A UDP-glycosyltransferase CsUGT74Y1 gene and its encoded protein and application

Abstract

The present invention discloses a UDP-glycosyltransferase CsUGT74Y1 gene and its encoded protein and application, and relates to the fields of bioengineering and enzyme engineering technology. The gene is isolated and obtained from fresh tea leaves, and its open reading frame sequence length is 1392bp, encoding a protein sequence containing 463 amino acids. By constructing a recombinant plasmid, the recombinant expression of the CsUGT74Y1 gene in Escherichia coli is achieved, and a purified recombinant protein is obtained, and by establishing the most suitable reaction conditions, the conditions for efficiently converting and generating flavonol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside, and coniferaldehyde-O-glucoside are obtained. The present invention provides a metabolic engineering basis for realizing the commercial production of efficient synthesis of flavonol glycosides, phenolic acid glycosides, plant hormone glycosides, and lignin glycosides.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering and enzyme engineering, and in particular to a UDP-glycosyltransferase CsUGT74Y1 gene and a coded protein and application thereof. Background Art

[0002] Tea trees have many secondary metabolites, including flavonoids, phenolic acids, lignin, plant hormones, etc. Among them, flavonoids are characteristic substances that constitute the quality and functional components of tea, and are important compounds that make tea have health functions. Phenolic acids in tea mainly include gallic acid, caffeic acid, p-coumaric acid and its glycosylated derivatives, which are closely related to the umami intensity of tea soup. Plant hormones include auxin, cytokinin, ethylene, abscisic acid, jasmonic acid, salicylic acid, gibberellin, brassinolide, etc., which can regulate plant growth and development at extremely low levels. Lignin is a complex phenolic polymer formed by three alcohol monomers (p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol), and is one of the components of plant cell walls.

[0003] Flavonoids and phenolic acid compounds are one of the main active ingredients of medicinal plants, such as anti-mutagenic, anti-diabetic, antibacterial, anti-inflammatory, anti-hypertensive, anti-ultraviolet, weight control, and Parkinson's disease prevention and treatment, as well as protecting the liver, inhibiting cancer cell proliferation, and increasing the antioxidant activity of blood. They are a treasure trove of sources of active leads for new drug research and development. From the biological perspective of plant growth and development and stress resistance, lignin can promote the growth and development of tea trees and enhance the stress resistance of plants. However, from the perspective of tea beverage quality, excessive lignin content will lead to increased leaf hardness and reduced applicability. However, naturally occurring flavonoids, phenolic acids, plant hormones, and lignin compounds often have the disadvantages of poor water solubility, poor stability, multiple targets, poor selectivity, and poor efficacy, which brings considerable difficulties to the search and development of adaptive new drugs. Chemical modification is one of the effective ways to develop new drugs.

[0004] The secondary metabolites modified by glycosylation exist in the plant body in the form of glycosylation. The glycosylated compounds can participate in other metabolic pathways as an activated intermediate. Glycosylation is one of the effective ways to chemically modify secondary metabolites. It can improve the water solubility, stability, selectivity and efficacy of the compounds. UDP-glycosyltransferase is a glycosyltransferase (UDP-glycosyltransferases, UGTs) that depends on uridine diphosphate (UDPG). It can specifically catalyze the glycosylation of the hydroxyl group of flavonoids on the A ring and efficiently form flavonoid-O-glucoside ( Figure 1-3), thus affecting the taste quality of tea. Phenolic acid compounds are converted into glycosides or sugar esters after being catalyzed by UDP-glycosyltransferase, thus providing precursor substances for downstream secondary metabolic pathways such as lignin and flavonoids ( Figure 4 After being modified by glycosylation, plant hormones form bound glycoside compounds in the plant body, thereby promoting the growth and development of the plant ( Figure 5 ). In addition, glycosyltransferases can also catalyze the formation of lignin glycosides from lignin monomers. These glycoside compounds are potential intermediates in the lignin synthesis pathway ( Figure 6 ).

