A flavone 6-c-glycosyltransferase mutant for improving the production of isoobtusoside

Abstract

The application discloses a flavone 6-C-glycosyltransferase mutant for improving the yield of isohyduloside, and provides a mutant protein of GyCGT1, which comprises the following steps: mutating the amino acid residues at positions 7, 8, 9, 18, 205, 258, 280 and 417 of SEQ ID NO: 2, and keeping the amino acid residues at other positions unchanged, so that a protein with the same function is obtained; or the mutant protein of GyCGT1 comprises the following steps: mutating the amino acid residues at positions 7, 9 and 205 of SEQ ID NO: 2, and keeping the amino acid residues at other positions unchanged, so that a protein with the same function is obtained. The glycosyltransferase GyCGT1 mutant is obtained through the method, and the yield of isohyduloside is increased by 8 times in the fermentation of the constructed Escherichia coli recombinant strain in a 50mL shaking flask system, so that the mutant has a good prospect for industrial production application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a flavonoid 6-C-glycosyltransferase mutant that increases the yield of isopropargyl glycoside. Background Technology

[0002] Isophoroside is an important flavonoid-C-glycoside compound and is a traditional Chinese medicine, gentian (Gentiana scabra). Gentiana scabra Isohexin is one of the main active ingredients in corn. Recent studies have revealed its broad pharmacological activities, including significant antioxidant and anti-inflammatory effects, particularly demonstrating good therapeutic potential in relieving allergic asthma. Furthermore, isohexin also exhibits unique value in agriculture, such as inhibiting weed growth and enhancing corn's resistance to corn borers, making it a natural product with great development potential. However, the natural sources of isohexin are limited. Direct extraction from plants faces problems such as high resource consumption, long cycles, and low content, resulting in high costs and difficulty in meeting market demand. Chemical synthesis, on the other hand, is limited in its large-scale application due to cumbersome steps, poor regional selectivity, use of harmful reagents, and environmental pollution. Therefore, developing green and efficient biosynthetic methods has become a key approach to solving the supply bottleneck of isohexin.

[0003] Biosynthesis, especially enzyme-catalyzed synthesis, is considered an ideal strategy for producing high-value natural products due to its advantages such as mild reaction conditions, high selectivity, and environmental friendliness. Yunnan gentian ( Gentiana yunnanensis The discovery of the glycosyltransferase GyCGT1, derived from luteolin, provides a crucial tool for the biosynthesis of isoharmonin. This enzyme can efficiently and specifically catalyze the formation of isoharmonin using luteolin as a substrate, marking a breakthrough in the biosynthetic route of this compound. However, existing natural GyCGT1 enzymes still have significant drawbacks, restricting their industrial application: 1) Insufficient catalytic efficiency: The specific enzyme activity and conversion rate of wild-type GyCGT1 are insufficient to meet the economic requirements of industrial production, resulting in high production costs. 2) Low yield and productivity: In existing heterologous expression systems (such as E. coli and yeast), the yield, productivity, and production intensity of isoharmonin using natural GyCGT1 do not reach the threshold for industrial production. This directly leads to long fermentation cycles, low equipment utilization, and the inability to achieve large-scale, continuous industrial manufacturing.

[0004] Therefore, there is a need to seek more highly active GyCGT1 enzymes. Summary of the Invention

[0005] The technical problem solved by this invention is how to improve the activity of GyCGT1 enzyme.

[0006] To address this technical problem, the first aspect of the present invention provides a mutant protein of GyCGT1, which is any one of the following:

[0007] The amino acid sequence of the mutant protein shown in A1) includes the sequence obtained by mutating amino acid residues at positions 7, 8, 9, 18, 205, 258, 280 and 417 of SEQ ID NO:2, while keeping the amino acid residues at other positions unchanged.

