A synthesis method of quercetin-3,4′-O-diglucoside and the enzyme used therefor

Abstract

The present invention discloses a method for synthesizing quercetin-3,4'-O-diglucoside and the enzymes used therein. Taking UGT78D2 derived from Arabidopsis thaliana ( Arabidopsis thaliana ) and the mutant 73G1_V371A of UGT73G1 derived from onion ( Allium cepa ) as the research objects, protein engineering means are used to modify the regioselectivity of UGT78D2 and the soluble expression of 73G1_V371A. At the same time, sucrose synthase (SuSy) is introduced to form a coupling system with 73G1_V371A and UGT78D2 and its mutants respectively to realize the cyclic regeneration of UDPG, and a comparative study on the phosphorylation mutation of SuSy is carried out. Finally, a three-enzyme co-expression catalytic system S31D-F378S-V371A is constructed, and quercetin and sucrose are used as substrates to catalyze the synthesis of Q3,4'G. The amount of Q3,4'G converted and synthesized is 4.4 ± 0.03 g / L, and the yield of quercetin-3,4'-O-diglucoside is significantly higher than that of the original enzyme.

Description

Technical Field

[0001] The present invention relates to a glycosyltransferase mutant and its application in using an enzymatic catalysis method to generate a Q3,4′G glycoside product with a relatively high concentration using quercetin as a substrate, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Quercetin is one of the most common and widely distributed flavonoids in human plant-based foods and pharmaceutical ingredients. It is a flavonol compound and often exists widely in the flowers, fruits, and leaves of various plants in the form of quercetin glycosides. It is the aglycone of many other flavonoid glycosides. Quercetin has been proven to have various biological activities such as anti-inflammatory, anti-cancer, diabetes prevention, nerve and heart protection, osteoporosis prevention, and anti-allergy. Research shows that the antioxidant potential of quercetin in vitro and in vivo in rodent models is achieved by increasing mitochondrial activity and inhibiting atrophy factors. The antioxidant activity of quercetin mainly comes from the catechol group and a large number of free hydroxyl groups in its structure. Quercetin is also widely used as a dietary supplement. In the daily diet, nearly 2 / 3 of the flavonoids ingested by people every day are quercetin and its related derivatives. In addition, quercetin is added to functional foods.

[0003] Research shows that compared with quercetin, quercetin diglycosides have stronger biological activities in many aspects. For example, quercetin diglycosides are superior to quercetin in inhibiting the proliferation activity of malignant tumors, inhibiting the activities of HIV-1 and HSV-1 / 2, and are superior to quercetin in inhibiting acetylcholinesterase activity. Therefore, quercetin diglycosides have great application potential in anti-cancer, anti-Alzheimer's disease, anti-HIV, etc. Secondly, quercetin diglycosides have better bioavailability. They have appropriate molecular polarity, better water solubility, and higher intestinal epithelial absorption rate compared with quercetin.

[0004] Quercetin-3,4′-O-diglucoside (Q3,4′G) is one of the most studied flavonoid diglucosides. It is more soluble in water than quercetin or quercetin monoglycosides. A large number of studies show that Q3,4′G has antioxidant and neuroprotective effects, and it may be of great significance in maintaining neuron survival and scavenging free radicals. In addition, it also has pharmacological functions such as antibacterial and anti-inflammatory, blood pressure lowering, and antiviral. However, its content in nature is low. If traditional extraction methods are used, not only the yield is low, the cost is high, but also the pollution is large, which is not conducive to sustainable development. There is a large amount of quercetin in nature, but due to its poor water solubility, its bioavailability is low, making it unable to be fully utilized. If quercetin can be used as a raw material to transform and synthesize Q3,4′G, it can not only make full use of the existing quercetin resources, but also generate quercetin derivatives with higher biological utilization value.

[0005] There have been reports on the synthesis of quercetin 3-O-glycoside acylation derivatives, specifically including in vitro enzymatic synthesis and whole-cell biotransformation methods. In vitro enzymatic synthesis uses biocatalysts as biological enzymes to carry out enzymatic synthesis in a controllable manner in vitro, which is the main method for biosynthesizing quercetin 3-O-glycoside esters. Currently, four tool enzymes capable of performing in vitro acylation modification of quercetin 3-O-glycosides have been developed, namely acyltransferase, lipase, protease, and esterase. Among them, hydrolases can be used as better catalysts for the in vitro acylation modification of quercetin 3-O-glycosides due to their easy availability, economy, broad regioselectivity, good solvent tolerance, and the absence of the need for cofactors. Although the research on the in vitro enzymatic synthesis of quercetin 3-O-glycoside esters by hydrolases such as lipase has been relatively extensive, the acylation reaction mediated by lipase often needs to be carried out in organic solvents, which is likely to cause environmental pollution and is not conducive to coupling with other water-soluble pathway enzymes. Whole-cell biocatalysts can solve the above problems to a certain extent and are therefore an important means of obtaining quercetin 3-O-glycoside esters. However, in the operation process, the choice of solvent is a key factor in non-aqueous phase biotransformation, which not only affects the solubility of the substrate but also affects the catalytic activity of the whole cell. Quercetin 3-O-glycoside has a high polarity, so organic solvents with stronger polarity are needed to increase its solubility. However, strong polar organic solvents will interact with the bound water in the protein active center, causing the protein to lose its activity. In addition, the use of organic solvents will also change the integrity of the cell membrane and cell morphology.

[0006] When Lim et al. used quercetin and UDPG as substrates to explore the catalytic ability of the glycosyltransferase family UGT from Arabidopsis thaliana, they found that UGT78D2 had a high activity for synthesizing isoquercetin and showed good substrate regioselectivity for quercetin, with its glycosylation product being only isoquercetin. Pei et al. coupled UGT78D2 with the soybean-derived sucrose synthase GmSUS to catalyze quercetin. At different times, 1.25 mM quercetin substrate and fresh enzyme solution (70 mU / mL UGT78D2 and 120 mU / mL GmSUS) were added in a fed-batch manner, and finally 3830 mg / L of isoquercetin was produced, with a molar conversion rate reaching 94.3%. In addition, to catalyze the synthesis of astragalin (kaempferol-3-O-glucoside) from kaempferol, they constructed an engineered strain BL21-II capable of effectively synthesizing UDPG by introducing sucrose permease, sucrose phosphorylase, and uridyltransferase. After optimizing the catalytic reaction conditions and performing fed-batch fermentation and fed-batch addition of substrate in a 1 L bioreactor, finally 3600 mg / L of astragalin (molar conversion rate of 91.9%) was produced. Additionally, Kim et al. used an Escherichia coli engineered strain co-expressing UGT78D2 and UGT89C1. The former catalyzed the synthesis of isoquercetin from quercetin and UDPG, and the latter transferred the rhamnosyl group from UDP-rhamnose to the 7-OH of isoquercetin to synthesize 67 mg / L of quercetin 3-O-glucoside-7-O-rhamnoside.

[0007] Kramer et al. isolated the glycosyltransferase UGT73G1 from the epidermal layer of yellow onions. Through in vitro enzymatic property characterization, they found that a variety of different flavonoid compounds could serve as its substrates, including flavonoids not naturally present in onions. Among them, this enzyme could catalyze the C3, C7, and C 4′ hydroxyl groups of the quercetin structure to generate 3 kinds of monoglucosides and 2 kinds of diglucoside products, namely 3-O-glucoside, 7-O-glucoside, 4′-O-glucoside, 3,7-O-diglucoside, and Q3,4′G. Cai et al. constructed a fusion protein of UGT73G1 with a histidine tag and co-expressed it with a molecular chaperone, which improved the soluble expression of UGT73G1 in Escherichia coli. In vitro specific enzyme activity showed that the dual-terminal histidine tag contributed more to soluble expression than the molecular chaperone; for the pure enzyme of UGT73G1, the catalytic performance study found that Mn 2+ and Mg 2+ significantly promoted its activity, while Cu 2+ strongly inhibited its activity. There has been no article reporting the synthesis of Q3,4′G yet. Summary of the Invention

[0008] The object of the present invention is to carry out molecular modification on the regioselectivity of glycosyltransferase UGT78D2 and the soluble expression of mutant glycosyltransferase UGT73G1 (73G1_V371A), mutate the phosphorylation sites of sucrose synthase McSuSy, prepare recombinant enzymes by co-expressing three enzymes, and optimize the initial quercetin substrate concentration, substrate ratio, and DMSO concentration to increase the accumulation of the product Q3,4′G.

