Sculellaria barbata-sourced flavonoid glycosyltransferase and application thereof

Abstract

The invention discloses a sculellaria barbata-sourced flavonoid glycosyl transferase and application thereof, the flavonoid glycosyl transferase can take flavonoid compounds (apigenin, baicalein, scutellarein and the like) as a substrate, and simultaneously catalyzes the 5-OH site and the 7-OH site of the substrate to carry out an oxygen glycosylation reaction, so that the flavonoid compounds (apigenin, baicalein, scutellarein and the like) can be obtained. The enzyme comprises an amino acid sequence shown in SEQ ID No.2 or variants with the same activity. Besides, the mutant is obtained through site-specific mutagenesis (for example, threonine at the 276th site is mutated into serine), the 5-O-glycosylation activity is remarkably enhanced, an important enzyme element is provided for efficient synthesis of rare flavone 5-O glycoside, and the mutant has wide application prospects in synthetic biology and drug development.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and in particular to a flavonoid glycosyltransferase with dual 5-O and 7-O glycosylation activity derived from Scutellaria barbata and its applications. Background Technology

[0002] Flavonoids are important secondary metabolites widely found in plants, possessing various biological activities such as antioxidant, anti-inflammatory, and antitumor activity. Among them, flavonoid glycosides, due to their enhanced water solubility, improved stability, and enhanced bioavailability after glycosylation modification, have significant application value in drug development and functional foods. Glycosylation modification is mainly catalyzed by UDP-glycosyltransferase (UGT), which can introduce glycosyl groups at different hydroxyl sites on the flavonoid skeleton, forming structurally diverse glycoside derivatives.

[0003] Scutellaria barbata D. Don, a traditional Chinese medicine, contains various flavonoids with significant anti-tumor activity, especially 5-O-glycosylated flavonoids (such as apigenin-5-O-glucoside), which have shown inhibitory effects on various cancer cells in in vitro experiments. However, the content of these compounds in the plant is extremely low, and due to the intramolecular hydrogen bond between the 5-hydroxyl group and the 4-carbonyl group, their chemical synthesis is difficult and inefficient, severely limiting their further development and application.

[0004] Furthermore, although the flavonoid synthesis pathway in Scutellaria baicalensis has been preliminarily elucidated, the key enzyme genes involved in the downstream glycosylation modification process, especially 5-O-glycosylation, remain unknown. Current technologies lack systematic research on 5-O-glycosyltransferases in Scutellaria barbata, hindering the efficient heterologous biosynthesis of these compounds and restricting their industrialization.

[0005] Therefore, identifying the UGT gene in Scutellaria barbata that specifically catalyzes 5-O-glycosylation of flavonoids and elucidating its catalytic mechanism has become an urgent need in the fields of natural product synthetic biology and drug development. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the prior art by providing a flavonoid glycosyltransferase with dual 5-O and 7-O glycosylation activity derived from Scutellaria barbata and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a flavonoid glycosyltransferase capable of catalyzing oxyglycosylation at both the 5-OH and 7-OH sites of a flavonoid compound as a substrate; the flavonoid glycosyltransferase comprises a protein as shown in (a) or (b) below: (a) A protein with the amino acid sequence shown in SEQ ID No. 2; (b) A protein having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the amino acid sequence shown in SEQ ID No. 2 and having dual catalytic activity of flavonoid-5-O and 7-O-glycosylation.

[0008] MKKVELVFIPSPGLSHLVSTVEAAKLLLDRDDRLSITVLIMSLRKDTIVDNYTQKTTSNLNASHRLRFINLPTQDHTPASSAYAQFNQINDQITLVRDAISDLINHSQLAGLVLDMFCI NFMDVADEFCIPAYVFYTANASALGLFLHLTSLKLEHNQDLTQYRDSDVELSVPCFSIPVPARVLPDVLIDGTPMADLLMNYHKRISETKGIMVNSFYELETYAIESLLSDAKTPKVYPV GPILGFDQSQRSFGDDVIKKWLDDQPESSVVFLCFGTMGSFSEGQVKEIALALEKSGNRFLWSLRKKVDNVKVTEYENFEEVLPQGFLERTKELGKVIGWAPQAAVLSHPAVGGFISHC GWNSTLESLWFGVPMATFPLYAEQQMNAFKLVKEQGIAEMIRLDYNMDFTGERPPEIVGWEEIEAAIRRLMAEKGESGVRHKVKEMQNKARSALVEGGSSYNAQCFFIEDVIRNIA (SEQ ID No.2).

