A glycosyltransferase mutant and use in synthesis of rebaudioside

Abstract

This invention discloses a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Based on the amino acid sequence of the glycosyltransferase shown in SEQ ID NO:1, this invention performs single-point and multi-point mutations to obtain mutants with enhanced catalytic activity. The catalytic activity, substrate specificity, and / or substrate specificity of the glycosyltransferase mutant are altered; mutations at specific sites can significantly improve the enzyme's catalytic activity for specific substrates. The mutant enzyme is obtained by inducing expression and purifying the protein from the obtained mutant sequence. Using the mutant enzyme as a catalyst and UDPG as a glycosyl donor, the catalytic reaction efficiency for the substrates steviol glycoside ST, rebaudioside A (RebA), and rebaudioside D (RebD) can be significantly improved. The glycosyltransferase UGT76G1 mutant constructed in this study improves its catalytic activity, and the efficient production of rebaudioside M is achieved through optimization of the reaction system.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Background Technology

[0002] Sweeteners, as one of the most commonly used ingredients in the food, beverage, and confectionery industries, can be added during the production process, or used alone as a tabletop sweetener with appropriate dilution. Sweeteners include natural sweeteners (such as steviol glycosides, mogrosides, sweet protein, sucrose, corn syrup, molasses, maple syrup, and honey) and artificial sweeteners (such as aspartame, saccharin, and sucralose).

[0003] Stevia glycosides are diterpenoid glycosides extracted and purified from stevia leaves. The main components are steviol glycoside, rebaudioside A (Reb A), rebaudioside B (Reb B), rebaudioside D (Reb D), rebaudioside E (Reb E), and rebaudioside M (Reb M). The differences between various steviol glycosides lie in the number and linkage of sugar groups at positions 13 and 19 of the steviol molecule. Rebaudioside M has a higher sweetness than other steviol glycosides, approximately 250-300 times that of sucrose, while its calorific value is only 1 / 300th that of sucrose. It also lacks a noticeable bitter aftertaste and has a taste closer to sucrose, making it the ideal sucrose substitute. Stevia glycosides have been approved as safe food additives by the food safety authorities of the United States, Brazil, South Korea, Japan, and the European Union. Besides its use as a sweetener in the food industry, rebaudioside M is also used in the pharmaceutical industry, exhibiting effects in lowering blood pressure and blood sugar, and showing good therapeutic effects for patients with hyperlipidemia.

[0004] Rebaudioside M is present in very low amounts in the original plant, and its extraction and purification from plants is extremely costly, with current production far from meeting market demand. According to reports, rebaudioside M is produced from rebaudioside D via transglycosylation catalyzed by the glycosyltransferase UGT76G1. However, the low catalytic activity and poor substrate specificity of the plant-derived glycosyltransferase UGT76G1 severely restrict the industrial production of rebaudioside M. Researchers such as Kim Olsson modified the glycosyltransferase UGT76G1 to obtain the optimal mutants UGT76G1-T146G and UGT76G1-H155L (Olsson et al.). et al. Microbial production of next-generation stevia sweeteners, Microb Cell Fact (2016) 15:207 However, its activity is still insufficient for the production of rebaudioside M. Therefore, there is an urgent need in the field to explore methods to improve the substrate specificity and catalytic activity of UGT76G1. Summary of the Invention

[0005] Based on the needs of existing technologies, the purpose of this invention is to modify glycosyltransferase UGT76G1 to obtain a mutant with higher catalytic activity, improve its efficiency in catalyzing the synthesis of rebaudioside, solve the problem of insufficient activity in the current enzymatic synthesis of rebaudioside M, and thus provide a method for catalyzing the synthesis of rebaudioside M using a glycosyltransferase mutant.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0007] This invention first analyzes the protein sequence of glycosyltransferase UGT76G1 using homologous sequence alignment, identifying five key amino acid sites: F46L, N138G, S280A, G405E, and V429E, and performing single-point and multi-point mutations. Therefore, this invention provides a glycosyltransferase mutant obtained by single-point or multi-point mutation of amino acids 20, 46, 138, 155, 199, 200, 280, 405, and 429 of the amino acid sequence of glycosyltransferase UGT76G1 shown in SEQ ID NO.1.

[0008] Preferably, the glycosyltransferase UGT76G1 mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following:

[0009] (1) The proline at position 46 is mutated to leucine (F46L);

[0010] (2) The asparagine at position 138 is mutated to glycine (N138G);

[0011] (3) The serine at position 280 is mutated to alanine (UGT76G1-M1:S280A);

[0012] (4) Glycine at position 405 is mutated to glutamic acid (G405E);

[0013] (5) Valine at position 429 is mutated to glutamic acid (V429E);

[0014] (6) Serine at position 280 is mutated to alanine and proline at position 46 is mutated to leucine (S280A-F46L);

[0015] (7) Serine at position 280 was mutated to alanine and asparagine at position 138 was mutated to glycine (S280A-N138G);

[0016] (8) Serine at position 280 was mutated to alanine and glycine at position 405 was mutated to glutamic acid (UGT76G1-M2: S280A-G405E);

[0017] (9) Serine at position 280 is mutated to alanine and valine at position 429 is mutated to glutamic acid (S280A-V429E);

[0018] (10) Serine at position 280 is mutated to alanine, glycine at position 405 is mutated to glutamic acid, and proline at position 46 is mutated to leucine (S280A-G405E-F46L);

[0019] (11) Serine at position 280 was mutated to alanine, glycine at position 405 was mutated to glutamic acid, and asparagine at position 138 was mutated to glycine (UGT76G1-M3: S280A-G405E-N138G);

[0020] (12) Serine at position 280 is mutated to alanine, glycine at position 405 is mutated to glutamic acid, and valine at position 429 is mutated to glutamic acid (S280A-G405E-V429E);

[0021] (13) Serine at position 280 was mutated to alanine, glycine at position 405 was mutated to glutamic acid, asparagine at position 138 was mutated to glycine, and proline at position 46 was mutated to leucine (S280A-G405E-N138G-F46L).

[0022] (14) Serine at position 280 is mutated to alanine, glycine at position 405 is mutated to glutamic acid, asparagine at position 138 is mutated to glycine, and valine at position 429 is mutated to glutamic acid (S280A-G405E-N138G-V429E).

[0023] (15) Serine at position 280 is mutated to alanine, glycine at position 405 is mutated to glutamic acid, asparagine at position 138 is mutated to glycine, proline at position 46 is mutated to leucine, and valine at position 429 is mutated to glutamic acid (S280A-G405E-N138G-F46L-V429E).

[0024] The present invention further obtains the three-dimensional protein structure of glycosyltransferase UGT76G1-M3 (S280A-G405E-N138G) through homology modeling, and selects amino acid residues L200, H155, I199, and V20 located near the active site for single and multiple mutations. Preferably, the UGT76G1 mutant of the glycosyltransferase is obtained by mutating the amino acid sequence of UGT76G1-M3 (S280A-G405E-N138G) to one of the following:

[0025] (1) The valine at position 20 is mutated to alanine (V20A).

[0026] (2) The phenylalanine at position 22 is mutated to alanine (F22A).

[0027] (3) The phenylproline at position 84 is mutated to alanine (P84A).

[0028] (4) The leucine at position 85 is mutated to alanine (L85A).

[0029] (5) The glycine at position 87 is mutated to alanine (G87A).

[0030] (6) The methionine at position 88 is mutated to alanine (M88A).

[0031] (7) The isoleucine at position 90 is mutated to alanine (I90A).