[0009] Modify the regioselectivity of glycosyltransferase UGT78D2, select two sites to be mutated, His 154 and Phe 378, for mutation, and find that the effect of the mutation site Phe 378 is better than that of the mutation site His 154. Further study the different amino acid mutations of the mutation site Phe 378, and finally obtain the single mutant 78D2-F378S with the mutated Phe 378 site. The sequence of the glycosyltransferase UGT78D2 mutant 78D2_F378S is shown in SEQ ID NO.1.

[0010] Carry out soluble expression modification on the mutant glycosyltransferase UGT73G1 73G1_V371A. Select the best mutant Pd5 obtained by the Pross algorithm for 73G1_V371A, subclone Pd5 and 73G1_V371A into pCDFDuet-1 respectively, carry out single-point and multi-point back mutations at the sites of Ser 96, Gln 205, Gln 18, and Ala 288 in Pd5, and compare the specific enzyme activity and soluble expression ability with 73G1_V371A. The sequence of the glycosyltransferase UGT73G1 mutant Pd5 is shown in SEQ ID NO.5.

[0011] Mutate the phosphorylation sites of sucrose synthase McSuSy, mutate Ser31 to Asp or Glu to obtain Mc_S31D and Mc_S31E, and find that after mutating Ser31 of SuSy in McSuSy-F378S (pET) to Asp, that is, Mc_S31D, the enzyme activities of both SuSy and UGT will be improved. The sequence of the sucrose synthase mutant Mc_S31D is shown in SEQ ID NO.3.

[0012] Applications of the glycosyltransferase UGT8D2 mutant 78D2_F378S, the glycosyltransferase UGT73G1 mutant Pd5, and the sucrose synthase mutant Mc_S31D in the synthesis of quercetin-3,4′-O-diglucoside.

[0013] The above three enzymes can synthesize quercetin-3,4′-O-diglucoside alone or in combination. For example, a dual-bacteria three-enzyme system (S31D-F378S&S31D-V371A) or a three-enzyme co-expression system (S31D-F378S-V371A) can be constructed to catalyze the reaction of quercetin.

[0014] The above-mentioned dual-bacteria three-enzyme system refers to that after the glycosyltransferase UGT78D2 and sucrose synthase McSuSy are codon-optimized and synthesized, they are subcloned into the expression vector pETDuet-1. The coding gene of McSuSy is inserted between the NcoI and EcoRI sites of the expression vector, and the coding gene of UGT78D2 is inserted between the NdeI and XhoI sites to obtain the recombinant plasmid pET-McSuSy-78D2. Then, primers are designed according to the sequences near the mutation sites and mutated into pET-S31D-F378S and pET-S31D-V371A in the same principle. The three-enzyme co-expression system is prepared by the CaCl2 method to make the engineering strain S31D-F378S into E. coli BL21(DE3)_S31D-F378S competent cells, and then the recombinant plasmid pRSF-S31D-V371A is transformed into the competent cells to obtain the engineering strain S31D-F378S-V371A.

[0015] Single colonies on the above-mentioned plate are picked and inoculated into a shake tube containing 5 mL of liquid medium (containing 50 mg / L kanamycin) of LB and activated in a shaker at 37°C and 200 rpm for 12 h as the seed solution. It is transferred to a 100 mL LB flask containing the corresponding resistance according to an inoculation amount of 2% (v:v) for induction. The induced bacterial solution is frozen, centrifuged, and disrupted, and the obtained supernatant is the crude enzyme solution.

[0016] A method for synthesizing quercetin-3,4′-O-diglucoside uses quercetin as the sugar acceptor, sucrose as the sugar donor, and a buffer solution with a pH of 7.0-8.0 as the reaction medium, and reacts to synthesize quercetin-3,4′-O-diglucoside under the catalytic action of a mutant of glycosyltransferase UG78D2, a mutant of glycosyltransferase UGT73G1, and a coupled mutant of sucrose synthase.

[0017] The glycosyltransferase UGT78D2 mutant is the mutant 78D2_F378S obtained by mutating the Phe378 site of glycosyltransferase UGT78D2, and its sequence is as shown in SEQ ID NO.1; the glycosyltransferase UGT73G1 mutant is 73G1_V371A, and its sequence in the single-enzyme expression vector is as shown in SEQ ID NO.4, and its sequence in the SuSy-UGT co-expression vector is as shown in SEQ ID NO.2; the mutant Pd5 in the soluble modification of glycosyltransferase UGT73G1 has a sequence as shown in SEQ ID NO.5. The sucrose synthase mutant is Mc_S31D obtained by mutating the Ser31 site of sucrose synthase McSuSy to Asp, and its sequence is as shown in SEQ ID NO.3.

[0018] In the said reaction, the concentration ratio of quercetin to sucrose is 1:(3 - 40), the quercetin concentration is 3 - 50 g / L, the crude enzyme solution concentration is 0.3 - 10 mg / mL, the reaction temperature is 35 - 45 °C, and the reaction time is 2 - 30 h.

[0019] Further preferably, in the reaction, the concentration ratio of quercetin to sucrose is 1:30, the quercetin concentration is 10 g / L, the sucrose concentration is 300 g / L, the crude enzyme solution concentration is 10 mg / mL, the reaction temperature is 35 °C, and the reaction time is 24 h.

[0020] In the present invention, the reactions of catalyzing quercetin by the dual-bacteria triple-enzyme system S31D-F378S&S31D-V371A (a dual-bacteria triple-enzyme system composed of the co-expressed enzyme of glycosyltransferase mutant 78D2_F378S and sucrose synthase mutant Mc_S31D, and the co-expressed enzyme of sucrose synthase mutant Mc_S31D and glycosyltransferase mutant 73G1_V371A) and the triple-enzyme co-expression system S31D-F378S-V371A were compared, and it was found that the yields of Q3,4′G synthesized by the catalytic systems were similar.

[0021] The enzyme production operation of the engineering bacteria using triple-enzyme co-expression is relatively simple, and the S31D-F378S-V371A system is preferably selected.

[0022] In the present invention, factors such as the initial concentration of quercetin, the concentration ratio of quercetin to sucrose, and the DMSO concentration were respectively investigated for their effects on the catalytic synthesis of Q3,4′G by S31D-F378S-V371A, and the preferred reaction conditions were obtained as follows: the initial concentration of quercetin is 10 g / L, the concentration ratio of quercetin to sucrose is 1:30, and the DMSO concentration is 20%. Reacting for 24 h under these conditions, the concentration of the transformed and synthesized Q3,4′G is 4.4 ± 0.03 g / L.

[0023] The said mutation is related to the yield of quercetin-3,4′-O-diglucoside produced by glycosyltransferase.

[0024] The glycosyltransferase UGT78D2 mutant is coupled with sucrose synthase, and the role of sucrose synthase here is to achieve the recycling of UDPG. Similarly, the coupling of UGT73G1 with sucrose synthase also serves this purpose. UGT78D2 catalyzes quercetin to produce isoquercitrin, and UGT73G1 uses this isoquercitrin to regenerate Q3′4G. However, some by-product Q3,7G is generated during the production of isoquercitrin by UGT78D2, which will affect the production of Q3′4G by UGT73G1 using isoquercitrin. Therefore, in this experiment, regional selectivity modification of UGT78D2 is carried out to reduce its by-products and improve the production efficiency of the final product.

[0025] The preparation method of the recombinant plasmid of the glycosyltransferase UGT78D2 mutant and the glycosyltransferase UGT73G1 mutant includes the following steps:

[0026] (1) Determine the mutation sites based on the amino acid sequence of the glycosyltransferase; design mutant primers for site-directed mutagenesis, and perform site-directed mutagenesis using the vector carrying the glycosyltransferase gene as a template; construct a plasmid vector containing the mutant.