[0009] The second aspect is to provide an isolated polynucleotide for encoding the flavonoid glycosyltransferase described in the first aspect.

[0010] Furthermore, the nucleotide sequence of the polynucleotide is shown in SEQ ID No. 1.

[0011]

[0012] The third aspect is to provide a primer composition for amplifying the full sequence or partial fragment of the polynucleotide described in the second aspect; the primer composition comprises one or more of the primer pairs consisting of the sequences shown in SEQ ID No. 3 and SEQ ID No. 4, the primer pairs consisting of the sequences shown in SEQ ID No. 5 and SEQ ID No. 6, and the primer pairs consisting of the sequences shown in SEQ ID No. 7 and SEQ ID No. 8.

[0013] The fourth aspect is to provide a recombinant vector comprising the polynucleotides described in the second aspect.

[0014] The fifth aspect is to provide a host cell containing the recombinant vector described in the fourth aspect, or having the polynucleotides described in the second aspect integrated into its genome.

[0015] The sixth aspect is to provide a flavonoid glycosyltransferase mutant, wherein the mutant is based on the amino acid sequence shown in SEQ ID No. 2, with a site-directed mutation of alanine at position 138 and / or threonine at position 276.

[0016] Furthermore, the mutant contains a mutation in which threonine at position 276 is changed to serine, thereby giving the mutant enhanced flavonoid-5-O-glycosylation activity.

[0017] The seventh aspect is to provide a method for synthesizing flavonoid-5-O-glycosides and / or flavonoid-7-O-glycosides, the method comprising: contacting a flavonoid substrate with a substance selected from the flavonoid glycosyltransferases described in the first aspect, the mutants described in the sixth aspect, and the host cells described in the fifth aspect, under conditions that allow glycosylation to occur.

[0018] Furthermore, the flavonoid substrate is selected from apigenin, baicalin, or baicalin.

[0019] Compared with the prior art, the present invention adopts the above technical solution and has the following technical effects: The present invention identifies SbarF5GT from Scutellaria barbata for the first time. Its single enzyme molecule can simultaneously and efficiently catalyze the generation of flavonoid-5-O-glycoside and 7-O-glycoside, realizing the one-step synthesis of two glycoside products with different sites in a single reaction system, avoiding the need to use multiple enzymes or complex chemical steps, which is a fundamental breakthrough in its catalytic function.

[0020] This invention, through further design, precisely modifies the enzyme molecule by mutating threonine at position 276 to serine (T276S), significantly enhancing its 5-O-glycosylation activity and increasing the yield of target products (such as apigenin-5-O-glucoside). This provides an important enzyme element for the efficient synthesis of rare flavonoid 5-O glycosides and has broad application prospects in synthetic biology and drug development. Attached Figure Description

[0021] Figure 1 The in vitro enzyme activity was used to synthesize apigenin-5-O-glucoside; (A) schematic diagram of SbarF5GT catalytic activity; (B) total ion chromatogram of SbarF5GT catalytic activity; (C) secondary mass spectra of standard and enzyme activity product.

[0022] Figure 2 The activity of the SbarF5GT mutant protein was measured. (A) Catalytic activity diagram of the mutation of alanine (A) at position 138 of SbarF5GT amino acid to serine (S); (B) Catalytic activity diagram of the mutation of threonine (T) at position 276 of SbarF5GT amino acid to serine (S).