[0008] The amino acid sequence of the mutant protein shown in A2) includes the sequence obtained by mutating amino acid residues at positions 7, 9 and 205 of SEQ ID NO:2, while keeping the amino acid residues at other positions unchanged;

[0009] A3) Proteins that, except for the mutated amino acid residues, have more than 80% identity with the mutant proteins shown in A1) or A2) and have the same function;

[0010] The mutant protein shown in A4) is a protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins shown in A1)-A3).

[0011] In some embodiments, the amino acid sequence of the mutant protein shown in A1) is obtained by mutating amino acid residues at positions 7, 8, 9, 18, 205, 258, 280 and 417 of SEQ ID NO:2, while leaving the amino acid residues at other positions unchanged.

[0012] In some embodiments, the amino acid sequence of the mutant protein shown in A2) is obtained by mutating the amino acid residues at positions 7, 9 and 205 of SEQ ID NO:2, while leaving the amino acid residues at other positions unchanged.

[0013] In the above text, the position of each mutation site is indicated by the amino acid residue number corresponding to the N-terminus of SEQ ID NO:2. For example, the 7th amino acid residue is indicated by the 7th amino acid residue corresponding to the 1st amino acid residue starting from the N-terminus of SEQ ID NO:2, and so on.

[0014] Among the mutant proteins mentioned above,

[0015] The mutation is as follows:

[0016] The 7th amino acid residue is mutated from N to S;

[0017] The 8th amino acid residue is mutated from D to Q;

[0018] The 9th amino acid residue was mutated from N to K;

[0019] The 18th amino acid residue was mutated from I to P;

[0020] The amino acid residue at position 205 is mutated from T to E.

[0021] The 258th amino acid residue was mutated from N to S;

[0022] The 280th amino acid residue is mutated from S to N;

[0023] The 417th amino acid residue is mutated from I to V.

[0024] Among the mutant proteins mentioned above,

[0025] The amino acid sequence of the mutant protein shown in A1) includes SEQ ID NO:4;

[0026] The amino acid sequence of the mutant protein shown in A2) includes SEQ ID NO:6.

[0027] In some embodiments, the amino acid sequence of the mutant protein shown in A1) is SEQ ID NO:4;

[0028] In some embodiments, the amino acid sequence of the mutant protein shown in A2) is SEQ ID NO:6.

[0029] In a second aspect, the present invention provides biological materials related to the mutant protein described in the first aspect, which are any one of B1) to B4) below:

[0030] B1) A nucleic acid molecule encoding the mutant protein described in the first aspect;

[0031] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0032] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0033] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0034] Among the biomaterials mentioned above,

[0035] B1) The nucleic acid molecule is any one of the following:

[0036] b1) The nucleotide sequence of the nucleic acid molecule shown includes SEQ ID NO:3;

[0037] b2) The nucleotide sequence of the nucleic acid molecule shown includes SEQ ID NO:5;

[0038] b3) has more than 75% identity with any of the defined nucleotide sequences in b1)-b2) and encodes a cDNA molecule or DNA molecule that encodes the mutant protein described in the first aspect;

[0039] b4) Hybridizes under stringent conditions to any of the defined nucleotide sequences in b1)-b2) and to a cDNA molecule or DNA molecule encoding the mutant protein described in the first aspect.

[0040] In some embodiments, the nucleotide sequence of the nucleic acid molecule shown in b1) is SEQ ID NO:3.

[0041] In some embodiments, the nucleotide sequence of the nucleic acid molecule shown in b2) is SEQ ID NO:5;

[0042] Thirdly, the present invention provides the mutant protein described in the first aspect for any of the following applications:

[0043] C1) is a glycosyltransferase;

[0044] C2) catalyzes the formation of isopropanol from luteolin;

[0045] C3) Preparation of isopropargyl glycoside;

[0046] C4) to prepare the product that catalyzes the conversion of luteolin to isorhamnetin.