[0027] (2) Construct a recombinant plasmid, which is ligated with the above expression gene and the sucrose synthase gene.

[0028] (3) Add DpnI restriction enzyme to the recombinant plasmid to digest the parental template;

[0029] (4) Transform the recombinant plasmid into an E.coli DH5α plate, pick a single colony on the plate, inoculate it into an LB liquid medium containing 50 mg / L kanamycin, culture at 37 °C and 200 rpm for 12 h, then extract the plasmid and identify it by sequencing;

[0030] (5) Transform the plasmid with correct sequencing into E.coli BL21(DE3), and culture it with shaking at 37 °C and 200 rpm until the OD 600 is about 0.6, induce at 16 °C with 0.1 mM IPTG for 36 h to obtain the bacterial cells containing the enzyme.

[0031] (6) Freeze and centrifuge. In the bacterial cells containing the enzyme, resuspend the bacterial cells once with 100 mM (pH 7.2) potassium phosphate buffer solution, centrifuge at 4 °C and 6000 rpm for 3 min to collect the bacterial cells, then add an appropriate amount of 100 mM potassium phosphate buffer solution (pH 7.2) to resuspend the bacterial cells, and the OD of the resuspension 600 ≤100, break the cell wall with a cell ultrasonic disruptor to obtain a crude enzyme solution, and detect the expression by polyacrylamide gel electrophoresis.

[0032] Beneficial effects:

[0033] Based on the glycosyltransferase UGT78D2 derived from Arabidopsis thaliana, through bioinformatics analysis, site-directed mutagenesis was carried out on the key amino acids in its active center. After screening mutant strains, a glycosyltransferase mutant was obtained. This glycosyltransferase mutant was co-expressed with the glycosyltransferase UGT73G1 mutant and sucrose synthase. Using quercetin as a substrate and adding an appropriate amount of sucrose, the system was optimized to catalytically produce quercetin-3,4′-O-diglucoside. This mutant is simple to prepare and has a large yield, achieving an increase in the yield of quercetin-3,4′-O-diglucoside. Under the same conditions, the amount of Q3,4′G transformed and synthesized is 4.4 ± 0.03 g / L, and the yield of quercetin-3,4′-O-diglucoside is significantly higher than that of the original enzyme.

[0034] In the present invention, molecular modification was carried out on the regioselectivity of UGT78D2 and the soluble expression of 73G1_V371A, and a multi-enzyme complex system mainly composed of UGT and supplemented by SuSy was constructed; phosphorylation mutagenesis of McSuSy in the coupling system was carried out, and after determining the enzyme production modes of two groups of UGT-SuSy, the best mutant strain S31D-F378S-V371A for establishing the conversion reaction was obtained; the accumulation amount of quercetin synthesized into Q3,4′G was increased by optimizing the reaction conditions. Brief Description of the Drawings

[0035] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of this application, and do not limit the embodiments of the present invention. In the drawings:

[0036] Figure 1 Mc-78D2 catalyzes the synthesis of quercetin glycoside, UGT78D2: 9975.66 mU / mL; McSuSy: 422.94 mU / mL;

[0037] Figure 2 SDS-PAGE analysis of the recombinant expression of Mc-78D2 and Mc-F378S. Note: M: Protein molecular weight Marker; Lanes 1–2: Mc-78D2 supernatant / precipitate; Lanes 3–4: Mc-F378S supernatant / precipitate;

[0038] Figure 3 The effects of UGT78D2 and its mutant 78D2_F378S on the conversion and synthesis of isoquercetin, Mc-78D2: 11094.55 mU / mL UGT78D2, 379.15 mU / mL McSuSy; Mc-F378S: 3222.73 mU / mL 78D2_F378S, 339.95 mU / mL McSuSy;

[0039] Figure 4SDS-PAGE analysis of the recombinant expression of 73G1_V371A and Pd5; Note: M: Protein molecular weight Marker; Lanes 1–2: Supernatant / precipitate of 73G1_V371A; Lanes 3–4: Supernatant / precipitate of Pd5;

[0040] Figure 5 Effect of different expression vectors on the conversion and synthesis of isoquercetin;

[0041] Figure 6 Effect of the coupling of McSuSy and its mutant enzymes with 78D2_F378S on the conversion and synthesis of isoquercetin;

[0042] Figure 7 Specific enzyme activity of 73G1_V371A and SuSy in different engineered bacteria;

[0043] Figure 8 Effect of the coupling of McSuSy and its mutant enzymes with 73G1_V371A on the conversion and synthesis of Q3,4′G;

[0044] Figure 9 Effect of different enzyme production methods on the catalytic synthesis of quercetin glycosides;

[0045] Figure 10 Effect of the initial concentration of quercetin on the catalytic synthesis of quercetin glycosides by S31D-F378S-V371A;

[0046] Figure 11 Effect of the concentration ratio of quercetin to sucrose on the catalytic synthesis of quercetin glycosides by S31D-F378S-V371A;

[0047] Figure 12 Effect of the concentration of DMSO on the catalytic synthesis of quercetin glycosides by S31D-F378S-V371A, (a) 10% DMSO; b) 20% DMSO. Specific implementation mode

[0048] The following further illustrates the content of the present invention in conjunction with embodiments, but does not limit the present invention.

[0049] The detection methods adopted in the following examples are as follows:

[0050] The contents of quercetin, isoquercetin, quercetin-3,7-O-diglucoside and quercetin-3,4′-O-diglucoside in the final reaction system were detected by HPLC.

[0051] LC-MS detection was performed using an Agilent 6530 Q-TOF liquid chromatography-mass spectrometry system and an electrospray mass spectrometer.

[0052] Liquid phase detection conditions: Use an Agilent TC-C18 chromatographic column (250 mm × 4.6 mm). The mobile phase, column temperature, and UV detection wavelength are the same as those in the above HPLC detection method. Gradient detection conditions: 0–8 min, 90–75% phase B; 8–14 min, 75% phase B; 14–24 min, 75–15% phase B; 24–29 min, 15–90% phase B; 29–31 min, 90% phase B. The sample injection volume is 20 μL, and the flow rate is 1 mL / min.

[0053] Mass spectrometry detection conditions: Electrospray ionization ESI(+), reverse blow gas temperature 350 °C, reverse blow gas flow rate 5 mL / min, sprayer pressure 45 psi, sheath gas temperature 350 °C, sheath gas flow rate 5 mL / min, capillary voltage 4 kV, fragmentation voltage 175 V, cone voltage 65 V, ion energy -0.3, proton number / charge number 50–2000.

[0054] Example 1 Regional selectivity modification of glycosyltransferase UGT78D2

[0055] (1) Mc-78D2 catalyzes the synthesis of quercetin glycoside

[0056] Construct engineering bacterium Mc-78D2:

[0057] After codon optimization and synthesis of glycosyltransferase UGT78D2 and sucrose synthase McSuSy, insert the McSuSy coding gene with the sequence of SEQ ID NO.6 between the NcoI and EcoRI sites of the expression vector pETDuet-1, and insert the UGT78D2 coding gene with the sequence of SEQ ID NO.7 between the NdeI and XhoI sites to obtain the recombinant plasmid pET-McSuSy-78D2, namely Mc-78D2(pET). Transform the recombinant plasmid pET-McSuSy-78D2 into E. coli DH5α. Pick a single colony on the plate, inoculate it into an LB liquid medium containing 50 mg / L kanamycin, culture it at 37 °C and 200 rpm for 12 h, then extract the plasmid and send it to Chuzhou General Biology Company for sequencing and identification. In the construction of pRSF-McSuSy-78D2, the expression vector is changed to Prsf-Duet-1, and the remaining construction steps are the same as those of pET-McSuSy-78D2. Transform the plasmid with correct sequencing into E. coli BL21(DE3) to obtain an engineering strain containing the recombinant plasmid. Pick a single colony on the above plate, inoculate it into a shake tube containing 5 mL of LB liquid medium (containing 50 mg / L kanamycin), and activate it in a shaker at 37 °C and 200 rpm for 12 h as the seed liquid. Transfer it to a 100 mL LB shake flask containing the corresponding resistance according to an inoculation amount of 2% (v:v). Place it in a shaker at 37 °C and oscillate at 200 rpm. Wait for OD600 When it reaches about 0.6, adjust the temperature to 16 °C, then add 0.1 mM IPTG and induce for 36 h. Centrifuge the induced bacterial solution by freezing (4 °C, 7500 rpm, 5 min), remove the above culture medium, resuspend and wash the bacterial cells once with 100 mM potassium phosphate buffer solution (pH 7.2), and centrifuge at 4 °C, 6000 rpm for 3 min to collect the bacterial cells. According to the OD 600 value of the above bacterial solution, add an appropriate amount of 100 mM potassium phosphate buffer solution (pH 7.2) to resuspend the bacterial cells, and the OD 600 of the resuspended solution ≤ 100. Place the well-mixed bacterial cells in an ice bath and disrupt the cells with an ultrasonic disruptor, setting the instrument parameters as Ф6, 300 W, 15 min. After disruption, centrifuge at 8000 rpm and 4 °C for 25 min, and the obtained supernatant is the crude enzyme solution. Establish a 10 mL reaction system (the reaction system is 3 g / L quercetin, 400 g / L sucrose, 2 mM UDP, 3 mM MgCl2, 10% DMSO (v:v), 10 mg / mL crude enzyme solution, and make up the remaining volume with 100 mM potassium phosphate buffer solution (pH 7.2)), the catalytic reaction conditions are 35 °C and 200 rpm, react for 12 h, sample 30 μL at certain time intervals, add 870 μL methanol to terminate the reaction, centrifuge at 12000 rpm for 1 min, take the supernatant and place it in a new EP tube, filter through an organic filter membrane (0.45 μm) and then put it into a liquid phase sample bottle, and detect it by HPLC.

[0058] The results are as Figure 1 shown. At 4 h, isoquercetin had accumulated 873.85 mg / mL, but its accumulation amount gradually decreased after 4 h of reaction, while the yield of Q3,7G continued to increase slowly within 4 - 12 h. It may be because the catalytic reaction uses the isoquercetin generated during this time period as a substrate and continues to add a glycosyl group at its 7-OH position, thus generating Q3,7G. And the production amount of Q3,7G gradually approaches the accumulation amount of isoquercetin, which may affect the subsequent cascade catalytic reaction with 73G1_V371A, resulting in that the isoquercetin catalyzed by UGT78D2 cannot be fully provided to 73G1_V371A as a substrate, affecting the production of Q3,4′G.

[0059] (2) Regional selectivity modification of UGT78D2

[0060] The sequences of UGT78D2 and UGT74AC2 were aligned using ClustalW and ESPript3, and potential mutation sites His154 and Phe378 in UGT78D2 were found. Software such as YASARA and Rosetta 3.10 were used to perform homology modeling of UGT78D2, molecular docking of the enzyme with substrates (quercetin and UDPG), and combined virtual mutation and analysis of potential mutation sites. These two residues were set to be mutated into any standard amino acid residue. A reasonable conformation was selected based on the total energy score, interface energy score, and enzyme catalytic mechanism of the obtained conformations, which was 78D2-F378S.

[0061] (3) Primer design

[0062] Primers were designed according to the sequences near the mutation sites, as shown in Table 1.

[0063] Table 1 Primer sequences of gene mutation sites for regional selective modification of UGT78D2

[0064]

[0065] (4) Site-directed mutagenesis

[0066] The method of site-directed mutagenesis referred to the instruction manual of Mut Express II Fast Mutagenesis Kit V2.

[0067] Using the recombinant plasmid pRSF-McSuSy-78D2 as a template, PCR (Polymerase Chain Reaction) amplification was carried out using the primers shown in Table 1. After verifying the success of the PCR reaction by agarose gel electrophoresis, DpnI restriction enzyme was added to the PCR reaction product to digest the parental template. Then, the digested PCR reaction product was transformed into E. coli DH5α. Finally, single colonies on the plate were picked and inoculated into LB liquid medium containing 50 mg / L kanamycin, cultured at 37 °C and 200 rpm for 12 h, and then the plasmid was extracted and sent to Chuzhou General Biology Company for sequencing and identification. The plasmid with correct sequencing was transformed into E. coli BL21(DE3) to obtain an engineered strain containing the mutant plasmid.

[0068] The PCR reaction system was always: 25 μL 2×Max Buffer, 1 μL dNTP Mix, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL template DNA, 1 μL each primer, and appropriate amount of ddH2O.

[0069] The PCR conditions were as follows: pre-denaturation at 95°C for 30 s; followed by 30 cycles (denaturation at 95°C for 15 s, annealing at 70°C for 15 s, extension at 72°C for 8 min); extension at 72°C for 5 min.

[0070] DpnI restriction enzyme was added to the PCR reaction product to digest the parental template. Then the digested PCR reaction product was transformed into E. coli DH5α. Finally, single colonies on the plate were picked and inoculated into LB liquid medium containing 50 mg / L kanamycin, cultured at 37°C and 200 rpm for 12 h, and then the plasmid was extracted. The mutant plasmid was transformed into competent cells of the expression host Escherichia coli BL21(DE3), and all mutant plasmids were sequenced correctly.

[0071] The specific steps of the heat shock transformation method are as follows:

[0072] 10 μL of the homologous recombination product was introduced into 100 μL of BL21(DE3) competent cells; ice bath for 30 - 40 min; heat shock in a 42°C water bath for 90 s, and then quickly placed on ice and allowed to stand in the ice bath for 3 - 5 min; 800 μL of antibiotic-free LB medium was added and mixed evenly, cultured at 37°C and 200 rpm for 30 min; centrifuged at 5000 rpm for 2 min to collect the bacteria; the supernatant was removed, and the remaining 100 - 200 μL was pipetted and mixed evenly and spread onto a kanamycin-resistant plate containing 50 μg / mL, and cultured at 37°C for about 12 h. Single colonies were picked and cultured in LB containing 50 μg / mL kanamycin at 200 rpm and 37°C for 12 h, and then sent to the company for sequencing. Those with correct sequencing were positive transformants.

[0073] (5) Induction expression of recombinant protein

[0074] Single colonies on the above plate were picked and inoculated into a shake tube containing 5 mL of LB liquid medium (10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, made up to volume with distilled water: containing 50 mg / L kanamycin), and activated at 37°C and 200 rpm in a shaker for 12 h as the seed solution. It was transferred to a 100 mL LB flask containing the corresponding resistance according to an inoculation amount of 2% (v:v). It was placed in a 37°C shaker and cultured with shaking at 200 rpm. When the OD 600 reached about 0.6, the temperature was adjusted to 16°C, and then 0.1 mM IPTG was added for induction culture for 36 h.

[0075] (6) Preparation of crude enzyme solution

[0076] The induced bacterial liquid was centrifuged by freezing (4°C, 7500 rpm, 5 min) to remove the above culture solution, and the cells were resuspended and rinsed once with 100 mM potassium phosphate buffer solution (pH 7.2), and the cells were collected by centrifugation at 4°C and 6000 rpm for 3 min. According to the OD of the above bacterial liquid600 The cells were resuspended in an appropriate amount of 100 mM potassium phosphate buffer (pH 7.2), and the OD of the resuspended solution 600 ≤100. The mixed cells were placed in an ice bath and disrupted by an ultrasonic crusher with the instrument parameters set as Ф6, 300 W, and 15 min. After disruption, centrifugation was performed at 8000 rpm and 4 °C for 25 min, and the resulting supernatant was the crude enzyme solution, which was stored at 4 °C.