[0005] In summary, how to achieve the large-scale commercial production of simultaneous biosynthesis of flavonoids, phenolic acids, phytohormones, and lignin glucosides can be of great significance to the regulation of plant growth and development, as well as the biosynthesis of medicinal compounds. Summary of the invention

[0006] The object of the present invention is to provide a UDP-glycosyltransferase CsUGT74Y1 gene and its encoded protein and application, aiming to provide a metabolic engineering basis for achieving the simultaneous and efficient commercial production of naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0008] The first object of the present invention is to provide a UDP-glycosyltransferase CsUGT74Y1 gene, wherein the UDP-glycosyltransferase CsUGT74Y1 gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0009] The second object of the present invention is to provide an application of the UDP-glycosyltransferase CsUGT74Y1 gene as described above in catalyzing the biosynthesis of multi-substrate glycoside compounds, wherein the protein encoded by the CsUGT74Y1 gene is used to simultaneously catalyze naringenin, apigenin, kaempferol, p-coumaric acid, IAA, and coniferyl aldehyde in vitro and convert them into corresponding naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside, and coniferyl aldehyde-O-glucoside.

[0010] The third object of the present invention is to provide a protein encoded by the UDP-glycosyltransferase CsUGT74Y1 gene as described above.

[0011] The fourth object of the present invention is to provide a use of the encoded protein as described above in catalyzing the biosynthesis of multi-substrate glycoside compounds.

[0012] The fifth object of the present invention is to provide a recombinant protein, which contains the encoded protein as described above and has an amino acid sequence as shown in SEQ ID NO.2.

[0013] The sixth object of the present invention is to provide a recombinant plasmid, which contains the nucleotide sequence of the UDP-glycosyltransferase CsUGT74Y1 gene that catalyzes the glycosylation of multiple substrates as described above, and the construction method is to connect the UDP-glycosyltransferase CsUGT74Y1 gene that catalyzes the glycosylation of multiple substrates to the multiple cloning site of the pMal-c2X vector to obtain the recombinant plasmid, and the recombinant plasmid is named pMal-c2X-CsUGT74Y1.

[0014] The seventh object of the present invention is to provide a genetically modified engineered bacterium, which contains the recombinant plasmid as described above, or the exogenous nucleotide sequence of the CsUGT74Y1 gene as described in claim 1 is integrated into its genome. The genetically modified engineered bacterium is an Escherichia coli Novablue (DE3) strain containing the recombinant plasmid, or the Escherichia coli Novablue (DE3) strain having the nucleotide sequence of the UDP-glycosyltransferase CsUGT74Y1 gene that catalyzes multi-substrate glycosylation integrated into its genome.

[0015] The eighth object of the present invention is to use the recombinant protein as described above in catalyzing the biosynthesis of multi-substrate glycoside compounds, wherein UDP-glucose is used as a sugar donor, naringenin, apigenin, kaempferol, p-coumaric acid, IAA and coniferyl aldehyde are used as sugar acceptors, and the recombinant protein is used as a catalytic enzyme, and catalytic enzyme reaction conditions are established to synthesize naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside, and coniferyl aldehyde-O-glucoside.

[0016] The ninth object of the present invention is to further improve that the enzyme reaction conditions of naringenin-7-O-glucoside, apigenin-7-O-glucoside and kaempferol-7-O-glucoside are reaction temperature of 20-35°C and reaction pH of 8.0-8.5; the optimal enzyme reaction conditions for the synthesis of p-coumaric acid-O-glucoside are reaction temperature of 20-35°C and reaction pH of 6.5-7.5; the optimal enzyme reaction conditions for the synthesis of IAA-O-glucoside and coniferyl aldehyde-O-glucoside are reaction temperature of 20-35°C and reaction pH of 6.0-7.0.

