Connecting peptide and its application in promoting glucose methylation modification of compounds

Abstract

The application discloses a connecting peptide and application thereof in promoting glucose methylation modification of a compound. Glycosyltransferase-methyltransferase fusion protein genes connected by connecting peptides, genes LtLasS1 and LtLasS2 encoding benzenediol lactone synthetase are co-expressed, and it is determined through liquid phase and mass spectrum analysis of fermentation products that the fusion protein can realize glucose methylation modification on benzenediol lactone. The conversion efficiency of the fusion protein using DL-4 connecting peptide on benzenediol lactone is the highest, and the relative conversion rate is 65.54+ / -3.75%. In addition, the glucose methylation conversion efficiency of the artificial fusion protein constructed by feeding the substrate kaempferol is tested, and the conversion efficiency of the fusion protein using DL-4 connecting peptide on the substrate is 6 times that of the conversion efficiency of the substrate by using a single glycosyltransferase and methyltransferase respectively, and the relative conversion rate is increased to 60.12+ / -4.79%.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology and biotechnology, and specifically relates to a linker peptide and its application in promoting the methylation modification of hydroquinone lactone and kaempferol glucose. Background Technology

[0002] In the field of biotechnology, the design and optimization of fusion proteins is a crucial step. Fusion proteins are typically composed of two or more functional proteins linked by a linker peptide, which plays a key role in this process. It not only connects the two functional proteins but also allows 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 peptides. For example, in the production of β-lactam antibiotics, rigid linker peptide (GGGGS)2 is used to fuse the GST tag with penicillin acylase to achieve efficient purification and catalyze the generation of penicillin G into the intermediate 6-aminopenicillin alkyl acid. However, with the development of biotechnology bringing new application scenarios, the design and optimization of fusion proteins urgently need to explore more diverse new sequences.

[0004] Multidomain proteins connect two or more functional domains via linker peptides. These linker peptides, in addition to their basic linking function, also perform other functions, such as maintaining interdomain interactions and preserving the biological activity of each domain. The linker peptide sequences in bifunctional proteins have evolved over a long period, exhibiting high stability and biocompatibility. Furthermore, linker peptide sequences can better maintain the synergistic effects between the functional domains of fusion proteins, thereby improving the overall performance of the fusion protein. Research on the structure and function of linker peptides in multidomain proteins can provide a reference for the design of linker peptides in recombinant fusion proteins. Summary of the Invention

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

[0006] A linker peptide, wherein the linker peptide is DL-1, DL-2, DL-3, and DL-4, and the amino acid sequences thereof are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0007] An artificial fusion protein comprising the linker peptide, wherein the fusion protein is composed of the linker peptide linked to glycosyltransferase BbGT and methyltransferase BbMT.

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

[0009] A vector containing the gene sequence encoding the fusion protein.

[0010] The linker peptide is used to promote glucose methylation modification of compounds.

[0011] The compound is either hydroquinone lactone or kaempferol.

[0012] The beneficial effects of this invention are: the linker peptide sequence described in this invention can tandemly link two independently functional modifying enzymes to form an artificial fusion protein. In *Saccharomyces cerevisiae* BJ5464-NpgA, the gene encoding a fusion protein containing a glycosyltransferase (BbGT)-methyltransferase (BbMT) tandemly linked by a linker peptide, as well as the genes encoding hydroquinone lactone synthases hrPKS and nrPKS, are included. LtLasS1 and LtLasS2 Co-expression was performed, and liquid chromatography and mass spectrometry analysis of the fermentation products confirmed that the fusion protein could achieve glucose methylation modification of resorcinolone. Based on the three-dimensional protein structure of the linker peptide, the linker peptide sequence was further truncated and optimized to obtain four truncated linker peptides. Among them, the fusion protein using DL-4 linker peptide showed the highest conversion efficiency for demethyl-lasiodiplodin, with a relative conversion rate of 65.54±3.75%. In addition, the glucose methylation conversion efficiency of the artificial fusion proteins constructed from the four linker peptides was tested by feeding them with the substrate kaempferol. The conversion efficiency of the fusion protein using DL-4 linker peptide was 6 times that of the conversion efficiency using single glycosyltransferases (BbGT) and methyltransferases (BbMT), respectively, with a relative conversion rate increased to 60.12±4.79%. Attached Figure Description

[0013] Figure 1 The structural formulas of compound 1 (hydroquinone lactone) and compound 2.

[0014] Figure 2 Transformed LtLasS1 , LtLasS2 The crude extract of yeast fermentation followed by extraction of fusion protein encoding genes linked by AoiQ-Linker and DL-1, respectively, is chromatographically analyzed at 300 nm using liquid chromatography.

