Mutant of flavone-5-O-glycosyl transferase ZmDo and application thereof

Abstract

The invention discloses a mutant of flavone-5-O-glycosyl transferase ZmDo and application of the mutant of flavone-5-O-glycosyl transferase ZmDo. Glucose donor preference key amino acid site mutation is carried out on flavone-5-O-glycosyl transferase ZmDo obtained by structural domain recombination, namely, the 142 amino acid T is mutated into V. According to the novel flavone-5-O-glycosyltransferase mutant, the glucose donor preference is obviously changed, luteolin can be specifically converted into luteolin 5-O-xyloside, compared with a wild type, the conversion rate of the generated luteolin 5-O-xyloside is reduced to 30%, the conversion rate of the generated luteolin 5-O-xyloside is increased by more than 3 times, and the novel flavone-5-O-glycosyltransferase mutant has the advantages of high yield, high yield and the like. The yield under suitable conditions can reach 4450 mg / L, and the conversion efficiency reaches 90%. The novel xylose glycosyl transferase obtained by the invention is strong in specificity and high in directional conversion efficiency, and a non-natural flavone glycoside compound luteolin 5-O-xyloside and a preparation process thereof are created.

Description

Technical Field

[0001] The invention relates to a mutant of flavonoid-5-O-glycosyltransferase ZmDo and application thereof, belonging to the field of flavonoid-5-O-glycosyltransferase. Background Art

[0002] Glycosyltransferases (GTs, EC 2.4.xy) are enzyme systems that specifically catalyze the activation of sugar donors, allowing them to precisely attach to different acceptor molecules, thereby generating glycosylated products. These enzymes have a wide range of substrates, including sugars, proteins, lipids, flavonoids, terpenes, steroids, and other bioactive substances. Among the numerous glycosyltransferases, uridine diphosphate glycosyltransferases (UDP-glycosyltransferases, UGTs)—glycosyltransferases that utilize sugars activated by uridine diphosphate (UDP) as glycosyl donors—are the largest superfamily of glycosyltransferases in plants. Notably, many glycosyltransferases closely related to plant hormone regulation and glycosylation of secondary metabolites belong to this superfamily. Glycosyltransferases can be further divided into subclasses based on the specific glycosylation site on the acceptor molecule, including O-, N-, C-, and S-glycosyltransferases. Among them, O-glycosyltransferases play a particularly critical role in nature. They are the main enzymes for synthesizing natural flavonoid glycosides.

[0003] Flavonoid glycosides are diverse in type. The same flavonoid aglycone can be attached to various types and numbers of glycosyl donors at different substitution positions. During biosynthesis, these glycosylation modifications are catalyzed by a variety of glycosyltransferases and sugar donors. Common sugar donors include UDP-glucose, UDP-glucuronic acid, UDP-xylose, UDP-rhamnose, UDP-arabinose, UDP-galactose, UDP-galacturonic acid, and UDP-mannose. UDP-glucose is the most widely studied sugar donor for flavonoid glycosylation. However, diverse glycosyl moieties facilitate the construction of diverse flavonoid-O-glycoside structures and hold promise for the discovery of flavonoids with enhanced bioactivity. For example, puerarin 6″-O-xyloside exhibits superior antitumor activity compared to puerarin glucoside; similarly, quercetin-3-O-β-D-xyloside exhibits significantly superior antioxidant activity compared to quercetin-3-O-glucoside.

[0004] Apigenin, a natural flavonoid widely distributed in plants such as the Thymelaeaceae, Verbenaceae, and Selaginellaceae, exhibits diverse pharmacological activities, including antitumor, cardiovascular, antiviral, and antibacterial properties. Compared to other flavonoids, such as quercetin and kaempferol, luteolin has attracted considerable attention due to its low toxicity and lack of mutagenicity. However, luteolin's low water solubility and poor oral bioavailability significantly limit its extensive application in animal pharmacology research and clinical practice. Glycosylation modification is an effective means of improving luteolin's bioavailability. However, common glycosyltransferases have low catalytic efficiency and poor conversion capacity, and most are limited to catalyzing UDP-glucose. This significantly limits the expansion of luteolin glycosides and the improvement of glycosylation efficiency. Consequently, research and development efforts are generally focused on identifying and screening new glycosyltransferases in the hope of overcoming the bottlenecks of existing technologies, but achieving the desired results has been difficult. With the development of modern biotechnology and bioinformatics, precise and targeted modification of glucosyltransferases with excellent properties or potential transxylosylation capabilities is expected to change the sugar donor preference of glycosyltransferases, thereby creating new glycosyltransferases with strong specificity, which is of great significance for promoting the development and application of luteolin and its related flavonoids. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a new mutant of flavonoid-5-O-glycosyltransferase ZmDo that can efficiently convert luteolin into luteolin 5-O-xyloside and its application.