[0077] (7) Protein concentration determination and SDS-PAGE analysis

[0078] The protein concentration was detected according to the Coomassie brilliant blue method (Brandford method). The expression of the recombinant protein was analyzed using 12% polyacrylamide gel electrophoresis (SDS-PAGE). The theoretical protein molecular weights of McSuSy, UGT78D2, and 78D2_F378S were predicted using the ExPaSy's Compute pI / Mw (https: / / web.expasy.org / compute_pi / ) online tool, which were 97.6 kDa, 52.6 kDa, and 52.6 kDa, respectively. After the engineering strains Mc-78D2 and Mc-F378S were cultured and induced for expression, SDS-PAGE analysis was performed on the supernatant and precipitate after ultrasonic disruption. As Figure 2 shown, target bands were observed near 44.3 kDa and 97.2 kDa, which were consistent with the predicted theoretical values of UGT78D2, 78D2_F378S, and McSuSy, indicating that the recombinant enzymes were successfully expressed. Comparing the protein expression in the supernatant and precipitate, in the supernatant (lanes 1 and 3), the target bands of UGT78D2, 78D2_F378S, and McSuSy could be clearly observed, but the inclusion body band of McSuSy in lanes 2 and 4 was deeper, indicating that its soluble expression level was low, and inclusion bodies also existed in UGT78D2 and 78D2_F378S, but relatively less.

[0079] (8) Enzyme activity assay

[0080] UDP-glycosyltransferase enzyme activity assay

[0081] In a 660 μL enzyme activity assay system, add 0.5 mM quercetin, 5 mM UDPG, 3 mM MgCl2, and 0.005 mg / mL of crude enzyme, and then make up the remaining volume with 100 mM potassium phosphate buffer (pH 7.2). After mixing evenly, aliquot it into 3 1.5 mL EP tubes. The reaction is carried out in a shaker at 30 °C and 200 rpm, and the sampling times are 0 min, 10 min, and 20 min. Sample treatment: Add 180 μL of methanol to the EP tube to terminate the reaction. After centrifuging at 12000 rpm for 1 min, take the supernatant, filter it through an organic filter membrane (0.45 μm), and put it into a liquid phase sample vial for analysis by high performance liquid chromatography (HPLC).

[0082] The enzyme activity of UDP - glycosyltransferase is defined as: The amount of enzyme required to convert quercetin to produce 1 μmol of isoquercetin within 1 minute is 1 enzyme activity unit (U).

[0083] Determination of sucrose synthase enzyme activity

[0084] In a 3 mL enzyme activity assay system, add 500 mM sucrose, 10 mM UDP, and 3 mg of crude enzyme, and then make up the remaining volume with 100 mM potassium phosphate buffer (pH 7.2). After mixing evenly, aliquot it into 3 1.5 mL EP tubes. The reaction is carried out in a shaker at 30 °C and 200 rpm, and the sampling times are 0 min, 15 min, and 30 min. Sample treatment: Place the EP tube in a water bath at 95 °C and heat it for 10 min to terminate the reaction. Then, place the EP tube in a centrifuge (12000 rpm, 1 min), take the supernatant and transfer it to a new EP tube. Pipette 250 μL of the supernatant, add 250 μL of distilled water and mix well. Then add 1 mL of DNS reagent, boil for 2 min, cool, and add 3.5 mL of distilled water. Measure the absorbance at a wavelength of 540 nm, and calculate the fructose content by referring to the DNS standard curve.

[0085] The enzyme activity of sucrose synthase is defined as: The amount of enzyme required to convert sucrose to produce 1 μmol of fructose within 1 minute is 1 enzyme activity unit (U).

[0086] (9) Catalytic reaction of the recombinant enzyme

[0087] In a catalytic reaction system (10 mL, named after the strain name), 3 g / L quercetin, 400 g / L sucrose, 2 mM UDP, 3 mM MgCl2, 10% DMSO (v:v), and 10 mg / mL crude enzyme solution were added, and the remaining volume was made up with 100 mM potassium phosphate buffer solution (pH 7.2). The catalytic reaction conditions were 35 °C and 200 rpm for 12 h. Samples of 30 μL were taken at certain time intervals, 870 μL of methanol was added to terminate the reaction, and the mixture was centrifuged at 12000 rpm for 1 min. The supernatant was transferred to a new EP tube, filtered through an organic filter membrane (0.45 μm), and placed in a liquid phase sample bottle for HPLC detection.

[0088] (10) Detection of quercetin glycosides by HPLC

[0089] (11) Result analysis

[0090] As Figure 3 shown, the enzyme activity of 78D2_F378S using quercetin as a substrate was 322.27 mU / mg, which was only 29% of the wild type. However, Mc-F378S could generate a single isoquercetin product, which improved the regioselectivity of UGT78D2 for the glycosyl acceptor, resulting in the fact that isoquercetin was no longer glycosylated continuously. After 12 h, it could catalyze the synthesis of 1296.10 mg / L of isoquercetin, which was 1.4 times the accumulation amount of isoquercetin in Mc-78D2. And the total yields of quercetin glycosides generated in the two coupling systems were similar. Among them, Mc-78D2 would continue to add glycosyl groups to isoquercetin to generate Q3,7G (497.57 mg / L), while the isoquercetin in Mc-F378S was not further transformed and was thus used to generate Q3,4′G to increase the yield.

[0091] Example 2 Soluble expression modification of glycosyltransferase 73G1_V371A

[0092] (1) Selection of Pd5:

[0093] The three-dimensional structure model of 73G1_V371A was constructed using the YASARA homology modeling method and submitted to the Pross online website, generating 9 Pross mutant design schemes. The soluble expression and stability of these 9 mutants and 73G1_V371A were evaluated. According to the predicted results of the soluble expression and stability of 73G1_V371A and 9 Pross mutants listed in Table 2, Pross mutant 5 (Pd5) with the best ccSOL score and ΔΔG score was considered a potential design with soluble expression and good stability, so Pd5 was selected for experimental verification.

[0094] Table 2 Prediction of solubility and stability of 73G1_V371A and 9 Pross mutants

[0095]

[0096] (2) Recombinant plasmid construction

[0097] The single-point mutants 73G1_V371A of glycosyltransferase UGT73G1 or the Pross mutant of 73G1_V371A were codon-optimized and synthesized, and the sequences were shown as SEQ ID NO.4 and 5 respectively, and then subcloned into the expression vector pCDFDuet-1. The above UGT encoding genes (sequences shown as SEQ ID NO.4 and 5) were inserted between the NdeI and XhoI sites of the expression vector to obtain recombinant plasmids pCDF-V371A and pCDF-Pd5.

[0098] (3) Engineering strain construction

[0099] The recombinant plasmids pCDF-V371A and pCDF-Pd5 were respectively added to the competent cells of Escherichia coli E.coli BL21(DE3). The recombinant plasmids and the competent cells were gently flicked and mixed evenly, and then ice-bathed at 0 °C for 30 min, and then heat-shocked in a 42 °C water bath for 90 s. After ice-bathing for 2 min, 400 μL of antibiotic-free LB liquid medium was added, and the mixture was pipetted and mixed evenly and then placed in a 37 °C shaker for activation for 30 min. 70 μL of the mixture was taken and spread on an LB plate containing kanamycin resistance, and cultured in a 37 °C incubator for 12 h to obtain engineering strains pCDF-V371A and pCDF-Pd5.

[0100] (4) Induced expression of recombinant protein

[0101] Single colonies on the above plate were picked and inoculated into a shake tube containing 5 mL of LB liquid medium (10 g / L NaCl, 5 g / L yeast powder, 10 g / L peptone, made up to volume with distilled water: containing 50 mg / L kanamycin), and activated in a 37 °C, 200 rpm shaker for 12 h as the seed solution. It was transferred to a 100 mL LB shake flask containing the corresponding resistance according to an inoculation amount of 2% (v:v). It was placed in a 37 °C shaker and cultured with shaking at 200 rpm. When the OD 600 reached about 0.6, the temperature was adjusted to 16 °C, and then 0.1 mM IPTG was added for induced culture for 36 h.

[0102] (5) Preparation of crude enzyme solution

[0103] The induced culture broth was frozen and centrifuged (4 °C, 7500 rpm, 5 min) to remove the above culture broth, and the cells were resuspended and washed once with 100 mM potassium phosphate buffer solution (pH 7.2), and the cells were collected by centrifugation at 4 °C, 6000 rpm for 3 min. According to the OD of the above broth 600The cells were resuspended in an appropriate amount of 100 mM potassium phosphate buffer (pH 7.2), and the OD of the resuspension 600 ≤ 100. The mixed cells were placed in an ice bath and disrupted by an ultrasonic crusher with the instrument parameters set as Ф6, 300 W, and 15 min. After disruption, centrifugation was performed at 8000 rpm and 4 °C for 25 min, and the resulting supernatant was the crude enzyme solution, which was stored at 4 °C.

