Glycosyltransferase mutants and their use in biosynthesis of rebaudioside m

Abstract

The present application obtains a mutant through directed evolution of a glycosyltransferase UGT76G1, the mutant contains at least one site mutation of H82, L85, M88, L126, T146, N196, I199, L200, I203, T284 on the basis of the amino acid sequence of the glycosyltransferase UGT76G1 shown in SEQ ID NO: 1. The glycosyltransferase mutant improves the enzyme activity and catalytic efficiency, and a cascade reaction is carried out by using the mutant and sucrose synthase AtSuSy to synthesize Reb M with Reb D as a substrate, the yield of Reb M is improved, and the catalytic synthesis yield can reach more than 90%, which improves the basis for the industrial production of Reb M.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to glycosyltransferase mutants and their application in the biosynthesis of rebaudioside M. Background Technology

[0002] With the improvement of living standards, healthy eating has become one of the daily pursuits of people today. However, excessive sugar intake places a heavy burden on the body, leading to various chronic diseases. To reduce the intake of high-calorie sugars, low-calorie or zero-calorie sweeteners are increasingly being used in the food industry, allowing people to enjoy delicious food while maintaining a healthy sweetness.

[0003] Stevia is characterized by its high sweetness and low calorie content. Its sweetness is 200-300 times that of sucrose, while its caloric value is only 1 / 300th that of sucrose. It does not affect blood sugar levels or interfere with insulin, providing diabetics with more flexibility in budgeting their total calorie intake. Furthermore, steviol glycosides are non-toxic, have no side effects, and contain no carcinogens, making them safe for consumption and an ideal sweetener to replace sucrose. Although rebaudioside A and stevia are highly sweet, they still have a bitter aftertaste compared to sucrose. There are many components of steviol glycosides, the most common being steviol glycosides, rebaudioside A (Reb A), rebaudioside D (Reb D), and rebaudioside M (Reb M). Among them, rebaudioside M and rebaudioside D are the sweetest (350 times sweeter than sucrose) and have less bitterness than rebaudioside A. Rebaudioside M, in particular, tastes most similar to regular sucrose. However, the proportion of rebaudioside M in stevia leaves is very small (accounting for 0.4 to 0.5% of the dry weight of the leaves), so it cannot be mass-produced using traditional plant extraction methods.

[0004] The biosynthetic preparation of steviol glycosides has been a key research focus in recent years. Current technologies often employ UDP-glycosyltransferases as enzymes to catalyze the production of rebaudioside M. In stevia leaves, steviol, under the action of different UDP-glycosyltransferases, binds different numbers of glucosyl and sucrose groups at the C-19 and C-13 positions, respectively, to form SS glycosides, RA glycosides, etc., with varying sweetness and flavor. For example, Chinese invention patent application CN201510139428.9 discloses an enzymatic method for preparing rebaudioside M, which utilizes tomato UDP-glycosyltransferase and potato sucrose synthase, using rebaudioside A and sucrose as raw materials to produce rebaudioside M. Chinese invention patent application CN201811228403.6 discloses an enzymatic method for preparing rebaudioside M. This method uses steviol glycosides as raw materials, ethyl acetate and β-cyclodextrin as glycosyl donors, and palmitoyltransferase, cyclodextrin glycosyltransferase, and UDP-glycosyltransferase as the main enzymes to generate rebaudioside M. Chinese invention patent application CN201810684818.8 discloses a steviol glycoside complex and its preparation method. This method uses rebaudioside A (RebA), the most abundant steviol glycoside, as the starting material. Under certain process conditions, a one-pot method using glucosyltransferase is employed to obtain a steviol glycoside complex containing both rebaudioside D (RebD) and rebaudioside M (RebM), both with excellent taste.

[0005] UDP-glycosyltransferases may recognize certain specific groups on receptor molecules, exhibiting specificity. Richman et al. isolated three UDP-glycosyltransferases from stevia leaves: UGT85C2, UGT74G1, and UGT76G1. UGT85C2 catalyzes the reaction of steviol to steviol monoglycosides, while UGT74G1 primarily catalyzes the glycosylation of steviol diglycosides to produce SS glycosides. The conversion of SS glycosides to RA glycosides is completed in a one-step glycosylation reaction by UGT76G1. Modern research has further discovered that Reb M can be produced by the UDP-dependent glycosyltransferase (UGT) SrUGT76G1 of RebD. Plant Communications published online the collaborative research findings of the research groups of Wang Yong and Zhang Peng at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, entitled "Structural Insights into the Catalytic Mechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1". This study elucidates in detail the molecular mechanism of substrate recognition and catalysis of the diterpenoid glycosyltransferase SrUGT76G1. It found that this enzyme can recognize various diterpenes and other small molecules to generate corresponding glycosylated products. However, SrUGT76G1 can adapt to various steviol substrates, such as steviol glycosides, rebaudioside A, and rebaudioside D, but with low selectivity, due to its large glycosyl receptor binding port (glycobinding capsule). This prior art also reveals that Thr284 is the key site determining substrate recognition specificity and product specificity. The mutant SrUGT76G1-T284S can selectively catalyze the synthesis of rebaudioside M from rebaudioside A via the intermediate rebaudioside D. The mutant UGT76G1-T284S showed an approximately 1-1.5-fold increase in its activity towards rebaudioside D. However, although the mutant UGT76G1-T284S improved activity to some extent, it still cannot reach the level required for large-scale production. Summary of the Invention

[0006] To further modify the glycosyltransferase UGT76G1 to improve its yield in the biocatalytic synthesis of rebaudioside D to rebaudioside M, a method for the biosynthesis of rebaudioside M is provided. This method has a high conversion rate of rebaudioside M and is suitable for the industrial production of rebaudioside M.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A glycosyltransferase mutant, said mutant being a mutation containing at least one of the following sites based on the amino acid sequence of glycosyltransferase UGT76G1 shown in SEQ ID NO:1: H82, L85, M88, L126, T146, N196, I199, L200, I203, T284.