[0032] (8) The proline at position 91 is mutated to alanine (P91A)

[0033] (9) The leucine at position 126 is mutated to alanine (L126A).

[0034] (10) The serine at position 147 is mutated to alanine (S147A).

[0035] (11) Histidine at position 155 is mutated to alanine (H155A)

[0036] (12) The serine at position 195 is mutated to alanine (S195A).

[0037] (13) The aspartic acid at position 196 is mutated to alanine (N196A).

[0038] (14) The isoleucine at position 199 is mutated to alanine (I199A).

[0039] (15) The 200th leucine residue is mutated to alanine (L200A).

[0040] (16) The isoleucine at position 203 is mutated to alanine (I203A).

[0041] (17) The leucine at position 204 is mutated to alanine (L204A).

[0042] (18) Tyrosine at position 284 is mutated to alanine (T284A)

[0043] (19) Leucine at position 379 is mutated to alanine (L379A).

[0044] This invention involves a single-point saturation mutagenesis of amino acid residue L200 located near the active site. Preferably, the glycosyltransferase UGT76G1 mutant is one in which the amino acid sequence of UGT76G1-M3 (S280A-G405E-N138G) is mutated to one of the following:

[0045] (1) The 200th leucose serine residue is mutated to arginine (L200R).

[0046] (2) The leucose serine at position 200 is mutated to asparagine (L200N).

[0047] (3) The 200th leucose serine residue is mutated to aspartic acid (L200D).

[0048] (4) The 200th leucose serine residue is mutated to cysteine ​​(L200C).

[0049] (5) The 200th leucose serine residue is mutated to glutamine (L200Q).

[0050] (6) The 200th leucose serine residue is mutated to glutamic acid (L200E).

[0051] (7) The 200th leucose serine residue is mutated to glycine (L200G).

[0052] (8) The 200th leucose serine residue is mutated to histidine (L200H).

[0053] (9) The 200th leucose serine residue is mutated to isoleucine (L200I).

[0054] (10) The 200th leucose serine residue is mutated to serine (L200S).

[0055] (11) The 200th leucose serine residue is mutated to lysine (L200K).

[0056] (12) The 200th leucose serine residue is mutated to methionine (L200M).

[0057] (13) The 200th leucose serine residue is mutated to phenylalanine (L200F).

[0058] (14) The 200th leucose serine residue is mutated to proline (L200P).

[0059] (15) The 200th leucose serine residue is mutated to threonine (L200T).

[0060] (16) The 200th leucose serine residue is mutated to tryptophan (L200W).

[0061] (17) The 200th leucose serine residue is mutated to tyrosine (L200Y).

[0062] (18) The 200th leucose serine is mutated to valine (L200V).

[0063] This invention involves a single-point saturation mutagenesis of amino acid residue H155 located near the active site. Preferably, the glycosyltransferase UGT76G1 mutant is one in which the amino acid sequence of UGT76G1-M4 (S280A-G405E-N138G-L200S) is mutated to one of the following:

[0064] (1) Histidine at position 155 is mutated to alanine (H155A)

[0065] (2) The histidine at position 155 is mutated to asparagine (H155N).

[0066] (3) Histidine at position 155 is mutated to aspartic acid (H155D)

[0067] (4) Histidine at position 155 is mutated to cysteine ​​(H155C)

[0068] (5) Histidine at position 155 is mutated to glutamine (H155Q)

[0069] (6) Histidine at position 155 is mutated to glutamate (H155E)

[0070] (7) Histidine at position 155 is mutated to glycine (H155G)

[0071] (8) Histidine at position 155 is mutated to arginine (H155R)

[0072] (9) The histidine at position 155 is mutated to isoleucine (H155I).

[0073] (10) Histidine at position 155 is mutated to leucine (H155L)

[0074] (11) The histidine at position 155 is mutated to lysine (H155K).

[0075] (12) Histidine at position 155 is mutated to methionine (H155M)

[0076] (13) The histidine at position 155 is mutated to phenylalanine (H155F).

[0077] (14) Histidine at position 155 is mutated to proline (H155P)

[0078] (15) The histidine at position 155 is mutated to serine (H155S).

[0079] (16) Histidine at position 155 is mutated to threonine (H155T)

[0080] (17) Histidine at position 155 is mutated to tryptophan (H155W)

[0081] (18) The histidine at position 155 is mutated to tyrosine (H155Y).

[0082] (19) Histidine at position 155 is mutated to valine (H155V).

[0083] This invention involves a single-point saturation mutagenesis of amino acid residue I199 located near the active site. Preferably, the glycosyltransferase UGT76G1 mutant is one in which the amino acid sequence UGT76G1-M5(S280A-G405E-N138G-L200S-H155V) is mutated to one of the following:

[0084] (1) The isoleucine at position 199 is mutated to alanine (I199A).

[0085] (2) The isoleucine at position 199 is mutated to arginine (I199R).

[0086] (3) The isoleucine at position 199 is mutated to asparagine (I199N).

[0087] (4) The isoleucine at position 199 is mutated to aspartic acid (I199D).

[0088] (5) The isoleucine at position 199 is mutated to cysteine ​​(I199C).

[0089] (6) The isoleucine at position 199 is mutated to glutamine (I199Q).

[0090] (7) The isoleucine at position 199 is mutated to glutamic acid (I199E).

[0091] (8) The isoleucine at position 199 is mutated to glycine (I199G)

[0092] (9) The isoleucine at position 199 is mutated to histidine (I199H).

[0093] (10) The isoleucine at position 199 is mutated to tyrosine (I199Y).

[0094] (11) The isoleucine at position 199 is mutated to leucine (I199L).

[0095] (12) The isoleucine at position 199 is mutated to lysine (I199K).

[0096] (13) The isoleucine at position 199 is mutated to methionine (I199M).

[0097] (14) The isoleucine at position 199 is mutated to phenylalanine (I199F).

[0098] (15) The isoleucine at position 199 is mutated to proline (I199P).

[0099] (16) The isoleucine at position 199 is mutated to serine (I199S).

[0100] (17) The isoleucine at position 199 is mutated to threonine (I199T).

[0101] (18) The isoleucine at position 199 is mutated to tryptophan (I199W)

[0102] (19) The isoleucine at position 199 is mutated to valine (I199V).

[0103] This invention involves a single-point saturation mutagenesis of amino acid residue V20 located near the active site. Preferably, the glycosyltransferase UGT76G1 mutant is one in which the amino acid sequence of UGT76G1-M6 (S280A-G405E-N138G-L200S-H155V-I199Q) is mutated to one of the following:

[0104] (1) The valine at position 20 is mutated to alanine (V20A).

[0105] (2) The valine at position 20 is mutated to arginine (V20R).

[0106] (3) The valine at position 20 is mutated to asparagine (V20N).

[0107] (4) The valine at position 20 is mutated to aspartic acid (V20D).

[0108] (5) The valine at position 20 is mutated to cysteine ​​(V20C).

[0109] (6) The valine at position 20 is mutated to glutamine (V20Q).

[0110] (7) The valine at position 20 is mutated to glutamic acid (V20E).

[0111] (8) The valine at position 20 is mutated to glycine (V20G).

[0112] (9) The valine at position 20 is mutated to histidine (V20H).

[0113] (10) The valine at position 20 is mutated to isoleucine (V20I).

[0114] (11) The valine at position 20 is mutated to leucine (V20L).

[0115] (12) The valine at position 20 is mutated to lysine (V20K).