[0006] Technical solution: To solve the above technical problems, the present invention provides a mutant of flavonoid-5-O-glycosyltransferase ZmDo, wherein the mutant is obtained by replacing the threonine at position 142 of the wild-type flavonoid-5-O-glycosyltransferase ZmDo with valine; the amino acid sequence of the wild-type flavonoid-5-O-glycosyltransferase ZmDo is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding a mutant of the flavonoid-5-O-glycosyltransferase ZmDo.

[0008] Among them, its nucleotide sequence is shown as SEQ ID NO.3.

[0009] The present invention also provides a method for constructing the glycosyltransferase mutant ZmDo-T142V, comprising the following steps: using inverse PCR with the recombinant plasmid pET-28a-ZmDo as a template, mutating the threonine at position 142 to valine to obtain the recombinant plasmid pET-28a-ZmDo-T142V; the nucleotide sequence of the recombinant plasmid pET-28a-ZmDo-T142V is shown in SEQ ID NO.3; the recombinant plasmid pET-28a-ZmDo is synthesized by synthesizing the wild-type glycosyltransferase gene ZmDo between the BamH I and Xho I cloning sites in pET28a.

[0010] The present invention also provides a recombinant plasmid, a recombinant cell or a recombinant bacterium containing the gene.

[0011] The host bacteria includes one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and Bacillus subtilis. Preferably, the host bacteria is Escherichia coli.

[0012] The present invention also provides the use of the mutant, gene or recombinant plasmid, recombinant cell or recombinant bacteria of the flavonoid-5-O-glycosyltransferase ZmDo in the production of luteolin 5-O-xyloside.

[0013] The present invention also provides the use of the mutant, gene or recombinant plasmid, recombinant cell or recombinant bacteria of the flavonoid-5-O-glycosyltransferase ZmDo in improving the yield of converting luteolin to luteolin 5-O-xyloside.

[0014] Wherein, the mutant of flavonoid-5-O-glycosyltransferase ZmDo is purified by imidazole.

[0015] Wherein, the concentration of the imidazole is 0~50 mM.

[0016] Preferably, the concentration of the imidazole is 20-50 mM.

[0017] The present invention also provides a method for producing luteolin 5-O-xyloside or improving the yield of luteolin converted into luteolin 5-O-xyloside, comprising the following steps: adding IPTG to the cultured recombinant bacteria, inducing, adding cellobiose and luteolin; and conducting shaker fermentation culture.

[0018] The concentration of IPTG is 0.01-1 mM, the induction temperature is 16-37° C., the fermentation temperature is 20-40° C., the concentration of luteolin is 2.5-3.5 g / L, and the concentration of cellobiose is 0-20 g / L.

[0019] Preferably, the concentration of IPTG is 0.01-0.5 mM, the induction temperature is 16-30° C., the fermentation temperature is 25-40° C., the concentration of luteolin is 2.75-3.5 g / L, and the concentration of cellobiose is 5-20 g / L.

[0020] Preferably, the concentration of IPTG is 0.01-0.25 mM, the induction temperature is 20-30° C., the fermentation temperature is 30-40° C., the concentration of luteolin is 3-3.5 g / L, and the concentration of cellobiose is 10-20 g / L.

[0021] Preferably, the concentration of IPTG is 0.01-0.1 mM, the induction temperature is 25-30° C., the fermentation temperature is 30-35° C., the concentration of luteolin is 3-3.25 g / L, and the concentration of cellobiose is 10-15 g / L.

[0022] Preferably, the concentration of IPTG is 0.01-0.05 mM.

[0023] Preferably, the concentration of IPTG is 0.025~0.05 mM.

[0024] Wherein, the fermentation culture time is 12~72 hours.

[0025] Preferably, the fermentation culture time is 12 to 60 hours.

[0026] Preferably, the fermentation culture time is 12 to 48 hours.