[0023] Figure 3 To synthesize apigenin-5-O-glucoside in yeast; (A) Extraction ion chromatogram of enzyme activity results in SbarF5GT yeast; (B) Secondary mass spectra of standard and enzyme activity product. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0025] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0026] All reagent kits and enzymes used in the following examples were purchased from Tiangen Biotech Co., Ltd., Thermo Fisher Scientific, NEB, Takara Bio Inc., and Nanjing Novizan Biotechnology Co., Ltd. Plasmid pK7WG2R was purchased from Thermo Fisher Scientific, and plasmid pDoner207 was purchased from Thermo Fisher Scientific.

[0027] Example 1: Cloning of SbarF5GT and Construction of Corresponding Heterologous Expression Vectors (1) Design and synthesis of primers for the SbarF5GT gene First, the genome of *Scutellaria barbata* was downloaded from NCBI. The UGT gene family was identified using a hidden Markov model. Then, a phylogenetic tree was constructed using the protein sequence of the *Arabidopsis thaliana* UGT gene and the sequence in *Scutellaria barbata*. Members of the same subfamily as sequences with similar functions reported in previous studies were selected to obtain the relevant gene sequences in *Scutellaria barbata*. Full-length primers were designed using Primer Premier 5.0, and Gateway recombination sites (underlined) were added to the primers.

[0028] attB1-SbarF5GT-F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGAAGAAAGTTGAATTGGTATTCATCC(SEQ IDNo.3); attB2-SbarF5GT-R: GGGGACCACTTTGTACAAGAAAGCTGGGTT CTAGGCAATGTTTCTGATAACATCCT(SEQ IDNo.4); Primers were synthesized by Shanghai Sangon Biotech.

[0029] (2) Cloning of SbarF5GT Using Scutellaria barbata root as material, total RNA was extracted from the root according to the instructions of the RNA extraction kit, and cDNA from the root was obtained by reverse transcription using a reverse transcription kit.

[0030] Using cDNA as a template and attB1-SbarF5GT-F and attB2-SbarF5GT-R as primers, the SbarF5GT gene was obtained through amplification.

[0031] PCR reaction system: cDNA 1μL, upstream primer 2.5μL, downstream primer 2.5μL, phusion HF buffer 10μL, 10mM dNTP mix 1μL, phusion enzyme 0.5μL, ddH2O 32.5μL.

[0032] The PCR program was set as follows: 1) 98℃, 3 min; 2) 98℃, 10 s; 3) 58℃, 20 s; 4) 72℃, 1 min; 5) 72℃, 5 min. The cycle was from 2) to 4), and the number of cycles was 30.

[0033] After the PCR reaction was completed, the product was separated by agarose gel electrophoresis, and 1425bp SbarF5GT was obtained.

[0034] (3) Constructing the pDonar207-SbarF5GT vector The specific bands of the PCR products from step (2) were recovered using the Tiangen gel extraction kit, and the concentration of the recovered products was determined.

[0035] The BP reaction was then performed using the recovered product of SbarF5GT. BP reaction: 100-200 ng (3 μL) of PCR product, 100 ng (1 μL) of pDonar207, 0.5 μL of 1×TE, and 0.5 μL of BP Clonase enzyme; incubated at 25°C for 5 h, then 0.5 μL of proteinase K was added and incubated at 37°C for 10 min to terminate the BP reaction.

[0036] The reaction product was transferred into *E. coli* DH5α and plated on LB agar containing 40 mg / L gentamicin. The culture was incubated at 37°C for 12 h. The following day, single colonies were picked for colony PCR verification, and positive clones were sent to a biotechnology company for sequencing. Based on the sequencing results, the plasmid pDonar207-SbarF5GT was extracted.

