Connecting peptide and application thereof in promoting compound glucose methylation modification

Abstract

The invention discloses a connecting peptide and application thereof in promoting compound glucose methylation modification. A glycosyl transferase-methyltransferase fusion protein gene and genes LtLasS1 and LtLasS2 coded by benzenediol lactone synthetase, which are connected in series by connecting peptides, are co-expressed, and liquid phase and mass spectrometry of a fermentation product are carried out to determine that the fusion protein can realize glucose methylation modification by hydroquinone lactone. Wherein the conversion efficiency of the fusion protein hydroquinone lactone using the DL-4 connecting peptide is the highest, and the relative conversion rate is 65.54 + / -3.75%. Besides, by feeding a substrate kaempferol and testing the glucose methylation conversion efficiency of the artificial fusion protein constructed by the connecting peptide, the conversion efficiency of the fusion protein using the DL-4 connecting peptide to the substrate is 6 times of the conversion efficiency of the fusion protein using a single glycosyl transferase and a single methyltransferase to the substrate, and the relative conversion rate is increased to 60.12 + / -4.79%.

Description

Technical Field

[0001] The invention belongs to the fields of synthetic biology and biotechnology, and particularly relates to a connecting peptide and application thereof in promoting the methylation modification of diol lactone and kaempferol glucose. Background Art

[0002] In the field of biotechnology, the design and optimization of fusion proteins is one of the key links. Fusion proteins are usually composed of two or more functional proteins connected by a linker peptide. The linker peptide plays a key role in this process, not only connecting the two functional proteins but also allowing them to maintain their respective structures and functions.

[0003] Currently, the design of linker peptides is mostly based on artificial synthesis or known flexible / rigid peptide segments. For example, in the production of β-lactam antibiotics, a rigid linker peptide (GGGGS)2 is used to fuse the GST tag with penicillin acylase to achieve efficient purification and catalyze penicillin G to produce the intermediate 6-aminopenicillinanoic acid. However, with the development of biotechnology bringing new application scenarios, the design and optimization of fusion proteins urgently need to discover more diverse new sequences.

[0004] Multidomain proteins connect two or more functional domains via linkers. Beyond their fundamental role as connectors, these linkers also possess additional functions, such as maintaining interdomain interactions and preserving the biological activities of each domain. Linker sequences in bifunctional proteins have evolved over time to possess high stability and biocompatibility. Furthermore, linker sequences can better maintain synergistic interactions between the functional domains of a fusion protein, thereby enhancing the overall performance of the fusion protein. Research on the structure and function of linkers in multidomain proteins can provide insights into the design of linkers in recombinant fusion proteins. Summary of the Invention

[0005] The purpose of the present invention is to provide a connecting peptide and its application in promoting the glucose methylation modification of compounds.

[0006] A connecting peptide, wherein the connecting peptides are DL-1, DL-2, DL-3, and DL-4, and their amino acid sequences are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0007] The invention relates to an artificial fusion protein comprising the connecting peptide, wherein the fusion protein connects the glycosyltransferase BbGT and the methyltransferase BbMT via the connecting peptide.

[0008] A DNA base sequence encodes the fusion protein.

[0009] A vector comprising the coding gene sequence of the fusion protein.

[0010] The application of the connecting peptide in promoting glucose methylation modification of compounds.

[0011] The compound is diol lactone or kaempferol.

[0012] The beneficial effects of the present invention are as follows: the connecting peptide sequence of the present invention can connect two independently functional modification enzymes in series to form an artificial fusion protein. In the yeast BJ5464-NpgA, the genes encoding the glycosyltransferase (BbGT)-methyltransferase (BbMT) fusion protein and the genes encoding the hydroxybenzene lactone synthases hrPKS and nrPKS connected in series by a connecting peptide were connected. LtLasS1 and LtLasS2 Co-expression of the fusion proteins and liquid chromatography-mass spectrometry analysis of the fermentation products confirmed their ability to methylate demethylated quinol lactone. Based on the three-dimensional protein structure of the linker peptide, the linker peptide sequence was further truncated and optimized, resulting in four truncated linker peptides. The fusion protein using the DL-4 linker peptide exhibited the highest conversion efficiency for demethyl-lasiodiplodin, with a relative conversion rate of 65.54±3.75%. Furthermore, the glucose methylation conversion efficiency of the artificial fusion proteins constructed using the four linker peptides was tested by feeding the substrate kaempferol. The fusion protein using the DL-4 linker peptide exhibited a six-fold increase in conversion efficiency compared to that achieved using either the individual glycosyltransferases (BbGT) or methyltransferases (BbMT), with a relative conversion rate of 60.12±4.79%. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Structural formulas of compound 1 hydroquinone lactone and compound 2.