[0027] Based on ZmDo, the present invention mutated the T at position 142 to V, successfully altering its sugar donor preference. This mutant can specifically convert luteolin to luteolin-5-O-xyloside. Compared with the original enzyme, the conversion rate to luteolin-5-O-glucoside was reduced to 30%, while the conversion rate to luteolin-5-O-xyloside was increased by more than threefold. This provides a theoretical basis for the analysis of key sites in the sugar donor preference of GT1 family glycosyltransferases. The present invention established an enzymatic conversion of luteolin to luteolin-5-O-xyloside with high conversion efficiency. After 48 hours of shaker fermentation, the recombinant bacteria achieved a conversion rate of greater than 90% to luteolin-5-O-xyloside, with a yield of 4450 mg / L.

[0028] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: 1. The glycosyltransferase ZmDo is mutated at the key site of glycosyl donor preference, and the mutant is used to efficiently convert luteolin to produce luteolin 5-O-xyloside; the glycosyltransferase mutant ZmDo-T142V obtained by the present invention has good activity, and the enzyme activity is highest under the conditions of 30°C and pH 9.5; the enzyme has high enzyme activity in the temperature range of 20~40°C and pH 8.0-9.5. 2. The glycosyltransferase mutant ZmDo-T142V obtained by the present invention significantly changes its sugar donor preference, converting from UDP-glucose to UDP-xylose. It is highly specific and can efficiently and specifically convert luteolin to luteolin 5-O-xyloside. The conversion rate of luteolin-5-O-glucoside is reduced to 30%, while the conversion rate of luteolin-5-O-xyloside is increased by more than 3 times. Under suitable conditions, the yield can reach 4450 mg / L, with a conversion efficiency of over 90%. 3. The novel xylose glycosyltransferase obtained by the present invention has strong specificity and high directional conversion efficiency. It also creates a non-natural flavonoid glycoside compound, luteolin 5-O-xyloside, and its preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the SDS-PAGE image of glycosyltransferase ZmDo; Figure 2 is the SDS-PAGE image of glycosyltransferase ZmDo-T142V; Figure 3 The enzymatic properties of glycosyltransferase ZmDo-T142V are shown in Figure 1 (A. Optimum temperature; B. Optimum pH); Figure 4 This is a comparative diagram of the liquid phase analysis of glycosyltransferase ZmDo and its mutant ZmDo-T142V; Figure 5 Optimization diagram of expression conditions for the glycosyltransferase mutant ZmDo-T142V (A. Optimal IPTG concentration; B. Induction temperature; C. Fermentation temperature; D. Cellobiose concentration; E. Substrate concentration); Figure 6 This is the reaction curve of luteolin conversion by the glycosyltransferase mutant ZmDo-T142V over time. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0032] The DNA polymerase (Prime STAR HS DNA Polymerase), deoxyribonucleic acid (dNTP), ligase (T4 DNA ligase), phosphorylase (T4 Polynucleotide Kinase) and related buffers used in the examples were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. (TaKaRa).

[0033] Example 1. Cloning, expression and enzyme activity determination of glycosyltransferase ZmDo gene 1. Cloning and plasmid construction of glycosyltransferase gene ZmDo

[0034] The gene of flavonoid-5-O-glycosyltransferase ZmDo (the amino acid sequence of the encoded protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2) was synthesized in the pET28a plasmid. BamH I and Xho

[0035] 2. Expression and purification of glycosyltransferase ZmDo

[0036] 1 μL of the recombinant plasmid pET28a-ZmDo solution was added to Escherichia coli BL21 (DE3) competent cells and mixed. The cells were placed on ice for 10 min, incubated in a 42°C water bath for 2 min, and then placed on ice for 5 min. 1 mL of SOC medium was added to the cells and cultured at 37°C for 1 h. The cells were then spread on LB plates containing kanamycin (the final concentration of kanamycin was 50 mg / L) to screen for transformants. Several single colonies were picked and cultured in LB liquid medium (with kanamycin added to a final concentration of 50 mg / L) for 8-10 h. 1 mL of the bacterial solution was then taken for sequencing to confirm that the Escherichia coli BL21 (DE3) strain contained the pET28a-ZmDo recombinant plasmid.