[0037] (4) Construct a heterologous expression vector for the SbarF5GT gene The *E. coli* and yeast expression vectors used for SbarF5GT were pYesDEST17 and pYesDEST52, respectively. After determining the concentration of the plasmid obtained from the BP reaction in (3), the LR reaction was performed. LR reaction: pDonar207-SbarF5GT 100-200 ng (3 μL), pYesDEST17 (or pYesDEST52) 100 ng (1 μL), 1×TE 0.5 μL, LR Clonase enzyme 0.5 μL; incubate at 25℃ for 5 h, add 0.5 μL proteinase K, incubate at 37℃ for 10 min, and terminate the LR reaction.

[0038] The reaction product was transferred into *E. coli* DH5α and plated on LB agar containing 50 mg / L ampicillin. The culture was incubated at 37°C for 12 h. The next day, single clones were picked for colony PCR verification. Plasmids were extracted from the positive clones to obtain the *E. coli* expression vector pYesDEST17-SbarF5GT or the yeast expression vector pYesDEST52-SbarF5GT containing SbarF5GT.

[0039] Example 2: Site-directed amino acid mutation of SbarF5GT protein (1) Design and synthesis of primers for site-directed mutagenesis of SbarF5GT gene Primers were designed using Primer Premier 5.0. The corresponding replacement bases were found according to the required mutated amino acid. A pair of completely complementary primers were designed. The original bases at the mutation site were replaced with the target bases on the primers. The sequence on the left and right of the mutation site was 15-20 bp consistent with the template. The underlined bases were the mutated bases.

[0040] SbarF5GT138-F: GCATACGTTTTCTACACT T CCAACGCGTCTGCACTTG (SEQ ID No.5) SbarF5GT138-R: CAAGTGCAGACGCGTTGG A AGTGTAGAAAACGTATGC (SEQ ID No. 6) SbarF5GT276-F: GTTTTCTTGTGCTTTGGG T CCATGGGAAGCTTCAGTG (SEQ ID No.7) SbarF5GT276-R: CACTGAAGCTTCCCATGG A CCCAAAGCACAAGAAAAC (SEQ ID No.8) Primers were synthesized by Shanghai Sangon Biotech.

[0041] (2) Site-directed mutagenesis and sequencing of the SbarF5GT gene Using the E. coli expression vector pYesDEST17-SbarF5GT from Example 1 as a template, and using SbarF5GT138-F and SbarF5GT138-R (or SbarF5GT276-F and SbarF5GT276-R) as primers, an amplification reaction was carried out.

[0042] PCR reaction system: cDNA 1μL, upstream primer 2.5μL, downstream primer 2.5μL, phusion HF buffer 10μL, 10 mM dNTP mix 1μL, phusion enzyme 0.5μL, ddH2O 32.5μL.

[0043] The PCR program was set as follows: 1) 98℃, 3 min; 2) 98℃, 10 s; 3) 58℃, 20 s; 4) 72℃, 30 s / kb; 5) 72℃, 5 min. The number of cycles was 30, from 2) to 4).

[0044] After the PCR reaction is complete, the remaining template plasmid is digested with the restriction endonuclease Dpn I.

[0045] Enzyme digestion system: 10 μL of the above PCR product, 2 μL of 10x buffer, 2 μL of Dpn 1 enzyme, and 6 μL of ddH2O. Incubate at 37°C for 3 hours.

[0046] The reaction product was then transferred into *E. coli* DH5α, plated on LB agar containing 100 mg / L ampicillin, and incubated at 37°C for 12 h, followed by incubation at 4°C for 10–20 min. White colonies were selected for colony PCR detection, and positive clones were sent to a biotechnology company for sequencing. Based on the sequencing results, the plasmid pYesDEST17-SbarF5GT was extracted. A138S and pYesDEST17-SbarF5GT T276S .