[0014] Figure 2 Converted LtLasS1 、 LtLasS2 Liquid chromatogram at 300 nm of the crude extract extracted after fermentation of yeast encoding genes of fusion proteins connected with AoiQ-Linker and DL-1 respectively.

[0015] Figure 3 Converted LtLasS1 、 LtLasS2 Liquid chromatogram at 300 nm of crude extracts extracted after fermentation of yeast encoding genes for BbGT-BbMT fusion proteins connected with linker peptides of different lengths.

[0016] Figure 4 Mass spectra of compound 1 and compound 2.

[0017] Figure 5 Bar graph showing the conversion efficiency of compound 1 by BbGT-BbMT fusion proteins constructed using linker peptides of different lengths.

[0018] Figure 6 The structural formulas of compound 3 kaempferol and compound 4.

[0019] Figure 7 Liquid chromatograms at 300 nm of crude extracts from yeast transformed with separate BbGT and BbMT encoding genes after fermentation with different concentrations of kaempferol.

[0020] Figure 8 Mass spectra of compound 3 kaempferol and compound 4.

[0021] Figure 9 Bar graph of the conversion efficiency of BbGT-BbMT non-fusion protein to different concentrations of kaempferol.

[0022] Figure 10 Liquid chromatogram at 300 nm of crude extracts from yeast transformed with BbGT-BbMT encoding genes linked by different length linker peptides and fermented with 1 mg / mL kaempferol.

[0023] Figure 11 Bar graph showing the conversion efficiency of 1 mg / mL kaempferol by BbGT-BbMT fusion proteins constructed using linker peptides of different lengths.

[0024] Figure 12 SDS-PAGE gel electrophoresis of the purified DL-4-linked fusion protein. DETAILED DESCRIPTION

[0025] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0026] The strains, vectors and plasmids used in the following examples were all from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0027] The expression plasmids Pxw06F_LtLasS1, pXW30F_LtLasS2, pRS425m-BbGT, pXW06F-BbMT, and glycosyltransferase encoding genes used in the following examples are BbGT , methyltransferase encoding gene BbMT,Xie, L., Zhang, L., Wang, C., Wang, X., Xu, Y. (2018). Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis. Proceedings of the National Academy of Sciences, 115(22), E4980–E4989. In the following examples, a reverse transcription kit (PrimeScript TM RT reagent kit with gDNAEraser was purchased from Takara, restriction endonucleases were purchased from NEB, seamless cloning kit (In-Fusion Kit), Escherichia coli competent Fast-T1, E. coli BL21 (DE3) and 2× Phanta Flash Master Mix (Dye Plus) were purchased from Novozymes, plasmid miniprep kit and universal DNA purification gel recovery kit were purchased from Tiangen, 10× TE buffer, 1 M lithium acetate and one-step protein fast staining solution were purchased from Coolaber, 5× SDS-PAGE loading buffer was purchased from Kangwei Century, and Spectra TM Multicolor Broad Protein Ladder was purchased from Thermo Fisher Scientific, and SurePAGE TM Protein precast gels were purchased from GenScript, and imidazole was purchased from Sigma.

[0028] The culture medium and reagent formulas used in the following examples are as follows: PDB medium (1 L): purchased from Difco, BD Biosciences. TM Potato Dextrose Broth powder 24 g, distilled water to 1 L; LB medium (1 L): 25 g of LB Broth purchased from Sangon Biotech Co., Ltd. was diluted to 1 L with distilled water. If solid, 20 g of agar was added. 2% YPD medium (1 L): 10 g yeast powder, 20 g glucose, 20 g peptone, distilled water to 1 L, add 20 g agar if solid; 1% YPD medium (1 L): 10 g yeast powder, 10 g glucose, 20 g peptone, and distilled water to 1 L; 1 M Sorbitol solution (1 L): 182 g sorbitol, dilute to 1 L with distilled water; SC 3- (Leu - Trp - Ura - ) Defective medium (1 L): 6.7 g Difo Yeast Nitrogen Base, 10 g glucose, 1.17 g DO Supplement-Leu / -Trp / -Ura (purchased from Takara), distilled water to 1 L, pH adjusted to 5.8, and 20 g agar if solid; SC 2- (Leu - Trp - ) Defective medium (1 L): 6.7 g Difo Yeast Nitrogen Base, 10 g glucose, 0.64 g DO Supplement-Leu / -Trp (purchased from Takara), distilled water to 1 L, add 20 g agar if solid; SC - (Leu - ) Defective medium (1 L): 6.7 g Difo Yeast Nitrogen Base, 10 g glucose, 0.70 g DO Supplement-Leu (purchased from Takara), distilled water to 1 L, add 20 g agar if solid; DNA extraction: 1.25% SDS, 100 mM Tris-HCl (pH 7.5), 50 mM EDTA (pH 8); Ampicillin (Amp): purchased from Sigma, stock solution concentration is 100 mg / mL, working concentration is 100 μg / mL, stored at -20°C; Kanamycin (Kan): purchased from Sigma, stock solution concentration is 50 mg / mL, working concentration is 50 μg / mL, stored at -20°C; PB buffer: 150 mM PB, 150 mM NaCl, pH = 7.6.