[0009] Furthermore, the mutant is based on the amino acid sequence of the glycosyltransferase UGT76G1 shown in SEQ ID NO:1 and contains mutations at at least one of the following sites: L85, M88, L126, N196, I199, L200, T284.

[0010] Preferably, the mutant is based on the amino acid sequence of glycosyltransferase UGT76G1 shown in SEQ ID NO:1 and contains mutations at at least one of the following sites: M88, L126, I199, L200, T284, and at least one of the mutation sites is T284.

[0011] Furthermore, the mutation at at least one of the sites H82, L85, M88, L126, T146, N196, I199, L200, I203, and T284 is selected from the substitution of residues at at least one of the sites H82A, L85A, M88L, L126F, T146S, N196A, I199F, L200A, I203A, and T284S.

[0012] According to an embodiment of the present invention, the mutant has more than 70% homology with the amino acid sequence shown in SEQ ID NO:1.

[0013] The present invention also provides an expression gene that encodes the above-mentioned glycosyltransferase mutant.

[0014] The present invention also provides a recombinant vector in which the above-mentioned expression gene is linked.

[0015] Furthermore, the recombinant vector connects the aforementioned expression gene and the sucrose synthase gene.

[0016] The present invention also provides a method for biosynthesizing rebaudioside M, using rebaudioside D as a substrate and a recombinant strain as a transformation strain to biosynthesize rebaudioside M.

[0017] The recombinant strain is a recombinant strain obtained by transforming a recombinant vector containing a glycosyltransferase mutant gene into competent cells.

[0018] Alternatively, it can be a recombinant strain obtained by transforming a recombinant vector containing a glycosyltransferase mutant gene and a sucrose synthase gene into competent cells.

[0019] The recombinant vector is a plasmid, preferably pET28a or pETDuet.

[0020] The competent cells are E. coli BL21 (DE3) or E. coli Rosetta (DE3), preferably E. coli BL21 (DE3).

[0021] Furthermore, the method for biosynthesizing rebaudioside M specifically includes the following steps:

[0022] 1) The recombinant vector was transformed into competent cells to obtain recombinant bacterial strains;

[0023] 2) The recombinant strain obtained in step 1) was induced to express in liquid culture medium, and the bacterial cells were collected;

[0024] 3) The bacterial cells obtained in step 2) are added to a catalytic reaction system containing rebaudioside D for transformation to obtain a transformation solution. The transformation solution is then separated and purified to obtain the product rebaudioside M.

[0025] Preferably, the inducing agent used for the induced expression is isopropyl-β-d-thiogalactoside (IPTG).

[0026] In one embodiment of the present invention, the reaction system comprises: 50-60 mM Tris-HCl, pH 7.5-8.5, 0.1-1.0 mM UDP, 200-900 mM sucrose, 5-60 g / L Reb D, 20-120 g / L bacterial cells, and reacted at 25-45°C for 1-24 h.

[0027] Preferably, the reaction system comprises: 55 mM Tris-HCl, pH 8.0, 0.8 mM UDP, 500 mM sucrose, 20 g / L Reb D, 50 g / L bacterial cells, and reacted at 30°C for 24 h.

[0028] Furthermore, in step (3), rebaudioside D is added in an intermittent manner, with 5 g / L of rebaudioside D added at 0 h, 0.5 h, 1.5 h and 2.5 h respectively.

[0029] Beneficial effects:

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] This invention obtains a mutant by directed evolution of glycosyltransferase UGT76G1, which improves enzyme activity and catalytic efficiency. The mutant and sucrose synthase AtSuSy are then used in a cascade reaction to synthesize Reb M using Reb D as a substrate, which improves the yield of Reb M. The catalytic synthesis yield can reach more than 90%, laying the foundation for the industrial production of Reb M. Attached Figure Description

[0032] Figure 1 This is a comparison chart of the conversion rates of some mutants. Detailed Implementation

[0033] This invention is based on in-depth and creative research, targeting key amino acid residues in the substrate-binding pocket of UGT76G1 to study UGT76G1 mutants that enhance its catalytic activity. A set of mutants with specific amino acid residue mutations is disclosed. The "substrate-binding pocket" refers to the location in the spatial structure of the glycosyltransferase UGT76G1 where it interacts with the substrate.

[0034] The glycosyltransferase mutant of the present invention is based on the amino acid sequence of glycosyltransferase UGT76G1, containing mutations at at least one of the following sites: H82, L85, M88, L126, T146, N196, I199, L200, I203, T284. The amino acid sequence of glycosyltransferase UGT76G1 is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2.

[0035] In one embodiment of the present invention, the amino acid sequence of the mutant contains a mutation at any one of the following sites: H82, L85, M88, L126, T146, N196, I199, L200, I203, and T284. Preferably, the amino acid sequence contains a mutation at any one of the following sites: L85, M88, L126, N196, I199, L200, and T284.