[0015] Figure 3 Transformed LtLasS1 , LtLasS2 Liquid chromatography at 300 nm for crude extracts of yeast fermentation containing BbGT-BbMT fusion protein encoding genes linked by peptides of different lengths.

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

[0017] Figure 5 A bar chart showing the conversion efficiency of compound 1 for the BbGT-BbMT fusion protein constructed using linker peptides of different lengths.

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

[0019] Figure 7 The crude extracts of yeast transformed with individual BbGT and BbMT encoding genes were fermented after being fed with different concentrations of kaempferol and then liquid chromatograms were obtained at 300 nm.

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

[0021] Figure 9 Bar chart showing the conversion efficiency of the BbGT-BbMT nonfusion protein for different concentrations of kaempferol.

[0022] Figure 10 Yeast transformed with BbGT-BbMT encoding genes linked by peptides of different lengths was fermented under 1 mg / mL kaempferol, and the crude extract was extracted and chromatogram was obtained at 300 nm.

[0023] Figure 11 A bar chart showing the conversion efficiency of BbGT-BbMT fusion protein with 1 mg / mL kaempferol using linker peptides of different lengths.

[0024] Figure 12 SDS-PAGE gel electrophoresis image of the purified DL-4-linked fusion protein. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description will be provided below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

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

[0027] The expression plasmids Pxw06F_LtLasS1, pXW30F_LtLasS2, pRS425m-BbGT, and pXW06F-BbMT used in the following examples, and the glycosyltransferase encoding gene are... BbGT The gene encoding methyltransferase is BbMT,This is documented in the following reference: 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.

[0028] The following examples use a reverse transcription kit (PrimeScript). TM The RT reagent kit with gDNA Eraser was purchased from Takara, restriction endonucleases were purchased from NEB, and the In-Fusion Kit, E. coli competent cells Fast-T1, and other reagents were also used. E. coli BL21 (DE3) and 2×Phanta Flash Master Mix (Dye Plus) were purchased from Novizan Biotech; plasmid miniprep kit and universal DNA purification gel extraction kit were purchased from Tiangen Biotech; 10×TE buffer, 1M lithium acetate and one-step protein rapid staining solution were purchased from Coolaber Biotech; 5×SDS-PAGE loading buffer was purchased from Kangwei Century Biotech; Spectra... TM Multicolor Broad Protein Ladder was purchased from Thermo Fisher Scientific, and SurePAGE... TM The protein pregel was purchased from GenScript and the imidazole was purchased from Sigma-Aldrich.

[0029] The culture medium and reagent formulations used in the following examples are as follows:

[0030] PDB medium (1 L): purchased from BD Difco TM 24 g of Potato Dextrose Broth powder, diluted to 1 L with distilled water;

[0031] LB medium (1 L): 25 g of LB Broth purchased from Sangon Biotech, diluted to 1 L with distilled water; if it is solid, add 20 g of agar.

[0032] 2% YPD medium (1 L): 10 g yeast extract, 20 g glucose, 20 g peptone, distilled water to a final volume of 1 L; if solid, add 20 g agar.

[0033] 1% YPD medium (1 L): 10 g yeast extract, 10 g glucose, 20 g peptone, distilled water to a final volume of 1 L;

[0034] 1 M Sorbitol solution (1 L): 182 g sorbitol, diluted to 1 L with distilled water;

[0035] SC 3- (Leu) - Trp - Ura - Deficiency-type culture 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 a final volume of 1 L, pH adjusted to 5.8, or 20 g agar if solid.

[0036] SC 2- (Leu) - Trp - Deficiency-type culture 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 a final volume of 1 L; if solid, add 20 g agar.

[0037] SC - (Leu) - Deficiency-type culture 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 a final volume of 1 L; if solid, add 20 g agar.

[0038] DNA extraction: 1.25% SDS, 100 mM Tris-HCl (pH 7.5), 50 mM EDTA (pH 8);

[0039] Ampicillin (Amp): Purchased from Sigma, stock solution concentration was 100 mg / mL, working concentration was 100 μg / mL, stored at -20℃;

[0040] Kanamycin (Kan): Purchased from Sigma, stock solution concentration was 50 mg / mL, working concentration was 50 μg / mL, stored at -20℃;

[0041] PB buffer: 150 mM PB, 150 mM NaCl, pH=7.6.