[0047] Example 3 Synthesis of apigenin-5-O-glucoside using an in vitro enzymatic method (1) Expression and purification of SbarF5GT and its mutant proteins The chemical transformation method was used to transform pYesDEST17-SbarF5GT from Example 1 or the mutant pYesDEST17-SbarF5GT from Example 2. A138T and pYesDEST17-SbarF5GT T276SThe vector was transformed into the *E. coli* expression strain BL21(DE3). Multiple single colonies were picked and inoculated into 50 mL of LB medium containing the corresponding ampicillin (Amp) and cultured overnight with shaking. 25 mL of this bacterial culture was transferred to 500 mL of LB medium containing Amp and cultured at 37°C until the OD600 reached 0.8-1.0. Then, 1 mM IPTG was added to a final concentration, and protein expression was induced for 16 h at 16°C and 120 rpm. The bacterial cells were collected by centrifugation at 4000 rpm for 15 minutes. The bacterial cells were resuspended in 50-100 mL of pre-chilled 1×Ni-NTA binding buffer (50 mM NaH2PO4 (pH 8.0) + 300 mM NaCl + 2 mM β-mercaptoethanol + 0.5 mM PMSF + 10 mM imidazole) and disrupted using an autoclave (800-900 bar). The lysate was centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant was gently mixed with the equilibrated Ni-NTA His Band resin suspension and incubated at 4°C for 60 minutes. After incubation, the resin was centrifuged at 700 rpm for 5 minutes at 4°C. The supernatant was carefully discarded, and the resin was gently resuspended in 10 mL of 1×Ni-NTA wash buffer (50 mM NaH2PO4 (pH 8.0) + 300 mM NaCl + 2 mM β-mercaptoethanol + 0.5 mM PMSF + 20 mM imidazole). The suspension was centrifuged again, the supernatant was discarded, and the wash was repeated once more. The resin suspension was transferred to a pretreated filter column, and the target protein was eluted with 2 mL of 1×Ni-NTA elution buffer (50 mM NaH2PO4 (pH 8.0) + 300 mM NaCl + 2 mM β-mercaptoethanol + 0.5 mM PMSF + 250 mM imidazole). 60% pre-chilled glycerol was added to the eluent to a final concentration of 40%. The eluent was aliquoted and stored at -80°C. Finally, the protein was detected by 10% Precast-GLgel Tris-Glycine gel electrophoresis, and the protein concentration was determined using the Bradford method.

[0048] (2) In vitro enzyme activity In vitro enzyme activity was performed using a 100 μL system containing: 100 mM Tris-HCl (pH 6.8), 5 μg purified protein, 0.1 mM apigenin, and 0.5 mM UDP-glucose, with the final volume made up to 100 μL using ddH2O. The system was incubated at 37°C for 2 h. The reaction was terminated by adding pre-cooled methanol at a final concentration of 70%, centrifuged at 12000 rpm for 2 min, and the supernatant was filtered through a 0.22 μm nylon membrane for LC-MS analysis.

[0049] (3) Detection method The product was determined by Agilent 1260 Infinity II HPLC. A Phenomenex Luna C18 (2) column (100 mm × 2 mm, 3 μm) was used. The flow rate was 0.4 mL / min, the column temperature was 35 °C, the detection wavelength was 340 nm, and the injection volume was 20 μL. The mobile phases were 0.1% (v / v) formic acid / water (A) and 0.1% (v / v) formic acid + acetonitrile / methanol (1:1) (B). The gradient elution conditions were: 0–3 min, 20% B; 20 min, 50% B; 20–30 min, 50% B; 36 min, 30% B; 37 min, 20% B; 37–43 min, 20% B. The concentration of the enzyme activity product peak was calculated based on the standard curve.

[0050] LC-MS / MS was performed using Thermo Q Exactive Plus. Using the same column and gradient elution conditions as described above, mass spectrometry ion fragments were obtained in negative ion mode using a heated ESI source with the following parameters: gas heater 350℃; sheath gas flow rate 40 L / min; spray voltage 3.5 kV; capillary temperature 320℃. Full scan / data-dependent secondary scan was performed using automatic gain control (AGC) with a target of 1×10⁻⁶. 5 and 1×10 6 The m / z recording range is 50-750, and the resolution is 1750.

[0051] Figure 1 The results showed that, in the in vitro reaction, the purified SbarF5GT protein could use apigenin as a substrate to simultaneously generate apigenin-5-O-glucoside and apigenin-7-O-glucoside, and the product structure was verified by secondary mass spectrometry, confirming its dual catalytic function.