[0116] (13) Valine at position 20 is replaced by methionine (V20M)

[0117] (14) The valine at position 20 is mutated to phenylalanine (V20F).

[0118] (15) The valine at position 20 is mutated to proline (V20P).

[0119] (16) The valine at position 20 is mutated to serine (V20S).

[0120] (17) Valine at position 20 is mutated to tryptophan (V20W)

[0121] (18) The valine at position 20 is mutated to tyrosine (V20Y).

[0122] (19) Valine at position 20 is mutated to threonine (V20T).

[0123] This invention also relates to the coding gene of the glycosyltransferase UGT76G1 mutant, a recombinant vector containing the coding gene, and a recombinant genetically engineered bacterium containing the recombinant vector. The base vector of the recombinant vector can be pET32a, and the host bacterium of the recombinant genetically engineered bacterium can be... E .coli BL21(DE3).

[0124] This invention provides a preferred method for constructing the recombinant genetically engineered bacteria: using plasmid pET32a-UGT76G1 as a template, full-plasmid PCR amplification is performed using primers; 0.5 μL of DpnI enzyme is directly added to the PCR product, and the mixture is reacted at 37°C for 1 h to digest the plasmid; 5 μL of the digested PCR product is then directly transformed... E The recombinant genetically engineered bacteria were obtained by using .coli BL21(DE3) competent cells.

[0125] This invention provides a method for synthesizing rebaudioside M using a glycosyltransferase mutant, comprising the following steps:

[0126] 1) The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain containing the recombinant gene;

[0127] 2) The recombinant strain was induced to express by adding an inducer in LB liquid medium, and the bacterial cells were collected by low-temperature centrifugation. The bacterial cells were resuspended by adding an appropriate buffer, and the bacterial solution was ultrasonically disrupted and centrifuged to collect the supernatant, which is the crude enzyme solution.

[0128] 3) Add rebaudioside D as substrate, UDPG and crude enzyme solution to the catalytic reaction system, react at an appropriate temperature, add sulfuric acid to terminate the reaction, and centrifuge to obtain the supernatant as the product rebaudioside M.

[0129] Preferably, the induction and culture method of the recombinant genetically engineered bacteria containing the glycosyltransferase UGT76G1 mutant is as follows:

[0130] 1) Inoculate the recombinant genetically engineered bacteria with glycosyltransferase UGT76G1 mutant into LB liquid medium containing 100 μg / mL ampicillin (Amp) and incubate overnight at 37°C and 220 rpm.

[0131] 2) Transfer the seed culture at a volume concentration of 1-2% (preferably 2%) to LB liquid medium containing 100 μg / mL ampicillin (Amp), and culture at 37°C and 220 rpm for 2.0-2.5 hours until the OD value of the fermentation broth reaches 0.6-0.8. Then add IPTG to a final concentration of 0.5 mM and continue induction culture at 25°C for 20 hours.

[0132] This invention provides a method for synthesizing rebaudioside A using a glycosyltransferase mutant, comprising the following steps:

[0133] 1) The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain containing the recombinant gene;

[0134] 2) The recombinant strain was induced to express by adding an inducer in LB liquid medium, and the bacterial cells were collected by low-temperature centrifugation. The bacterial cells were resuspended by adding an appropriate buffer, and the bacterial solution was ultrasonically disrupted and centrifuged to collect the supernatant, which is the crude enzyme solution.

[0135] 3) Add stevioside ST as substrate, UDPG and crude enzyme solution to the catalytic reaction system, react at an appropriate temperature, add sulfuric acid to terminate the reaction, and centrifuge to obtain the supernatant, which is the product lebodiin A.

[0136] Preferably, the induction and culture method of the recombinant genetically engineered bacteria containing the glycosyltransferase UGT76G1 mutant is as follows:

[0137] 1) Inoculate the recombinant genetically engineered bacteria with glycosyltransferase UGT76G1 mutant into LB liquid medium containing 100 μg / mL ampicillin (Amp) and incubate overnight at 37°C and 220 rpm.

[0138] 2) Transfer the seed culture at a volume concentration of 1-2% (preferably 2%) to LB liquid medium containing 100 μg / mL ampicillin (Amp), and culture at 37°C and 220 rpm for 2.0-2.5 hours until the OD value of the fermentation broth reaches 0.6-0.8. Then add IPTG to a final concentration of 0.5 mM and continue induction culture at 25°C for 20 hours.

[0139] This invention provides a method for synthesizing rebaudioside I using a glycosyltransferase mutant, comprising the following steps:

[0140] 1) The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain containing the recombinant gene;

[0141] 2) The recombinant strain was induced to express by adding an inducer in LB liquid medium, and the bacterial cells were collected by low-temperature centrifugation. The bacterial cells were resuspended by adding an appropriate buffer, and the bacterial solution was ultrasonically disrupted and centrifuged to collect the supernatant, which is the crude enzyme solution.

[0142] 3) Add rebaudioside A as substrate, UDPG and crude enzyme solution to the catalytic reaction system, react at an appropriate temperature, add sulfuric acid to terminate the reaction, and centrifuge to obtain the supernatant as the product rebaudioside I.

[0143] Preferably, the induction and culture method of the recombinant genetically engineered bacteria containing the glycosyltransferase UGT76G1 mutant is as follows:

[0144] 1) Inoculate the recombinant genetically engineered bacteria with glycosyltransferase UGT76G1 mutant into LB liquid medium containing 100 μg / mL ampicillin (Amp) and incubate overnight at 37°C and 220 rpm.

[0145] 2) Transfer the seed culture at a volume concentration of 1-2% (preferably 2%) to LB liquid medium containing 100 μg / mL ampicillin (Amp), and culture at 37°C and 220 rpm for 2.0-2.5 hours until the OD value of the fermentation broth reaches 0.6-0.8. Then add IPTG to a final concentration of 0.5 mM and continue induction culture at 25°C for 20 hours.

[0146] This invention relates to the modification of the glycosyltransferase UGT76G1. The UGT76G1 sequence was analyzed using homologous sequence alignment, and selected amino acid sites were mutated to amino acids with higher conservation in the homologous sequence. The crude enzyme activity of the UGT76G1 mutants was measured, and the selected mutants were F46L, N138G, S280A, G405E, and V429E. Multiple rounds of PCR were used to stack the selected mutations, and the crude enzyme activity of the multi-site mutated UGT76G1 mutants was measured. Finally, the mutant with increased enzyme activity, S280A-G405E-N138G, was selected. Based on this, homology modeling was performed on glycosyltransferase UGT76G1 to obtain its three-dimensional protein structure. By analyzing the amino acids at the substrate binding site of its protein structure, the amino acids around the substrate binding site were mutated, and the final glycosyltransferase UGT76G1 mutant was optimized to S280A-G405E-N138G-L200S-H155V-I199Q-V20L.

[0147] The wild-type glycosyltransferase UGT76G1 showed an enzyme activity of 5720 U / mg for steviol glycoside ST and a Kcat / Km ratio of 500.97 mM. -1 S -1 The glycosyltransferase UGT76G1 mutant UGT76G1 (S280A-G405E-N138G-L200S-H155V-I199Q-V20L) showed an enzyme activity of 6414 U / mg for steviol glycoside ST and a Kcat / Km ratio of 564.84 mM. -1 S -1 .