[0029] Example 1 Construction of fusion protein expression vector from B. bassianaAmplified from ATCC7159 cDNA BbGT and BbMT The coding gene fragment from D. phaseolorumd The connecting peptide sequences DL-1, DL-2, DL-3, and DL-4 were amplified from the gDNA of YX39261. A.oryzae of AoiQ The coding gene sequence fragments were synthesized by BGI, and the AoiQ-Linker sequence was amplified from them. Using the In-Fusion Kit, the above gene fragments were combined and ligated into the pRS425m vector to obtain plasmids pRS425m-BbGT-DL-1-BbMT, pRS425m-BbMT-DL-1-BbGT, pRS425m-BbGT-DL-2-BbMT, pRS425m-BbGT-DL-3-BbMT, pRS425m-BbGT-DL-4-BbMT, pRS425m-BbGT-AoiQ-Linker-BbMT, and pRS425m-BbMT-AoiQ-Linker-BbGT.

[0030] Extraction by Trizol method B. bassiana ATCC7159 RNA was reverse transcribed, and gDNA was extracted using the phenol-chloroform method.

[0031] Construction of pRS425m-BbGT-DL-1-BbMT: Nde I and Pme Ⅰ Double enzyme digestion of shuttle vector PRS425m was performed and a linear vector fragment of 7555 bp in size was recovered. B. bassiana ATCC7159 cDNA was used as a template and the gene was amplified using primers pRS425m-BbGT-F / BbGT-DL-1-R. BbGT The gene length is 1423 bp and the gene was amplified using primers DL-1-BbMT-F / BbMT-pRS425m-R BbMT , the gene length is 784 bp, D. phaseolorum The gDNA of YX39261 was used as a template, and the DL-1 sequence was amplified using primers DL-1-F / DL-1-R. The sequence length was 150 bp. After the fragment was recovered, the above fragment was ligated with the pRS425m linearized vector using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened using Amp-resistant LB solid medium, and the plasmid was extracted. Not Ⅰ and BamHIdentification by enzyme digestion with Ⅰ yielded two fragments of 6627 bp and 3224 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0032] Construction of pRS425m-BbMT-DL-1-BbGT: B. bassiana ATCC7159 cDNA was used as a template and the gene was amplified using primers pRS425m-BbMT-F / BbMT-DL-1-R. BbMT The gene length is 782 bp and the gene was amplified using primers DL-1-BbGT-F / BbGT-pRS425m-R BbGT , the gene length is 1425 bp, D. phaseolorum The gDNA of YX39261 was used as a template, and the DL-1 sequence was amplified using primers DL-1-F / DL-1-R. The sequence length was 150 bp. After the fragment was recovered, the above fragment was ligated with the pRS425m linearized vector using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened using Amp-resistant LB solid medium, and the plasmid was extracted. Not Ⅰ and BamH Identification by enzyme digestion with Ⅰ yielded two fragments of 6627 bp and 3224 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0033] Construction of pRS425m-BbGT-DL-2-BbMT: B. bassiana ATCC7159 cDNA was used as a template and the gene was amplified using primers pRS425m-BbGT-F / BbGT-R. BbGT The gene length is 1404 bp, and the gene was amplified using primers BbMT-F / BbMT-pRS425m-R BbMT , the gene length is 768 bp, D. phaseolorum The gDNA of YX39261 was used as a template, and the DL-2 sequence was amplified using primers BbGT-DL-2-3-F / DL-2-BbMT-R. The sequence length was 184 bp. After the fragment was recovered, the above fragment was ligated with the pRS425m linearized vector using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened using Amp-resistant LB solid medium, and the plasmid was extracted. Not Ⅰ and BamH Identification by enzyme digestion with Ⅰ yielded two fragments of 6627 bp and 3210 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0034] Construction of pRS425m-BbGT-DL-3-BbMT: B. bassiana ATCC7159 cDNA was used as a template and the gene was amplified using primers pRS425m-BbGT-F / BbGT-R. BbGT The gene length is 1404 bp, and the gene was amplified using primers BbMT-F / BbMT-pRS425m-R BbMT , the gene length is 768 bp, D. phaseolorum The gDNA of YX39261 was used as a template, and the DL-3 sequence was amplified using primers BbGT-DL-2-3-F / DL-3-BbMT-R. The sequence length was 168 bp. After the fragment was recovered, the above fragment was ligated with the pRS425m linearized vector using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened using Amp-resistant LB solid medium, and the plasmid was extracted. Not Ⅰ and BamH Identification by enzyme digestion with Ⅰ yielded two fragments of 6627 bp and 3192 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0035] Construction of pRS425m-BbGT-DL-4-BbMT: B. bassiana The cDNA of ATCC7159 was used as a template, the DNA sequence of DL-4 was designed into the primers BbGT-DL-4-R and DL-4-BbMT-F, and the gene was amplified using primers pRS425m-BbGT-F / BbGT-DL-4-R. BbGT The gene length is 1444 bp, and the gene was amplified using primers DL-4-BbMT-F / BbMT-pRS425m-R BbMT The gene length is 807 bp. After the fragments were recovered, the above fragments were connected to the pRS425m linearized vector using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strains were screened using Amp-resistant LB solid medium and the plasmid was extracted. Not Ⅰ and BamH Identification by enzyme digestion with Ⅰ yielded two fragments of 6627 bp and 3138 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0036] Construction of pRS425m-BbGT-AoiQ-Linker-BbMT: B. bassiana ATCC7159 cDNA was used as a template and the gene was amplified using primers pRS425m-BbGT-F / BbGT-AoiQ-Linker-R BbGTThe gene length is 1420 bp and the gene was amplified using primers AoiQ-Linker-BbMT-F / BbMT-pRS425m-R BbMT , the gene length is 786 bp, synthesized AoiQ The AoiQ-Linker sequence was amplified using the AoiQ-Linker-F / AoiQ-Linker-R primers. The length of the AoiQ-Linker sequence was 234 bp. After the fragment was recovered, the seamless cloning kit (In-Fusion Kit) was used to connect the above fragment to the pRS425m linear vector and transform it into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened with Amp-resistant LB solid medium and the plasmid was extracted. Not Ⅰ and BamH Identification by enzyme digestion with Ⅰ yielded two fragments of 6627 bp and 3312 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0037] Construction of pRS425m-BbMT-AoiQ-Linker-BbGT: B. bassiana ATCC7159 cDNA was used as a template and the gene was amplified using primers pRS425m-BbMT-F / BbMT-AoiQ-Linker-R BbMT The gene length is 783 bp and the gene was amplified using primers AoiQ-Linker-BbGT-F / BbGT-pRS425m-R BbGT , the gene length is 1423 bp, synthesized AoiQ The AoiQ-Linker sequence was amplified using the AoiQ-Linker-F / AoiQ-Linker-R primers. The length of the AoiQ-Linker sequence was 234 bp. After the fragment was recovered, the seamless cloning kit (In-Fusion Kit) was used to connect the above fragment to the pRS425m linear vector and transform it into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened with Amp-resistant LB solid medium and the plasmid was extracted. Not Ⅰ and BamH I enzyme digestion identified two fragments of 6627 bp and 3312 bp, which, combined with plasmid sequencing results, indicated that the plasmid was constructed correctly. The primers used to construct the fusion gene heterologous expression vector are as follows:

[0038] The PCR reaction system is: 2×Phanta Flash Max Master Mix 25 μL ddH2O 20.5 μL Upstream / downstream primer (10 μM) 2 μL each Template 0.5 μL PCR program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; annealing at 60°C for 15 s; extension at 72°C; extension rate of 5 s / kb, extension time determined by fragment size, 34 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0039] Example 2 Co-expression of fusion gene and hydroquinone lactone expression gene and product extraction Preparation and transformation of electrocompetent cells of Saccharomyces cerevisiae BJ5464-NpgA (1) Preparation of seed solution Saccharomyces cerevisiae BJ5464-NpgA was streaked and activated on 2% YPD solid medium. After culturing at 30°C for 2 days, a single colony was picked and inoculated into 5 mL of 2% YPD liquid medium and cultured overnight at 30°C and 220 rpm.

[0040] (2) Preparation of electroporation competent cells of Saccharomyces cerevisiae BJ5464-NpgA The seed solution was transferred to 50 mL of 2% YPD liquid medium (inoculation size was 0.15 OD / mL), shaken at 30°C and 220 rpm for 6 h, and cultured until OD600 was 0.9. The cells were collected by centrifugation at 4°C and 3000 rpm for 3 min, resuspended with 50 mL of pre-cooled sterile water, centrifuged at 4°C and 3000 rpm for 3 min, resuspended with 50 mL of pre-cooled 1 M Sorbitol solution, centrifuged at 4°C and 3000 rpm for 3 min, resuspended with 40 mL of pre-cooled 1 M Sorbitol solution, first added 5 mL of 10×TE buffer to mix, then added 5 mL of 1 M lithium acetate, shaken at 30°C and 220 rpm for 30 min, added 500 μL of 1 M DTT solution, shaken at 30°C and 220 rpm for 15 min, centrifuged at 4°C and 3000 rpm for 3 min, and resuspended with 50 mL of pre-cooled 1 M The cells were washed twice with Sorbitol solution, and finally resuspended in 2 mL of pre-cooled 1 M Sorbitol solution. The cells were aliquoted into 1.5 mL sterile EP tubes at 100 μL / tube and stored at -80°C.

