is a glucosyl group which forms a beta (1-2) glycosidic bond with the 1-glucosyl group, and the 1-glucosyl group is a glucosyl group which directly forms a beta-glycosidic bond with C13-hydroxyl or C19-carboxyl.