[0037] The recombinant E. coli was streaked onto an LB plate containing kanamycin (the final concentration of kanamycin was 50 mg / L) for activation and cultured at 37°C for 12 h. After a single colony grew, it was inoculated into 50 mL of LB liquid medium and cultured at 37°C with a shaker at 180 rpm for 3-4 h until the OD 600 =0.4-0.6, IPTG was added to induce the expression at a final concentration of 0.05 mM, and expression was continued at 20°C for 7-8 hours. Escherichia coli BL21(DE3) containing the empty plasmid pET28a was also used as a negative control. 1 mL of bacterial suspension was aspirated and centrifuged at 5000 rpm for 3 minutes to collect the cells. The supernatant was discarded. The cells were washed twice with 1 mL of PBS buffer, resuspended in 500 µL of PBS, and sonicated for 50 cycles of 3 s, 5 s intervals. After centrifugation at 12000 rpm for 10 minutes, 15 µL of supernatant was aspirated and set aside. The crude enzyme solution was filtered through a 0.22 μm filter and purified using a Ni affinity column to obtain the purified glycosyltransferase ZmDo. Take 15 μL of crude enzyme solution expressed by empty transformants without ZmDo gene, crude enzyme solution expressed by transformants with ZmDo gene, and glycosyltransferase ZmDo eluted with 20, 50, 100, 200, or 400 mM imidazole solution, add 15 μL of 5×SDS loading buffer, mix well, boil for 5 min, and take 10 μL of each for SDS-PAGE electrophoresis to identify the expressed protein ( Figure 1 The results showed that when different concentrations of imidazole were used for elution, when the imidazole concentration was controlled within the range of 50 mM, high-purity glycosyltransferase ZmDo could be obtained, and its electrophoresis pattern showed a single protein band. Figure 1Middle bands 1-8 are marker, crude enzyme solution, protein after passing through Ni-NTA column, protein eluted with 20 mM imidazole, protein eluted with 50 mM imidazole, protein eluted with 100 mM imidazole, protein eluted with 200 mM imidazole, and protein eluted with 400 mM imidazole.

[0038] 3. Determination of glycosyltransferase ZmDo activity

[0039] Fifty micrograms of crude enzyme expressed from the transformant containing the ZmDo gene obtained above was reacted with 0.5 mM luteolin as a substrate at 35°C, pH 9.5 for 30 min. The reaction was terminated with 400 μL of methanol. The product and substrate contents were measured at a wavelength of 340 nm using HPLC (1260 system (Agilent, USA) on an Xbridge C18 reversed-phase column (5 μm, 250 cm × 4.6 nm), with a mobile phase consisting of methanol (A) and ultrapure water containing 0.1% formic acid (B) in a ratio of 55:45, a flow rate of 0.8 mL / min, a column temperature of 30°C, and an injection volume of 10 μL). The enzyme activity was calculated to be 800 mU / mg.

[0040] The reaction system is 200 μL, and the proportions of each substance are shown in Table 1: Table 1 Glycosyltransferase activity assay system

[0041] The enzyme activity unit (U) is defined as the amount of enzyme required to synthesize 1 μmol of the product luteolin-5-O-glucoside per minute under the optimal temperature and pH conditions for the enzyme.

[0042] Example 2. Determination of the mutation site of glycosyltransferase ZmDo and construction of its recombinant plasmid

[0043] Research has shown that the catalytic activity of glycosyltransferases is closely linked to key sites surrounding the active pocket within the protein structure. Homology modeling revealed the three-dimensional structure of the glycosyltransferase ZmDo. Molecular docking was used to identify mutation sites for investigation. Saturation mutagenesis of site T142V ultimately identified the mutation as ZmDo-T142V.

[0044] Mutation primer design: Using bioinformatics analysis and homology modeling, we altered some amino acids in the glycosyltransferase ZmDo molecule to design mutation sites. Mutation sequences were obtained using inverse PCR, and two forward and reverse oligonucleotide sequences were designed for each mutation site. Mutation sites used codons preferred by E. coli. Mutation primers are listed in Table 2.

[0045] Table 2 Site-directed mutagenesis primers

[0046] Recombinant plasmid pET-28a -ZmDo Using the mutant gene as a template, T142 was further mutated by inverse PCR to obtain the recombinant plasmid pET-28a-ZmDo-T142V carrying the mutant gene.

[0047] Table 3 Preparation of inverse PCR reaction solution (50 μL in total)

[0048] Table 4 Reaction conditions for inverse PCR

[0049] The 5' end phosphorylation reaction of PCR fragments was prepared as shown in Table 5 and the reaction was carried out at 37°C for 1 h.

