A glycosyltransferase mutant, gene thereof, engineered bacteria and preparation method thereof

Abstract

This invention provides a glycosyltransferase mutant, its gene, engineered bacteria, and a preparation method. Relating to the field of bioengineering technology, this glycosyltransferase mutant is obtained by subjecting a wild-type glycosyltransferase to one or both mutations including G15Q or G295W, wherein the amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO.1. The obtained mutant G15Q / G295W and its encoding gene yjiCmG15Q / G295W simultaneously achieve efficient preparation of the glycosyltransferase.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically to a glycosyltransferase mutant and its gene, engineered bacteria, and preparation method. Background Technology

[0002] Glycosyltransferases play a crucial role in various metabolic and regulatory pathways by catalyzing the transfer of glycosyl groups to different receptor substrates, making them important targets for disease treatment research and drug development. Among them, UDP-glucose-dependent glycosyltransferases (UGTs), using uridine diphosphate glucose (UDPG) as a glycosyl donor, have become key biocatalysts in the fields of drug and food additive development due to their ability to selectively catalyze the glycosylation reactions of natural and non-natural compounds, generating glycosylated products with diverse biological activities and physicochemical properties. However, with the deepening of UGT research and the increasing difficulty in discovering novel enzyme resources, wild-type UGTs with low catalytic efficiency and narrow substrate specificity are significantly limited in their application for glycosylation modification of natural and non-natural compounds. Therefore, random mutagenesis and directed evolution strategies have become effective ways to improve the catalytic performance of UGTs, expand their substrate spectrum, and design new artificial enzymes to overcome the above limitations.

[0003] Irrational protein molecular design involves inducing numerous mutations in a gene under certain conditions without knowing the protein's three-dimensional structure and mechanism of action, followed by targeted selection of mutants with desired characteristics through multiple rounds of high-throughput screening. Rational molecular design, on the other hand, involves targeted mutations of a gene sequence most likely to affect a protein's function and properties, based on known three-dimensional structure and function. This aims to intentionally alter one or two amino acid residues or modules of the protein, thereby constructing new protein molecules. Compared to irrational design, rational design has the advantages of requiring less work and more readily obtaining effective mutants.

[0004] Therefore, there is an urgent need for a glycosyltransferase with high enzyme activity to meet the needs of current development.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a glycosyltransferase mutant, its gene, engineered bacteria, and preparation method. This glycosyltransferase mutant is obtained by generating G15Q and G295W from a wild-type glycosyltransferase. The preparation method is simple, and the resulting glycosyltransferase mutant exhibits high enzyme activity.

[0007] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0008] This invention provides a glycosyltransferase mutant obtained by causing one or both of the following mutations, namely G15Q or G295W, based on a wild-type glycosyltransferase, wherein the amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO.1.

[0009] The amino acid sequence of the wild-type glycosyltransferase, SEQ ID NO.1, is:

[0010] MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDD QPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDR FCFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ.

[0011] Preferably, the mutant has a G15Q / G295W double mutation, and the amino acid sequence of the mutant is shown in SEQ ID NO.2.

[0012] MKKYHISMINIPAYQHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDD QPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDR FCFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGWMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ.

[0013] The present invention provides a polynucleotide encoding the above-mentioned glycosyltransferase mutant, the polynucleotide sequence of which is shown in SEQ ID NO.3.

[0014]

[0015] Preferably, the encoding gene is yjiCmG15Q or G295W.

[0016] The present invention provides a recombinant plasmid comprising the above-mentioned polynucleotides.

[0017] Preferably, the expression vector of the recombinant plasmid is either pET-28a(+) or pBSA43.

[0018] The present invention provides a host cell comprising the above-mentioned polynucleotides or the above-mentioned recombinant plasmids.

[0019] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0020] Preferably, the host cell is any one of Escherichia coli, Bacillus amyloliquefaciens, or Bacillus licheniformis.

[0021] Preferably, the host cell is Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218, or Bacillus licheniformis 2709.

[0022] The present invention provides a genetically engineered bacterium containing the above-mentioned polynucleotides or the above-mentioned recombinant plasmids.