[0148] The wild-type glycosyltransferase UGT76G1 exhibited an enzyme activity of 11.04 U / mg for rebaudioside A, with a Kcat / Km ratio of 37.53 mM. -1 S -1 The glycosyltransferase mutant UGT76G1 (S280A-G405E-N138G-L200S-H155V-I199Q-V20L) exhibited an enzyme activity of 441.67 U / mg for steviol glycoside ST and a Kcat / Km ratio of 3791.6 mM. -1 S -1 .

[0149] The wild-type glycosyltransferase UGT76G1 showed an activity of 390 U / mg for rebaudioside D and a Kcat / Km ratio of 31.48 mM. -1 S -1 The glycosyltransferase UGT76G1 mutant UGT76G1 (S280A-G405E-N138G-L200S-H155V-I199Q-V20L) showed an enzyme activity of 10486 U / mg for steviol glycoside ST and a Kcat / Km ratio of 740.97 mM. -1 S -1 .

[0150] The glycosyltransferase UGT76G1 mutant obtained in this invention (S280A-G405E-N138G-L200S-H155V-I199Q-V20L) showed a 1.12-fold increase in ST enzyme activity for the substrate steviol glycoside and a 1.13-fold increase in catalytic efficiency.

[0151] The glycosyltransferase UGT76G1 mutant obtained in this invention (S280A-G405E-N138G-L200S-H155V-I199Q-V20L) showed a 40.01-fold increase in enzyme activity and a 101.03-fold increase in catalytic efficiency for the substrate rebaudioside A.

[0152] The glycosyltransferase UGT76G1 mutant (S280A-G405E-N138G-L200S-H155V-I199Q-V20L) obtained in this invention exhibits a 26.89-fold increase in enzyme activity and a 23.54-fold increase in catalytic efficiency for the substrate rebaudioside D. Combined with the kinetic parameters, the mutant is advantageous for catalyzing the synthesis of rebaudioside M and rebaudioside I, which will lay an excellent foundation for the industrialization of glycosyltransferases and their application in the food industry.

[0153] This invention utilizes directed evolution to molecularly modify the glycosyltransferase UGT76G1, improving its enzyme activity and catalytic efficiency. The mutant was then used to achieve highly efficient catalytic synthesis of rebaudioside M. This provides a guiding method for the modification of glycosyltransferases. Attached Figure Description

[0154] Figure 1 Sequence conservation analysis of glycosyltransferase UGT76G1.

[0155] Figure 2 : Sequence alignment of glycosyltransferase UGT76G1 and relative activity results of mutations.

[0156] Figure 3 : Relative activity results of mutation at the active site of glycosyltransferase UGT76G1.

[0157] Figure 4 : Relative activity results of L200 mutation at the UGT76G1 site of glycosyltransferase.

[0158] Figure 5 : Relative activity results of H155 mutation at the UGT76G1 site of glycosyltransferase.

[0159] Figure 6 : Relative activity results of the I199 mutation at the UGT76G1 site of glycosyltransferase.

[0160] Figure 7 : Relative activity results of the V20 mutation at the UGT76G1 site of glycosyltransferase. Detailed Implementation

[0161] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0162] Example 1: Homologous sequence alignment and single-point mutation sites

[0163] 1.1 Homologous sequence alignment

[0164] Ten glycosyltransferase sequences, namely UGT91D2, UGT79A2, UGT76G1, UGT85C1, UGT85A8, UGT89B2, UGT73E1, UGT73B2, UGT88B1, and UGT71E1, which are similar to the UGT76G1 glycosyltransferase sequence (amino acid sequence shown in SEQ ID NO:1), were selected for sequence alignment. The alignment results are as follows: Figure 1 As shown. Further mutations were performed at different sites on the glycosyltransferase UGT76G1 to obtain more conserved amino acids. The selected mutants were F46L, N138G, S280A, G405E, and V429E.

[0165] 1.2 Construction of the glycosyltransferase UGT76G1 mutant

[0166] Using PCR amplification technology, with the wild-type pET32a-UGT76G1 recombinant plasmid as a template, corresponding forward primer F and reverse primer R were designed to perform site-directed amino acid mutagenesis.

[0167] PCR amplification reaction system: 1 μL each of 10 μM primer F and primer R; 0.5 μL template plasmid; 2 PCR mix 25 μL, add sterile water (ddH2O) to make up to 50 μL. PCR amplification reaction conditions: 98℃ pre-denaturation for 30 s; 30 cycles (98℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 1 min); 72℃ complete extension for 7 min. PCR products were detected by nucleic acid electrophoresis on agarose gel.

[0168] Add 0.5 μL DpnI enzyme to the PCR product and incubate at 37℃ for 1 h to digest the plasmid template. Transform 5 μL of the digested PCR product into E. coli BL21(DE3) competent cells, place on ice for 30 min, heat at 42℃ for 90 s, place on ice for 2 min, add 600 μL LB medium (formulation: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L), incubate at 37℃ with shaking at 220 rpm for 60 min, and then spread the entire bacterial culture evenly on LB plates containing ampicillin resistance (Amp 100 μg / mL) and incubate overnight at 37℃. Pick 1–10 single colonies from the plate, inoculate with 200 μL of LB liquid medium for 3 h, perform bacterial PCR verification and sequencing, and inoculate the bacterial culture with correct sequencing results into a test tube containing 3 mL of LB (Amp 100 μg / mL) medium. Incubate overnight at 37°C and 220 rpm for 12–16 h, then preserve the bacteria and proceed with subsequent expression.

[0169] 1.3 Induced expression of glycosyltransferase UGT76G1 mutant

[0170] Take 400 μL of fresh bacterial culture of wild-type and mutant UGT76G1 glycosyltransferase culture that has been cultured overnight and inoculate it into a shake flask containing 20 mL of LB liquid (Amp 100 μg / mL). Place the shake flask in a shaker at 37℃ and 220 rpm for about 2.5 h. When the OD600 of the bacterial culture reaches 0.6-0.8, lower the shaker temperature to 25℃ and add IPTG to a final concentration of 0.5 mM to induce expression for 16-20 h. Take 4 mL of the cultured bacterial culture and centrifuge at 4℃ and 12000 rpm for 10 min to collect the bacterial cells. Resuspend the cells in 1 mL of PBS buffer (50 mM, pH 7.5) and sonicate them. After lysis, centrifuge the cells again using a refrigerated centrifuge at 4℃, 12000 rpm for 10 min. Collect the supernatant as the crude enzyme solution and store it at 4℃ for later use.

[0171] 1.4 Determination of crude enzyme activity of glycosyltransferase UGT76G1 mutant

[0172] The enzyme activity assay reaction system consisted of 4 mM RebD, 4 mM UDPG, 20–100 μL of crude enzyme solution, 20 μL of 100 mM potassium phosphate buffer (pH 7.0), and water to a final volume of 200 μL. The reaction was carried out at 40℃ and 800 rpm in a metal bath for 4–12 h. After the reaction, 200 μL of methanol was added to terminate the reaction. The reactants were incubated at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and then placed in a liquid chromatography sample vial for HPLC analysis.

[0173] The conversion result is as follows Figure 2 As shown in Figure a, the optimal glycosyltransferase single mutant of UGT76G1 is UGT76G1-M1 (S280A), which has a 1.74-fold increase in catalytic activity compared with the wild type. The mutant G405E has a 1.67-fold increase in catalytic activity compared with the wild type, the mutant V429E has a 1.27-fold increase in catalytic activity compared with the wild type, the mutant F46L has a 1.16-fold increase in catalytic activity compared with the wild type, and the mutant N138G has a 1.14-fold increase in catalytic activity compared with the wild type.