[0050] Table 5 Preparation of phosphorylation reaction solution

[0051] After phosphorylation at 37°C, 1 μL of T4 ligase was added and ligation was carried out at 16°C for 12 h. Plasmid DNA was used for transformation, and mutants were selected and identified by sequencing. The nucleotide sequence of ZmDo-T142V is shown in SEQ ID NO. 3.

[0052] Example 3. Expression and purification of glycosyltransferase ZmDo mutants and enzyme activity determination 1. Expression and purification of glycosyltransferase ZmDo-T142V

[0053] 1 μL of the recombinant plasmid pET28a-ZmDo-T142V was added to the competent Escherichia coli BL21 (DE3) cells, mixed well, and placed on ice for 10 min. The cells were incubated in a 42°C water bath for 2 min and then placed on ice for 5 min. 1 mL of SOC medium was added to the cells, and the cells were cultured at 37°C for 1 h. The cells were then spread on LB plates containing kanamycin (the final concentration of kanamycin was 50 mg / L) to screen for transformants. Several single colonies were picked and cultured in LB liquid medium (with kanamycin added to a final concentration of 50 mg / L) for 12 h. 1 mL of the bacterial solution was then taken for sequencing to confirm the Escherichia coli BL21 (DE3) strain containing the pET28a-ZmDo-T142V recombinant plasmid.

[0054] The recombinant Escherichia coli BL21 (DE3) was streaked onto LB plates (final concentration of kanamycin was 50 mg / L) for activation and cultured at 37°C for 12 h until a single colony grew. The colony was then inoculated into 50 mL LB liquid medium and cultured at 37°C with a shaker at 180 rpm for 3-4 h until the OD 600= 0.6-0.8, IPTG was added to induce the expression at a final concentration of 0.01 mM, and expression was continued at 20°C for 12 h. Escherichia coli BL21(DE3) containing the empty plasmid pET28a was also used as a negative control. 1 mL of bacterial culture was aspirated and centrifuged at 12,000 rpm for 3 min to collect the cells. The supernatant was discarded, and the cells were washed twice with 1 mL of PBS buffer, resuspended in 500 µL of PBS, and lysed by sonication. The working conditions were: 3 s for 50 cycles with 4 s intervals. Centrifuge at 12000 r / min for 10 minutes, aspirate 15 μL of supernatant for later use, and filter the remaining crude enzyme solution through a 0.22 μm filter membrane. Purify the remaining crude enzyme solution using a Ni-NTA affinity column to obtain the purified glycosyltransferase ZmDo-T142V. Take 30 μL of each of the transformant expressing whole cells containing the ZmDo-T142V gene, the crude enzyme solution expressed by the transformant containing the ZmDo-T142V gene, and the purified glycosyltransferase ZmDo-T142V eluted with different concentrations of imidazole, add 6 μL of 5×SDS loading buffer, mix well, boil for 5 minutes, and take 10 μL of each for SDS-PAGE electrophoresis to identify the expressed protein. Figure 2 As shown, using different concentrations of imidazole for elution, when the imidazole concentration is controlled within the range of 50mM, high-purity glycosyltransferase ZmDo-T142V can be obtained, and its electrophoresis pattern shows a single protein band. Figure 2 The middle band M is a marker; bands 1-7 are crude enzyme solution; protein after passing through the Ni-NTA column; protein eluted with 20mM imidazole; protein eluted with 50mM imidazole; protein eluted with 100mM imidazole; protein eluted with 200mM imidazole; protein eluted with 400mM imidazole.

[0055] 2. Determination of enzyme activity of glycosyltransferase ZmDo-T142V

[0056] 50 μg of crude enzyme solution expressed by the transformant containing the ZmDo-T142V gene was taken and reacted with 0.5 mM luteolin as a substrate at 30°C and pH 9.5 for 30 min. The reaction was terminated with 400 μL of methanol. The product and substrate contents were detected by HPLC (detection conditions were the same as those for the enzyme activity determination of glycosyltransferase ZmDo) at a wavelength of 340 nm, and the enzyme activity was calculated to be 650 mU / mg.

[0057] The reaction system was 100 μL, and the proportions of each substance were shown in Table 6.

[0058] Table 6 Glycosyltransferase activity assay system

[0059] The enzyme activity unit (U) is defined as the amount of enzyme required to synthesize 1 μmol of the product luteolin-5-O-xyloside per minute under the optimal temperature and pH conditions for the enzyme.