[0041] (3) Yeast competent electroporation

[0042] Transform according to the above plasmid combination. Add 2 μL of each plasmid for transformation to 100 μL of yeast competent medium, transfer the mixture to a 2 mm electric shock cup, and perform pulse transformation at 1.5 KV, 25 μF, and 200 Ω. Transfer the transformed cells to a 12 mL shaking tube, add 3 mL of recovery solution (1 M Sorbitol: 2% YPD = 1:1), and recover at 30°C and 220 rpm for 5 hours. Centrifuge at 3000 rpm for 3 minutes, remove the supernatant, and use 3 mL of SC 3- (Leu - Trp - Ura - ) Wash twice with defective liquid culture medium and apply to SC 3- (Leu - Trp - Ura - ) on defective solid culture medium.

[0043] Fermentation and product extraction (1) Pick positive transformants and culture them in SC 3- (Leu - Trp - Ura - ) Defective medium was streaked for expansion culture, and after culturing at 30℃ for 24 h, the bacteria were picked and placed in 15 mL of SC 3- (Leu - Trp - Ura - ) The defective liquid culture medium was cultured to an OD600 of 1.0, and then cultured at 30°C, 220 rpm in a shaking incubator for 12 h. Then, an equal volume of 1% YPD liquid culture medium was added and cultured at 30°C, 220 rpm in a shaking incubator for 48 h.

[0044] An equal volume of ethyl acetate was added to the fermentation broth for extraction. The ethyl acetate layer was dried using a rotary evaporator and dissolved in 500 μL of chromatographic-grade methanol. The solution was filtered through a 0.22 μm organic filter membrane. The product was detected by liquid chromatography-mass spectrometry (LC-MS). The detection program was set as follows: 5%-95% acetonitrile-water gradient elution for 15 min, 95% acetonitrile-water isocratic elution for 3 min, and 95%-5% acetonitrile-water gradient elution for 2 min. The flow rate was 0.35 mL / min and UV full-wavelength scanning was used.

[0045] The transformed LtLasS1 、 LtLasS2 The crude extract was extracted after fermentation of yeast encoding BbGT-BbMT fusion protein gene connected with different linker peptides (DL-1, DL-2, DL-3, DL-4). LtLasS1 、 LtLasS2 The product peak of compound 1 (demethyl-lasiodiplodin, diphenol lactone) was obtained from the crude extract after yeast fermentation. LtLasS1 、 LtLasS2 In addition to the product peak of compound 1, another product peak, compound 2 ( Figure 1 、 Figure 4 ). By comparing the relative molecular masses of the two compounds through mass spectrometry analysis, it was found that the relative molecular mass of compound 2 was 176 greater than that of compound 1, indicating that there was an extra methylated glucose molecule on the molecule of compound 2. Based on the molecular weight of compound 2, it was determined that it was a glucose methylation product, indicating that after the conversion of LtLasS1 、 LtLasS2 Compound 1 was mostly transformed in yeast expressing the BbGT-BbMT fusion protein encoding gene connected with different linker peptides (DL-1, DL-2, DL-3, DL-4).

[0046] The high performance liquid chromatography of combination 1-6 ( Figure 2 ) The results showed that BbGT-BbMT / BbMT-BbGT connected to DL-1 through AoiQ-Linker can both perform glucose methylation modification on compound 1, and the order of gene connection (N-terminus and C-terminus) has no effect on the conversion.

[0047] By comparing the high performance liquid chromatography (HPLC) of the crude extracts after fermentation of combinations 1, 2, 5, 7, 8, and 9 Figure 3 ) was used to calculate the relative conversion rate of compound 1 by BbGT-BbMT under different length linker peptides (DL-1, DL-2, DL-3, DL-4). The conversion efficiency of compound 1 by the fusion proteins connected by the three linker peptides after truncation and optimization based on DL-1 gradually increased. Among them, the fusion protein connected by DL-4 had the best effect and the highest relative conversion rate of compound 1, which was 31.24±3.75% higher than that of the fusion protein connected by DL-1 ( Figure 5 ).

[0048] The above proves that the fusion protein connected by the connecting peptide AoiQ-Linker, DL-1, DL-2, DL-3, and DL-4 can achieve glucose methylation modification of compound 1, and the order of gene connection (N-terminus and C-terminus) has no effect on the conversion. Among the DLs truncated and optimized based on DL-1, the fusion protein connected by DL-4 has the highest relative conversion rate for compound 1.