[0174] Example 2: Combinatorial Mutation of Glycosyltransferase UGT76G1 Mutant

[0175] The beneficial mutations F46L, N138G, S280A, G405E, and V429E of the glycosyltransferase UGT76G1 obtained in Example 1 were used for combined mutation studies. The specific methods are as follows:

[0176] 2.1 Construction of the UGT76G1 combinatorial mutant of glycosyltransferase

[0177] Using plasmid pET32a-UGT76G1(S280A) as a template, full plasmid PCR amplification was performed using primers to obtain UGT76G1(S280A-F46L), UGT76G1(S280A-N138G), UHT76G1(S280A-G405E), and UGT76G1(S280A-V429E) mutants.

[0178] Using plasmid pET32a-UGT76G1(S280A-G405E) as a template, full plasmid PCR amplification was performed using primers F1 / R1, F2 / R2, and F5 / R5 from Example 1 to obtain UGT76G1(S280A-G405E-F46L), UGT76G1(S280A-G405E-N138G), and UGT76G1(S280A-G405E-V429E) mutants.

[0179] Using plasmid pET32a-UGT76G1(S280A-N138G-G405E) as a template, full plasmid PCR amplification was performed using primers to obtain UGT76G1(S280A-N138G-G405E-F46L), UGT76G1(S280A-N138G-G405E-V429E), and UGT76G1(S280A-N138G-G405E-F46L-V429E) mutants.

[0180] 2.2 Determination of crude enzyme activity of the glycosyltransferase UGT76G1 combinatorial mutant

[0181] The enzyme activity assay reaction system consisted of 4 mM RebD, 4 mM UDPG, 20–100 μL of crude enzyme solution, 20 μL of 100 mM potassium phosphate buffer (pH 7.0), and water to a final volume of 200 μL. The reaction was carried out at 40℃ and 800 rpm in a metal bath for 4–12 h. After the reaction, 200 μL of methanol was added to terminate the reaction. The reactants were incubated at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and then placed in a liquid chromatography sample vial for HPLC analysis.

[0182] The transformation results of the double mutant are as follows Figure 2 As shown in Figure b, the optimal double mutant of the glycosyltransferase UGT76G1 is UGT76G1-M2 (S280A-G405E), which exhibits a 2.09-fold increase in catalytic activity compared to the wild type. The transformation results of the triple mutant are shown below. Figure 2As shown in Figure c, the optimal triple mutant of the glycosyltransferase UGT76G1 is UGT76G1-M3 (S280A-G405E-N138G), which exhibits a 2.20-fold increase in catalytic activity compared to the wild type. The transformation results of the quadruple mutant are shown below. Figure 2 As shown in Figure d, the optimal glycosyltransferase UGT76G1 quadruple mutant is UGT76G1(S280A-G405E-N138G-V429E), which exhibits a 2.13-fold increase in catalytic activity compared to the wild type. The pentamutant UGT76G1(S280A-G405E-N138G-F46L-V429E) shows a 2.11-fold increase in activity compared to the wild type.

[0183] Example 3: Molecular modification based on the protein structure of glycosyltransferase UGT76G1

[0184] Based on the structure of the glycosyltransferase UGT76G1 protein, amino acids near the substrate binding site were selected for mutation. The selected amino acid sites were V20, F22, P84, L85, G87, M88, I90, P91, L126, S147, H155, S195, N196, I199, L200, I203, L204, T284, and L379.

[0185] 3.1 Construction of the glycosyltransferase UGT76G1 mutant

[0186] Based on the gene sequence of the glycosyltransferase UGT76G1 mutant UGT76G1-M3, we designed and synthesized primers with forward primer F and reverse primer R for the following mutants: V20, F22, P84, L85, G87, M88, I90, P91, L126, S147, H155, S195, N196, I199, L200, I203, L204, T284, and L379. Using the recombinant plasmid pET32a-UGT76G1(S280A-N138G-G405E) obtained in Example 2 as a template, we amplified the plasmid using whole plasmid PCR amplification technology, transformed it into E. coli BL21(DE3), and verified it by sequencing.

[0187] PCR amplification reaction system: 1 μL each of 10 μM primer F and primer R; 0.5 μL template plasmid; 2 Add 25 μL of PCR mix and sterile water (ddH2O) to bring the total volume to 50 μL.

[0188] PCR amplification conditions: 98℃ pre-denaturation for 30 s; 30 cycles (98℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 1 min); 72℃ complete extension for 7 min. PCR products were detected by agarose gel electrophoresis.

[0189] Add 0.5 μL DpnI enzyme to the PCR product and incubate at 37℃ for 1 h to digest the plasmid template. Transform 5 μL of the digested PCR product into E. coli BL21(DE3) competent cells, place on ice for 30 min, heat at 42℃ for 90 s, place on ice for 2 min, add 600 μL LB medium (formulation: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L), incubate at 37℃ with shaking at 220 rpm for 60 min, and then spread the entire bacterial culture evenly on LB plates containing ampicillin resistance (Amp 100 μg / mL) and incubate overnight at 37℃. Pick 1–10 single colonies from the plate, inoculate with 200 μL of LB liquid medium for 3 h, perform bacterial PCR verification and sequencing, and inoculate the bacterial culture with correct sequencing results into a test tube containing 3 mL of LB (Amp 100 μg / mL) medium. Incubate overnight at 37°C and 220 rpm for 12–16 h, then preserve the bacteria and proceed with subsequent expression.

[0190] 3.2 Determination of crude enzyme activity of the glycosyltransferase UGT76G1 combinatorial mutant

[0191] The enzyme activity assay reaction system consisted of 4 mM RebD, 4 mM UDPG, 100 μL crude enzyme solution, 20 μL 100 mM potassium phosphate buffer (pH 7.0), and water to a final volume of 200 μL. The reaction was carried out at 40℃ and 800 rpm in a metal bath for 12 h. After the reaction, 200 μL of methanol was added to terminate the reaction. The reactants were incubated at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and placed in a liquid chromatography sample vial for HPLC analysis.

[0192] The conversion result is as follows Figure 3 As shown, the catalytic activity of the glycosyltransferase UGT76G1 mutant L200A was increased by 3.81 times compared with the control group, the catalytic activity of mutant H155A was increased by 1.82 times compared with the control group, the catalytic activity of mutant I199A was increased by 1.49 times compared with the control group, the catalytic activity of mutant G87A was increased by 1.43 times compared with the control group, and the catalytic activity of mutant V20A was increased by 1.36 times compared with the control group.

[0193] Example 4: Construction and screening of an optimized glycosyltransferase UGT76G1 mutant

[0194] 4.1 Construction of the 200th leucine mutant of glycosyltransferase UGT76G1

[0195] Based on the gene sequence of the glycosyltransferase UGT76G1 mutant UGT76G1-M3, forward primer F and reverse primer R to introduce the L200 mutation were designed and synthesized. Using the recombinant plasmid pET32a-UGT76G1(S280A-N138G-G405E) obtained in Example 2 as a template, the plasmid was amplified using whole plasmid PCR amplification technology, transformed into E. coli BL21(DE3), and sequenced for verification, obtaining the L200 site saturated mutant.

[0196] PCR amplification reaction system: 1 μL each of 10 μM primer F and primer R; 0.5 μL template plasmid; 2 Add 25 μL of PCR mix and sterile water (ddH2O) to bring the total volume to 50 μL.