[0060] Example 4. Enzymatic Properties Analysis of Glycosyltransferase ZmDo Mutants 1. Determination of the optimal reaction pH of the mutant enzyme ZmDo-T142V

[0061] The enzyme activity of the mutant enzyme ZmDo-T142V was measured in KH2PO4-Na2HPO4 buffer solutions at pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, and glycine-NaOH solutions at pH 8.0, 8.5, 9.0, 9.5, and 10.0, respectively, in a 35°C water bath according to the standard enzyme activity assay method. The highest enzyme activity value measured was taken as 100%, and the relative enzyme activity of the mutant enzyme ZmDo-T142V at each pH was calculated to determine the effect of reaction pH on the activity of the mutant enzyme ZmDo-T142V. The pH of 9.5, at which the enzyme activity reached its highest, was the optimal reaction pH for ZmDo-T142V ( Figure 3 B).

[0062] 2. Determination of the optimal reaction temperature of the mutant enzyme ZmDo-T142V

[0063] The enzyme activity of the mutant enzyme ZmDo-T142V was measured in a water bath at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C at pH 9.0 according to the standard enzyme activity assay method. The highest enzyme activity measured was taken as 100%, and the relative enzyme activity of the mutant enzyme ZmDo-T142V at each reaction temperature was calculated to determine the effect of reaction temperature on the activity of the mutant enzyme ZmDo-T142V. The reaction temperature of 30°C, at which the enzyme activity reached the highest, was the optimal reaction temperature for the recombinant enzyme ZmDo ( Figure 3 A).

[0064] Example 5. Determination of the performance of glycosyltransferase ZmDo and its mutants in converting luteolin in vitro

[0065] Luteolin and its glycosylated derivatives were detected using a high-performance liquid chromatography (HPLC) 1260 system (Agilent, USA) using an Xbridge C18 reversed-phase column (5 µm, 250 cm × 4.6 nm). The mobile phases were methanol (A) and ultrapure water containing 0.1% formic acid (B), with an A:B ratio of 55:45. The flow rate was 0.8 mL / min. The detection wavelength was 340 nm. The column temperature was 30°C. The injection volume was 10 µL.

[0066] The luteolin concentration was 0.5 mM, and conversion was carried out using ZmDo and its mutants at 30°C and pH 9.5 for 0.5 h (reaction systems are shown in Tables 1 and 6, respectively). HPLC analysis showed that after 0.5 h, the conversion rate of luteolin to luteolin-5-O-xyloside by ZmDo-T142V was 60%. The conversion rate of luteolin to luteolin-5-O-xyloside by ZmDo was 20%.

[0067] Example 6. Application of glycosyltransferase ZmDo and its mutants in the conversion of luteolin

[0068] The gene of cellobiose synthase Cep (nucleotide sequence is shown in SEQ ID NO.4, enzyme cleavage site is BamH I and EcoR I) and uridine diphosphotransferase UgpA genes (nucleotide sequence is shown in SEQ ID NO.5, enzyme cleavage site is Nde I and Xho I) was synthesized on the pACYCDuet plasmid to obtain the recombinant plasmid pACYCDuet-Cep-UgpA. The gene of xylose synthase Peuxs1 (nucleotide sequence shown in SEQ ID NO.6, restriction site is BamH I and EcoR I) and glucose dehydrogenase Ecugd genes (nucleotide sequence shown in SEQ ID NO.7, enzyme cleavage site is Nde I and Xho I) was synthesized on the pCDFDuet plasmid to obtain the recombinant plasmid pCDFDuet-Peuxs1-Ecugd.