[0042] Example 1: Construction of a fusion protein expression vector

[0043] from B. bassiana Amplification of ATCC7159 cDNA BbGT and BbMT The coding gene fragment, from D. phaseolorumd The sequences of linker peptides DL-1, DL-2, DL-3, and DL-4 were amplified from the gDNA of YX39261, derived from... A.oryzae of AoiQ The coding gene sequence fragments were synthesized by BGI Genomics, from which the AoiQ-Linker sequence was amplified. Using an In-Fusion Kit, the gene fragments were 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.

[0044] Extraction using the Trizol method B. bassiana ATCC7159 RNA was reverse transcribed, and gDNA was extracted using the phenol-chloroform extraction method.

[0045] Construction of pRS425m-BbGT-DL-1-BbMT: using Nde I and Pme I. The shuttle vector PRS425m was double-digested and the 7555 bp linear vector fragment was recovered. B. bassiana Using ATCC7159 cDNA as a template, the gene was amplified using primers pRS425m-BbGT-F / BbGT-DL-1-R. BbGT The gene is 1423 bp in length and was amplified using primers DL-1-BbMT-F / BbMT-pRS425m-R. BbMT The gene length is 784 bp. D. phaseolorumUsing YX39261 gDNA as a template, the DL-1 sequence was amplified using primers DL-1-F / DL-1-R, with a sequence length of 150 bp. After fragment recovery, the fragment was ligated into the pRS425m linearized vector using an In-Fusion Kit, and transformed into E. coli competent cells Fast-T1 using the heat shock method. Transformed strains were screened using Amp-resistant LB solid medium, and plasmids were extracted. Not I and BamH I. Enzyme digestion and identification yielded two fragments, 6627 bp and 3224 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0046] Construction of pRS425m-BbMT-DL-1-BbGT: B. bassiana Using ATCC7159 cDNA as a template, the gene was amplified using primers pRS425m-BbMT-F / BbMT-DL-1-R. BbMT The gene is 782 bp in length and was amplified using primers DL-1-BbGT-F / BbGT-pRS425m-R. BbGT The gene length is 1425 bp. D. phaseolorum Using YX39261 gDNA as a template, the DL-1 sequence was amplified using primers DL-1-F / DL-1-R, with a sequence length of 150 bp. After fragment recovery, the fragment was ligated into the pRS425m linearized vector using an In-Fusion Kit, and transformed into E. coli competent cells Fast-T1 using the heat shock method. Transformed strains were screened using Amp-resistant LB solid medium, and plasmids were extracted. Not I and BamH I. Enzyme digestion and identification yielded two fragments, 6627 bp and 3224 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0047] Construction of pRS425m-BbGT-DL-2-BbMT: B. bassiana Using ATCC7159 cDNA as a template, the gene was amplified using primers pRS425m-BbGT-F / BbGT-R. BbGT The gene is 1404 bp in length and was amplified using primers BbMT-F / BbMT-pRS425m-R. BbMT The gene length is 768 bp. D. phaseolorumUsing YX39261 gDNA as a template, the DL-2 sequence was amplified using primers BbGT-DL-2-3-F / DL-2-BbMT-R. The sequence length was 184 bp. After fragment recovery, the fragment was ligated into the pRS425m linearized vector using an In-Fusion Kit, and transformed into E. coli competent cells Fast-T1 using the heat shock method. Transformed strains were screened using Amp-resistant LB solid medium, and plasmids were extracted. Not I and BamH I. Enzyme digestion and identification yielded two fragments, 6627 bp and 3210 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0048] Construction of pRS425m-BbGT-DL-3-BbMT: B. bassiana Using ATCC7159 cDNA as a template, the gene was amplified using primers pRS425m-BbGT-F / BbGT-R. BbGT The gene is 1404 bp in length and was amplified using primers BbMT-F / BbMT-pRS425m-R. BbMT The gene length is 768 bp. D. phaseolorum Using YX39261 gDNA as a template, the DL-3 sequence was amplified using primers BbGT-DL-2-3-F / DL-3-BbMT-R. The sequence length was 168 bp. After fragment recovery, the fragment was ligated into the pRS425m linearized vector using an In-Fusion Kit, and transformed into E. coli competent cells Fast-T1 using the heat shock method. Transformed strains were screened using Amp-resistant LB solid medium, and plasmids were extracted. Not I and BamH I. Enzyme digestion and identification yielded two fragments, 6627 bp and 3192 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0049] Construction of pRS425m-BbGT-DL-4-BbMT: B. bassiana Using ATCC7159 cDNA as a template, the DNA sequence of DL-4 was designed into 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 is 1444 bp in length and was amplified using primers DL-4-BbMT-F / BbMT-pRS425m-R. BbMTThe gene length was 807 bp. After fragment recovery, the fragment was ligated into the pRS425m linearized vector using an In-Fusion Kit, and then transformed into *E. coli* competent cells Fast-T1 using the heat shock method. Transformed strains were screened using Amp-resistant LB agar, and plasmids were extracted. Not I and BamH I. Enzyme digestion and identification yielded two fragments, 6627 bp and 3138 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0050] Construction of pRS425m-BbGT-AoiQ-Linker-BbMT: B. bassiana Using ATCC7159 cDNA as a template, the gene was amplified using primers pRS425m-BbGT-F / BbGT-AoiQ-Linker-R. BbGT The gene is 1420 bp in length and was amplified using primers AoiQ-Linker-BbMT-F / BbMT-pRS425m-R. BbMT The gene is 786 bp in length and is synthesized using... AoiQ Using the gene as a template, the AoiQ-Linker sequence was amplified using primers AoiQ-Linker-F / AoiQ-Linker-R, with a sequence length of 234 bp. After fragment recovery, the fragment was ligated into the pRS425m linearized vector using an In-Fusion Kit, and transformed into E. coli competent cells Fast-T1 using the heat shock method. Transformed strains were screened using Amp-resistant LB solid medium, and plasmids were extracted. Not I and BamH I. Enzyme digestion and identification yielded two fragments, 6627 bp and 3312 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0051] Construction of pRS425m-BbMT-AoiQ-Linker-BbGT: B. bassiana Using ATCC7159 cDNA as a template, the gene was amplified using primers pRS425m-BbMT-F / BbMT-AoiQ-Linker-R. BbMT The gene length is 783 bp. The gene was amplified using primers AoiQ-Linker-BbGT-F / BbGT-pRS425m-R. BbGT The gene is 1423 bp in length and is synthesized using... AoiQUsing the gene as a template, the AoiQ-Linker sequence was amplified using primers AoiQ-Linker-F / AoiQ-Linker-R, with a sequence length of 234 bp. After fragment recovery, the fragment was ligated into the pRS425m linearized vector using an In-Fusion Kit, and transformed into E. coli competent cells Fast-T1 using the heat shock method. Transformed strains were screened using Amp-resistant LB solid medium, and plasmids were extracted. Not I and BamH I. Enzyme digestion and identification yielded two fragments, 6627 bp and 3312 bp, respectively. Combined with plasmid sequencing results, this confirmed the correct plasmid construction. The primers used for constructing the fusion gene heterologous expression vector are as follows:

[0052]

[0053] The PCR reaction system is as follows:

[0054] 2×Phanta Flash Max Master Mix 25 μL

[0055] ddH2O 20.5 μL

[0056] 2 μL each of upstream and downstream primers (10 μM)

[0057] Template 0.5 μL

[0058] PCR program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 60℃ annealing for 15 s; 72℃ extension; extension rate of 5 s / kb, extension time determined by fragment size, 34 cycles; final extension at 72℃ for 5 min, store at 4℃.

[0059] Example 2: Co-expression of fusion gene and hydroquinone lactone expression gene and product extraction

[0060] Preparation and transformation of electrocompetent cells of Saccharomyces cerevisiae BJ5464-NpgA

[0061] (1) Preparation of seed liquid

[0062] Saccharomyces cerevisiae BJ5464-NpgA was activated by streaking 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. The culture was then carried out overnight at 30°C and 220 rpm.

[0063] (2) Preparation of electrocompetent cells of Saccharomyces cerevisiae BJ5464-NpgA

[0064] The seed culture was transferred to 50 mL of 2% YPD liquid medium (inoculum size 0.15 OD / mL), and cultured at 30°C with shaking at 220 rpm for 6 h until the OD600 reached 0.9. Cells were collected by centrifugation at 3000 rpm for 3 min at 4°C, resuspended in 50 mL of pre-chilled sterile water, and then resuspended in 50 mL of pre-chilled 1 M Sorbitol solution. The cells were then resuspended in 40 mL of pre-chilled 1 M Sorbitol solution, mixed with 5 mL of 10×TE buffer, and then 5 mL of 1 M lithium acetate was added. The mixture was incubated at 30°C with shaking at 220 rpm for 30 min. 500 μL of 1 M DTT solution was added, and the mixture was incubated at 30°C with shaking at 220 rpm for 15 min. Cells were then collected by centrifugation at 3000 rpm for 3 min at 4°C. The cells were then resuspended in 50 mL of pre-chilled 1 M Sorbitol solution. Wash twice with Sorbitol solution, and finally resuspend the cells in 2 mL of pre-cooled 1 M Sorbitol solution. Aliquot the cells into 1.5 mL sterile EP tubes at 100 μL / tube and store at -80°C.