[0052] Figure 2 The results showed that the mutant SbarF5GT obtained through site-directed mutagenesis... T276S (Threonine at position 276 is mutated to serine) Compared with the wild type, the yield of apigenin-5-O-glucoside is significantly increased, indicating that this mutant has enhanced flavonoid-5-O-glycosylation activity.

[0053] Example 4 Synthesis of apigenin-5-O-glucoside in yeast The pYesDEST52 vector constructed in Example 1 was transformed into WAT11 competent yeast cells using the ZYMO yeast transformation kit. The cells were then plated on SD / -Ura plates containing 2% glucose and cultured at 28°C for 3 days. After colony growth, several single colonies were picked and inoculated into 20 mL of SD / -Ura liquid medium containing 2% glucose, and cultured at 28°C and 220 rpm for 24 h. The cells were collected by centrifugation at 4000 rpm for 10 min, resuspended in 20 mL of SD / -Ura medium containing 2% galactose, and apigenin was added to a final concentration of 50 μM. Induction culture was continued at 220 rpm for 2 days (with 2 mL of galactose added after 24 h of culture). After induction, the cells were collected by centrifugation at 12000 rpm for 5 min and then extracted by sonication using 2 mL of 70% methanol solution for 2 h.

[0054] (2) Detection method Same as step (3) in Example 3.

[0055] Figure 3 The results showed that SbarF5GT was able to successfully synthesize apigenin-5-O-glucoside in vivo using intracellularly provided UDP-glucose and exogenously added apigenin substrate in the yeast expression system, demonstrating its potential in practical biomanufacturing applications.

[0056] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A flavonoid glycosyltransferase, characterized in that, The flavonoid glycosyltransferase is capable of using flavonoids as substrates and simultaneously catalyzing oxyglycosylation at both the 5-OH and 7-OH sites of the substrates; the flavonoid glycosyltransferase comprises a protein as shown in (a) or (b) below: (a) a protein with the amino acid sequence shown in SEQ ID No. 2; (b) a protein having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID No. 2 and possessing dual catalytic activity of flavonoid-5-O and 7-O-glycosylation.

2. An isolated polynucleotide, characterized in that, The polynucleotide is used to encode the flavonoid glycosyltransferase as described in claim 1.

3. The polynucleotide according to claim 2, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID No.

1.

4. A primer composition, characterized in that, The primer composition is used to amplify the full sequence or partial fragment of the polynucleotide of claim 2 or 3; the primer composition comprises one or more of the primer pairs consisting of the sequences shown in SEQ ID No. 3 and SEQ ID No. 4, the primer pairs consisting of the sequences shown in SEQ ID No. 5 and SEQ ID No. 6, and the primer pairs consisting of the sequences shown in SEQ ID No. 7 and SEQ ID No.

8.

5. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide as described in claim 2 or 3.

6. A host cell, characterized in that, The host cell contains the recombinant vector of claim 5, or has the polynucleotide of claim 2 or 3 integrated into its genome.

7. A flavonoid glycosyltransferase mutant, characterized in that, The mutant is a site-directed mutation of alanine at position 138 and / or threonine at position 276, based on the amino acid sequence shown in SEQ ID No.

2.

8. The mutant according to claim 7, characterized in that, The mutant contains a mutation that changes threonine at position 276 to serine, thereby giving the mutant enhanced flavonoid-5-O-glycosylation activity.

9. A method for synthesizing flavonoid-5-O-glycoside and / or flavonoid-7-O-glycoside, characterized in that, The method comprises contacting a flavonoid substrate with a substance selected from the flavonoid glycosyltransferase of claim 1, the mutant of claim 7 or 8, or the host cell of claim 6, under conditions that allow glycosylation to occur.

10. The method according to claim 9, characterized in that, The flavonoid substrates are selected from apigenin, baicalin, or baicalin.

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