[0023] This invention also provides the application of the above-mentioned glycosyltransferase mutant in any one of the following: cosmetic preparation, pharmaceutical and health product preparation, and industrial production.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] Based on the wild-type glycosyltransferase, mutations including G15Q and G295W were introduced to obtain the mutant G15Q / G295W and the encoding gene yjiCmG15Q / G295W. The enzyme activity of G15Q / G295W was increased by 47% compared with the wild-type glycosyltransferase (WT). This also achieved efficient preparation of the glycosyltransferase. Attached Figure Description

[0026] Figure 1 : This is an electrophoresis diagram of PCR amplification of the yjiC mutant G15Q / G295W gene of the present invention, where M is DNA Marker and 1 is the yjiC mutant G15Q / G295W gene;

[0027] Figure 2 : This is the enzyme digestion verification diagram of the recombinant plasmid pET-28a(+)-yjiCmG15Q / G295W of the present invention, where M is DNA Marker and 1 is the double enzyme digestion electrophoresis diagram of the recombinant plasmid pET-28a(+)-yjiCmG15Q / G295W.

[0028] Figure 3 : This is an SDS-PAGE image of the purified wild-type yjiC and yjiC mutant G15Q / G295W of this invention, where: M is the protein marker and 1 is the purified sample of mutant G15Q / G295W. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0030] The solutions and culture media used in the embodiments of this invention are as follows:

[0031] Lysis buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 20.

[0032] Wash buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 100.

[0033] Elution Buffer (mM): Tris 20, NaCl 500, dithiothreitol 1, imidazole 500.

[0034] LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, the remainder being water. Solid medium supplemented with 2% agar.

[0035] Example 1: Synthesis of the wild-type yjiC encoding gene yjiC

[0036] The yjiC gene (GenBank: WP_003232783.1) was submitted to Genewiz for synthesis and cloning on plasmid pET-28a(+). The recombinant plasmid pET-yjiC was obtained. This recombinant plasmid was then transformed into *E. coli* BL21 competent cells, resulting in the recombinant strain BL21 / pET-yjiC.

[0037] Example 2: Construction of glycosyltransferase mutants

[0038] 1. Construction of yjiC mutant

[0039] Design amplification primers for the gene encoding yjiC of the mutant yjiC, with the following sequences:

[0040] Upstream P1:

[0041] 5'-ATGAAAAAGTACCATATTTCGATGAT-3'

[0042] Downstream P2:

[0043] 5'-TTACTGCGGGACAGCG-3'

[0044] The 15th and 295th amino acids were selected for mutation, and the mutation primers were designed according to the mutation sites as shown in Table 1 below.

[0045] Table 1 lists the mutation primers as follows:

[0046]

[0047] Site-directed mutagenesis was performed using overlap PCR to construct the glycosyltransferase mutant G15Q / G295W.

[0048] First, construct the mutant G15Q:

[0049] In the first step of the overlap PCR reaction system, P1 and 15-F were used as upstream and downstream primers, respectively, and P2 and 15-R were used as upstream and downstream primers, respectively. Using plasmid pET-yjiC as a template, PCR1 reaction was performed to obtain the upstream and downstream fragments, respectively.

[0050] The reaction system for upstream fragment amplification is shown in Table 2.

[0051] Table 2: Reaction system for upstream fragment amplification

[0052]

[0053] The reaction system for downstream fragment amplification is shown in Table 3.

[0054] Table 3: Downstream Fragment Amplification Reaction System

[0055]

[0056] The amplification program was as follows: 98 °C pre-denaturation for 30 s; 98 °C denaturation for 10 s, 55 °C annealing for 20 s, 72 °C extension for 5 s, for 30 cycles; 72 °C extension for 10 min.

[0057] 2. After gel extraction and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system is shown in Table 4.

[0058] Table 4: PCR 2 reaction system after gel extraction and recovery of upstream and downstream fragments.

[0059]

[0060] The amplification program was as follows: 98 °C pre-denaturation for 30 s; 98 °C denaturation for 10 s, 54 °C annealing for 20 s, 72 °C extension for 7 s, for 5 cycles; 72 °C extension for 10 min.