[0065] (3) Electroconversion of yeast competent cells

[0066]

[0067] Transformation was performed according to the plasmid combination described above. 2 μL of each plasmid was added to 100 μL of competent yeast cells. The mixture was transferred to a 2 mm electroporation cuvette and pulsed transformation was performed at 1.5 KV, 25 μF, and 200 Ω. The transformed cells were then transferred to 12 mL shake tubes, and 3 mL of recovery solution (1 M Sorbitol: 2% YPD = 1:1) was added. The cells were incubated at 30℃ and 220 rpm for 5 h. After centrifugation at 3000 rpm for 3 min, the supernatant was discarded, and the cells were treated with 3 mL of SC... 3- (Leu) - Trp - Ura - The defective liquid culture medium was washed twice and then spread onto SC. 3- (Leu) - Trp - Ura - On defective solid culture media.

[0068] Fermentation and Product Extraction

[0069] (1) Selecting positive transformants in SC 3- (Leu) - Trp - Ura -) Defective culture medium was streaked for expansion culture. After incubation at 30°C for 24 h, bacterial cells were picked and transferred to 15 mL of SC medium. 3- (Leu) - Trp - Ura - The culture was carried out in the defective liquid medium until the OD600 reached 1.0. After 12 h of culture in a shaker at 30℃ and 220 rpm, an equal volume of 1% YPD liquid medium was added, and the culture was carried out in a shaker at 30℃ and 220 rpm for 48 h.

[0070] An equal volume of ethyl acetate was added to the fermentation broth for extraction. The ethyl acetate layer was evaporated to dryness using a rotary evaporator, dissolved in 500 μL of chromatographic grade methanol, and filtered through a 0.22 μm organic filter membrane. The product was detected by liquid chromatography-mass spectrometry (LC-MS) with the following detection program: gradient elution of 5%-95% acetonitrile-water for 15 min, isocratic elution of 95% acetonitrile-water for 3 min, and gradient elution of 95%-5% acetonitrile-water for 2 min, at a flow rate of 0.35 mL / min, using UV full-wavelength scanning.

[0071] The transformation was analyzed by high performance liquid chromatography. LtLasS1 , LtLasS2 Crude extracts were extracted from yeast fermented with the gene encoding the BbGT-BbMT fusion protein linked by different linker peptides (DL-1, DL-2, DL-3, DL-4), and then transformed with... LtLasS1 , LtLasS2 The product peak of compound 1 (demethyl-lasiodiplodin, a benzyl lactone) was obtained from the crude extract after yeast fermentation. After conversion... LtLasS1 , LtLasS2 In the crude extract of yeast fermentation containing the gene encoding the BbGT-BbMT fusion protein linked with different linking peptides (DL-1, DL-2, DL-3, DL-4), in addition to the product peak of compound 1, another product peak of compound 2 was obtained. Figure 1 , Figure 4 Mass spectrometry analysis revealed that compound 2 had a molecular weight 176 greater than compound 1, indicating that compound 2 contained an additional methylated glucose molecule. Based on the molecular weight of compound 2, it was confirmed to be a glucose methylation product, indicating that after conversion... LtLasS1 , LtLasS2 In yeast containing the gene encoding the BbGT-BbMT fusion protein linked by different linking peptides (DL-1, DL-2, DL-3, DL-4), compound 1 was mostly transformed.

[0072] Among them, combinations 1-6 are high performance liquid chromatography (HPLC) Figure 2The results showed that BbGT-BbMT / BbMT-BbGT linked to DL-1 via AoiQ-Linker could both modify compound 1 by glucose methylation, and the order of gene linking (N-terminus and C-terminus) had no effect on transformation.

[0073] By comparing the high-performance liquid chromatography (HPLC) of the crude extracts after fermentation from combinations 1, 2, 5, 7, 8, and 9 (…), Figure 3 The relative peak area ratio of the conversion product to the substrate was used to calculate the relative conversion efficiency of BbGT-BbMT for compound 1 under different lengths of linker peptides (DL-1, DL-2, DL-3, DL-4). The conversion efficiency of the fusion proteins linked by the three optimized linker peptides (DL-1, DL-2, DL-3, DL-4) gradually increased. Among them, the DL-4-linked fusion protein showed the best effect and the highest relative conversion efficiency for compound 1, which was 31.24 ± 3.75% higher than that of the DL-1-linked fusion protein. Figure 5 ).