[0017] The tenth object of the present invention is that, based on the optimization of reaction conditions, under the condition of a fixed reaction pH of 7.5, the conversion rate of kaempferol synthesized into kaempferol-7-O-glucoside at 30°C is 82%; the conversion rate of p-coumaric acid synthesized into p-coumaric acid-O-glucoside at 25°C is 76%; the conversion rate of IAA synthesized into IAA-O-glucoside at 25°C is 38%; the conversion rate of coniferaldehyde synthesized into coniferaldehyde-O-glucoside at 30°C is 40%;

[0018] The eleventh object of the present invention is that, based on the optimization of reaction conditions, under the condition of a fixed reaction temperature of 35, the conversion rate of kaempferol synthesized into kaempferol-7-O-glucoside at a reaction pH of 8.5 is 86%; the conversion rate of p-coumaric acid synthesized into p-coumaric acid-O-glucoside at a reaction pH of 7.0 is 72%; the conversion rate of IAA synthesized into IAA-O-glucoside at a reaction pH of 6.5 is 39%; the conversion rate of coniferaldehyde synthesized into coniferaldehyde-O-glucoside at a reaction pH of 7.0 is 41.2%;

[0019] The present invention provides the following beneficial effects:

[0020] (1) The present invention cloned and verified for the first time the function of the glycosyltransferase gene CsUGT74Y1 that catalyzes multi-substrate glycoside compounds, and also provided a recombinant plasmid, a transgenic engineering bacterium and a recombinant protein containing the CsUGT74Y1 gene, and also provided a method for producing naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside using the engineering bacteria.

[0021] (2) Compared with traditional separation and extraction, chemical synthesis and other methods, the advantages of the present invention are that the raw materials are easy to obtain, the method is simple, the production cost is low, and the amount of reagents used is small.

[0022] (3) The present invention provides a metabolic engineering basis for the efficient commercial production of naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The catalytic flow chart and chemical structure of the production of naringenin-7-O-glucoside and UDP by catalysis of CsUGT74Y1 recombinant protein (hereinafter referred to as rCsUGT74Y1) using naringenin as a sugar acceptor and UDP-glucose as a sugar donor;

[0024] Figure 2 The catalytic flow chart and chemical structure of the production of apigenin-7-O-glucoside and UDP by rCsUGT74Y1 using apigenin as the sugar acceptor and UDP-glucose as the sugar donor;

[0025] Figure 3 The catalytic flow chart and chemical structure of the production of kaempferol-7-O-glucoside and UDP by rCsUGT74Y1 using kaempferol as the sugar acceptor and UDP-glucose as the sugar donor;

[0026] Figure 4 The catalytic flow chart and chemical structure of the production of p-coumaric acid-O-glucoside and UDP by rCsUGT74Y1 using p-coumaric acid as the sugar acceptor and UDP-glucose as the sugar donor;

[0027] Figure 5 The catalytic flow chart and chemical structure of the production of IAA-O-glucoside and UDP by rCsUGT74Y1 using IAA as the sugar acceptor and UDP-glucose as the sugar donor;

[0028] Figure 6 With coniferyl aldehyde as the sugar acceptor and UDP-glucose as the sugar donor, coniferyl aldehyde-O-glucoside and UDP were generated by rCsUGT74Y1 catalysis.

[0029] Figure 7 The SDS-PAGE protein electrophoresis analysis diagram of rCsUGT74Y1; (wherein, M is the protein marker; 1 is before the induction of the recombinant plasmid; 2 is after the induction of the recombinant plasmid; 3 is the supernatant after the induction and fragmentation; 4 is the precipitate after the induction and fragmentation; 5 is the purified protein);

[0030] Figure 8 The results of HPLC analysis of the enzyme activity products catalyzed by rCsUGT74Y1 (wherein, (a)-(f) are HPLC spectra with UDP-glucose as the sugar donor, and naringenin, apigenin, kaempferol, p-coumaric acid, IAA and coniferyl aldehyde as the sugar acceptors, respectively);