[0047] Fourthly, the present invention provides the biomaterials described in the second aspect for any of the following applications:

[0048] D1) Preparation of glycosyltransferases;

[0049] D2) Catalyzes the formation of isopropanol from luteolin;

[0050] D3) Preparation of isopropargyl glycoside;

[0051] D4) Prepare the product that catalyzes the conversion of luteolin to isorhodin.

[0052] Fifthly, the present invention provides a method for preparing isoharonidine, comprising the following steps: using the mutant protein described in the first aspect, with the assistance of UDPG, to catalyze the formation of isoharonidine from luteolin.

[0053] In a sixth aspect, the present invention provides a method for preparing isoharonidine, comprising the following steps: using the recombinant microorganisms or their cultures described in the second aspect to catalyze the generation of isoharonidine from luteolin.

[0054] In the above text, the culture is the IPTG-induced culture product of the recombinant microorganisms described in the second aspect.

[0055] In a seventh aspect, the present invention provides a product for preparing isopropionol, comprising the mutant protein described in the first aspect or the biomaterial described in the second aspect, luteolin, and UDPG.

[0056] Experiments of this invention demonstrate that the modified glycosyltransferase GyCGT1 mutant obtained by this invention exhibits higher activity in vitro compared to the wild type. When this mutant was used to construct a recombinant E. coli strain and fermented together with WT in a 50 mL shake flask, the yield increased by 8 times, showing promising potential for industrial application. The recombinant strain with mutant M3 (134.33 mg / L) also showed a 2.75-fold increase in yield in a 50 mL shake flask compared to the WT strain. Attached Figure Description

[0057] Figure 1 SDS-PAGE gel images of WT, M3, and M8 proteins.

[0058] Figure 2 Yields of WT, M3 and M8 (E. coli 50 mL shake flask experiment). Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0062] pCDFDuet-1 plasmid was purchased from huaaobio (catalog number: Trans 23-31). pCDFDuet-1 is a dual expression vector for E. coli proteins, vector size: 3781bp, vector tags: N-His, CS, vector resistance: streptomycin.

[0063] The *E. coli* DH5α competent cells were preserved in the laboratory and are commercially available (Vazyme, C502-02). *E. coli* BL21(DE3) competent cells were purchased from Beijing TransGen Biotech Co., Ltd., catalog number: CD601-02.

[0064] The luteolin standard was purchased from Shaanxi Kangzhou Biotechnology Co., Ltd. (product number: KZ2025051303-01), purity: HPLC≥98%, appearance: light yellow to yellow powder. The CAS of luteolin is 491-70-3, the molecular formula is C15H10O6, and the English name is Luteolin. Before the experiment, luteolin was dissolved in 100% DMSO to prepare a 200 mM stock solution.

[0065] The isoorientin standard was purchased from Yuanye Bio-Technology Co., Ltd. (product number: B21528-10mg), purity: HPLC≥98%, appearance: yellow powder. The CAS of isoorientin is 4261-42-1, the molecular formula is C21H20O11, and the English name is Isoorientin.

[0066] UDPG, uridine diphosphate glucose, biotechnology grade, 98%, product number: S18074-1g, CAS number: 28053-08-9, brand: YUANYE / Yuanye Bio-Technology Co., Ltd.

[0067] Example 1. Obtaining of glycosyltransferase GyCGT1 mutants

[0068] I. Glycosyltransferase GyCGT1 mutants

[0069] 1. Construction of a recombinant vector expressing glycosyltransferase GyCGT1

[0070] The amino acid sequence of the natural glycosyltransferase GyCGT1 derived from Gentiana rigescens Franch. Gentiana yunnanensis (from Yunnan) is SEQ ID NO: 2, and the nucleotide sequence of the coding nucleic acid of the natural glycosyltransferase GyCGT1 is SEQ ID NO: 1.