[0049] Example 3 Determination of the Conversion Efficiency of Kaempferol by Combined Biosynthesis of Glycosyltransferase (BbGT) and Methyltransferase (BbMT) Yeast competent transformation The Saccharomyces cerevisiae BJ5464-NpgA electroporation competent medium prepared by the method in Example 2 was used, and the plasmids pRS425m-BbGT and pXW06F-BbMT were co-transformed into the yeast competent medium according to the following method.

[0050] 100 μL of competent yeast was added with 2 μL of each plasmid for transformation, and the mixture was transferred to a 2 mm electric shock cup. Pulse transformation was performed at 1.5 KV, 25 μF, and 200 Ω. The transformed cells were transferred to a 12 mL shaking tube, 3 mL of recovery solution was added, and the cells were recovered at 30°C and 220 rpm for 5 h. Centrifuged at 3000 rpm for 3 min, the supernatant was removed, and 3 mL of SC 2- (Leu - Trp - ) Wash twice with defective liquid culture medium and apply to SC 2- (Leu - Trp - ) on defective solid culture medium.

[0051] (2) Fermentation and product extraction Positive transformants were picked and plated on SC 2- (Leu - Trp - ) streak on the defective solid medium for expansion culture, and after culturing at 30℃ for 24 h, pick the bacteria and place them in 5 mL of SC 2- (Leu - Trp - ) The defective type cells were cultured in liquid culture medium until OD600 reached 1.0. After incubation at 30℃ and 220 rpm for 12 h, an equal volume of 1% YPD liquid culture medium was added. At the same time, kaempferol was fed at final concentrations of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, and 5 mg / mL. Three biological replicates were performed for each concentration group. The cells were cultured at 30℃ and 220 rpm for 48 h.

[0052] An equal volume of ethyl acetate was added to the fermentation broth for extraction. The ethyl acetate layer was dried using a rotary evaporator and dissolved in 500 μL of chromatographic-grade methanol. The solution was filtered through a 0.22 μm organic filter membrane. The product was detected by liquid chromatography-mass spectrometry (LC-MS). The detection program was set as follows: 5%-95% acetonitrile-water gradient elution for 15 min, 95% acetonitrile-water isocratic elution for 3 min, and 95%-5% acetonitrile-water gradient elution for 2 min. The flow rate was 0.35 mL / min and UV full-wavelength scanning was used.

[0053] Different concentrations of kaempferol (compound 3) were fed to yeast transformed with separate glycosyltransferase (BbGT) and methyltransferase (BbMT) encoding genes, and the fermentation products were analyzed by high performance liquid chromatography ( Figure 7 ), it was found that in addition to the peak of the starting substrate compound 3, another product peak, namely compound 4, was obtained, indicating that compound 3 was converted. After mass spectrometry analysis, the relative molecular weights of the two compounds were compared and it was found that the relative molecular weight of compound 4 was 176 ( Figure 6 、 Figure 8 ), indicating that there is an extra methylated glucose molecule in the molecule of compound 4.

[0054] The relative conversion rates of kaempferol by the glycosyltransferase (BbGT) and methyltransferase (BbMT) alone were calculated by comparing the relative peak area ratios of the conversion products and substrates in HPLC under the conditions of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, and 5 mg / mL kaempferol concentrations. It was found that with the increase of the kaempferol concentration, the conversion rate of kaempferol by the combination of glycosyltransferase (BbGT) and methyltransferase (BbMT) alone decreased gradually. When the kaempferol concentration was 1 mg / mL and approached saturation ( Figure 9 ).

[0055] The above demonstrates that the combination of separate glycosyltransferase (BbGT) and methyltransferase (BbMT) can achieve glucose methylation modification of kaempferol, and the conversion efficiency tends to saturation when the kaempferol concentration is 1 mg / mL.

[0056] Example 4 Determination of the Conversion Efficiency of GT-MT Fusion Protein to Kaempferol According to the results in Example 3 that the glycosyltransferase (BbGT) and methyltransferase (BbMT) alone reached saturation at a kaempferol concentration of 1 mg / mL, 1 mg / mL of kaempferol was used to feed the yeast transformed with the fusion gene.

[0057] The Saccharomyces cerevisiae BJ5464-NpgA prepared by the method in Example 2 was electroporated into competent cells, and pRS425m-BbGT-DL-1-BbMT, pRS425m-BbGT-DL-2-BbMT, pRS425m-BbGT-DL-3-BbMT pRS425m-BbGT-DL-4-BbMT were transformed into competent yeast cells.