[0069] To determine the effect of glycosyltransferase ZmDo and its mutants on sugar donor preference, the recombinant plasmids pACYCDuet-Cep-UgpA, pCDFDuet-Peuxs1-Ecugd and the glycosyltransferase recombinant plasmid pET28a-ZmDo or mutant plasmid pET28a-ZmDo-T142V were co-transformed into Escherichia coli BL21 (DE3) competent cells, mixed well, and placed on ice for 10 min. The cells were incubated in a 42°C water bath for 2 min and then placed on ice for 5 min. 1 mL of SOC medium was added to the cells and cultured at 37°C for 1 h. The cells were then spread on LB plates containing kanamycin, spectinomycin, and chloramphenicol (final concentrations of kanamycin, spectinomycin, and chloramphenicol were 50 mg / L, 50 mg / L, and 34 mg / L) to screen for transformants. Single colonies were picked and cultured in LB liquid medium supplemented with kanamycin, spectinomycin, and chloramphenicol resistance for 12 h, and then inoculated on 5 mL fresh LB liquid medium, incubate at 37°C, 180 rpm shaker for 1-2 h until OD 600 = 0.6~0.8, IPTG was added to a final concentration of 0.01 mM for induction, and expression was performed at 20°C for 12 h. 10 g / L cellobiose and 500 mg / L luteolin were added thereto, and cultured on a shaker at 30°C and 180 rpm for 48 h. Samples were then taken for HPLC analysis (analysis conditions were the same as in Example 5). The results showed that ( Figure 4 ), after a single point mutation, the conversion rate of luteolin to luteolin-5-O-xyloside by ZmDo-T142V increased by more than 3 times.

[0070] The recombinant bacteria containing the ZmDo-T142V mutant were inoculated into 5 mL of LB medium containing Kana+CM+Spec (the final concentration of kanamycin was 50 mg / L, the final concentration of spectinomycin was 50 mg / L, and the final concentration of chloramphenicol was 34 mg / L). The activated bacterial solution was transferred to 5 mL of fresh TB medium at a 1% inoculum volume. The cells were grown at 37°C until the OD 600 The final concentration of IPTG (0-1.0 mM) was added to the fermentation broth, and the expression was induced at 20°C for 12 h. Then, 10 g / L cellobiose and 500 mg / L luteolin were added. The fermentation was carried out at 30°C and the results were tested at 30 h and 60 h. Figure 5 As shown in A, the yield of luteolin-5-O-xyloside was the highest when the IPTG concentration was 0.05 mM.

[0071] The recombinant bacteria containing the ZmDo-T142V mutant were inoculated into 5 mL of LB medium containing Kana+CM+Spec (the final concentration of kanamycin was 50 mg / L, the final concentration of spectinomycin was 50 mg / L, and the final concentration of chloramphenicol was 34 mg / L). The activated bacterial solution was transferred to 5 mL of fresh TB medium at a 1% inoculum volume. The cells were grown at 37°C until the OD 600 The expression of the protein was detected by adding 0.05 mM IPTG to the fermentation broth and inducing expression at different temperatures (16-37 °C) for 12 h. Then, 10 g / L cellobiose and 500 mg / L luteolin were added and fermented at 30 °C. The results were tested at 30 h and 60 h. Figure 5 As shown in B, the yield of luteolin-5-O-xyloside was the highest when the induction temperature was 25°C.

[0072] The recombinant bacteria containing the ZmDo-T142V mutant were inoculated into 5 mL of LB medium containing Kana+CM+Spec (the final concentration of kanamycin was 50 mg / L, the final concentration of spectinomycin was 50 mg / L, and the final concentration of chloramphenicol was 34 mg / L). The activated bacterial solution was transferred to 5 mL of fresh TB medium at a 1% inoculum volume. The cells were grown at 37°C until the OD 600 The expression of the protein was detected at 0.6~0.8. 0.05mM IPTG was added to the fermentation broth, and the expression was induced at 25℃ for 12 hours. Then 10 g / L cellobiose and 500 mg / L luteolin were added. The fermentation was carried out at different temperatures (20~40℃) and the results were tested at 30 hours and 60 hours. Figure 5 As shown in Figure C, the yield of luteolin-5-O-xyloside was the highest when the fermentation temperature was 30°C.

[0073] The recombinant bacteria containing the ZmDo-T142V mutant were inoculated into 5 mL of LB medium containing Kana+CM+Spec (the final concentration of kanamycin was 50 mg / L, the final concentration of spectinomycin was 50 mg / L, and the final concentration of chloramphenicol was 34 mg / L). The activated bacterial solution was transferred to 5 mL of fresh TB medium at a 1% inoculum volume. The cells were grown at 37°C until the OD 600 The expression of 0.6~0.8 was reached by adding 0.05mM IPTG to the fermentation broth and inducing expression at 25℃ for 12 hours. Then, different concentrations (0-20 g / L) of cellobiose and 500 mg / L luteolin were added and fermented at 30℃. The results were tested at 30 hours and 60 hours. Figure 5 As shown in Figure D, the yield of luteolin-5-O-xyloside was the highest when the cellobiose concentration was 10 g / L.