[0061] 3. After PCR 2, add 2 μL each of primers P1 and P2 to the system. The PCR 3 amplification program is as follows: 98 ℃ pre-denaturation for 30 s; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 20 s, 72 ℃ extension for 6 s, for 5 cycles; 72 ℃ extension for 10 min. The PCR amplification products are subjected to 0.8% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit to obtain the site-directed mutagenesis glycosyltransferase encoding gene yjiCmG15Q.

[0062] 4. The site-directed mutant gene yjiCmG15Q of glycosyltransferase was ligated into the expression vector pET-28a(+) and transformed into JM109. The plasmid was then extracted to obtain the recombinant plasmid pET-yjiCmG15Q. The recombinant plasmid pET-yjiCmG15Q was then transformed into Escherichia coli BL21. Single colonies of the mutant were picked and inoculated into liquid LB medium containing Amp resistance. The plasmid was extracted and sequenced to confirm the G15Q mutant gene yjiCmG15Q.

[0063] 5. Based on the site-directed mutagenesis of G15Q, a novel glycosyltransferase was constructed by site-directed mutagenesis of G295W using overlap PCR technology.

[0064] In the first step of the overlap PCR reaction system, P1 and 295-F were used as upstream and downstream primers, respectively, and P2 and 295-R were used as upstream and downstream primers, respectively, to perform PCR 4 reaction and obtain upstream and downstream fragments.

[0065] The reaction system for upstream fragment amplification is shown in Table 6.

[0066] Table 5: Reaction system for upstream fragment amplification

[0067]

[0068] The reaction system for downstream fragment amplification is shown in Table 7.

[0069] Table 6: Reaction system for downstream fragment amplification

[0070]

[0071] The amplification program was as follows: 98 °C pre-denaturation for 30 min; 98 °C denaturation for 10 s, 55 °C annealing for 20 s, 72 °C extension for 5 s for 30 cycles; 72 °C extension for 10 min.

[0072] 6. After gel extraction and recovery of upstream and downstream fragments, PCR 5 was performed. The reaction system is shown in Table 7.

[0073] Table 7: PCR reaction system after gel extraction and recovery of upstream and downstream fragments.

[0074]

[0075] The amplification program was as follows: 98 °C pre-denaturation for 30 s; 98 °C denaturation for 10 s, 55 °C annealing for 20 s, 72 °C extension for 5 s, for 5 cycles; 72 °C extension for 10 min.

[0076] After PCR 5, 2 μL each of primers P1 and P2 were added to the system. The PCR 3 amplification program was as follows: 98 ℃ pre-denaturation for 30 s; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 20 s, 72 ℃ extension for 6 s, for 5 cycles; 72 ℃ extension for 10 min. The PCR amplification products were subjected to 0.8% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit to obtain the gene encoding the glycosyltransferase mutant G15Q / G295W, yjiCmG15Q / G295W.

[0077] 7. The site-directed mutant gene yjiCmG15Q / G295W of glycosyltransferase was obtained and ligated into the vector pET-28a(+), then transformed into JM109 and its plasmid was extracted to obtain the recombinant plasmid pET-yjiCmG15Q / G295W. The recombinant plasmid pET-yjiCmG15Q / G295W was then transformed into Escherichia coli BL21. Single colonies of the mutant were picked and inoculated into liquid LB medium containing Amp resistance for culture. The plasmid was extracted and sent for sequencing to determine the sequence of the mutant G15Q / G295W gene yjiCmG15Q / G295W.

[0078] Example 3: Expression, purification, activity determination, and sample preparation of glycosyltransferases

[0079] 1. Induced expression of glycosyltransferase strains

[0080] (1) On an LB plate, pick a single colony of BL21 / pET-yjiC and BL21 / pET-yjiCmG15Q / G295W, inoculate it into a 5 mL LB tube (containing 50 µg / mL Amp), and place it in a shaker at 37 ℃ for 10 h.

[0081] (2) Transfer the recombinant bacterial culture to 250 mL LB medium (final concentration of 50 µg / mL Amp) and place it in a shaker at 37 ℃ for 2-2.5 h.