[0036] In another embodiment of the present invention, the amino acid sequence of the mutant contains mutations at any two sites selected from H82, L85, M88, L126, T146, N196, I199, L200, I203, and T284. Preferably, the amino acid sequence contains mutations at any two sites selected from L85, M88, L126, N196, I199, L200, and T284. Further, among the above mutation sites, one of the mutation sites is either T284 or L200. Preferably, the amino acid sequence of the mutant contains mutations at the following two sites: T85 / T284, M88 / T284, L126 / T284, T146 / T284, I203 / T284, T284 / L200, M88 / L200, or I199 / L200. Furthermore, the amino acid sequence of the mutant contains mutations at the two sites mentioned above, and one of the mutation sites is T284. Preferably, the amino acid sequence of the mutant contains mutations at the following two sites: M88 / T284, L126 / T284, I203 / T284, or T284 / L200.

[0037] In another embodiment of the present invention, the amino acid sequence of the mutant contains mutations at any three sites selected from H82, L85, M88, L126, T146, N196, I199, L200, I203, and T284. Preferably, the amino acid sequence contains mutations at any three sites selected from M88, L126, N196, I199, L200, and T284. Further, among the above-mentioned mutation sites, one of the mutation sites is either T284 or L200. Preferably, the amino acid sequence of the mutant contains mutations at the following three sites: L85 / M88 / T284, M88 / T146 / T284, M88 / L126 / T284, M88 / I203 / T284, M88 / L200 / T284, M88 / I199 / T284, I199 / L200 / T284, L126 / I199 / L200, or I199 / I203 / L200. More specifically, the amino acid sequence of the mutant contains mutations at the above three sites, and one of the mutation sites is T284. Preferably, the amino acid sequence of the mutant contains mutations at the following two sites: M88 / T146 / T284, M88 / L126 / T284, M88 / I203 / T284, M88 / L200 / T284, or M88 / I199 / T284.

[0038] In another embodiment of the present invention, the amino acid sequence of the mutant contains mutations at any four sites selected from H82, L85, M88, L126, T146, N196, I199, L200, I203, and T284. Preferably, the amino acid sequence contains mutations at any four sites selected from M88, L126, N196, I199, L200, and T284. Further, among the above mutation sites, one of the mutation sites is either T284 or L200. Preferably, the amino acid sequence of the mutant contains mutations at the following three sites: M88 / L126 / T146 / T284, M88 / L126 / I199 / T284, M88 / L126 / I203 / T284, L126 / N196 / L200 / T284, or L126 / N196 / I199 / L200.

[0039] Furthermore, the mutation at at least one of the sites H82, L85, M88, L126, T146, N196, I199, L200, I203, and T284 is selected from the substitution of residues at at least one of the sites H82A, L85A, M88L, L126F, T146S, N196A, I199F, L200A, I203A, and T284S.

[0040] In some specific implementations, the mutants are: UGT76G1-L85A, UGT76G1-M88L, UGT76G1-L126F, UGT76G1-I199F, UGT76G1-L200A, UGT76G1-I203A, UGT76G1-T284S; UGT76G1-L85A / T284S, UGT76G1-M88L / T284S, UGT76G1-I199F / T284S, UGT76G1-M88L / L200A; UGT76G1-L85A / M88L / T284S, UGT76G1-M88L / L126F / T284S, UGT76G1- M88L / L126F / L200A, UGT76G1-M88L / L200A / T284S, UGT76G1-L126F / I199F / L200A; UGT76G1-M88L / L126F / T146S / T284S, UGT76G1-M88L / L126F / I199F / T284S, UGT76G1-L126F / N196A / L200A, etc.

[0041] According to an embodiment of the present invention, the mutant has more than 70% homology with the amino acid sequence shown in SEQ ID NO:1. In a specific embodiment, the homology or sequence similarity can be more than 90%, preferably more than 95%.

[0042] In this paper, mutation sites and their substitutions are expressed by their position numbers and the types of amino acids at those sites. For example, M88L indicates that, compared with SEQ ID NO:1, methionine at position 88 of SEQ ID NO:1 is replaced by leucine. In this invention, " / " is used to indicate combinations of mutation sites. For example, "M88L / L200A" indicates that methionine at position 88 is replaced by leucine and leucine at position 200 is replaced by alanine, containing two mutation sites, which is a double mutant. Similarly, "M88L / L126F / L200A" indicates that the corresponding three sites are mutated simultaneously, which is a triple mutant.

[0043] The present invention also provides an expression gene that encodes the above-mentioned glycosyltransferase mutant.

[0044] In this invention, the glycosyltransferase mutant expression gene can be inserted into a recombinant vector. Commonly used recombinant vectors are all suitable for this invention. In this invention, the recombinant vector is a plasmid, preferably pET28a or pETDuet.

[0045] The present invention also provides a recombinant vector in which the above-mentioned expression gene is linked.

[0046] Furthermore, the recombinant vector connects the aforementioned expression gene and sucrose synthase gene.

[0047] In a specific embodiment of the present invention, the glycosyltransferase mutant was expressed and purified in *E. coli*: the wild-type UGT76G1 gene was amplified from the cloning vector and subcloned into the protein expression vector pETDuet-1 by PCR. Using the UGT76G1 gene expression vector as a template, a mutation was introduced by PCR primers, and the mutant protein expression vector was constructed by PCR amplification. The correctly sequenced mutant was heterologously expressed in *E. coli* BL21(DE3). The enzyme activity detection method for the glycosyltransferase mutant can utilize techniques known in the art. In a specific embodiment of the present invention, the in vitro enzyme activity of different mutant proteins was tested using an enzyme reaction system under the same conditions. After the reaction, the product generation was detected by HPLC. The catalytic activity of the mutant was analyzed by comparing the conversion rate of lebodiin D by the mutant and wild-type glycosyltransferase UGT76G1.