[0104] (6) Protein concentration determination and SDS-PAGE analysis

[0105] The protein concentration was detected according to the Coomassie Brilliant Blue method (Brandford method). The expression of the recombinant protein was analyzed by 12% polyacrylamide gel electrophoresis (SDS-PAGE).

[0106] (7) Enzyme activity determination

[0107] In a 660 μL enzyme activity assay system, 0.5 mM quercetin or isoquercetin, 5 mM UDPG, 3 mM MgCl2, and 0.3 mg / mL of the crude enzyme were added, and the remaining volume was made up with 100 mM potassium phosphate buffer (pH 7.2). For the rest, see 2.3.8.1 for details.

[0108] The enzyme activity was defined as: the amount of enzyme required to convert the substrate (quercetin or isoquercetin) to produce 1 μmol of the corresponding product (isoquercetin or Q3,4′G) within 1 minute was 1 enzyme activity unit (U).

[0109] (8) Analysis of soluble expression results

[0110] As Figure 4 shown by the SDS-PAGE analysis results, the target band in lane 3 was significantly darker than the corresponding band in lane 1, and the inclusion body band in the Pd5 precipitate also became significantly lighter (lane 4). Combining with the gray scale analysis of the Quantity One gel analysis software, it was estimated that the soluble expression level of Pd5 was about 17.6 times higher than that of 73G1_V371A. Therefore, the Pross algorithm provided an effective mutation scheme for improving the soluble expression of heterologous proteins in Escherichia coli.

[0111] Example 3 Selection of expression vectors for McSuSy and UGT78D2 / 78D2_F378S

[0112] (1) Construction of recombinant plasmids

[0113] After codon optimization and synthesis, the glycosyltransferase UGT78D2 (whose sequence is SEQ.NO.7) and the sucrose synthase McSuSy (whose sequence is SEQ.NO.6) were subcloned into the expression vector pETDuet-1. The McSuSy coding gene was inserted between the NcoI and EcoRI sites of the expression vector, and the UGT78D2 coding gene was inserted between the NdeI and XhoI sites to obtain the recombinant plasmid pET-McSuSy-78D2.

[0114] During the construction of pRSF-McSuSy-78D2 and pRSF-McSuSy-V371A, the expression vector was changed to Prsf-Duet-1, and the principle of the remaining construction steps was the same as that of pET-McSuSy-78D2.

[0115] (2) Induced expression of recombinant protein

[0116] Pick a single colony on the above plate and inoculate it into a shaking tube containing 5 mL of liquid medium LB (10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, made up to volume with distilled water: containing 50 mg / L kanamycin). Incubate at 37 °C and 200 rpm in a shaker for 12 h as the seed culture. Transfer it to a 100 mL LB shake flask containing the corresponding antibiotic at an inoculation amount of 2% (v:v). Place it in a shaker at 37 °C and shake at 200 rpm. When the OD 600 reaches about 0.6, adjust the temperature to 16 °C, and then add 0.1 mM IPTG for induced culture for 36 h.

[0117] (3) Preparation of crude enzyme solution

[0118] The induced bacterial solution was subjected to freezing centrifugation (4 °C, 7500 rpm, 5 min) to remove the above culture medium, and the cells were resuspended and rinsed once with 100 mM potassium phosphate buffer (pH 7.2). The cells were collected by centrifugation at 4 °C and 6000 rpm for 3 min. An appropriate amount of 100 mM potassium phosphate buffer (pH 7.2) was added to resuspend the cells according to the OD 600 value of the above bacterial solution, and the OD 600 of the resuspended solution was ≤100. The mixed cells were placed in an ice bath and broken by an ultrasonic crusher with the instrument parameters set as Ф6, 300 W, and 15 min. After breaking, centrifugation was carried out at 8000 rpm and 4 °C for 25 min, and the obtained supernatant was the crude enzyme solution, which was stored at 4 °C.

[0119] (4) Protein concentration determination and SDS-PAGE analysis

[0120] The protein concentration was detected according to the Coomassie brilliant blue method (Brandford method). The expression of the recombinant protein was analyzed using 12% polyacrylamide gel electrophoresis (SDS-PAGE).

[0121] (5) Result analysis

[0122] We constructed the coding genes of McSuSy and UGT78D2 / 78D2_F378S into the pETDuet-1 vector to obtain the engineering strains Mc-78D2(pET) and Mc-F378S(pET). Using the engineering strains Mc-78D2 and Mc-F378S with the vector backbone of pRSFDuet-1 as controls, after preparing the corresponding recombinant enzymes, the enzyme activities and catalytic reactions were compared.

[0123] Table 3 Specific enzyme activities of UGT and SuSy in different expression vectors

[0124]

[0125] As shown in the specific enzyme activity results in Table 3, compared with Mc-78D2, the specific enzyme activity of McSuSy in Mc-78D2(pET) decreased by about 3.7 times, while the enzyme activity of UGT78D2 towards quercetin increased by 19%.

[0126] The results of the catalytic reaction are as Figure 5 shown. The yields of Q3,7G catalyzed by Mc-78D2 and Mc-78D2(pET) are comparable, while the yields of isoquercetin they accumulated are 627.49 mg / L and 379.60 mg / L respectively. When comparing the catalytic results of Mc-F378S and Mc-F378S(pET), the yields of isoquercetin synthesized by the two are 1075.66 mg / L and 876.42 mg / L respectively. It is speculated that after being constructed into the pETDuet-1 expression vector, the decrease in the enzyme activity of McSuSy led to a reduction in the subsequent catalytic reaction products.

[0127] In this study, it was considered that the enzyme activity of 78D2_F378S would affect the synthesis of the intermediate product isoquercetin, thereby affecting the synthesis of the final target product Q3,4′G by 73G1_V371A. Moreover, in the two engineering strains containing the recombinant plasmids pET-McSuSy-F378S and pRSF-McSuSy-V371A or in the strain with co-expression of three enzymes, the total expression level of McSuSy would be higher than that in the Mc-F378S(pET) system, which could reduce its enzyme activity loss to a certain extent. Therefore, Mc-F378S(pET) was selected for further study later.

[0128] Example 4 Effect of phosphorylated mutant enzyme of McSuSy on the catalytic synthesis of quercetin glycosides

[0129] (1) Recombinant plasmid construction

[0130] After codon optimization and synthesis, the glycosyltransferase UGT78D2 and sucrose synthase McSuSy were subcloned into the expression vector pETDuet-1. The McSuSy coding gene was inserted between the NcoI and EcoRI sites of the expression vector, and the UGT78D2 coding gene was inserted between the NdeI and XhoI sites to obtain the recombinant plasmid pET-McSuSy-78D2. When constructing the pRSF-McSuSy-78D2 expression vector with Prsf-Duet-1, the remaining construction steps were the same as those of pET-McSuSy-78D2. When constructing pRSF-McSuSy-V371A, after codon optimization and synthesis, the glycosyltransferase UGT73G1-V371A and sucrose synthase McSuSy were subcloned into the expression vector Prsf-Duet-1. The McSuSy coding gene was inserted between the NcoI and EcoRI sites of the expression vector, and the UGT73G1-V371A coding gene was inserted between the NdeI and XhoI sites. We respectively carried out the mutation of the SuSy phosphorylation site in the two recombinant plasmids of pET-McSuSy-F378S and pRSF-McSuSy-V371A to test the effect of the McSuSy mutant enzyme on the conversion and synthesis of quercetin glycosides. Through phosphorylation site prediction, Ser31 of McSuSy was respectively mutated to Asp and Glu, and mutation primers were designed according to the mutation sites to mutate McSuSy to S31D and S31E. The primer design is shown in Table 4 below:

[0131] Table 4 Primer sequences for the mutation of the McSuSy phosphorylation site

[0132]

[0133] (2) Engineering strain construction

[0134] The above recombinant plasmids were respectively transformed into Escherichia coli BL21(DE3) competent cells to obtain the engineering strains Mc-F378S(pET), S31D-F378S, S31E-F378S, Mc-V371A, S31D-V371A, and S31E-V371A.