[0082] (3) Add 125 µL of IPTG (final concentration 0.5 mmol / L) and incubate in a shaker at 16 °C for 16-20 h;

[0083] (4) The fermentation broth was purified to prepare wild-type glycosyltransferase and glycosyltransferase mutant G15Q / G295W.

[0084] 2. Ni column purification of recombinant proteins

[0085] (1) Broken bacterial cells

[0086] Collect the fermentation broth using a centrifuge cup, centrifuge at 10,000 rpm for 15 min, discard the supernatant, add 20 mL of Lysisbuffer to aspirate the bacterial cells, and use ultrasound to break down the bacterial cells, destroy the cell wall and release the intracellular proteins.

[0087] After disruption, the bacterial culture was poured into a 50 mL centrifuge tube and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was then collected.

[0088] bonded with nickel pillars

[0089] a. Before performing the nickel column purification process, add an appropriate amount of ddH2O to the purification column and add twice the column volume of Lysis buffer to balance the resin.

[0090] b. Mix the supernatant of the equilibrated resin and bacterial cells, place them in a magnetic stirrer, and combine at 80-100 r / min for 1 h, while maintaining a low temperature (4 °C) throughout the process.

[0091] (3) Protein purification

[0092] a. Add the binding buffer to the purification column in 2-3 fractions in the chromatography cabinet;

[0093] b. After the binding buffer has been completely filtered out, add 10 mL of wash buffer to elute any extraneous proteins bound to the resin;

[0094] c. Finally, add 10 mL of pre-cooled Elution Buffer to the purification column to elute all the target protein bound to the resin and collect the filtrate;

[0095] d. Transfer all eluent to an ultrafiltration centrifuge tube and centrifuge until 1 mL of solution remains. Add pre-cooled 50 mM Tris-HCl buffer (pH 7.0) and repeat the replacement process twice. The purified protein is then obtained.

[0096] 3. Glycosyltransferase activity assay

[0097] 3.1 Synthesis of nerol glycosides catalyzed by YjiC glycosyltransferase

[0098] A mixture of 50 mM Tris-HCl buffer (pH 8.0), 2 mM nerol, 5 mM UDP-glucose, and an appropriate amount of enzyme was prepared and catalyzed at 40 °C and 220 rpm for 15 min. The reaction was terminated by adding 4 times the volume of methanol, and the results were analyzed by high-performance liquid chromatography (HPLC). Enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μM nerol glycoside per minute under standard assay conditions. HPLC detection was performed using a C18 column (ZORBAX SB-C18, Agilent, USA) at 30 °C and a flow rate of 1 mL / min. The detection wavelength was 275 nm. Mobile phase A consisted of acetonitrile containing 0.1% formic acid, and mobile phase B consisted of water containing 0.1% formic acid. The flow rate was 1 mL / min. The column temperature and injection volume were set to 30 °C and 5 μL, respectively. Samples were detected by the HPLC system. The elution program was set as follows: 0-20 minutes, gradient elution with mobile phase A (10% to 90%); 20-22 minutes, isocratic elution (10% A); flow rate 1 mL / min; the conversion rate of nerol glycosides was calculated using the following formula:

[0099]

[0100] Where M t and M t0 These represent the final molar concentration of nerol glycoside and the initial molar concentration of nerol, respectively. The conversion rate of nerol glycoside is calculated by dividing the final molar concentration of nerol glycoside by the initial molar concentration of nerol.