[0031] Fig. 9 The secondary mass spectra of the products synthesized by rCsUGT74Y1 protein are naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside; (wherein, (a)-(f) are the secondary mass spectra of naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside, respectively);

[0032] Fig.10 The gradient curve diagram of the optimal reaction temperature for the synthesis of kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside catalyzed by rCsUGT74Y1 protein. (Wherein, (a)-(d) are the gradient curve diagrams of the optimal reaction temperature for the synthesis of kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside, respectively);

[0033] Fig.11 The gradient curve of the optimal reaction pH for the synthesis of kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside catalyzed by rCsUGT74Y1 protein. (Wherein, (a)-(d) are the gradient curves of the optimal reaction pH for the synthesis of kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside, respectively);

[0034] Fig.12 This is the plasmid map of pMal-c2X. DETAILED DESCRIPTION

[0035] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0036] 1. Materials and Reagents

[0037] (1) Tea variety: Camellia sinensis (L.) O. Kuntze. var. sinensis cultivar Qiancha 1, harvested from the tea resource garden of Guizhou Tea Research Institute. Fresh tea leaves were quickly frozen with liquid nitrogen after collection, and then used to extract total RNA from tea trees, which was reverse transcribed into cDNA for gene cloning;

[0038] (2) Synthesis of gene cloning primers: The gene cloning primers used in the present invention were synthesized by General Biotechnology (Anhui) Co., Ltd.;

[0039] (3) Protein expression vector pMal-c2X: This vector was purchased from NEB. The plasmid map of pMal-c2X is as follows: Fig.10 As shown;

[0040] (4) Escherichia coli Novablue (DE3) expression host bacteria: purchased from Shanghai Beino Biotechnology Co., Ltd.;

[0041] (5) LB medium: weigh 10 g of NaCl, 5 g of yeast extract, and 10 g of tryptone, add 950 mL of ultrapure water and stir to dissolve, adjust the pH to 7.0 with 1 mol / L NaOH, add water to make up to 1000 mL, and sterilize with high pressure steam for 15 min to obtain LB liquid medium. LB solid medium is prepared by adding 15 g of agar powder to LB liquid medium;

[0042] (6) 40% glucose solution: weigh 40 g of glucose, add ultrapure water to dissolve and stir evenly, dilute to 100 mL, and sterilize at 110°C for 10 min;

[0043] (7) Ampicillin stock solution (Amp+, 100 mg / mL): Weigh 1 g of ampicillin Amp, dissolve in 10 mL of sterile water, filter and sterilize, divide into small tubes, and store at -20°C;

[0044] (8) 1 mol / L IPTG (isopropylthio-β-D-galactoside): weigh 2.383 g IPTG, dissolve in sterile ultrapure water, dilute to 10 mL, filter sterilize, aliquot and store at -20°C;

[0045] (9) Protein purification buffer: Column buffer: Weigh 0.37 g EDTA, 11.67 g NaCl, 2.42 g Tris, and 0.15 g DTT in sufficient pure water and stir to mix thoroughly. Adjust the pH to 7.4 with dilute hydrochloric acid and make the volume to 1 L to obtain column buffer. Elution buffer: Add 3.60 g maltose to 1 L column buffer and dissolve and stir evenly.

[0046] (10) 100 mM Tris-HCl buffer solution, pH 7.5: weigh 1.1214 g Tris, add water to 90 mL, stir to dissolve evenly, add diluted HCl to adjust the pH to 7.5, and add water to make up to 100 mL;

[0047] (11) 1% by volume acetic acid: Use a pipette to measure 10 mL of chromatography-grade acetic acid solution into a 1 L volumetric flask and dilute to 1 L with ultrapure water.

[0048] No specific experimental method is specified in this example, and all experimental methods can be carried out according to conventional methods. The prokaryotic expression and functional verification technical means used in this example are commonly used or fully understandable by ordinary technicians in the field. The materials, reagents, etc. used, unless otherwise specified, can be obtained through commercial channels.