[0074] The above demonstrates that fusion proteins linked by the linker peptides AoiQ-Linker, DL-1, DL-2, DL-3, and DL-4 can achieve glucose methylation modification of compound 1, and the order of gene linking (N-terminus and C-terminus) has no effect on transformation. Among the DL-1-linked optimized DL-1, the fusion protein linked by DL-4 has the highest relative conversion rate for compound 1.

[0075] Example 3: Determination of the conversion efficiency of kaempferol by combined biosynthesis of glycosyltransferase (BbGT) and methyltransferase (BbMT).

[0076] yeast competent transformation

[0077] Using the Saccharomyces cerevisiae BJ5464-NpgA electrotransfer competent cells prepared by the method in Example 2, plasmids pRS425m-BbGT and pXW06F-BbMT were co-transformed into the yeast competent cells according to the following method.

[0078] Add 2 μL of plasmid to each of 100 μL competent yeast cells for transformation. Transfer the mixture to a 2 mm electroporation cuvette and perform pulsed transformation at 1.5 KV, 25 μF, and 200 Ω. Transfer the transformed cells to 12 mL shake tubes, add 3 mL of resuscitation solution, and incubate at 30°C and 220 rpm for 5 h. Centrifuge at 3000 rpm for 3 min, discard the supernatant, and use 3 mL of SC... 2- (Leu) - Trp - The defective liquid culture medium was washed twice and then spread onto SC. 2- (Leu) - Trp -On defective solid culture media.

[0079] (2) Fermentation and product extraction

[0080] Selecting positive transformants in SC 2- (Leu) - Trp - ) Defective solid culture medium was streaked for expansion culture. After incubation at 30°C for 24 h, bacterial cells were picked and transferred to 5 mL of SC medium. 2- (Leu) - Trp - The culture medium was cultured to an OD600 of 1.0 at 30°C and 220 rpm for 12 h. Then, an equal volume of 1% YPD liquid medium was added. At the same time, the culture was fed with kaempferol at final concentrations of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL and 5 mg / mL. Each concentration was repeated three times. The culture was carried out at 30°C and 220 rpm for 48 h.

[0081] An equal volume of ethyl acetate was added to the fermentation broth for extraction. The ethyl acetate layer was evaporated to dryness using a rotary evaporator, dissolved in 500 μL of chromatographic grade methanol, and filtered through a 0.22 μm organic filter membrane. The product was detected by liquid chromatography-mass spectrometry (LC-MS) with the following detection program: gradient elution of 5%-95% acetonitrile-water for 15 min, isocratic elution of 95% acetonitrile-water for 3 min, and gradient elution of 95%-5% acetonitrile-water for 2 min, at a flow rate of 0.35 mL / min, using UV full-wavelength scanning.

[0082] Different concentrations of kaempferol (compound 3) were fed into yeast transformed with separate genes encoding glycosyltransferase (BbGT) and methyltransferase (BbMT). The fermentation products were analyzed by high-performance liquid chromatography. Figure 7 In addition to the peak of the starting substrate compound 3, another product peak, compound 4, was observed, indicating that compound 3 was transformed. Mass spectrometry analysis revealed that compound 4 had a molecular weight 176 greater than that of compound 3. Figure 6 , Figure 8 This indicates that compound 4 has an additional methylated glucose molecule.

[0083] The relative conversion rates of kaempferol by individual glycosyltransferases (BbGT) and methyltransferases (BbMT) at four concentrations (0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, and 5 mg / mL) were calculated by comparing the relative peak area ratios of the conversion product and the substrate in high-performance liquid chromatography (HPLC). The results showed that the conversion rate of kaempferol by the combination of BbGT and BbMT gradually decreased with increasing substrate concentration, and approached saturation at a concentration of 1 mg / mL. Figure 9 ).

[0084] The above demonstrates that combining individual glycosyltransferases (BbGT) and methyltransferases (BbMT) can achieve glucose methylation modification of kaempferol, and the conversion efficiency tends to saturate when the concentration of kaempferol is 1 mg / mL.

[0085] Example 4: Determination of kaempferol conversion efficiency of GT-MT fusion protein

[0086] Based on the results in Example 3 that the individual glycosyltransferase (BbGT) and methyltransferase (BbMT) reached saturation at a kaempferol concentration of 1 mg / mL, the yeast transformed with the fusion gene was fed with 1 mg / mL of kaempferol.

[0087] Using the Saccharomyces cerevisiae BJ5464-NpgA electrocompetent states prepared by the method in Example 2, pRS425m-BbGT-DL-1-BbMT, pRS425m-BbGT-DL-2-BbMT, and pRS425m-BbGT-DL-3-BbMT were transferred to the cells.