[0101] 3.2 Synthesis of Rhodioloside Catalyzed by YjiC Glycosyltransferase

[0102] A mixture of 50 mM Tris-HCl buffer (pH 8.0), 2 mM tyrosol, 5 mM UDP-glucose, and an appropriate amount of enzyme was prepared and catalyzed at 40 °C and 220 rpm for 15 min. The reaction was terminated by adding 4 times the volume of methanol, and the mixture was analyzed by high-performance liquid chromatography (HPLC). One unit of enzyme activity (U) was defined as the amount of enzyme required to generate 1 μM rhodioloside per min under the above conditions. The specific enzyme activity was defined as the enzyme activity units contained in each mg of protein, and the crude enzyme activity was defined as the enzyme activity units contained in each mL of enzyme solution. Rhodioloside was quantified and analyzed using HPLC. A C18 column (250 mm × 4.6 mm, 5 μm) was used. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was 100% methanol. The flow rate was 1 mL / min. The gradient elution conditions were: 0–25 min 80% mobile phase A and 20% mobile phase B; 26–40 min 80% mobile phase A and 20% mobile phase B gradually transitioning to 100% mobile phase B; 41–45 min 100% mobile phase B, flow rate 1 mL / min. The UV detection wavelength was 224 nm, the column temperature was 35 ℃, and the injection volume was 10 μL. Rhodioloside standard solutions of different concentrations were prepared, and a standard curve was generated. The R² value of the standard curve was >0.999.

[0103]

[0104] Where M t and M t0 These represent the final molar concentration of rhodioloside and the initial molar concentration of tyrosol, respectively. The conversion rate of nerol glycosides is calculated by dividing the final molar concentration of rhodioloside by the initial molar concentration of tyrosol.

[0105] The yields of nerol glycosides and rhodiolosides synthesized by the glycosyltransferase mutants were significantly increased compared to the wild-type glycosyltransferases. The experimental results are shown in Tables 8-9. The results show that the activity of G15Q / G295W was significantly improved compared to the wild-type. The glycosyltransferases and their mutants were purified using the method described in Example 3, and their specific enzyme activities were determined using the same method. The results showed that the specific enzyme activity of the mutant G15Q / G295W in nerol was 60% higher than that of the wild-type; and the specific enzyme activity of the mutant G15Q / G295W in rhodiolosides was 80% higher than that of the wild-type, further indicating that the activity of the mutant G15Q / G295W was significantly improved.

[0106] Table 8: The ratio of the final nerol activity of wild-type yjiC to that of the G15Q / G295W mutant is as follows:

[0107]

[0108] Table 9: The activity ratios of rhodiolosides obtained from wild-type yjiC and G15Q / G295W mutants are as follows:

[0109]

[0110] 3.3 Synthesis of rosel glucoside, geraniol glucoside, and menthol glucoside catalyzed by glycosyltransferase mutants

[0111] The glycosyltransferase mutant G15Q / G295W was used to catalyze the glycosylation reaction of UDP-glucose with rose alcohol, geraniol, and menthol to synthesize rose alcohol glucoside, geraniol glycoside, and menthol glycoside.

[0112] 1. Enzyme-catalyzed synthesis

[0113] Reaction system:

[0114] In 20 mL of 50 mM Tris-HCl buffer (pH 7.5), equimolar amounts of rose alcohol, geraniol, and menthol (with 5% v / v DMSO for solubilization) were added, along with UDP-glucose and purified glycosyltransferase mutant G15Q / G295W enzyme solution (10 mg / mL). The reaction was carried out overnight at 40 °C with shaking (220 rpm) for 24 h, followed by heating at 90 °C for 5 min to terminate enzyme activity. The enzyme activity was analyzed by liquid chromatography-mass spectrometry. The amount of enzyme required to generate 1 μM of rose alcohol glycoside, geraniol glycoside, and menthol glycoside per min under the above conditions was defined as 1 enzyme activity unit (U). The specific enzyme activity was defined as the enzyme activity unit contained in 1 mg of protein, and the crude enzyme activity was defined as the enzyme activity unit contained in 1 mL of enzyme solution. Mobile phase A: 0.1% ammonia solution (1 mL of ammonia solution was dissolved in 999 mL of ultrapure water, mixed, and filtered through a membrane). Mobile phase B: pure methanol, flow rate: 0.2 mL / min, mass spectrometry detection selected negative ion mode, chromatographic column specification: Alphasil VC-C18, 2.5 μm column, injection volume: 10 μL.