[0049] 2. Methods

[0050] 1. Cloning of CsUGT74Y1 gene

[0051] (1) According to the instructions of TaKaRa RNAiso kit and RNAiso Plus kit, RNA was extracted from fresh leaves of tea variety Shucha Zao and reverse transcribed into cDNA;

[0052] (2) Design specific primers for the CsUGT74Y1 gene and clone the 1392 bp open reading frame nucleic acid sequence of the gene from tea plant cDNA. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4:

[0053] SEQ ID NO.3: Forward primer: 5'-ATGGGTAGTAGTAGTGAAAC-3'

[0054] SEQ ID NO.4: Reverse primer: 5'-TTATGCATTCAGAATTTCTG-3'

[0055] (3) The reverse transcribed cDNA was used as a template and amplified with primers SEQ ID NO: 3 and SEQ ID NO: 4. The amplification procedure was 94°C pre-denaturation for 30 s, 94°C denaturation for 10 s, 62°C annealing for 20 s, 72°C extension for 50 s, 30 cycles, and 72°C extension for 10 min. The obtained PCR product was stored at 4°C.

[0056] (4) The PCR product was purified using a PCR purification kit and connected to pMD19-T Simple Vector for colony PCR verification to obtain positive colonies. The colony plasmid was extracted to obtain the T vector containing the CsUGT74Y1 gene. The bacterial solution was sent to Shenzhen BGI for sequencing. The nucleotide sequence of the CsUGT74Y1 gene is shown in SEQ ID NO.1, and the encoded protein of the CsUGT74Y1 gene has the amino acid sequence shown in SEQ ID NO.2.

[0057] (5) Constructing the CsUGT74Y1 gene into the expression vector pMal-c2X: Designing specific primers with multiple cloning restriction sites of the expression vector pMal-c2X, the primer sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6:

[0058] SEQ ID NO.5: Forward primer: 5'-GGATTTCAGAATTCGGATCCATGGGTAGTAGTA GTGAAAC-3';

[0059] SEQ ID NO. 6: Reverse primer: 5'-CAGGTCGACTCTAGAGGATCCTTATGCATTCAG AATTTCTG-3'.

[0060] 2. Prokaryotic expression and functional verification of CsUGT74Y1 gene

[0061] The prokaryotic expression and function verification techniques used in this example are commonly used or fully understandable to those skilled in the art.

[0062] (1) The pMal-c2X plasmid was cut with restriction endonuclease BamHI, and the pMal-c2X digested DNA fragment was obtained by gel recovery. The CsUGT74Y1 gene containing the pMal-c2X vector adapter primer was connected to the pMal-c2X vector recovered after digestion using recombinase (Nanjing Novogene Biotechnology Co., Ltd.) to obtain the pMal-c2X-CsUGT74Y1 recombinant plasmid;

[0063] (2) The pMal-c2X-CsUGT74Y1 recombinant plasmid was transformed into the Escherichia coli Novablue (DE3) expression host bacteria, inoculated into 100 μL of LB liquid culture medium, and cultured at 37°C, 180 r / min for 45 to 60 min; 100 μL of the bacterial solution was spread on an LB plate containing 100 μg / mL Amp+ and cultured inverted at 37°C.

[0064] (3) After colony PCR verification, positive colonies were picked and inoculated into 100 mL of sterilized LB liquid culture medium containing 2 g / L of 1% 2H2O, and cultured at 37°C and 200 rpm until OD600≈0.6 to obtain genetically modified engineered bacteria.