[0088] pRS425m-BbGT-DL-4-BbMT were transformed into competent yeast cells.

[0089] Add 2 μL of plasmid for transformation to 100 μL of competent yeast cells. Transfer the mixture to a 2 mm electroporation cuvette and perform pulse transformation at 1.5 KV, 25 μF, and 200 Ω. Transfer the transformed cells to 12 mL shake tubes, add 3 mL of resuscitation solution, and incubate at 30°C and 220 rpm for 5 h. Centrifuge at 3000 rpm for 3 min, discard the supernatant, and use 3 mL of SC... - (Leu) - The defective liquid culture medium was washed twice and then spread onto SC. - (Leu) - On defective solid culture media.

[0090] Fermentation and product extraction:

[0091] Selecting positive transformants in SC - (Leu)- ) Defective solid culture medium was streaked for expansion culture. After incubation at 30°C for 24 h, bacterial cells were picked and transferred to 5 mL of SC medium. - (Leu) - The cells were cultured in the defective liquid medium until the OD600 reached 1.0. They were then cultured at 30°C and 220 rpm for 12 h on a shaker. An equal volume of 1% YPD liquid medium was added, and the cells were fed with kaempferol at a final concentration of 1 mg / mL. Three biological replicates were performed, and the cells were cultured at 30°C and 220 rpm for 48 h on a shaker.

[0092] An equal volume of ethyl acetate was added to the fermentation broth for extraction. The ethyl acetate layer was evaporated to dryness using a rotary evaporator, dissolved in 500 μL of chromatographic grade methanol, and filtered through a 0.22 μm organic filter membrane. The product was detected by liquid chromatography-mass spectrometry (LC-MS) with the following detection program: gradient elution of 5%-95% acetonitrile-water for 15 min, isocratic elution of 95% acetonitrile-water for 3 min, and gradient elution of 95%-5% acetonitrile-water for 2 min, at a flow rate of 0.35 mL / min, using UV full-wavelength scanning.

[0093] Kaempferol (compound 3) at a final concentration of 1 mg / mL was fed into yeast transformed with a gene encoding the BbGT-BbMT fusion protein linked by different linker peptide sequences (DL-1, DL-2, DL-3, DL-4). High-performance liquid chromatography (HPLC) analysis of the fermentation products revealed an additional peak besides the peak of the starting substrate compound 3, indicating that compound 3 was transformed. Mass spectrometry analysis showed similar results. Figure 8 As shown, comparing the relative molecular masses of the two compounds reveals that compound 4 has a relative molecular mass 176 greater than that of compound 3, indicating that compound 4 has an additional methylated glucose molecule. Figure 9 ).

[0094] The relative conversion rates of compound 3 by glycosyltransferase (BbGT)-methyltransferase (BbMT) linked with different lengths of peptides (DL-1, DL-2, DL-3, DL-4) to glycosyltransferase (BbGT)-methyltransferase (BbMT) were calculated by comparing the relative peak area ratios of the conversion product and the substrate in high-performance liquid chromatography (HPLC). It was found that under feeding conditions of 1 mg / mL kaempferol, the relative conversion rate of the fusion protein to kaempferol gradually increased with optimization of DL truncation. The DL-4 linked fusion protein showed the best effect, with a relative conversion rate of 60.12 ± 4.80%, which is 6 times the conversion efficiency of the substrate using a single enzyme combination. Figure 10 ).

[0095] The above demonstrates that glycosyltransferase (BbGT)-methyltransferase (BbMT) fusion proteins linked by peptide sequences of different lengths (DL-1, DL-2, DL-3, DL-4) can achieve glucose methylation modification of kaempferol, and the fusion protein linked by DL-4 exhibits the best catalytic activity.

[0096] Example 5: Purification and Validation of BbGT-BbMT Fusion Protein

[0097] Based on the above experimental results, a glycosyltransferase (BbGT)-methyltransferase (BbMT) linked by the linker peptide DL-4 was selected for further verification, confirming that the active protein is a fusion protein.