[0071] The recombinant vector pCDFDuet-1-GyCGT1 expressing the glycosyltransferase GyCGT1 coding gene is a vector obtained by replacing the fragment between the BamHⅠ and HindⅢ restriction enzyme sites of plasmid pCDFDuet-1 with the coding nucleic acid of GyCGT1. The N-His tag and the coding nucleic acid of GyCGT1 in this vector are co-expressed to obtain the recombinant protein GyCGT1.

[0072] 2. Construction of glycosyltransferase GyCGT1 mutants and recombinant vectors expressing them

[0073] 1) Glycosyltransferase GyCGT1 mutants

[0074] Based on the AI ​​large model (Pro-PRIME) targeted modification method, GyCGT1 was targeted modified. The top 50 single-point mutants ranked from highest to lowest were used for experimental verification. The mutants were purified and subjected to in vitro enzyme activity experiments. The experimental results were used as a new training set to fine-tune and optimize the model, thereby obtaining double-mutant to multi-mutant mutants. The purified mutants were subjected to in vitro enzyme activity experiments, and it was found that the enzyme activity of the 3-point mutant M3 and the 8-point mutant M8 was significantly improved.

[0075] The amino acid sequence of mutant M8 is SEQ ID NO:4, and the nucleotide sequence encoding the nucleic acid of mutant M8 is SEQ ID NO:3. Mutant M8 is formed by mutating SEQ ID NO:2 as follows: N at position 7 is mutated to S, D at position 8 is mutated to Q, N at position 9 is mutated to K, I at position 18 is mutated to P, T at position 205 is mutated to E, N at position 258 is mutated to S, S at position 280 is mutated to N, and I at position 417 is mutated to V, while the amino acid residues at other positions remain unchanged. The coding nucleic acid of mutant M8 is obtained by mutating SEQ ID NO: 1 as follows: aac at positions 19-21 is mutated to agc, cat at positions 22-24 is mutated to cag, aac at positions 25-27 is mutated to aaa, atc at positions 52-54 is mutated to cct, acc at positions 613-615 is mutated to gaa, AAC at positions 772-774 is mutated to tcg, AGC at positions 838-840 is mutated to aac, and ATC at positions 1249-1251 is mutated to gtg. The nucleotide residues at other positions remain unchanged.

[0076] The amino acid sequence of mutant M3 is SEQ ID NO:6, and the nucleotide sequence encoding the nucleic acid of mutant M3 is SEQ ID NO:5. Mutant M3 is formed by the following mutations in SEQ ID NO:2: N at position 7 is mutated to S, N at position 9 is mutated to K, and T at position 205 is mutated to E, while other amino acid residues remain unchanged. The nucleic acid encoding mutant M3 is formed by the following mutations in SEQ ID NO:1: aac at positions 19-21 bp is mutated to agc, aac at positions 25-27 is mutated to aaa, and acc at positions 613-615 is mutated to gaa, while other nucleotide residues remain unchanged.

[0077] 2) Recombinant vector expressing the GyCGT1 mutant glycosyltransferase

[0078] Based on the pCDFDuet-1-GyCGT1 plasmid sequence, primers for introducing mutation sites were designed and synthesized to perform site-directed mutagenesis on the gene sequence of flavonoid 6-C glycosyltransferase, and sequencing was performed to confirm whether the coding gene of the mutant was correct.

[0079] Primers used for point mutation of M8 mutant:

[0080] F789: CAACTCTagccagAAaCTGCACATTTTCCTGGT

[0081] R789: GtttctggctAGAGTTGTGGCTACCCATcgga

[0082] F18: TTCcctGGCCAGGGTGTTGTTAACC

[0083] R18: AACACCCTGGCCaggGAAGCAAACCAGGAAAATG

[0084] F205: GTTCTTCGGCgaaAACATCCTGGGCCAGTTT

[0085] R205: AGGATGTTttcGCCGAAGAACGGATACGG

[0086] F258: GCAGCtcgGGTATCACCGGTAACTTC

[0087] R258: CGGTGATACCcgaGCTGCTCGGGTCTTTCGG

[0088] F280: GCCGaacACCGTTGTTTACGTGAGC

[0089] R280: GTAAACAACGGTgttCGGCGGACGGGTGTCCAG

[0090] F417: GTATTgtgACCCGTGACGAAGTTGAA

[0091] R417: CGTCACGGGTcacAATACGGTTTTCCAGTTC

[0092] Primers used for point mutation of M3 mutant:

[0093] F79: CAACTCTagcGATaaaCTGCACATTTTCCTGGT

[0094] R79: tccgATGGGTAGCCACAACTCTagcGATaaaCTG

[0095] F205: GTTCTTCGGCgaaAACATCCTGGGCCAGTTT

[0096] R205: AGGATGTTttcGCCGAAGAACGGATACGG

[0097] The recombinant vector pCDFDuet-1-M8, expressing the GyCGT1 mutant M8, is obtained by replacing the fragment between the BamHI and HindIII restriction sites in plasmid pCDFDuet-1 with the coding nucleic acid of the GyCGT1 mutant M8. The N-His tag in this vector is co-expressed with the coding nucleic acid of M8 to obtain the recombinant protein M8.

[0098] The recombinant vector pCDFDuet-1-M3, expressing the GyCGT1 mutant M8, was obtained by replacing the fragment between the BamHI and HindIII restriction sites in plasmid pCDFDuet-1 with the coding nucleic acid of the GyCGT1 mutant M8. The N-His tag in this vector was co-expressed with the coding nucleic acid of M3 to obtain the recombinant protein M3.

[0099] The above-mentioned recombinant vector expressing the GyCGT1 mutant glycosyltransferase was prepared according to the following method:

[0100] (1) Amplification of plasmids: High-fidelity enzymes were used with pCDFDuet-1-GyCGT1 as a template and primers corresponding to different mutants were used for amplification. The amplification program and system are shown in Table 1 and Table 2.

[0101]

[0102]

[0103] (2) Digest the unmutated plasmid with DpnⅠ and digest it at 37 °C for 2 h.

[0104] (3) Transform DH5α Escherichia coli competent cells, select single clones, and perform DNA sequencing to determine the successfully mutated genes, and obtain the recombinant vector pCDFDuet-1-M8 expressing the glycosyltransferase GyCGT1 mutant M8 and the recombinant vector pCDFDuet-1-M3 expressing the glycosyltransferase GyCGT1 mutant M3.

[0105] II. Preparation of GyCGT1 mutant glycosyltransferase

[0106] 1. Preparation of recombinant bacteria

[0107] The recombinant vectors pCDFDuet-1-GyCGT1, pCDFDuet-1-M8, and pCDFDuet-1-M3 expressing the glycosyltransferase GyCGT1 and its mutants were transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3) / pCDFDuet-1-GyCGT1, BL21(DE3) / pCDFDuet-1-M8, and BL21(DE3) / pCDFDuet-1-M3.

[0108] 2. Expression and purification of glycosyltransferases and their mutants

[0109] Recombinant *E. coli* BL21(DE3) / pCDFDuet-1-GyCGT1, BL21(DE3) / pCDFDuet-1-M8, and BL21(DE3) / pCDFDuet-1-M3 were inoculated into LB liquid medium containing streptomycin and cultured at 37 °C until OD600 = 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM for induction, and the cells were cultured at 18 °C and 180 rpm for 16 h. The cells were collected by centrifugation at 7,000 rpm for 3 min. The cells were resuspended in washing buffer (30 mM imidazole, 20 mM Tris, 200 mM NaCl, and 2% glycerol (v / v)) and sonicated at 35-40% power for 3.3 s / 3.3 s for a total of 8-10 min. The cells were then centrifuged at 7,500 rpm at 4 °C for 45-50 min, and the supernatant was collected.