[0065] (4) IPTG was added to the genetically modified bacteria to a final concentration of 1 mmol / L, and the cells were cultured at 37°C overnight. The cells were collected, 10 mL of column buffer solution was added, the cells were fully suspended, and the cells were placed at -20°C overnight. The cells were thawed on ice, and after thawing, they were placed in an ultrasonic disruptor at 15% power for 10 min, and the supernatant was collected by centrifugation at 12000 rpm; the recombinant protein was purified using an amylase resin affinity column (affinity chromatography on an amylase resin, New England Biolabs, MA, USA), and the protein expression and purification effect were detected using the SDS-PAGE method commonly used in the art. The results are shown in Figure 2. Figure 7 shown.

[0066] Figure 7It can be seen that the pMal-c2X-CsUGT74Y1 recombinant plasmid was transformed into the expression host bacteria Novablue (DE3), and the recombinant protein was expressed after induction. The size of the recombinant protein band was consistent with the prediction. After adding the 42.5kDa maltose binding protein (MBP) recombinant tag, the protein size was 88.9kd, so there was an obvious recombinant protein band at 90kd in the SDS-PAGE protein electrophoresis analysis chart; after induction, the bacteria were ultrasonically crushed and centrifuged, and the induced recombinant protein was expressed in the supernatant, which can be used for further purification and analysis; after the supernatant protein was purified by amylose resin column, a relatively pure recombinant protein (lane 5) was obtained, and the purified protein can be used for further enzymatic analysis.

[0067] 3. Identification of rCsUGT74Y1 enzyme reaction products

[0068] (1) Analysis of the enzyme activity of rCsUGT74Y1 on different substrates

[0069] The total reaction system for enzyme activity detection is 50 μL, which contains a Tris-HCL buffer solution with a pH of 7.5 and a concentration of 100 mM, 5 mM glycosyl donors (including UDP-glucose, UDP-galactose, UDP-xylose, UDP-rhamnose), 200 μM glycosyl acceptors (kaempferol, naringenin, apigenin, IAA, coniferyl aldehyde), 10-25 μg of purified recombinant protein and 0.1% β-mercaptoethanol. All enzyme reaction systems were incubated in a 35°C water bath for 30 minutes and then an equal volume of methanol was added to terminate the reaction. The reaction was controlled with empty protein to obtain the enzyme reaction product.

[0070] (2) HPLC identification

[0071] The enzyme reaction product was identified by product standard combined with HPLC, and the HPLC chromatographic detection conditions were as follows: Thermo Fisher high performance liquid chromatograph; chromatographic column: Agilent C18 100*4.6mm (size: 5μm; dimensions, 4.6mm×100mm; Agilent Technologies); detector: Thermo Fisher adjustable wavelength ultraviolet detector; sensitivity: 0.01; detection wavelength: 280nm-350nm; flow rate: 0.4mL / min; injection volume was 5μL; mobile phase A was a solution containing 1% (v / v) acetic acid; mobile phase B was a 100% acetonitrile solution; for the detection of the compound, the HPLC gradient program was set to: 0-5min, 1-25%B; 5-14min, 25-45%B; 14-22min, 45-60%B; 22-24min, 60-1%B; 24-25min, 1-1%B.

[0072] The results are as follows Figure 8As shown in a-f, it can be found that rCsUGT74Y1 can specifically catalyze naringenin, apigenin, kaempferol, p-coumaric acid, IAA and coniferyl aldehyde to produce naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside and coniferyl aldehyde-O-glucoside.