[0115]

[0116] Where M t and M t0The values ​​represent the final molar concentrations of roseol glycosides, geraniol glycosides, and menthol glycosides, and the initial molar concentrations of roseol, geraniol, and menthol, respectively. The experimental results are shown in Table 10-12. The conversion rates of roseol glycosides, geraniol glycosides, and menthol glycosides were calculated by dividing the final molar concentrations of roseol glycosides, geraniol glycosides, and menthol glycosides by the initial molar concentrations of roseol, geraniol, and menthol, respectively. The experimental data in Table 10-12 show that the specific enzyme activities of the mutant G15Q / G295W were increased by 59%, 68%, and 57%, respectively. In the glycosylation reactions of roseol, geraniol, and menthol, the specific enzyme activities were significantly higher than those of the wild type. This indicates that the double mutant (G15Q / G295W) effectively enhances the catalytic efficiency of glycosyltransferases for various terpene alcohol substrates and has broad application potential.

[0117] Table 10: The activity ratios of the final wild-type yjiC and the G15Q / G295W mutant of roseol glycosides are as follows:

[0118]

[0119] Table 11: The activity ratios of geraniol glycosides obtained from wild-type yjiC and G15Q / G295W mutants are as follows:

[0120]

[0121] Table 12: The activity ratios of menthol glycosides obtained from wild-type yjiC and G15Q / G295W mutants are as follows:

[0122]

[0123] Example 4: Expression and preparation of glycosyltransferase mutant in Bacillus subtilis

[0124] The glycosyltransferase mutant G15Q / G295W encoding gene yjiCmG15Q / G295W and the wild-type glycosyltransferase encoding gene yjiC were respectively ligated with expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-yjiCmG15Q / G295W and pBSA43-yjiC.

[0125] The recombinant plasmids were transformed into Bacillus subtilis WB600, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, the mutant recombinant strain WB600 / pBSA43-yjiCmG15Q / G295W and the wild-type glycosyltransferase recombinant strain WB600 / pBSA43-yjiC were obtained.

[0126] Recombinant strains WB600 / pBSA43-yjiCmG15Q / G295W and WB600 / pBSA43-yjiC were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37 °C and 220 r / min. The inoculum was then transferred at a 2% inoculation rate to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37 °C and 220 r / min (fermentation medium (g / L): corn flour 64, soybean meal 40, with 2.7% amylase, 4% Na2HPO4, 0.3% KH2PO4, and the remainder water; incubated at 90 °C for 30 min and then sterilized at 121 °C for 20 min).

[0127] The enzyme activities of glycosyltransferases obtained from Bacillus subtilis fermentation, as described in Examples 3.2 and 3.3, were used to catalyze the synthesis of nerol glucoside, rhodioloside, rosel glucoside, geraniol glucoside, and menthol glucoside (enzyme activity was determined by centrifuging the fermentation broth and collecting the supernatant). The wild-type enzyme activities in Bacillus subtilis were 2.87 U / mL, 6.56 U / mL, 10.53 U / mL, 10.02 U / mL, and 11.72 U / mL, respectively. The fermentation enzyme activities of the mutants G15Q / G295W were 4.31 U / mL, 11.74 U / mL, 16.07 U / mL, 14.95 U / mL, and 18.05 U / mL, respectively.

[0128] Example 5: Expression and preparation of glycosyltransferase mutants in recombinant strains of Bacillus amyloliquefaciens

[0129] The glycosyltransferase mutant G15Q / G295W encoding gene yjiCmG15Q / G295W and the wild-type glycosyltransferase encoding gene yjiC were respectively ligated with expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-yjiCmG15Q / G295W and pBSA43-yjiC.

[0130] The recombinant plasmids were transformed into Bacillus amyloliquefaciens CGMCC No.11218, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, the mutant recombinant strain CGMCC No.11218 / pBSA43-yjiCmG15Q / G295W and the wild-type glycosyltransferase recombinant strain CGMCC No.11218 / pBSA43-yjiC were obtained.

[0131] Recombinant strains CGMCC No.11218 / pBSA43-yjiCmG15Q / G295W and CGMCC No.11218 / pBSA43-yjiC were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37 °C and 220 r / min. The inoculum was then transferred at a 2% inoculation rate to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37 °C and 220 r / min (fermentation medium (g / L): corn flour 64, soybean meal 40, with 2.7 g of amylase, 4 g of Na2HPO4, 0.3 g of KH2PO4, and the remainder water; incubated at 90 °C for 30 min and then sterilized at 121 °C for 20 min).