[0197] PCR amplification conditions: 98℃ pre-denaturation for 30 s; 30 cycles (98℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 1 min); 72℃ complete extension for 7 min. PCR products were detected by agarose gel electrophoresis.

[0198] Add 0.5 μL DpnI enzyme to the PCR product and incubate at 37℃ for 1 h to digest the plasmid template. Transform 5 μL of the digested PCR product into E. coli BL21(DE3) competent cells, place on ice for 30 min, heat at 42℃ for 90 s, place on ice for 2 min, add 600 μL LB medium (formulation: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L), incubate at 37℃ with shaking at 220 rpm for 60 min, and then spread the entire bacterial culture evenly on LB plates containing ampicillin resistance (Amp 100 μg / mL) and incubate overnight at 37℃. Pick 1–10 single colonies from the plate, inoculate with 200 μL of LB liquid medium for 3 h, perform bacterial PCR verification and sequencing, and inoculate the bacterial culture with correct sequencing results into a test tube containing 3 mL of LB (Amp 100 μg / mL) medium. Incubate overnight at 37°C and 220 rpm for 12–16 h, then preserve the bacteria and proceed with subsequent expression.

[0199] 4.2 Induction and expression of glycosyltransferase UGT76G1 mutant and enzyme activity assay

[0200] The enzyme activity assay reaction system consisted of: 4 mM RebD, 4 mM UDPG, 20–100 μL crude enzyme solution, 20 μL 100 mM potassium phosphate buffer (pH 7.0), and water to a final volume of 200 μL. The reaction was carried out at 40℃ and 800 rpm in a metal bath for 4–12 h. After the reaction, 200 μL of methanol was added to terminate the reaction. The reactants were incubated at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and then transferred to a liquid chromatography sample vial for HPLC analysis. The transformation results are as follows: Figure 4 As shown, the optimal glycosyltransferase, the UGT76G1 mutant L200S, exhibited a 5.66-fold increase in catalytic activity compared to the control group; mutant L200A showed a 3.81-fold increase; mutant L200C a 3.42-fold increase; mutant L200E a 3.36-fold increase; mutant L200M a 2.73-fold increase; mutant L200G a 2.59-fold increase; and mutant L200K a 2.26-fold increase. Compared with the control group, the catalytic activity of mutant L200Q increased by 2.13 times, mutant L200T by 1.85 times, mutant L200V by 1.42 times, mutant L200H by 1.36 times, mutant L200N by 1.23 times, mutant L200D by 1.21 times, mutant L200R by 1.23 times, and mutant L200I by 1.1 times.

[0201] Example 5: Construction and screening of another optimized glycosyltransferase UGT76G1 mutant

[0202] 5.1 Construction of Glycosyltransferase Mutants

[0203] Based on the gene sequence of the glycosyltransferase UGT76G1 mutant UGT76G1-M4 (S280A-G405E-N138G-L200S), forward primer F and reverse primer R, which introduce the R95 mutation, were designed and synthesized. Using the recombinant plasmid pET32a-UGT76G1 (S280A-N138G-G405E-L200S) obtained in Example 4 as a template, the plasmid was amplified using whole plasmid PCR amplification technology, transformed into E. coli BL21(DE3), and sequenced for verification, obtaining the H155 site saturated mutant.

[0204] PCR amplification reaction system: 1 μL each of 10 μM primer F and primer R; 0.5 μL template plasmid; 2 Add 25 μL of PCR mix and sterile water (ddH2O) to bring the total volume to 50 μL.

[0205] PCR amplification conditions: 98℃ pre-denaturation for 30 s; 30 cycles (98℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 1 min); 72℃ complete extension for 7 min. PCR products were detected by agarose gel electrophoresis.

[0206] Add 0.5 μL DpnI enzyme to the PCR product and incubate at 37℃ for 1 h to digest the plasmid template. Transform 5 μL of the digested PCR product into E. coli BL21(DE3) competent cells, place on ice for 30 min, heat at 42℃ for 90 s, place on ice for 2 min, add 600 μL LB medium (formulation: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L), incubate at 37℃ with shaking at 220 rpm for 60 min, and then spread the entire bacterial culture evenly on LB plates containing ampicillin resistance (Amp 100 μg / mL) and incubate overnight at 37℃. Pick 1–10 single colonies from the plate, inoculate with 200 μL of LB liquid medium for 3 h, perform bacterial PCR verification and sequencing, and inoculate the bacterial culture with correct sequencing results into a test tube containing 3 mL of LB (Amp 100 μg / mL) medium. Incubate overnight at 37°C and 220 rpm for 12–16 h, then preserve the bacteria and proceed with subsequent expression.

[0207] 5.2 Induction and expression of glycosyltransferase UGT76G1 mutant and enzyme activity assay

[0208] The enzyme activity assay reaction system consisted of 4 mM RebD, 4 mM UDPG, 20–100 μL of crude enzyme solution, 20 μL of 100 mM potassium phosphate buffer (pH 7.0), and water to a final volume of 200 μL. The reaction was carried out at 40℃ and 800 rpm in a metal bath for 4–12 h. After the reaction, 200 μL of methanol was added to terminate the reaction. The reactants were incubated at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and then placed in a liquid chromatography sample vial for HPLC analysis.

[0209] The conversion result is as follows Figure 5 As shown, the catalytic activity of the glycosyltransferase UGT76G1 mutant H155V was increased by 1.36 times compared with the control group, and the catalytic activity of the mutant H155A was increased by 1.01 times compared with the control group.

[0210] Example 6: Construction and screening of another optimized glycosyltransferase UGT76G1 mutant

[0211] 6.1 Construction of Glycosyltransferase Mutants

[0212] Based on the gene sequence of the glycosyltransferase UGT76G1 mutant UGT76G1-M5 (S280A-G405E-N138G-L200S-H155V), forward primer F and reverse primer R to introduce the I199 mutation were designed and synthesized. Using the recombinant plasmid pET32a-UGT76G1 (S280A-N138G-G405E-L200S-H155V) obtained in Example 5 as a template, the plasmid was amplified using whole plasmid PCR amplification technology, transformed into E. coli BL21(DE3), and sequenced for verification, obtaining the I199 site saturated mutant.

[0213] PCR amplification reaction system: 1 μL each of 10 μM primer F and primer R; 0.5 μL template plasmid; 2 Add 25 μL of PCR mix and sterile water (ddH2O) to bring the total volume to 50 μL.

[0214] PCR amplification conditions: 98℃ pre-denaturation for 30 s; 30 cycles (98℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 1 min); 72℃ complete extension for 7 min. PCR products were detected by agarose gel electrophoresis.

[0215] Add 0.5 μL DpnI enzyme to the PCR product and incubate at 37℃ for 1 h to digest the plasmid template. Transform 5 μL of the digested PCR product into E. coli BL21(DE3) competent cells, place on ice for 30 min, heat at 42℃ for 90 s, place on ice for 2 min, add 600 μL LB medium (formulation: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L), incubate at 37℃ with shaking at 220 rpm for 60 min, and then spread the entire bacterial culture evenly on LB plates containing ampicillin resistance (Amp 100 μg / mL) and incubate overnight at 37℃. Pick 1–10 single colonies from the plate, inoculate with 200 μL of LB liquid medium for 3 h, perform bacterial PCR verification and sequencing, and inoculate the bacterial culture with correct sequencing results into a test tube containing 3 mL of LB (Amp 100 μg / mL) medium. Incubate overnight at 37°C and 220 rpm for 12–16 h, then preserve the bacteria and proceed with subsequent expression.