[0073] (3) UPLC–MS / MS identification

[0074] The enzyme reaction product was identified by product standard combined with UPLC-MS, and the UPLC-MS detection conditions were as follows: Waters ACQUITY UPLC HSS T3 chromatographic column (150 mm×2.1 mm, 1.7 tzm); column temperature was 30°C; flow rate was 1 mL / min; injection volume was 5 μL; mobile phase A was a 1% (v / v) acetic acid solution; mobile phase B was a 100% acetonitrile solution; for the detection of flavonoid compounds, the UPLC gradient program was set as follows: 0-5 min, 10-15% B; 5-15 min, 15-40% B; 15-20 min, 40-60% B; 20-25 min, 60-80% B; 25-30 min, 80-10% B; 30-32 min, stop. The spectral detection wavelength scanning range was 200-550 nm. In the MS qualitative identification of the compound, an ESI electrospray ion source was used in negative ion mode; the capillary voltage was 3.5 kV, the ion source temperature was 350 °C, the nebulizer gas (nitrogen) flow rate was 6 L / min, the compound detection scanning mass-to-charge ratio range was set to m / z 100-1000, and the collision voltage was 45 V. The results are shown in Fig. 9 As shown in (af), the parent ion of the product naringenin-7-O-glucoside is 433.0989 and the daughter ion is 271.0173; the parent ion of apigenin-7-O-glucoside is 431.0957 and the daughter ion is 269.0591; the parent ion of kaempferol-7-O-glucoside is 447.0943 and the daughter ion is 217.0037; the parent ion of p-coumaric acid-O-glucoside is 325.1585 and the daughter ion is 265.1605; the parent ion of IAA-O-glucoside is 338.0861 and the daughter ion is 130.0174; and the parent ion of coniferyl aldehyde-O-glucoside is: 339.0952 and the daughter ion is 177.0617.

[0075] 4. Detection of reaction conditions for high conversion rate of rCsUGT74Y1

[0076] (1) Analysis of the optimal reaction temperature and conversion rate of rCsUGT74Y1 for different substrates

[0077] Each reaction was a 50 μL system, which contained a Tris-HCL buffer solution with a pH of 7.5 and a concentration of 100 mM, 5 mM UDP-glucose, 200 μM glycosyl receptor, 10 μg of purified recombinant protein and 0.1% β-mercaptoethanol. The four substrates of kaempferol, p-coumaric acid, IAA and coniferyl aldehyde were subjected to enzyme reactions according to the above conditions. 10 temperature gradients were set for each substrate. All enzyme reaction systems were reacted in a water bath for 15 minutes under different temperature conditions, and then an equal volume of methanol was added to terminate the reaction. The above reactions were all controlled with empty protein and were biologically repeated 3 times. After centrifugation of the above methanol-terminated enzyme reaction system at 13,000 g for 10 minutes, the supernatant was taken and HPLC was used to detect the enzyme reaction.

[0078] like Fig.10 As shown in (a)-(d), the optimal reaction temperature range of rCsUGT74Y1 for the four substrates of kaempferol, p-coumaric acid, IAA and coniferyl aldehyde is 20-35°C. As shown in Table 1, when the reaction pH is 7.5 and the temperature is 30°C, the conversion rate of p-kaempferol is the highest (82%); when the reaction pH is 7.5 and the temperature is 25°C, the conversion rate of p-coumaric acid is the highest (76%); when the reaction pH is 7.5 and the temperature is 25°C, the conversion rate of IAA is the highest (38%); when the reaction pH is 7.5 and the temperature is 30°C, the conversion rate of coniferyl aldehyde is the highest (40%).

[0079] Table 1 shows the conversion rate of CsUGT74Y1 recombinant protein to substrate at a reaction pH of 7.5 and different temperature gradient conditions.

[0080]

[0081] (2) Analysis of the optimal reaction pH and conversion rate of rCsUGT74Y1 for different substrates

[0082] The reaction system of this step is consistent with the analysis of the optimal temperature of the enzyme reaction. During the analysis of the optimal pH conditions, the reaction temperature was fixed at 35°C, and 10 reaction pH gradients were set for the four substrates of kaempferol, p-coumaric acid, IAA and coniferyl aldehyde. All enzyme reaction systems were reacted in a water bath at 35°C for 15 minutes, and then an equal volume of methanol was added to terminate the reaction. The above reactions were all controlled with empty protein and biologically repeated three times. After centrifugation of the above methanol-terminated enzyme reaction system at 13,000g for 10 minutes, the supernatant was taken for HPLC detection of the enzyme reaction.