[0132] The enzyme activities of glycosyltransferases obtained from Bacillus amyloliquefaciens fermentation, as described in Examples 3.2 and 3.3, were used to catalyze the synthesis of nerol glucoside, rhodioloside, rosel glucoside, geraniol glucoside, and menthol glucoside (enzyme activity was determined by centrifuging the fermentation broth and collecting the supernatant). The wild-type enzyme activities in Bacillus amyloliquefaciens were 5.44 U / mL, 12.96 U / mL, 20.19 U / mL, 19.57 U / mL, and 22.68 U / mL, respectively. The fermentation enzyme activities of the mutants G15Q / G295W were 8.97 U / mL, 22.38 U / mL, 33.76 U / mL, 32.40 U / mL, and 38.22 U / mL, respectively.

[0133] Example 6 Expression and preparation of glycosyltransferase mutants in recombinant strains of Bacillus licheniformis

[0134] The recombinant plasmids pBSA43-yjiCmG15Q / G295W and pBSA43-yjiC were transformed into Bacillus licheniformis TCCC2709, respectively. After screening for kanamycin (Kan) resistance and enzyme digestion verification, the wild-type recombinant strain TCCC2709 / pBSA43-yjiC and the mutant recombinant strain TCCC2709 / pBSA43-yjiCmG15Q / G295W were obtained.

[0135] The Bacillus licheniformis mutant recombinant strain TCCC2709 / pBSA43-yjiCmG15Q / G295W and the wild-type recombinant strain TCCC2709 / pBSA43-yjiC were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37 °C and 220 r / min. Then, at a 2% inoculum size, the culture was transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37 °C and 220 r / min. (Fermentation medium (g / L): corn flour 64, soybean meal 40, with 2.7 g / L amylase, 4 g / L Na₂HPO₄, 0.3 g / L KH₂PO₄, and the remainder water; incubated at 90 °C for 30 min, then sterilized at 121 °C for 20 min.)

[0136] The enzyme activities of glycosyltransferases obtained from Bacillus licheniformis fermentation, as described in Examples 3.2 and 3.3, were used to catalyze the synthesis of nerol glucoside, rhodioloside, rosel glucoside, geraniol glucoside, and menthol glucoside (enzyme activity was determined by centrifuging the fermentation broth and collecting the supernatant). The wild-type enzyme activities in Bacillus licheniformis were 7.42 U / mL, 17.38 U / mL, 26.27 U / mL, 26.11 U / mL, and 30.08 U / mL, respectively. The fermentation enzyme activities of the mutants G15Q / G295W were 11.97 U / mL, 30.38 U / mL, 38.82 U / mL, 41.10 U / mL, and 48.07 U / mL, respectively.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A glycosyltransferase mutant, characterized in that, A mutant obtained by mutation on the basis of a wild-type glycosyltransferase, wherein the amino acid sequence of the wild-type glycosyltransferase is shown as SEQ ID NO. 1; The mutant has G15Q and G295W double mutations, and the amino acid sequence of the mutant is shown as SEQ ID NO.

2.

2. A polynucleotide, comprising, The polynucleotide sequence encoding the glycosyltransferase mutant according to claim 1 is shown as SEQ ID NO.

3.

3. A recombinant plasmid, characterized in that, The polynucleotide according to claim 2.

4. The recombinant plasmid of claim 3, wherein, The expression vector of the recombinant plasmid is any one of pET-28a(+) or pBSA43.

5. A host cell, characterized in that, The polynucleotide according to claim 2 or the recombinant plasmid according to any one of claims 3-4.

6. The host cell of claim 5, wherein, The host cell is a prokaryotic cell or a eukaryotic cell.

7. The host cell of claim 6, wherein, The host cell is any one of Escherichia coli, Bacillus amyloliquefaciens or Bacillus licheniformis.

8. A genetically engineered bacterium, characterized by, The polynucleotide according to claim 2 or the recombinant plasmid according to any one of claims 3-4.

9. Use of the glycosyltransferase mutant according to claim 1 in industrial production or preparation of pharmaceuticals, health products or cosmetics.

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