[0058] 100 μL of competent yeast was added with 2 μL of plasmid for transformation, the mixture was transferred to a 2 mm electroporation cup, and pulsed with 1.5 KV, 25 μF, and 200 Ω for transformation. The transformed cells were transferred to a 12 mL shaking tube, 3 mL of recovery solution was added, and the cells were recovered at 30°C and 220 rpm for 5 h. Centrifuged at 3000 rpm for 3 min, the supernatant was removed, and 3 mL of SC - (Leu - ) Wash twice with defective liquid culture medium and apply to SC - (Leu - ) on defective solid culture medium.

[0059] Fermentation and product extraction: Positive transformants were picked and plated on SC - (Leu - ) streak on the defective solid medium for expansion culture, and after culturing at 30℃ for 24 h, pick the bacteria and place them in 5 mL of SC - (Leu - ) defective liquid culture medium was cultured to an OD600 of 1.0, and then cultured at 30°C, 220 rpm in a shaking incubator for 12 h. Then, an equal volume of 1% YPD liquid culture medium was added, and kaempferol was fed at a final concentration of 1 mg / mL. Three biological replicates were performed, and the cells were cultured at 30°C, 220 rpm in a shaking incubator for 48 h.

[0060] An equal volume of ethyl acetate was added to the fermentation broth for extraction. The ethyl acetate layer was dried using a rotary evaporator and dissolved in 500 μL of chromatographic-grade methanol. The solution was filtered through a 0.22 μm organic filter membrane. The product was detected by liquid chromatography-mass spectrometry (LC-MS). The detection program was set as follows: 5%-95% acetonitrile-water gradient elution for 15 min, 95% acetonitrile-water isocratic elution for 3 min, and 95%-5% acetonitrile-water gradient elution for 2 min. The flow rate was 0.35 mL / min and UV full-wavelength scanning was used.

[0061] Kaempferol (compound 3) at a final concentration of 1 mg / mL was fed to yeast transformed with the BbGT-BbMT fusion protein encoding gene connected by different connecting peptide sequences (DL-1, DL-2, DL-3, DL-4). The fermentation products were analyzed by high-performance liquid chromatography. In addition to the peak of the starting substrate compound 3, another peak was obtained, indicating that compound 3 was converted. Mass spectrometry analysis showed that Figure 8 As shown, the relative molecular mass of the two compounds is compared and it is found that the relative molecular mass of compound 4 is 176 larger than that of compound 3, indicating that there is an additional methylated glucose molecule on the molecule of compound 4 ( Figure 9 ).

[0062] The relative conversion rates of compound 3 by glycosyltransferase (BbGT)-methyltransferase (BbMT) linked by different length linker peptides (DL-1, DL-2, DL-3, and DL-4) were calculated by comparing the relative peak area ratios of the conversion products and substrates in high performance liquid chromatography. It was found that under the feeding condition of 1 mg / mL kaempferol, the relative conversion rate of the fusion protein to kaempferol gradually increased with the optimization of DL truncation. Among them, the fusion protein linked by DL-4 had the best effect, with a relative conversion rate of 60.12±4.80%, which was 6 times the conversion efficiency of the substrate using a single enzyme combination ( Figure 10 ).

[0063] The above results demonstrate that the glycosyltransferase (BbGT)-methyltransferase (BbMT) fusion protein connected by different length linker peptides (DL-1, DL-2, DL-3, DL-4) can achieve glucose methylation modification of kaempferol, and the fusion protein with DL-4 connection has the best catalytic activity.

[0064] Example 5: Purification and Verification of BbGT-BbMT Fusion Protein Based on the above experimental results, glycosyltransferase (BbGT)-methyltransferase (BbMT) linked by connecting peptide DL-4 was selected for subsequent verification, and it was determined that the active protein was a fusion protein.