[0083] like Fig.11As shown in (a)-(d), the optimal reaction pH range of rCsUGT74Y1 for kaempferol is 8.0-8.5, the optimal reaction pH range of p-coumaric acid is 6.5-7.5, the optimal reaction pH range of IAA is 6.0-7.0, and the optimal reaction pH range of coniferaldehyde is 6.0-7.0. In addition, as shown in Table 2, when the reaction temperature is 35°C and the pH is 8.5, the conversion rate of p-kaempferol is the highest (86%); when the reaction temperature is 35°C and the pH is 7.0, the conversion rate of p-coumaric acid is the highest (72%); when the reaction temperature is 35°C and the pH is 6.5, the conversion rate of IAA is the highest (39%); when the reaction temperature is 35°C and the pH is 7.0, the conversion rate of coniferaldehyde is the highest (41.2%).

[0084] Table 2 shows the conversion rate of CsUGT74Y1 recombinant protein to substrate at a fixed reaction temperature of 35°C and different pH gradient conditions.

[0085]

[0086] Therefore, by establishing the optimal reaction conditions, we obtained the conditions for efficient conversion to kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside, and coniferyl aldehyde-O-glucoside.

[0087] Conclusion

[0088] In summary, the present invention studies a UDP-glycosyltransferase CsUGT74Y1 gene that can encode catalytic multi-substrate glycosylation, obtains recombinant protein by constructing recombinant engineering bacteria, and provides a method for simultaneously producing flavonol-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside, and coniferaldehyde-O-glucoside using engineering bacteria. Compared with traditional separation and extraction, chemical synthesis and other methods, the present invention has the advantages of simple and easy-to-obtain raw materials, simple methods, low production costs, and small amounts of reagents. The present invention provides a metabolic engineering basis for the commercial production of naringenin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, p-coumaric acid-O-glucoside, IAA-O-glucoside, and coniferaldehyde-O-glucoside.

[0089] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A UDP-glycosyltransferase CsUGT74Y1 The use of a gene in catalyzing the biosynthesis of a multi-substrate glycoside compound is characterized in that: The UDP-glycosyltransferase CsUGT74Y1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The application is to utilize the CsUGT74Y1 The protein encoded by the gene catalyzes the conversion of naringenin, apigenin, kaempferol, p-coumaric acid, IAA and coniferyl aldehyde to the corresponding naringenin-7- O -Glucoside, apigenin-7- O -Glucoside, Kaempferol-7- O -Glucoside, p-coumaric acid- O -Glucoside, IAA- O -Glucoside and coniferyl aldehyde- O -Glucoside.

2. A UDP-glycosyltransferase CsUGT74Y1 The use of the encoded protein of the gene in catalyzing the biosynthesis of multi-substrate glycoside compounds is characterized in that: The UDP-glycosyltransferase CsUGT74Y1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. CsUGT74Y1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; The application is to utilize the CsUGT74Y1 The protein encoded by the gene catalyzes the conversion of naringenin, apigenin, kaempferol, p-coumaric acid, IAA and coniferyl aldehyde in vitro to produce the corresponding naringenin-7- O -Glucoside, apigenin-7- O -Glucoside, Kaempferol-7- O -Glucoside, p-coumaric acid- O -Glucoside, IAA- O -Glucoside and coniferyl aldehyde- O -Glucoside.

3. The use according to claim 2, characterized in that: Kaempferol-7- O The optimal enzyme reaction conditions for -glucoside are reaction temperature: 20-35°C, reaction pH: 8.0-8.5; the p-coumaric acid- O The optimal enzyme reaction conditions for the synthesis of IAA-glucosidase are reaction temperature: 20-35°C, reaction pH: 6.5-7.5; O -Glucoside and coniferyl aldehyde- O The optimal enzyme reaction conditions for -glucoside synthesis are reaction temperature: 20-35°C, reaction pH: 6.0-7.0.

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