[0110] The supernatant was added to a Ni-NTA-containing chromatography column (equilibrated with washing buffer beforehand), and washed twice to ensure the protein was fully bound to the nickel column. Impurities were eluted with washing buffer, and the target protein was eluted with elution buffer (300 mM imidazole, 20 mM Tris, 200 mM NaCl, and 2% glycerol (v / v)) to obtain GyCGT1 protein solution, mutant M8 protein solution, or mutant M3 protein solution.

[0111] Proteins were analyzed using SDS-PAGE: GyCGT1 (denoted as WT in the figure), mutant M8 (denoted as M8 in the figure), or mutant M3 (denoted as M3 in the figure). The results are as follows: Figure 1 As shown, the values ​​are all 53.9 kDa, consistent with expectations.

[0112] After centrifuging and concentrating each protein solution, the protein content was detected using a micro spectrophotometer to ensure that the concentration was 1 mg / mL.

[0113] Example 2: Functional verification of the glycosyltransferase GyCGT1 mutant

[0114] 1. In vitro experiments

[0115] One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 micromole of luteolin to isorhamnetin per minute under standard assay conditions.

[0116] 100 µL reaction system: 0.1 mM luteolin, 0.25 mM UDPG, 0.4 µg glycosyltransferase GyCGT1 (referred to as wild type) or its mutant (M8 or M3), with the remainder being 50 mM NaH2PO4-Na2HPO4 buffer at pH 8.

[0117] The formula for a NaH2PO4-Na2HPO4 buffer solution with a pH of 8 and a concentration of 50 mM is as follows: Mix approximately 94 mL of 50 mM NaH2PO4 solution with approximately 406 mL of 50 mM Na2HPO4 solution and bring the volume to 1 L. Then, precisely adjust the pH to 8.0 using a pH meter.

[0118] After reacting the above reaction system at 37 °C for 2 h, 200 μL of pre-cooled methanol was added and mixed well. The mixture was then centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for HPLC analysis.

[0119] The HPLC detection conditions are as follows:

[0120] HPLC was performed using an Agilent 1260 (Agilent, USA). The column was an Agilent Eclipse XDB-C18 (Agilent), 4.6 mm (inner diameter) × 150 mm (length); the packing pore size was 5 μm; the column temperature was 30 °C; the flow rate was 0.8 mL / min; the injection volume was 50 μL; and the detection wavelength was 353 nm. The mobile phase was: phase A was aqueous (containing 0.1% formic acid), and phase B was methanol (HPLC grade). The method involved a linear gradient of the mobile phase (0–5 min: 5–20% B, 5–20 min: 20–40% B, 20–30 min: 40–70% B, 30–42 min: 70–100% B).

[0121] Accurately weigh 5 mg each of isopropargyl glycoside and luteolin standards, dissolve them in chromatographic grade methanol using ultrasound to prepare a 100 mg / L standard solution; pipette 2 mL of each of the two standard solutions into a volumetric flask, shake to mix, and obtain a 50 mg / L mixed standard stock solution. The retention time of isopropargyl glycoside standard is 18 min, and the retention time of luteolin standard is 23 min.

[0122] The formation of isopropanol was determined by HPLC based on the comparison of retention times of the standard and the sample.

[0123] The peaks detected by HPLC are recorded as substrate peaks and product peaks.

[0124] Conversion rate = (Peak area of ​​product peak / (Peak area of ​​substrate peak + Peak area of ​​product peak)) * 100%

[0125] The ratio of the conversion rate of the relative enzyme activity mutant to the conversion rate of the wild type is used to represent this.

[0126] Results: The activities of mutants M3 and M8 in catalyzing the production of isorhodin from luteolin were 10.42 and 48.5 times that of the wild type, respectively.