[0216] 6.2 Induction and expression of glycosyltransferase UGT76G1 mutant and enzyme activity assay

[0217] The enzyme activity assay reaction system consisted of 4 mM RebD, 4 mM UDPG, 20–100 μL of crude enzyme solution, 20 μL of 100 mM potassium phosphate buffer (pH 7.0), and water to a final volume of 200 μL. The reaction was carried out at 40℃ and 800 rpm in a metal bath for 4–12 h. After the reaction, 200 μL of methanol was added to terminate the reaction. The reactants were incubated at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and then placed in a liquid chromatography sample vial for HPLC analysis.

[0218] The conversion result is as follows Figure 6 As shown, the catalytic activity of the glycosyltransferase UGT76G1 mutant I199Q was increased by 1.48 times compared with the control group, the catalytic activity of mutant I199L was increased by 1.38 times compared with the control group, the catalytic activity of mutant I199M was increased by 1.36 times compared with the control group, the catalytic activity of mutant I199K was increased by 1.32 times compared with the control group, and the catalytic activity of mutant I199A was increased by 1.23 times compared with the control group.

[0219] Example 7: Construction and screening of another optimized glycosyltransferase UGT76G1 mutant

[0220] 7.1 Construction of Glycosyltransferase Mutants

[0221] Based on the gene sequence of the glycosyltransferase UGT76G1 mutant UGT76G1-M6 (S280A-G405E-N138G-L200S-H155V-I1199Q), forward primer F and reverse primer R to introduce the V20 mutation were designed and synthesized. Using the recombinant plasmid pET32a-UGT76G1 (S280A-N138G-G405E-L200S-H155V-I199Q) obtained in Example 6 as a template, the plasmid was amplified using whole plasmid PCR amplification technology, transformed into E. coli BL21(DE3), and sequenced for verification, obtaining the V20 site saturated mutant.

[0222] PCR amplification reaction system: 1 μL each of 10 μM primer F and primer R; 0.5 μL template plasmid; 2 Add 25 μL of PCR mix and sterile water (ddH2O) to bring the total volume to 50 μL.

[0223] PCR amplification conditions: 98℃ pre-denaturation for 30 s; 30 cycles (98℃ denaturation for 10 s; 55℃ annealing for 10 s; 72℃ extension for 1 min); 72℃ complete extension for 7 min. PCR products were detected by agarose gel electrophoresis.

[0224] Add 0.5 μL DpnI enzyme to the PCR product and incubate at 37℃ for 1 h to digest the plasmid template. Transform 5 μL of the digested PCR product into E. coli BL21(DE3) competent cells, place on ice for 30 min, heat at 42℃ for 90 s, place on ice for 2 min, add 600 μL LB medium (formulation: NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L), incubate at 37℃ with shaking at 220 rpm for 60 min, and then spread the entire bacterial culture evenly on LB plates containing ampicillin resistance (Amp 100 μg / mL) and incubate overnight at 37℃. Pick 1–10 single colonies from the plate, inoculate with 200 μL of LB liquid medium for 3 h, perform bacterial PCR verification and sequencing, and inoculate the bacterial culture with correct sequencing results into a test tube containing 3 mL of LB (Amp 100 μg / mL) medium. Incubate overnight at 37°C and 220 rpm for 12–16 h, then preserve the bacteria and proceed with subsequent expression.

[0225] 7.2 Induction and expression of glycosyltransferase UGT76G1 mutant and enzyme activity assay

[0226] The enzyme activity assay reaction system consisted of 4 mM RebD, 4 mM UDPG, 20–100 μL of crude enzyme solution, 20 μL of 100 mM potassium phosphate buffer (pH 7.0), and water to a final volume of 200 μL. The reaction was carried out at 40℃ and 800 rpm in a metal bath for 4–12 h. After the reaction, 200 μL of methanol was added to terminate the reaction. The reactants were incubated at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and then placed in a liquid chromatography sample vial for HPLC analysis.

[0227] The conversion result is as follows Figure 7 As shown, the catalytic activity of the glycosyltransferase UGT76G1 mutant V20L was increased by 1.57 times compared with the control group, and the catalytic activity of the mutant V20I was increased by 1.27 times compared with the control group.

[0228] The optimal glycosyltransferase UGT76G1 mutant obtained through multiple rounds of screening is UGT76G1-M7 (S280A-G405E-N138G-L200S-H155V-I1199Q-V20).

[0229] Example 8: Purification of the UGT76G1 mutant glycosyltransferase protein

[0230] 8.1 Expression of the glycosyltransferase UGT76G1 mutant

[0231] The glycosyltransferase strain UGT76G1-WT and its mutant UGT76G1-M7, stored at -20℃, were inoculated into 20 mL of LB liquid (Amp 100 μg / mL) and cultured at 37℃ and 220 rpm for 12–16 h. 8 mL of the cultured bacterial suspension was then inoculated into 400 mL of LB liquid (Amp 100 μg / mL) and cultured at 37℃ and 220 rpm for 2–3 h until OD600 = 0.6–0.8. The culture was then cooled to 25℃, and IPTG was added to a final concentration of 0.5 mM for induction. After culturing at 25℃ and 220 rpm for 18–20 h, the bacterial cells were collected.

[0232] 8.2 Purification of the UGT76G1 mutant glycosyltransferase

[0233] The expressed bacterial cells were resuspended in Buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) at a concentration of 100–150 g / L. The resuspended bacterial solution was then homogenized using a high-pressure homogenizer at 4°C and 1200 Bar. The homogenized bacterial solution was centrifuged at 4°C and 16000 rpm for 90 min, the precipitate was removed, and the supernatant was filtered through a 0.22 μm filter. The filtrate was used as the loading solution for purification of the enzyme protein using a 5 mL nickel column. The specific steps are as follows:

[0234] 1) First, flush the pipeline with ultrapure water to remove impurities and air, and remove 20% anhydrous ethanol from the nickel column;

[0235] 2) Equilibration: Equilibrate the nickel column with 5-10 column volumes of Buffer A buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) to achieve baseline equilibration.

[0236] 3) Sample loading: Load the previously collected filtrate at a flow rate of 2 mL / min.

[0237] 4) Eluting out contaminating proteins: Elute contaminating proteins with 5-10 column volumes of Buffer A buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) at a flow rate of 2 mL / min until baseline equilibrium is reached, ensuring that contaminating proteins are completely washed away.

[0238] 5) Elution of the target protein: Elute the target protein with Buffer A (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5) at a flow rate of 2 mL / min. Monitor the eluent by observing the UV absorbance value of the detector. Collect the eluent in a test tube when the UV absorbance value rises relative to the baseline, and stop collecting when the UV absorbance value returns to the baseline. Store the collected eluent (i.e., the eluent containing the target protein) on ice.

[0239] 6) Dialysis: Place the eluent containing the target protein into a dialysis bag and dialyze at 4°C for 12-24 h in 20mM PBS solution (pH 7.5). The retentate after dialysis is the pure enzyme solution. Adjust the protein concentration of the pure enzyme solution to 1 mg / mL with 20mM PBS solution (pH 7.5). Store the purified protein at -80°C.