[0135] (3) Induced expression of recombinant protein

[0136] The recombinant Escherichia coli was first incubated in 5 mL of LB medium and incubated overnight at 37 °C with continuous shaking at 200 rpm. Then, 2% (v / v) of the overnight culture was cultured in a 100 mL LB medium shake flask containing 50 μg / mL kanamycin at 37 °C for about 2 h. When the culture turbidity (OD 600)When it reaches 0.5 - 0.6, add IPTG with a final concentration of 0.1 mM, and then continue culturing at 16 °C for 36 h.

[0137] (4) Preparation of crude enzyme solution

[0138] Centrifuge the induced bacterial solution by freezing (4 °C, 7500 rpm, 5 min), remove the above-mentioned culture solution, resuspend and wash the bacterial cells once with 100 mM potassium phosphate buffer solution (pH 7.2), centrifuge at 4 °C, 6000 rpm for 3 min to collect the bacterial cells. Resuspend again, place the mixed bacterial cells in an ice bath, and disrupt the cells with an ultrasonic disruptor. Set the instrument parameters as Ф6, 300 W, 15 min. After disruption, centrifuge at 8000 rpm and 4 °C for 25 min. The obtained supernatant is the crude enzyme solution and is stored at 4 °C.

[0139] (5) Protein concentration determination and SDS-PAGE analysis

[0140] Detect the protein concentration according to the Coomassie Brilliant Blue method (Brandford method). Analyze the expression of the recombinant protein using 12% polyacrylamide gel electrophoresis (SDS-PAGE).

[0141] (6) Result analysis

[0142] The specific enzyme activities of the recombinant enzymes in the Mc-F378S(pET) coupling system and its McSuSy mutant enzyme coupling system are shown in Table 4. The specific enzyme activities of the McSuSy mutant enzymes Mc_S31D and Mc_S31E are 38.80 mU / mg and 14.23 mU / mg respectively, which are 4.4 and 1.6 times that of the wild type (8.84 mU / mg). And the specific enzyme activity of 78D2_F378S co-expressed with Mc_S31D for quercetin is 921.92 mU / mg, which is 1.5 times that of the co-expression system with McSuSy; while the specific enzyme activity of 78D2_F378S in S31E-F378S decreases slightly. It can be seen that after mutating Ser31 of SuSy in McSuSy-F378S(pET) to Asp, the sequence is as shown in SEQ ID NO.5, and the enzyme activities of both SuSy and UGT will be improved.

[0143] Table 5 Specific enzyme activities of 78D2_F378S and SuSy in different engineered bacteria

[0144]

[0145] Subsequently, the studies on the synthesis of isoquercetin from quercetin catalyzed by Mc-F378S(pET), S31D-F378S, and S31E-F378S were carried out. In the catalytic reaction system (10 mL), 3 g / L quercetin, 400 g / L sucrose, 2 mM UDP, 3 mM MgCl2, 10% DMSO (v:v), and 10 mg / mL crude enzyme solution were added, and the remaining part was made up with 100 mM potassium phosphate buffer solution (pH 7.2). The catalytic reaction conditions were 35 °C and 200 rpm, and the reaction was carried out for 12–24 h. The results are as Figure 6 shown. After 12 h of reaction, the yields of isoquercetin generated by the coupling systems of S31D-F378S and S31E-F378S were 1083.84 mg / L and 836.42 mg / L in sequence, which were 119% and 92% of Mc-F378S(pET) respectively, and this was positively correlated with the enzyme activity of 78D2_F378S in the three coupling systems. Due to the advantages of the enzyme activity of the recombinant enzyme and the yield of isoquercetin in S31D-F378S, it was selected for subsequent research.

[0146] In the engineered strains co-expressing Mc-V371A and 73G1_V371A with the McSuSy mutant enzyme, the specific enzyme activities of the two SuSy mutants ( Figure 7 ) showed a similar improvement phenomenon as described above. The specific enzyme activities of Mc_S31D and Mc_S31E were 4.5 times and 1.8 times that of the wild type respectively. In addition, compared with the enzyme activity of 73G1_V371A in Mc-V371A, the enzyme activities of 73G1_V371A in S31D-V371A and S31E-V371A towards quercetin were similar, however, the enzyme activities towards isoquercetin decreased by 40% and 60% respectively. Therefore, after phosphorylating the mutation of SuSy in the Mc-V371A coupling system, although the enzyme activity of SuSy was improved, to a certain extent, the expression of 73G1_V371A was affected.

[0147] The catalytic reaction results of the three coupling systems of Mc-V371A, S31D-V371A, and S31E-V371A are as Figure 8 shown. The yields of Q3,4′G catalyzed by S31D-V371A and S31E-V371A were 744.77 mg / L and 524.29 mg / L respectively, which were 131% and 96% of Mc-V371A respectively. In addition, in terms of the yield of isoquercetin, S31E-V371A was only slightly higher than the other two groups. In summary, S31D-V371A with better yield of Q3,4′G was selected for subsequent research.

[0148] Example 5 Effects of Different Enzyme Production Methods on the Catalytic Synthesis of Q3,4′G

[0149] Construction of the Dual-Bacteria and Three-Enzyme System:

[0150] The double - bacterium and triple - enzyme system refers to glycosyltransferase UGT78D2 and sucrose synthase McSuSy. After codon optimization and synthesis, they are sub - cloned into the expression vector pETDuet - 1. The McSuSy - encoding gene is inserted between the NcoI and EcoRI sites of the expression vector, and the UGT78D2 - encoding gene is inserted between the NdeI and XhoI sites to obtain the recombinant plasmid pET - McSuSy - 78D2. Then, primers are designed according to the sequences near the mutation sites and mutated into pET - S31D - F378S respectively. The construction of pET - S31D - V371A is the same. The primer design is shown in Table 6 below:

[0151] Table 6 Primer sequences of the mutations

[0152] Table 6Primer sequences of the mutations

[0153]

[0154] The triple - enzyme co - expression system uses the CaCl2 method to prepare the engineered strain S31D - F378S into E. coli BL21(DE3)_S31D - F378S competent cells, and then transforms the recombinant plasmid pRSF - S31D - V371A into the competent cells to obtain the engineered strain S31D - F378S - V371A. Single colonies on the above - mentioned plate are picked and inoculated into a shaking tube containing 5 mL of liquid medium (LB containing 50 mg / L kanamycin), and activated in a shaker at 37 °C and 200 rpm for 12 h as the seed solution. It is transferred to a 100 - mL LB shake - flask containing the corresponding resistance according to an inoculation amount of 2% (v:v) for induction. The induced bacterial solution is frozen, centrifuged, and disrupted, and the obtained supernatant is the crude enzyme solution.

[0155] The reactions of the double - bacterium and triple - enzyme system (S31D - F378S&S31D - V371A) and the triple - enzyme co - expression system (S31D - F378S - V371A) catalyzing quercetin were compared. In the catalytic reaction system (10 mL), 3 g / L quercetin, 400 g / L sucrose, 2 mM UDP, 3 mM MgCl2, 10% DMSO (v:v), and 10 mg / mL crude enzyme solution were added, and the remaining part was made up with 100 mM potassium phosphate buffer solution (pH 7.2). The catalytic reaction conditions were 35 °C and 200 rpm, and the reaction was carried out for 12 - 24 h. The results are as Figure 9As shown, after 12 h of reaction, the yields of Q3,4′G and isoquercetin synthesized by the two systems were similar, and the concentrations of Q3,4′G were 733.14 mg / L and 789.55 mg / L, respectively. However, compared with S31D-V371A that only produced a small amount of non-target diglycoside Q3,7G in the same time, both the S31D-F378S-V371A catalytic system and the S31D-F378S&S31D-V371A catalytic system produced Q3,7G products with a relatively high proportion of total glycosides (about 300 mg / L). It is speculated that under the condition of higher sucrose concentration, 78D2_F378S can rapidly synthesize more isoquercetin in a short time. When the product accumulates to a higher concentration, although 73G1_V371A will glycosylate the 4′-OH of isoquercetin more, glycosylation of 7-OH will also occur. Since the yields of Q3,4′G synthesized by the above catalytic systems are similar, and the enzyme production operation of the engineered bacterium with co-expression of three enzymes is relatively simple, the S31D-F378S-V371A system was selected for optimization research subsequently.