[0048] The present invention also provides a method for biosynthesizing rebaudioside M, using rebaudioside D as a substrate and a recombinant strain as a transformation strain to biosynthesize rebaudioside M.

[0049] The recombinant strain is a recombinant strain obtained by transforming a recombinant vector containing a glycosyltransferase mutant gene into competent cells.

[0050] Alternatively, it can be a recombinant strain obtained by transforming a recombinant vector containing a glycosyltransferase mutant gene and a sucrose synthase gene into competent cells.

[0051] The competent cells are E. coli BL21 (DE3) or E. coli Rosetta (DE3), preferably E. coli BL21 (DE3).

[0052] Furthermore, the method for biosynthesizing rebaudioside M specifically includes the following steps:

[0053] 1) The recombinant vector was transformed into competent cells to obtain recombinant bacterial strains;

[0054] 2) The recombinant strain obtained in step 1) was induced to express in liquid culture medium, and the bacterial cells were collected;

[0055] 3) The bacterial cells obtained in step 2) are added to a catalytic reaction system containing rebaudioside D for transformation to obtain a transformation solution. The transformation solution is then separated and purified to obtain the product rebaudioside M.

[0056] Preferably, the inducing agent used for the induced expression is isopropyl-β-d-thiogalactoside (IPTG).

[0057] In one embodiment of the present invention, the reaction system comprises: 50-60 mM Tris-HCl, pH 7.5-8.5, 0.1-1.0 mM UDP, 200-900 mM sucrose, 5-60 g / L Reb D, 20-120 g / L bacterial cells, and reacted at 25-45°C for 1-24 h.

[0058] Preferably, the reaction system comprises: 55 mM Tris-HCl, pH 8.0, 0.8 mM UDP, 500 mM sucrose, 20 g / L Reb D, 50 g / L bacterial cells, and reacted at 30°C for 24 h.

[0059] Furthermore, in step (3), rebaudioside D is added in an intermittent manner, with 5 g / L of rebaudioside D added at 0 h, 0.5 h, 1.5 h and 2.5 h respectively.

[0060] In this article, "reb glycoside D" and "Reb D", "reb glycoside M" and "Reb M" have the same meaning and can be used interchangeably.

[0061] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0062] Example 1: Construction of wild-type UGT76G1 expression vector

[0063] Using a codon-optimized UGT76G1 gene cloning vector as a template, the UGT76G1 gene was amplified using specific primer pairs (loaded with BamHI and HindIII sites, Table 1). The PCR product was ligated into the BamHI / HindIII double-digested vector pET28a using T4 ligase, and the resulting expression vector pET28a-UGT76G1 was verified by sequencing.

[0064] Table 1. Primers used for constructing the wild-type UGT76G1 expression vector

[0065] 76G1F <![CDATA[CGC GGATCC ATGGAAAACAAACCGAA]]> 76G1R <![CDATA[CCC AAGCTT TTACAGAGAGAGATGTA]]>

[0066] Example 2 Construction of mutant expression vector

[0067] Using PCR amplification technology, site-directed mutagenesis was performed on the wild-type expression vector pET28a-UGT76G1 as a template, and the cells were transformed into DH5α competent cells. Clones were selected and sequenced for verification. The corresponding primers are shown in Table 2.

[0068] Table 2. Primers used for constructing mutant expression vectors

[0069]

[0070] Example 3 Expression and purification of mutant proteins

[0071] The sequenced mutant expression vectors prepared in Example 2 were transformed into *E. coli* BL21 (DE3) and cultured at 37°C in 2×YT medium containing 100 mg / L ampicillin. When the cell concentration reached OD600 0.6-0.8, protein expression was induced for 8 h at 18°C ​​using 0.1 mM isopropyl-β-d-thiogalactoside (IPTG). Cells were collected and lysed with a lysis buffer at a ratio of 0.1 g / mL. The lysis buffer system consisted of 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, and 10% glycerol. Cells were homogenized using an autoclave, and the crude sample was centrifuged at 40,000 × g for 30 min. Protein purification was performed using a Ni-NTA column and then concentrated to 10 mg / mL for later use.

[0072] Example 4: Determination of mutant enzyme activity

[0073] Determination of the catalytic activity of wild-type UGT76G1 glycosyltransferase and glycosyltransferase mutants:

[0074] The enzyme obtained in Example 3 and wild-type UGT76G1 glycosyltransferase were added separately to the reaction system, along with 2.5 mM of the enzyme and 2 mM of RebD. The mixture was incubated at 35°C for 10 min. The reaction system consisted of 5 mM UDPG, 50 mM Tris-HCl, pH 8.0, and 200 μL. After the reaction, the mixture was diluted with 10 times its volume of 20% acetonitrile aqueous solution, heated at 90°C for 2 min, centrifuged at 6000g for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The product formation was analyzed by HPLC. Based on the HPLC results, the catalytic activity of different mutants was compared. Figure 1 The conversion rates of some mutants with significant catalytic effects are compared.

[0075] The HPLC instrument was an Agilent 1260, and the column was a C18 column (Silgreen HPLC column 250×4.6mm). The HPLC conditions were: column temperature 40°C, flow rate 1 mL / min, mobile phase B: water, mobile phase C: acetonitrile, using a linear gradient elution method: 0 min 95% B + 5% C, 10 min 55% B + 45% C, 15 min 0% B + 100% C, 16 min 95% B + 5% C, 20 min 95% B + 5% C.