[0240] Example 9: Enzymatic study on the synthesis of rebaudioside A from steviol glycoside ST catalyzed by the glycosyltransferase UGT76G1 mutant

[0241] The final concentration of rebaudioside A was set to 100–1000 μM. Stevioside ST at different concentration gradients was mixed with appropriate amounts of purified wild-type glycosyltransferase UGT76G1 and mutant UGT76G1-M7, respectively. Enzyme activity was measured according to the method described above for glycosyltransferase purification. Km, Vmax, Kcat, and Kcat / Km of the purified wild-type and mutant were calculated using the Michaelis-Menten equation. The results are shown in Table 1. The enzyme activity of glycosyltransferase UGT76G1 for steviol glycoside ST was 5720 U / mg, and Kcat / Km was 500.97 mM. -1 S -1 The glycosyltransferase UGT76G1 mutant UGT76G1-M7 exhibits an enzyme activity of 6413.33 U / mg for steviol glycoside ST, with a Kcat / Km ratio of 564.84 mM. -1 S -1 .

[0242] Table 1. Enzymatic properties of glycosyltransferase UGT76G1 and its mutants

[0243]

[0244] Example 10: Enzymatic study on the synthesis of rebaudioside I from rebaudioside A catalyzed by the glycosyltransferase UGT76G1 mutant

[0245] The final concentration of steviol glycoside St was set to 100–1000 μM. Rebaudioside A at different concentration gradients was mixed with appropriate amounts of purified wild-type glycosyltransferases UGT76G1 and UGT76G1-M7, respectively. Enzyme activity was measured according to the method described above for glycosyltransferase purification. Km, Vmax, Kcat, and Kcat / Km of the purified wild-type and mutant enzymes were calculated using the Michaelis-Menten equation. The results are shown in Table 2. The enzyme activity of glycosyltransferase UGT76G1 for rebaudioside A was 11.04 U / mg, and Kcat / Km was 37.53 mM. -1 S -1 The glycosyltransferase mutant UGT76G1-M7 exhibits an enzyme activity of 441.67 U / mg for rebaudioside A and a Kcat / Km ratio of 3791.60 mM. -1 S -1 .

[0246] Table 2. Enzymatic properties of glycosyltransferase UGT76G1 and its mutants

[0247]

[0248] Example 11: Enzymatic study on the synthesis of rebaudioside M from rebaudioside D catalyzed by the glycosyltransferase UGT76G1 mutant

[0249] The final concentration of steviol glycoside ST was set to 100–1000 μM. Different concentration gradients of rebaudioside D were mixed with appropriate amounts of purified wild-type glycosyltransferases UGT76G1 and UGT76G1-M7, respectively. Enzyme activity was measured according to the enzyme activity assay method for purified glycosyltransferases described above. Km, Vmax, Kcat, and Kcat / Km of the purified wild-type and mutant enzymes were calculated using the Michaelis-Menten equation. The results are shown in Table 3.

[0250] Table 3. Enzymatic properties of glycosyltransferase UGT76G1 and its mutants

[0251]

[0252] The data in the table show that the enzyme activity of glycosyltransferase UGT76G1-WT for rebaudioside D is 390 U / mg, and the Kcat / Km ratio is 31.48 mM. -1 S -1 The glycosyltransferase mutant UGT76G1-M7 exhibits an enzyme activity of 10486.7 U / mg for rebaudioside D and a Kcat / Km ratio of 740.97 mM. -1 S -1 .

Claims

1. A glycosyltransferase mutant, characterized in that, The mutant is obtained by mutating any one of the following sites in the amino acid sequence shown in SEQ ID NO.1: (1) F46L; (2) S280A; (3) G405E; (4) V429E; (5)S280A-F46L; (6)S280A-N138G; (7)S280A-G405E; (8)S280A-V429E; (9) S280A-G405E-F46L; (10)S280A-N138G-G405E; (11) S280A-G405E-V429E; (12)S280A-N138G-G405E-F46L; (13)S280A-N138G-G405E-V429E; (13)S280A-N138G-G405E-F46L-V429E; (15)S280A-N138G-G405E-V20A; (16) S280A-N138G-G405E-G87A; (17) S280A-N138G-G405E-H155A; (18) S280A-N138G-G405E-I199A; (19) S280A-N138G-G405E-L200A; (20) S280A-N138G-G405E-L200R; (21) S280A-N138G-G405E-L200N; (22) S280A-N138G-G405E-L200D; (23) S280A-N138G-G405E-L200C; (24) S280A-N138G-G405E-L200Q; (25) S280A-N138G-G405E-L200E; (26) S280A-N138G-G405E-L200G; (27) S280A-N138G-G405E-L200H; (28) S280A-N138G-G405E-L200I; (20) S280A-N138G-G405E-L200K; (30) S280A-N138G-G405E-L200M; (31) S280A-N138G-G405E-L200S; (32) S280A-N138G-G405E-L200T; (33) S280A-N138G-G405E-L200V; (34) S280A-N138G-G405E-L200S-H155V; (35) S280A-N138G-G405E-L200S-H155V-I199A; (36) S280A-N138G-G405E-L200S-H155V-I199R; (37) S280A-N138G-G405E-L200S-H155V-I199D; (38) S280A-N138G-G405E-L200S-H155V-I199Q; (39) S280A-N138G-G405E-L200S-H155V-I199E; (40) S280A-N138G-G405E-L200S-H155V-I199H; (41) S280A-N138G-G405E-L200S-H155V-I199L; (42) S280A-N138G-G405E-L200S-H155V-I199K; (43) S280A-N138G-G405E-L200S-H155V-I199M; (44) S280A-N138G-G405E-L200S-H155V-I199S; (45) S280A-N138G-G405E-L200S-H155V-I199T; (46) S280A-N138G-G405E-L200S-H155V-I199W; (47) S280A-N138G-G405E-L200S-H155V-I199Y; (48) S280A-N138G-G405E-L200S-H155V-I199V; (49) S280A-N138G-G405E-L200S-H155V-I199Q-V20A; (50) S280A-N138G-G405E-L200S-H155V-I199Q-V20I; (51) S280A-N138G-G405E-L200S-H155V-I199Q-V20L.

2. The encoding gene of the glycosyltransferase mutant as described in claim 1.

3. A recombinant vector containing the encoding gene as described in claim 2.

4. The recombinant vector as described in claim 3, characterized in that, Its launch vehicle is pET32a.

5. Recombinant genetically engineered bacteria containing the recombinant vector as described in claim 4.

6. The recombinant genetically engineered bacteria as described in claim 5, characterized in that, It is Escherichia coli.

7. A method for synthesizing sweeteners using glycosyltransferase mutants, characterized in that, Includes the following steps: When the sweetener is rebaudioside M: using the glycosyltransferase mutant as described in claim 1 as a catalyst and rebaudioside D as a substrate, rebaudioside M is synthesized by catalysis. When the sweetener is rebaudioside A: using the glycosyltransferase mutant as described in claim 1 as a catalyst and stevioside ST as a substrate, rebaudioside A is synthesized by catalysis. When the sweetener is rebaudioside I: using the glycosyltransferase mutant as described in claim 1 as a catalyst and rebaudioside A as a substrate, rebaudioside I is synthesized by catalysis.

8. The method as described in claim 7, characterized in that, Cultivate the recombinant genetically engineered bacteria as described in claim 5 to prepare a crude enzyme solution as a catalyst; The catalytic reaction conditions were 35-45℃ and 500-1000 rpm in a metal bath for 4-12 h. After the reaction is complete, methanol is added to terminate the reaction, and then the product is collected.

9. The method as described in claim 8, characterized in that, The catalytic reaction system includes rebaudioside D, UDPG, and a catalyst; Or steviol ST, UDPG, and catalysts; Alternatively, rebaudioside A can be used as a substrate, UDPG, and a catalyst.

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