[0156] Example 6 studied the factors affecting the synthesis of Q3,4′G in the S31D-F378S-V371A system

[0157] (1) Effect of quercetin concentration on the catalytic synthesis of Q3,4′G

[0158] The catalytic reaction was carried out under the conditions of 400 g / L sucrose, 10% DMSO (v:v), and the addition amount of crude enzyme solution of 10 mg / ml. Different concentrations of quercetin (3 g / L, 10 g / L, 30 g / L, 50 g / L) were added and reacted for 12 h, and the rest was the same as in Example 1(7).

[0159] The results are as Figure 10 , with the increase of the initial quercetin concentration, the yields of the four quercetin glycosides all seemed to show a linear increase, indicating that in the presence of a high concentration of quercetin glycosyl acceptor, UDPG regenerated by Mc_S31D can be quickly supplied, continuously promoting the formation of quercetin glycoside derivatives.

[0160] Except for the reaction system with 50 g / L quercetin added, in other reaction systems, the accumulation amounts of quercetin glycosides were: Q3,4′G > Q3,7G > quercetin triglycoside > isoquercetin. At these four initial quercetin concentrations, after 12 h of reaction, the yields of Q3,4′G were 789.55, 1369.53, 2286.24, and 2916.71 mg / L, respectively, accounting for 49%, 46%, 35%, and 29% of the total quercetin glycoside yield. Considering the yield of Q3,4′G and its proportion in the total glycosides comprehensively, the initial quercetin concentration was set to 10 g / L.

[0161] (2) Influence of Substrate Ratio on the Catalytic Synthesis of Q3,4′G

[0162] The catalytic reaction was carried out under the conditions of 10 g / L quercetin, 10% DMSO (v:v), and the addition amount of crude enzyme solution was 10 mg / ml. The reaction was carried out for 12 h with different quercetin / sucrose concentration ratios (1:3, 1:5, 1:10, 1:20, 1:30, 1:40), and the rest was the same as in Example 1(7).

[0163] The results are as Figure 11 shown. It was found that under the conditions of each substrate concentration ratio, by-products Q3,7G and quercetin trisaccharide with a relatively high proportion of total glycosides were generated, but their concentrations tended to be stable when the substrate ratio was 1:20–1:40. When the concentration ratio of substrate quercetin to sucrose was 1:3–1:5, the yields of total quercetin glycosides and Q3,4′G in the catalytic synthesis were similar, about 1.3 g / L and 0.5 g / L respectively. With the increase of sucrose concentration, the yields of total quercetin glycosides and Q3,4′G also increased. When the substrate ratio was 1:30, the yield of Q3,4′G was 1524.92 mg / L, which was 3.1 times that of the substrate ratio of 1:3. And at this substrate ratio, the proportion of total glycosides of Q3,4′G was the highest (49%). However, when the substrate ratio was 1:40, the yields of total glycosides and Q3,4′G both decreased probably because high-concentration fructose inhibited the reaction. Therefore, the optimal concentration ratio of substrate quercetin to sucrose was 1:30.

[0164] (3) Influence of DMSO Concentration on the Catalytic Synthesis of Q3,4′G

[0165] The catalytic reaction was carried out under the conditions of 10 g / L quercetin and 300 g / L sucrose, and different concentrations of DMSO (10%, 20%) were added respectively for 30 h, and the rest was the same as in Example 1(7).

[0166] The results are as Figure 12 shown. Within 2–12 h of the reaction, the total amounts of quercetin glycosides generated in the two DMSO addition systems were similar, and the concentration of Q3,4′G showed a slow increase. In the addition system of 20% DMSO, more isoquercetin and quercetin trisaccharide were accumulated, and the concentration of Q3,4′G was slightly lower than that in the 10% DMSO system.

[0167] When the reaction proceeded for 12–24 h, different from the reaction system with 10% DMSO added, in the 20% DMSO system, the concentrations of Q3,4′G and the trisaccharide still increased linearly. And at 24 h, the yield of Q3,4′G was the highest, reaching 4.4 ± 0.03 g / L (the conversion rate of quercetin was 79.75%). In the 10% DMSO system, due to the low solubility of quercetin, the concentration level of the intermediate isoquercetin was also low, which affected the continuous production of Q3,4′G. However, after the reaction proceeded for 24 h, the accumulation of Q3,4′G in both systems decreased, probably due to the continued glycosylation of Q3,4′G and the decrease in the enzyme activity of the recombinant enzyme caused by the long-term extracellular environment. It can be seen that adding 20% DMSO can effectively promote the continuous production of Q3,4′G within 24 h.

[0168] The optimized reaction conditions were obtained as follows: the initial concentration of quercetin was 10 g / L, the concentration ratio of quercetin to sucrose was 1:30, and the DMSO concentration was 20%. Under these conditions, the reaction was carried out at 45 °C for 24 h, and the conversion synthesis of Q3,4′G was 4.4 ± 0.03 g / L, the yield of Q3,4′G was 44%, and the conversion rate of quercetin was 99.95%. This experiment greatly improved the conversion rate of quercetin, increased the yield of Q3,4′G, and also generated some beneficial by-products Q3,7G.

Claims

1. A method for synthesizing quercetin-3,4'- O -diglucoside, characterized in that, Using quercetin as the sugar receptor, sucrose as the sugar donor, and a buffer solution with a pH of 7.0 - 8.0 as the reaction medium, under the catalytic action of the glycosyltransferase UGT78D2 mutant, the glycosyltransferase UGT73G1 mutant, and the coupled sucrose synthase mutant, quercetin-3,4'- O -diglucoside is synthesized; the sequence of the glycosyltransferase UGT78D2 mutant is shown in SEQ ID NO.1; the sequence of the sucrose synthase mutant is shown in SEQ ID NO.3; the sequence of the glycosyltransferase UGT73G1 mutant is shown in SEQ ID NO.5; the reaction medium also includes DMSO, UDP, and MgCl2.

2. The synthesis method of quercetin-3,4'- O -diglucoside according to claim 1, characterized in that In the said reaction, the concentration ratio of quercetin to sucrose is 1:(3 - 40), the concentration of quercetin is 3 - 50 g / L, the concentration of the crude enzyme solution is 0.3 - 10 mg / mL, the reaction temperature is 35 - 45 °C, and the reaction time is 2 - 30 h.

3. The synthesis method of quercetin-3,4'- O -diglucoside according to claim 2, characterized in that The said crude enzyme solution is obtained by co-expressing the glycosyltransferase UGT78D2 mutant, the glycosyltransferase UGT73G1 mutant, and the coupled sucrose synthase mutant in pETDuet-1 to obtain the crude enzyme solution.

4. A glycosyltransferase UGT78D2 mutant 78D2_F378S, whose sequence is shown in SEQ ID NO.

1.

5. A sucrose synthase mutant Mc_S31D, whose sequence is shown in SEQ ID NO.

3.

6. A glycosyltransferase UGT73G1 mutant Pd5, whose sequence is shown in SEQ ID NO.

5.

7. Use of the glycosyltransferase UGT78D2 mutant 78D2_F378S according to claim 4, the sucrose synthase mutant Mc_S31D according to claim 5, and the glycosyltransferase UGT73G1 mutant Pd5 according to claim 6 in the synthesis of quercetin-3,4'- O -diglucoside.

8. The application according to claim 7, wherein The three enzymes can synthesize quercetin-3,4'- O -diglucoside alone or in combination. The combination is to construct a dual-bacteria three-enzyme system S31D-F378S&S31D-V371A or a three-enzyme co-expression system S31D-F378S-V371A to catalyze the reaction of quercetin.

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