[0076] Example 5 Construction of recombinant strains

[0077] Mutants with relatively high enzyme activity were selected as key mutants from mutants, double mutants, and triple mutants: UGT76G1-L126F, UGT76G1-L200A, UGT76G1-L126F / T284S, UGT76G1-M88L / T284S, UGT76G1-M88L / L126F / T284S, UGT76G1-M88L / I199F / T284S, and UGT76G1-M88L / L126F / I199F / T284S.

[0078] The amino acid and nucleotide sequences of the key mutants mentioned above are as follows:

[0079] UGT76G1-L126F, with the amino acid sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4;

[0080] UGT76G1-L200A, amino acid sequence as shown in SEQ ID NO: 5, nucleotide sequence as shown in SEQ ID NO: 6;

[0081] UGT76G1-L126F / T284S, amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 8;

[0082] UGT76G1-M88L / T284S, amino acid sequence as shown in SEQ ID NO: 9, nucleotide sequence as shown in SEQ ID NO: 10;

[0083] UGT76G1-M88L / L126F / T284S, amino acid sequence as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 12;

[0084] UGT76G1-M88L / I199F / T284S, amino acid sequence as shown in SEQ ID NO: 13, nucleotide sequence as shown in SEQ ID NO: 14;

[0085] UGT76G1-M88L / L126F / I199F / T284S, the amino acid sequence is shown in SEQ ID NO: 15, and the nucleotide sequence is shown in SEQ ID NO: 16.

[0086] Using pETDuet-1 as a vector, the aforementioned mutant gene was inserted into the BamHI / HindIII site of the vector to obtain pETDuet-UGT76G1. Then, the NdeI / XhoI site was introduced into the sucrose synthase AtSUS gene using PCR, and the resulting plasmid was inserted to form the recombinant expression plasmid pETDuet-UGT76G1-AtSUS. This recombinant plasmid was then introduced into *E. coli* BL21(DE3) for transformation to obtain the recombinant strain. The amino acid sequence of sucrose synthase AtSUS is shown in SEQ ID NO:17; the nucleotide sequence of sucrose synthase AtSUS is shown in SEQ ID NO:18.

[0087] Table 3. Primers used for constructing recombinant strains

[0088]

[0089] Example 6 Biosynthesis of Rebaudioside M

[0090] The recombinant strains prepared in Example 4 were inoculated into TB medium and cultured at 37°C for 2 h. Protein expression was induced for 12 h using 0.1 mM isopropyl-β-d-thiogalactoside (IPTG), and the bacterial cells were collected. The bacterial cells were added to a catalytic reaction system consisting of 55 mM Tris-HCl, pH 8.0, 0.8 mM UDP, 500 mM sucrose, 20 g / L RebD, and 50 g / L bacterial cells. The reaction was carried out at 30°C for 24 h to obtain the transformation solution. After the reaction, the transformation solution was diluted 20 times with 20% acetonitrile aqueous solution, heated at 90°C for 2 min, centrifuged at 6000 g for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The product formation was analyzed by HPLC.

[0091] The HPLC instrument was an Agilent 1260, and the column was a C18 column (Silgreen HPLC column 250×4.6mm). The HPLC conditions were: column temperature 40°C, flow rate 1 mL / min, mobile phase B: water, mobile phase C: acetonitrile, using a linear gradient elution method: 0 min 95% B + 5% C, 10 min 55% B + 45% C, 15 min 0% B + 100% C, 16 min 95% B + 5% C, 20 min 95% B + 5% C.

[0092]

[0093] Example 7: Intermittent feeding preparation of rebaudioside M

[0094] The recombinant strain containing the UGT76G1-M88L / L126F / T284S mutant, which showed the highest detection results in Example 6, was inoculated into TB medium and cultured at 37°C for 2 h. Protein expression was induced for 12 h using 0.1 mM isopropyl-β-d-thiogalactoside (IPTG), and the bacterial cells were collected. The bacterial cells were added to a catalytic reaction system consisting of 55 mM Tris-HCl, pH 8.0, 0.8 mM UDP, 500 mM sucrose, 30 g / L Reb D, and 50 g / L bacterial cells. The reaction was carried out at 30°C for 24 h to obtain the transformation solution. After the reaction, the transformation solution was diluted 20 times with 20% acetonitrile aqueous solution, heated at 90°C for 2 min, centrifuged at 6000 g for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The product formation was analyzed by HPLC using the same method as in Example 6.

[0095] The 30 g / L Reb D was added at 12 g / L, 8 g / L, 4 g / L, 3 g / L, 2 g / L, and 1 g / L respectively at 0 h, 1 h, 3 h, 5 h, 7.0 h, and 8 h.

[0096] The yield of Reb M was 32.161 g / L, with a yield of 93.75%.