[0127] 2. Escherichia coli in vivo experiment (50mL shake flask)

[0128] Recombinant Escherichia coli BL21(DE3) / pCDFDuet-1-GyCGT1, BL21(DE3) / pCDFDuet-1-M8, or BL21(DE3) / pCDFDuet-1-M3 were inoculated into LB liquid medium containing 100 μg / mL streptomycin and cultured overnight at 37°C to obtain seed culture. Take 100 μL of seed culture and add it to 20 mL of LB liquid medium containing streptomycin for propagation; take 1 mL of the propagated bacterial culture and add it to 10 mL of fermentation medium TB-Gly-Glc (tryptone: 12 g / L; yeast extract: 24 g / L; glycerol 10 g / L; KH2PO4: 2.32 g / L; K2HPO4: 12.54 g / L; glucose 10 g / L, the remainder being water) containing streptomycin and incubate at 37 °C and 200 rpm. When OD600 = 0.6-0.8, add 0.1 mM IPTG and induce culture at 20 °C and 180 rpm for 19 h, then add luteolin (concentration of 1 mM in the system) and incubate at 20 °C and 180 rpm for another 24 h, and collect the bacterial culture. 200 μL of bacterial culture was added to an equal volume of methanol to terminate the reaction. The mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was analyzed by HPLC (parameters were the same as in vitro experiments). Three biological replicates were performed for each assay, and the results are expressed as Mean ± SD.

[0129] Accurately weigh 5 mg of isoharmonic acid standard and dissolve it in chromatographic grade methanol using ultrasound to prepare a standard solution with a concentration of 1 mg / mL. Prepare working solutions for the isoharmonic acid standard curve by sequentially diluting with chromatographic grade methanol at concentration gradients of 10, 50, 100, 200, 300, 400, and 500 mg / L.

[0130] A standard curve was plotted using isoharmonic acid standard, and the standard curve for isoharmonic acid standard is y = 28.917x - 21.741 (R0). 2 =0.9994), where x is the concentration of isopropargyl glycoside and y is the peak area. The yield is calculated using this standard curve function.

[0131] The results are as follows Figure 2 As shown, the glycosyltransferase GyCGT1 (wild type) catalyzed the production of isopropionate from luteolin at a yield of 48.9 mg / L. The mutants M3 and M8 catalyzed the production of isopropionate from luteolin at yields of 134.3 mg / L and 414.4 mg / L, respectively, with activities (reflected by yield ratios) that were 2.75 times and 8.47 times that of the wild type, respectively.

[0132] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A mutant protein of GyCGT1, which is any one of the following: The amino acid sequence of the mutant protein shown in A1) is SEQ ID NO:4; The amino acid sequence of the mutant protein shown in A2) is SEQ ID NO:

6.

2. The biological material associated with the mutant protein of claim 1 is any one of B1) to B4) below: B1) A nucleic acid molecule encoding the mutant protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule is any one of the following: b1) The nucleotide sequence of the nucleic acid molecule shown is SEQ ID NO:3; The nucleotide sequence of the nucleic acid molecule shown in b2) is SEQ ID NO:

5.

4. The mutant protein of claim 1 in any of the following applications: C1) is a glycosyltransferase; C2) catalyzes the formation of isopropanol from luteolin; C3) Preparation of isopropargyl glycoside; C4) to prepare the product that catalyzes the conversion of luteolin to isorhamnetin.

5. The biomaterial of claim 2 or 3 may be used in any of the following applications: D1) Preparation of glycosyltransferases; D2) Catalyzes the formation of isopropanol from luteolin; D3) Preparation of isopropargyl glycoside; D4) Prepare the product that catalyzes the conversion of luteolin to isorhodin.

6. A method for preparing isoharonin, comprising the following steps: using the mutant protein of claim 1, with the assistance of UDPG, to catalyze the formation of isoharonin from luteolin.

7. A method for preparing isoharonidine, comprising the following steps: using the IPTG-induced culture of the recombinant microorganism of claim 2 to catalyze the generation of isoharonidine from luteolin.

8. A product for preparing isopropionyl glycoside, comprising the mutant protein of claim 1 or the biomaterial of claim 2, luteolin, and UDPG.

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