[0098] Construction of prokaryotic expression vector: Using 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 Example 1.7 was amplified. BbGT , BbMT , BbGT-DL-4-BbMT Gene fragments were ligated into the vector pET28a using an In-Fusion Kit to obtain the corresponding protein expression plasmids NTHLIC-BbGT, NTHLIC-BbMT, and NTHLIC-BbGT-DL-4-BbMT. The specific methods are as follows:

[0099] Building NTHLIC-BbGT

[0100] Using plasmid pRS425m-BbGT-DL-4-BbMT as a template, the fragment containing homologous arms was amplified using primers NTHLIC-BbGT-F / BbGT-NTHLIC-R. BbGT The fragment was 1420 bp in length. After recovery, the fragment was ligated into the pET28a linearized vector using the In-Fusion Kit and transformed into *E. coli* competent cells Fast-T1 via heat shock. Transformed strains were screened using Kan-resistant LB agar, and plasmids were extracted. Hind III and SnaB I. Enzyme digestion and identification yielded two fragments, 5407 bp and 1319 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0101] Building NTHLIC-BbMT

[0102] Using plasmid pRS425m-BbGT-DL-4-BbMT as a template, the fragment containing homologous arms was amplified using primers NTHLIC-BbMT-F / BbMT-NTHLIC-R. BbMTThe fragment length was 784 bp. After recovery, the fragment was ligated into the pET28a linearized vector using the In-Fusion Kit and transformed into *E. coli* competent cells Fast-T1 via heat shock. Transformed strains were screened using Kan-resistant LB agar, and plasmids were extracted. EcoR I and Pvu I. Enzyme digestion and identification yielded two fragments, 4949 bp and 1141 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0103] Construction of NTHLIC-BbGT-DL-4-BbMT

[0104] Using plasmid pRS425m-BbGT-DL-4-BbMT as a template, the fragment containing homologous arms was amplified using primers NTHLIC-BbGT-F / BbMT-NTHLIC-R. BbGT-DL-4-BbMT The fragment was 2227 bp in length. After recovery, the fragment was ligated into the pET28a linearized vector using the In-Fusion Kit and transformed into *E. coli* competent cells Fast-T1 via heat shock. Transformed strains were screened using Kan-resistant LB agar, and plasmids were extracted. EcoR I and SnaB I. Enzyme digestion and identification yielded two fragments, 5426 bp and 2107 bp. Combined with the plasmid sequencing results, this indicated that the plasmid was constructed correctly.

[0105] The primers used are as follows:

[0106]

[0107] The PCR reaction system is as follows:

[0108] 2×Phanta Flash Max Master Mix 25 μL

[0109] ddH2O 20.5 μL

[0110] 2 μL each of upstream and downstream primers (10 μM)

[0111] Template 0.5 μL

[0112] PCR program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 60℃ annealing for 15 s; 72℃ extension; extension rate of 5 s / kb, extension time determined by fragment size, 34 cycles; final extension at 72℃ for 5 min, store at 4℃.

[0113] Protein isolation and purification: The prokaryotic expression vector constructed above was transformed into coli competent cells BL21(DE3), and positive transformants were picked and cultured overnight at 37°C and 220 rpm in 10 mL of LB liquid medium containing 50 μg / mL kanamycin.

[0114] Transfer 10 mL of seed culture to 1 L of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C and 220 rpm for 4-5 h with shaking until the OD value reaches 0.6-0.8. Incubate on ice 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 cells by centrifugation at 4°C and 4000 rpm for 15 min. Resuspend the cells in 50 mL of PB buffer. Hypolyze the cells using a homogenizer at a pressure greater than 700 Pa for 3 cycles. Centrifuge the cell lysate at 4°C and 10000 rpm for 40 min to remove cell debris and collect the crude protein extract. Add 2 mL of protein packing material (Ni-NTA Superflow resin) to 50 mL of crude protein extract, incubate at 4°C for 45-60 min, pass through a chromatography column, collect the flow-through, and then elute the protein using a gradient of imidazole solutions (dissolved in PB buffer) at concentrations of 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 500 mM. Take 20 μL of crude protein extract, precipitate (resuspended in 20 μL of PB buffer), flow-through, and protein eluted with different concentrations of imidazole solutions, add 5 μL of 5×SDS-PAGE loading buffer, heat at 100°C for 5 min, load the sample, and perform SDS-PAGE gel electrophoresis at 150 V, 400 mA for 45 min. Stain the gel with protein staining solution, then destain with distilled water.

[0115] The predicted size of the BbGT protein is approximately 50.3 kDa, and the BbMT protein is approximately 28 kDa. SDS-PAGE gel electrophoresis results showed that the glycosyltransferase (BbGT) and methyltransferase (BbMT), linked by the DL-4 linker, fused into a single protein of 79.2 kDa. This indicates that the DL-4 linker can successfully tandemly link the independent enzymes BbGT and BbMT to form a bifunctional fusion enzyme. Figure 12 ).

[0116] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A connecting peptide, characterized in that The linker 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.

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