[0097] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims. sequence list <110> Sichuan Yingjia Hesheng Technology Co., Ltd. <120> Glycosyltransferase mutants and their application in the biosynthesis of rebaudioside M <130> 20210303 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 458 <212> PRT <213> Artificial Sequence <400> 1 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 2 <211> 1377 <212> DNA <213> Artificial Sequence <400> 2 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tatgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgctgtg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagatcctg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta cctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 3 <211> 460 <212> PRT <213> Artificial Sequence <400> 3 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Phe Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu Thr Ser 450 455 460 <210> 4 <211> 1377 <212> DNA <213> Artificial Sequence <400> 4 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tatgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgttctg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagatcctg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta cctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 5 <211> 458 <212> PRT <213> Artificial Sequence <400> 5 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Ala Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 6 <211> 1377 <212> DNA <213> Artificial Sequence <400> 6 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tatgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgctgtg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagatcgcg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta cctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 7 <211> 458 <212> PRT <213> Artificial Sequence <400> 7 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Phe Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Ser Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 8 <211> 1377 <212> DNA Artificial Sequence <400> 8 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tatgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgttctg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagatcctg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta gctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 9 <211> 458 <212> PRT <213> Artificial Sequence <400> 9 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Leu Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Ser Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 10 <211> 1377 <212> DNA <213> Artificial Sequence <400> 10 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tttgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgctgtg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagatcctg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta gctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 11 <211> 458 <212> PRT <213> Artificial Sequence <400> 11 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Leu Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Phe Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Ser Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 12 <211> 1377 <212> DNA <213> Artificial Sequence <400> 12 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tttgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgttctg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagatcctg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta gctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 13 <211> 458 <212> PRT <213> Artificial Sequence <400> 13 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Leu Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Phe Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Ser Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 14 <211> 1377 <212> DNA <213> Artificial Sequence <400> 14 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tttgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgctgtg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagttcctg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta gctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 15 <211> 458 <212> PRT <213> Artificial Sequence <400> 15 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Leu Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Phe Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Phe Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Ser Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 16 <211> 1377 <212> DNA <213> Artificial Sequence <400> 16 atggaaaaca aaaccgaaac caccgttcgt cgtcgtcgtc gtatcatcct gttcccggtt 60 ccgttccagg gtcacatcaa cccgatcctg cagctggcga acgttctgta ctctaaaggt 120 ttctctatca ccatcttcca caccaacttc aacaaaccga aaacctctaa ctacccgcac 180 ttcaccttcc gtttcatcct ggacaacgac ccgcaggacg aacgtatctc taacctgccg 240 acccacggtc cgctggcggg tttgcgtatc ccgatcatca acgaacacgg tgcggacgaa 300 ctgcgtcgtg aactggaact gctgatgctg gcgtctgaag aagacgaaga agtttcttgc 360 ctgatcaccg acgcgttctg gtacttcgcg cagtctgttg cggactctct gaacctgcgt 420 cgtctggttc tgatgacctc ttctctgttc aacttccacg cgcacgtttc tctgccgcag 480 ttcgacgaac tgggttacct ggacccggac gacaaaaccc gtctggaaga acaggcgtct 540 ggtttcccga tgctgaaagt taaagacatc aaatctgcgt actctaactg gcagttcctg 600 aaagaaatcc tgggtaaaat gatcaaacag accaaagcgt cttctggtgt tatctggaac 660 tctttcaaag aactggaaga atctgaactg gaaaccgtta tccgtgaaat cccggcgccg 720 tctttcctga tcccgctgcc gaaacacctg accgcgtctt cttcttctct gctggaccac 780 gaccgtaccg ttttccagtg gctggaccag cagccgccgt cttctgttct gtacgtttct 840 ttcggttcta gctctgaagt tgacgaaaaa gacttcctgg aaatcgcgcg tggtctggtt 900 gactctaaac agtctttcct gtgggttgtt cgtccgggtt tcgttaaagg ttctacctgg 960 gttgaaccgc tgccggacgg tttcctgggt gaacgtggtc gtatcgttaa atgggttccg 1020 cagcaggaag ttctggcgca cggtgcgatc ggtgcgttct ggacccactc tggttggaac 1080 tctaccctgg aatctgtttg cgaaggtgtt ccgatgatct tctctgactt cggtctggac 1140 cagccgctga acgcgcgtta catgtctgac gttctgaaag ttggtgttta cctggaaaac 1200 ggttgggaac gtggtgaaat cgcgaacgcg atccgtcgtg ttatggttga cgaagaaggt 1260 gaatacatcc gtcagaacgc gcgtgttctg aaacagaaag cggacgtttc tctgatgaaa 1320 ggtggttctt cttacgaatc tctggaatct ctggtttctt acatctcttc tctgtaa 1377 <210> 17 <211> 809 <212> PRT <213> Artificial Sequence <400> 17 Met Ala Asn Pro Lys Leu Thr Arg Val Leu Ser Thr Arg Asp Arg Val 1 5 10 15 Gln Asp Thr Leu Ser Ala His Arg Asn Glu Leu Val Ala Leu Leu Ser 20 25 30 Arg Tyr Val Asp Gln Gly Lys Gly