[0074] The recombinant bacteria containing the ZmDo-T142V mutant were inoculated into 5 mL of LB medium containing Kana+CM+Spec (the final concentration of kanamycin was 50 mg / L, the final concentration of spectinomycin was 50 mg / L, and the final concentration of chloramphenicol was 34 mg / L). The activated bacterial solution was transferred to 5 mL of fresh TB medium at a 1% inoculum volume. The cells were grown at 37°C until the OD 600 The expression of the enzyme reached 0.6~0.8. 0.05mM IPTG was added to the fermentation broth and the expression was induced at 25℃ for 12 hours. Then 10 g / L cellobiose and different concentrations (2.5-3.5 g / L) of luteolin were added and fermented at 30℃. The results were tested at 30 hours and 60 hours. Figure 5 As shown in Figure E, the yield of luteolin-5-O-xyloside was the highest when the luteolin concentration was 3.25 g / L.

[0075] Therefore, the conversion efficiency of luteolin by the recombinant bacteria was the highest when the induction temperature was 25°C, the IPTG concentration was 0.05 mM, the fermentation temperature was 30°C, the cellobiose concentration was 10 g / L, and the substrate concentration was 3.25 g / L.

[0076] Determination of the time-dependent response curve of ZmDo-T142V: The recombinant bacteria containing the ZmDo-T142V mutant were inoculated into 5 mL of LB medium containing Kana+CM+spec (the final concentrations of kanamycin were 50 mg / L, spectinomycin were 50 mg / L, and chloramphenicol were 34 mg / L). The activated bacterial solution was transferred to 5 mL of TB medium and the OD value was 0. 600 When the pH value reached 0.6-0.8, 0.05 mM IPTG was added and induced at 25°C for 12 h. Then, 10 g / L cellobiose and 3.25 g / L luteolin were added. The culture was shaken at 30°C and samples were taken at regular intervals to measure the OD value. 600 , until 72 h, HPLC analysis (conditions are the same as in Example 5), the results are shown in Figure 6 After 48 h of fermentation, the recombinant bacteria achieved the highest conversion of luteolin, reaching 4450 mg / L, with a conversion rate of over 90%. The yield of luteolin 5-O-xyloside in the fermentation broth did not increase significantly with the extension of fermentation time. ZmDo reaction over time determination: At 48 h, the yield of luteolin-5-O-xyloside reached 418 mg / L.

Claims

1. A mutant of flavonoid-5-O-glycosyltransferase ZmDo, characterized in that: The mutant is obtained by replacing the 142th threonine of the wild-type flavonoid-5-O-glycosyltransferase ZmDo with valine; the amino acid sequence of the wild-type flavonoid-5-O-glycosyltransferase ZmDo is shown in SEQ ID NO.

1.

2. A gene encoding the mutant of flavonoid-5-O-glycosyltransferase ZmDo according to claim 1.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

3.

4. A recombinant plasmid, recombinant cell or recombinant bacterium, characterized in that: It contains the gene according to claim 2 or 3.

5. Use of the mutant of flavonoid-5-O-glycosyltransferase ZmDo according to claim 1, the gene according to claim 2 or 3, or the recombinant plasmid, recombinant cell or recombinant bacterium according to claim 4 in the production of luteolin 5-O-xyloside.

6. Use of the mutant of flavonoid-5-O-glycosyltransferase ZmDo according to claim 1, the gene according to claim 2 or 3, or the recombinant plasmid, recombinant cell or recombinant bacterium according to claim 4 in improving the yield of luteolin converted to luteolin 5-O-xyloside.

7. A method for producing luteolin 5-O-xyloside or increasing the yield of luteolin converted into luteolin 5-O-xyloside, characterized in that: The method comprises the following steps: adding IPTG to the recombinant bacteria according to claim 4, inducing, adding cellobiose and luteolin; and fermenting and culturing on a shaking platform.

8. The method according to claim 7, characterized in that: The concentration of IPTG is 0.01-1 mM, the induction temperature is 16-37° C., the fermentation temperature is 20-40° C., and the concentration of luteolin is 2.5-3.5 g / L.

9. The method according to claim 7, characterized in that: The fermentation culture time is 12 to 72 hours.

10. The method according to claim 7, characterized in that: The concentration of the cellobiose is 0-20 g / L.

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