[0065] Construction of prokaryotic expression vector: By gene cloning technology, the protein expression vector pET28a containing the N-terminal His6 Tag was selected and the vector pRS425m-BbGT-DL-4-BbMT constructed in 1.7 of Example 1 was amplified. BbGT 、 BbMT 、 BbGT-DL-4-BbMT The gene fragment was ligated into the pET28a vector using an In-Fusion Kit to obtain the corresponding protein expression plasmids NTHLIC-BbGT, NTHLIC-BbMT, and NTHLIC-BbGT-DL-4-BbMT. The specific method is as follows: Construction of NTHLIC-BbGT Using plasmid pRS425m-BbGT-DL-4-BbMT as a template, primers NTHLIC-BbGT-F / BbGT-NTHLIC-R were used to amplify the fragment containing the homology arms. BbGT The fragment length was 1420 bp. After the fragment was recovered, the above fragment was connected to the pET28a linearized vector using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened using Kan-resistant LB solid medium and the plasmid was extracted. Hind III and SnaBIdentification by enzyme digestion with Ⅰ yielded two fragments of 5407 bp and 1319 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0066] Construction of NTHLIC-BbMT Using plasmid pRS425m-BbGT-DL-4-BbMT as a template, primers NTHLIC-BbMT-F / BbMT-NTHLIC-R were used to amplify the fragment containing the homology arms. BbMT The fragment length was 784 bp. After the fragment was recovered, the pET28a linearized vector was connected using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened using Kan-resistant LB solid medium and the plasmid was extracted. EcoR Ⅰ and Pvu Identification by enzyme digestion with I yielded two fragments of 4949 bp and 1141 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0067] Construction of NTHLIC-BbGT-DL-4-BbMT Using plasmid pRS425m-BbGT-DL-4-BbMT as a template, primers NTHLIC-BbGT-F / BbMT-NTHLIC-R were used to amplify the fragment containing the homology arms. BbGT-DL-4-BbMT The fragment length was 2227 bp. After the fragment was recovered, the above fragment was connected to the pET28a linearized vector using the seamless cloning kit (In-Fusion Kit) and transformed into the competent E. coli Fast-T1 by heat shock method. The transformed strain was screened using Kan-resistant LB solid medium and the plasmid was extracted. EcoR Ⅰ and SnaB Identification by enzyme digestion with Ⅰ yielded two fragments of 5426 bp and 2107 bp, which, combined with the plasmid sequencing results, indicated that the plasmid was constructed correctly.

[0068] The primers used were as follows:

[0069] The PCR reaction system is: 2×Phanta Flash Max Master Mix 25 μL ddH2O 20.5 μL Upstream / downstream primer (10 μM) 2 μL each Template 0.5 μL PCR program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; annealing at 60°C for 15 s; extension at 72°C; extension rate of 5 s / kb, extension time determined by fragment size, 34 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0070] Protein isolation and purification: The prokaryotic expression vector constructed above was transformed into the competent BL21 (DE3) colony lining culture medium, and positive transformants were picked and cultured in 10 mL of LB liquid medium containing 50 μg / mL kanamycin at 37°C with a shaker at 220 rpm overnight.

[0071] Transfer 10 mL of seed liquid to 1 L of LB liquid medium containing 50 μg / mL kanamycin, culture on a shaker at 37°C and 220 rpm for 4-5 h, culture to an OD value of 0.6-0.8, ice bath for 30 min, add 1 mL of 0.1 M IPTG and induce at 16°C and 220 rpm for 18-20 h, collect the bacteria by centrifugation at 4°C and 4000 rpm for 15 min, resuspend the bacteria in 50 mL of PB buffer, use a homogenizer to break the cells, set the pressure to greater than 700 Pa, and cycle lysis three times. Centrifuge the cell lysis solution at 4°C and 10000 rpm for 40 min to remove cell debris, and collect the crude protein extract. Add 2 mL of protein filler (Ni-NTA Superflow resin) to 50 mL of crude protein extract. Adsorb at 4°C for 45-60 min. Pass the column, collect the flow-through, and then elute the protein with a gradient of 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 500 mM imidazole (dissolved in PB buffer). Pipette 20 μL of crude protein extract, the pellet (resuspended in 20 μL of PB buffer), the flow-through, and the protein eluted with various imidazole concentrations. Add 5 μL of 5× SDS-PAGE loading buffer, heat at 100°C for 5 min, load the sample, and run SDS-PAGE gel at 150 V and 400 mA for 45 min. Stain the gel with protein staining solution and destain with distilled water.

[0072] The predicted size of BbGT protein is about 50.3 kDa, and the size of BbMT protein is about 28 kDa. The results of SDS-PAGE gel electrophoresis showed that the glycosyltransferase (BbGT) and methyltransferase (BbMT) connected by the linker peptide DL-4 were fused into a single protein with a size of 79.2 kDa, indicating that the linker peptide DL-4 can successfully connect the independent enzymes BbGT and BbMT in series to form a dual-functional fusion enzyme ( Figure 12 ).

[0073] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A connecting peptide, characterized in that The connecting peptides are DL-1, DL-2, DL-3, and DL-4, and their amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

2. An artificial fusion protein comprising the connecting peptide according to claim 1, characterized in that: The fusion protein is connected by a connecting peptide to the glycosyltransferase BbGT and the methyltransferase BbMT.

3. A DNA base sequence, characterized in that: Encodes the fusion protein according to claim 2.

4. A carrier, characterized in that It comprises the coding gene sequence of the fusion protein according to claim 3.

5. Use of the connecting peptide according to claim 1 in promoting glucose methylation modification of compounds.

6. Use of the connecting peptide according to claim 5 in promoting glucose methylation modification of compounds, characterized in that: The compound is diol lactone or kaempferol.

Images