Ile Leu Gln Pro His Asn Leu Ile 35 40 45 Asp Glu Leu Glu Ser Val Ile Gly Asp Asp Glu Thr Lys Lys Ser Leu 50 55 60 Ser Asp Gly Pro Phe Gly Glu Ile Leu Lys Ser Ala Met Glu Ala Ile 65 70 75 80 Val Val Pro Pro Phe Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val 85 90 95 Trp Glu Tyr Val Arg Val Asn Val Phe Glu Leu Ser Val Glu Gln Leu 100 105 110 Thr Val Ser Glu Tyr Leu Arg Phe Lys Glu Glu Leu Val Asp Gly Pro 115 120 125 Asn Ser Asp Pro Phe Cys Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala 130 135 140 Asn Val Pro Arg Pro Ser Arg Ser Ser Ser Ile Gly Asn Gly Val Gln 145 150 155 160 Phe Leu Asn Arg His Leu Ser Ser Val Met Phe Arg Asn Lys Asp Cys 165 170 175 Leu Glu Pro Leu Leu Asp Phe Leu Arg Val His Lys Tyr Lys Gly His 180 185 190 Pro Leu Met Leu Asn Asp Arg Ile Gln Ser Ile Ser Arg Leu Gln Ile 195 200 205 Gln Leu Ser Lys Ala Glu Asp His Ile Ser Lys Leu Ser Gln Glu Thr 210 215 220 Pro Phe Ser Glu Phe Glu Tyr Ala Leu Gln Gly Met Gly Phe Glu Lys 225 230 235 240 Gly Trp Gly Asp Thr Ala Gly Arg Val Leu Glu Met Met His Leu Leu 245 250 255 Ser Asp Ile Leu Gln Ala Pro Asp Pro Ser Ser Leu Glu Lys Phe Leu 260 265 270 Gly Met Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly 275 280 285 Tyr Phe Gly Gln Ala Asn Val Leu Gly Leu Pro Asp Thr Gly Gly Gln 290 295 300 Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Thr Glu Met Leu 305 310 315 320 Leu Arg Ile Lys Arg Gln Gly Leu Asp Ile Ser Pro Ser Ile Leu Ile 325 330 335 Val Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Thr Cys Asn Gln Arg 340 345 350 Leu Glu Arg Val Ser Gly Thr Glu His Thr His Ile Leu Arg Val Pro 355 360 365 Phe Arg Ser Glu Lys Gly Ile Leu Arg Lys Trp Ile Ser Arg Phe Asp 370 375 380 Val Trp Pro Tyr Leu Glu Asn Tyr Ala Gln Asp Ala Ala Ser Glu Ile 385 390 395 400 Val Gly Glu Leu Gln Gly Val Pro Asp Phe Ile Ile Gly Asn Tyr Ser 405 410 415 Asp Gly Asn Leu Val Ala Ser Leu Met Ala His Arg Met Gly Val Thr 420 425 430 Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser 435 440 445 Asp Ile Tyr Trp Lys Asp Phe Asp Asn Lys Tyr His Phe Ser Cys Gln 450 455 460 Phe Thr Ala Asp Leu Ile Ala Met Asn Asn Ala Asp Phe Ile Ile Thr 465 470 475 480 Ser Thr Tyr Gln Glu Ile Ala Gly Thr Lys Asn Thr Val Gly Gln Tyr 485 490 495 Glu Ser His Gly Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His 500 505 510 Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala 515 520 525 Asp Met Thr Ile Tyr Phe Pro Tyr Ser Glu Glu Thr Arg Arg Leu Thr 530 535 540 Ala Leu His Gly Ser Ile Glu Glu Met Leu Tyr Ser Pro Asp Gln Thr 545 550 555 560 Asp Glu His Val Gly Thr Leu Ser Asp Arg Ser Lys Pro Ile Leu Phe 565 570 575 Ser Met Ala Arg Leu Asp Lys Val Lys Asn Ile Ser Gly Leu Val Glu 580 585 590 Met Tyr Ser Lys Asn Thr Lys Leu Arg Glu Leu Val Asn Leu Val Val 595 600 605 Ile Ala Gly Asn Ile Asp Val Asn Lys Ser Lys Asp Arg Glu Glu Ile 610 615 620 Val Glu Ile Glu Lys Met His Asn Leu Met Lys Asn Tyr Lys Leu Asp 625 630 635 640 Gly Gln Phe Arg Trp Ile Thr Ala Gln Thr Asn Arg Ala Arg Asn Gly 645 650 655 Glu Leu Tyr Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Ala Gln Pro 660 665 670 Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys 675 680 685 Gly Leu Pro Thr Phe Ala Thr Cys His Gly Gly Pro Ala Glu Ile Ile 690 695 700 Glu His Gly Leu Ser Gly Phe His Ile Asp Pro Tyr His Pro Glu Gln 705 710 715 720 Ala Gly Asn Ile Met Ala Asp Phe Phe Glu Arg Cys Lys Glu Asp Pro 725 730 735 Asn His Trp Lys Lys Val Ser Asp Ala Gly Leu Gln Arg Ile Tyr Glu 740 745 750 Arg Tyr Thr Trp Lys Ile Tyr Ser Glu Arg Leu Met Thr Leu Ala Gly 755 760 765 Val Tyr Gly Phe Trp Lys Tyr Val Ser Lys Leu Glu Arg Arg Glu Thr 770 775 780 Arg Arg Tyr Leu Glu Met Phe Tyr Ile Leu Lys Phe Arg Asp Leu Val 785 790 795 800 Lys Thr Val Pro Ser Thr Ala Asp Asp 805 <210> 18 <211> 2430 <212> DNA <213> Artificial Sequence <400> 18 atggcaaacc ctaagctcac tagggttcta agcacaaggg atcgcgtgca agacacgctt 60 tccgctcacc gcaacgaact cgttgctctt ctctccaggt atgtggatca ggggaaaggg 120 attcttcaac cacataactt aattgacgaa ctcgaatctg ttatcggaga cgatgaaaca 180 aaaagagtc tctctgatgg tccttttgga gagatcctta aatcagcaat ggaagctata 240 gttgtaccac cttttgttgc gttagccgtt agaccaagac ctggtgtttg ggaatatgtt 300 360 aaaagaac tcgttgatgg acctaatagt gacccttttt gtcttgagct tgatttcgag 420 ccctttaacg caaacgtgcc acgtccttct cgttcgtctt cgattggtaa tggagttcag 480 tttctgaatc gtcacttgtc ttctgttatg ttccgtaaca aagattgctt ggagcctctg 540 cttgatttcc ttagagttca tagtacaaa ggtcatccgt tgatgttgaa tgatcggatt 600 660 tcacaagaaa ctccgttctc ggaattcgaa tacgcgttgc aaaggaatggg ttttgagaaa 720 ggatggggag ataccgcagg gagagttctt gaaatgatgc atcttctctc tgatattctt 780 caagctcctg atccttcgtc cttggagaag tttcttggga tggtaccaat ggttttcaac 840 gttgtgatct tatctccaca tggatatttc gggcaagcca atgttttagg cttacctgac 900 actggtggac aagttgtcta tattcttgac caagtccgtg cccttgagac tgaaatgctg ttgagaata agagacaggg gttggatata tcacctagta ttcttattgt aactaggttg ataccggatg ctaaaggaac tacgtgtaac cagcggttag agagagtcag cggaacagag catactcata ttctccgggt tccttttagg tctgagaaag gaatcctccg tagtggatt tcaagattcg acgtatggcc ttatctagag aactatgctc aggatgcagc aagcgagatt gtcggtgaat tgcaaggcgt accggacttt atcatcggta actatagtga cggaaacctt gttgcatcgt taatggcaca tagaatgggt gttacacaat gtactattgc acatgctttg gagaaacca agtatccaga ttcagacatt tactggaaag acttcgacaa caagtatcat ttctcttgtc aattcacagc tgatcttatc gcaatgaaca acgcagattt catcatcaca agcacttacc aagaaatcgc aggaacgaag aacaccgtcg gtcaatatga aagccacggg gcttttacgc tcccgggact atatagagta gtacacggca tcgatgtgtt tgatccgaag 1560. ttcaacatag tctcgcccgg tgcagacatg accatatt tcccgtattc cgaagaaact aggagactta cagctttaca tggttcaata gaggaaatgc tctatagccc tgaccagact 1680 gatgagcatg tcggtacact gagtgatcga tcaagccaa tactctctc tatggcgagg 1740 ctcgacaag tgagaacat ctccggttta gttgagatgt atagtagaa cacaagttg 1800 agggagctgg ttaatctggt tgtaatagct ggtaacattg atgtgaacha gtccaagat 1860 aggagaaaa tcgtagagat tgagaaaatg catacctta tgaagatta caagcttgat 1920 ggacagtttc gttggataac tgctcagact aaccgagctc gaatggtga gctttaccgc 1980 tacatcgcgg atacaagagg tgctttgct cagcctgcgt tcatcgaggc ttttggactt 2040 acggtagtgg aagcgatgac ttgcggctc ccgactttg ccacttgtca cggtggtcca 2100 gcagagatca tcgagcacgg gctctcggggt ttccacatcg atccatacca tcctgagcaa 2160 gcgggtaaca taatggctga ttctttgaa cgttgtagg aagatccaaa ccattggaag 2220 aaagtatcag acgctggtct ccaaggata tacgaaggt accatgga gatatactcg 2280 gagagattga tgacactagc tggtgtgtat ggttttctgga atacgtatc gaaattggag 2340 cgtcgtgaga ctcggcgata tcttgaaatg ttctacattc tcaaattccg cgacttggtg 2400 aaaactgttc cttcaaccgc cgatgactga 2430

Claims

1. A glycosyltransferase mutant, characterized in that, The mutant is a glycosyltransferase mutant obtained by M88L, I199F and T284S mutations based on the amino acid sequence of glycosyltransferase UGT76G1 shown in SEQ ID NO:

1.

2. A glycosyltransferase mutant, characterized in that, The mutant is a glycosyltransferase mutant obtained by M88L, L126F and T284S mutations based on the amino acid sequence of glycosyltransferase UGT76G1 shown in SEQ ID NO:

1.

3. An expressed gene, characterized in that, The gene encodes a glycosyltransferase mutant according to any one of claims 1-2.

4. A recombinant vector, characterized in that, The recombinant vector is linked to the expression gene of claim 3.

5. A recombinant vector as described in claim 4, wherein the recombinant vector is coupled with the expression gene and sucrose synthase gene as described in claim 3.

6. A method for biosynthesizing rebaudioside M, characterized in that, Using rebaudioside D as a substrate and a recombinant strain as a transformation strain, rebaudioside M is biosynthesized; the recombinant strain is a recombinant strain obtained by transforming the recombinant vector of claim 5 in competent cells.

7. The method for biosynthesizing rebaudioside M as described in claim 6, characterized in that, Includes the following steps: 1) The recombinant vector of claim 5 is transformed into competent cells to obtain a recombinant strain; 2) The recombinant strain obtained in step 1) was induced to express in liquid culture medium, and the bacterial cells were collected; 3) The bacterial cells obtained in step 2) are added to a catalytic reaction system containing rebaudioside D for transformation to obtain a transformation solution. The transformation solution is then separated and purified to obtain the product rebaudioside M.

8. The method for biosynthesizing rebaudioside M as described in claim 7, characterized in that, The reaction system comprises: 50-60 mM Tris-HCl, pH 7.5-8.5, 0.1-1.0 mM UDP, 200-900 mM sucrose, 5-60 g / L Reb D, 20-60 g / L bacterial cells, and reacted at 25-45℃ for 1-24 h.

9. The method for biosynthesizing rebaudioside M as described in claim 7, characterized in that, In step (3), Rebaddi glycoside D was added intermittently at 0 h, 0.5 h, 1.5 h and 2.5 h, respectively, at 5 g / L.

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