A high-throughput screening method for glycosaminoglycan backbone synthases

Abstract

The application relates to a high-throughput screening method of a glycosaminoglycan backbone synthase. The method comprises the following steps: (1) constructing a glycosaminoglycan backbone synthase library; (2) constructing a recombinant vector; (3) constructing a recombinant bacterium for screening the glycosaminoglycan backbone synthase; (4) culturing the recombinant bacterium; (5) after the bacterial liquid of the recombinant bacterium is analyzed or sorted through flow, the fluorescence is measured, and the target glycosaminoglycan backbone synthase is screened. In the application, the Bacillus subtilis BS168ASS is used as a host bacterium, the uridine diphosphate-glucose-6-dehydrogenase gene and the glycosaminoglycan backbone synthase gene are overexpressed, and the recombinant bacterium for screening the glycosaminoglycan backbone synthase is constructed. Then, the fluorescence of the strain is measured, the glycosaminoglycan backbone synthase strain is accurately screened in a high-throughput mode, the screening efficiency is improved, and a new starting point is provided for the discovery of new glycosaminoglycan backbone synthases in microorganism groups of new sources, especially in the microorganism groups that cannot be cultured in a metagenome library.

Description

Technical Field

[0001] This invention relates to a high-throughput screening method for glycosaminoglycan backbone synthases, specifically to the modification of the glycosaminoglycan backbone synthesis pathway of Bacillus subtilis by azidation and the fluorescence-activated cell sorting of glycosaminoglycan backbone synthases based on this strain, belonging to the field of biotechnology. Background Technology

[0002] Glycosaminoglycans (GAGs) are long, linear heteropolysaccharides widely found in the connective tissues of higher animals. They are composed of repeating disaccharide units of hexuronic acid or hexoses and hexosamines. Based on the type of monosaccharide residues, the type of inter-residue bonds, and the number and position of sulfate groups, glycosaminoglycans can be classified into: hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratin sulfate (KS), heparan sulfate (HS), and heparin (HP). Glycosaminoglycans can interact with growth factors, cytokines, chemokines, and enzymes, and have important effects on processes such as growth, inflammatory responses, coagulation, and tumor metastasis.

[0003] Uridine diphosphate glucose-6-dehydrogenase (tuaD), an enzyme found in the genomic DNA of Bacillus subtilis, catalyzes the conversion of uridine diphosphate glucose (UDP-Glucose) to uridine diphosphate glucuronic acid (UDP-GlcA). Existing studies have demonstrated that UDP-GlcA restricts the biosynthesis of glycosaminoglycan backbones; therefore, tuaD is crucial for high-level glycosaminoglycan backbone biosynthesis.

[0004] Glycosyltransferases (GTs) are enzymes that catalyze the regio- and stereo-oriented transfer of monosaccharide units from sugar donors to polysaccharides, providing a highly efficient method for synthesizing complex oligosaccharides. However, the limited number of available GTs, coupled with their instability and strict substrate specificity, severely hinders their widespread application. Therefore, obtaining glycosyltransferases capable of efficiently synthesizing specific oligosaccharides and glycoconjugates is of profound significance.

[0005] Bacillus subtilis is a species of Bacillus, a Gram-positive bacterium, and is considered GRAS (Generally recognized as safe) because it has good tolerance to the environment. Furthermore, according to existing research, the background of Bacillus subtilis regarding the glycosaminoglycan synthesis pathway is relatively clean.

[0006] Past research has achieved limited success in expanding substrate scope, altering substrate specificity, and enhancing GT activity through directed evolution via protein engineering, partly due to the lack of effective high-throughput screening methods. Assessing GT activity is extremely challenging because fluorescence or absorbance associated with glycosidic bond formation does not show significant changes. Furthermore, screening for ideal phenotypes is, in most cases, a stochastic process. Therefore, developing high-throughput, low-cost screening methods applicable to large GT libraries is of great significance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a high-throughput screening method for glycosaminoglycan backbone synthases. Specifically, it involves constructing a recombinant Bacillus subtilis strain that efficiently synthesizes an azide-modified glycosaminoglycan backbone, and then using this strain to perform high-throughput screening of glycosaminoglycan backbone synthases.

[0008] The technical solution of the present invention is as follows:

[0009] A high-throughput screening method for glycosaminoglycan backbone synthases includes the following steps:

[0010] (1) Using the BLASTp method, glycosaminoglycan backbone synthase genes were randomly obtained and glycosaminoglycan backbone synthase libraries were constructed.

[0011] (2) The glycosaminoglycan backbone synthase gene and the uridine diphosphate-glucose-6-dehydrogenase gene tuaD expression cassette in the library were respectively ligated into the pHCMC04 plasmid to obtain the recombinant vector pHCMC04-GTs-tuaD;

[0012] (3) The recombinant vector pHCMC04-GTs-tuaD was transformed into Bacillus subtilis BS168ASS, and positive recombinants were selected to obtain the recombinant strain GD for screening glycosaminoglycan backbone synthase.

[0013] (4) The recombinant bacteria GD, which screened for glycosaminoglycan backbone synthase, was inoculated into LB liquid medium containing special substrate at a volume ratio of 0.05-0.15% and cultured overnight at 35-40℃ and 200-250 rpm to obtain the bacterial culture of recombinant bacteria GD.

[0014] (5) Inoculate the recombinant bacterial GD culture into LB liquid medium containing a special substrate at a volume ratio of 0.5-1.5% and culture until OD. 600 The concentration of the glycosaminoglycan synthase was 0.6–0.8, and then the cells were transferred to M9 low-salt medium without glucose and cultured for 0.5–1.5 h. Xylose inducer with a final concentration of 15–25 g / L and N-azidoacetylglucosamine with a final concentration of 80–120 μg / mL were added, and the cells were induced to culture for 4–8 h. Then, the cells were reacted with luciferin in the dark for 0.5–1.5 h. When the number of genes in the glycosaminoglycan backbone synthase library was less than 20, the cells after fluorescence reaction were analyzed by flow cytometry, and the fluorescence intensity or the ratio of fluorescence intensity to absorbance was measured to screen for the target glycosaminoglycan backbone synthase. When the number of genes in the glycosaminoglycan backbone synthase library exceeded 20, the cells after fluorescence reaction were sorted by flow cytometry, and the fluorescence intensity was measured. The cells with the highest fluorescence intensity (0.5–2%) were collected to screen for the target glycosaminoglycan backbone synthase.

[0015] According to a preferred embodiment of the present invention, in step (1), the glycosaminoglycan backbone synthase is chondroitin sulfate backbone synthase, heparin backbone synthase or hyaluronic acid synthase.

[0016] According to a preferred embodiment of the present invention, in step (2), the NCBI database ID of the uridine diphosphate-glucose-6-dehydrogenase gene tuaD is 936766.

[0017] According to a preferred embodiment of the present invention, in step (3), the Bacillus subtilis BS168ASS is a Bacillus subtilis that blocks the endogenous uridine diphosphate-N-acetylglucosinolate (GlcNAc) synthesis pathway and expresses the heterologous uridine diphosphate-N-acetylglucosinolate salvage synthesis pathway.

[0018] According to a preferred embodiment of the present invention, the method for constructing Bacillus subtilis BS168ASS is as follows:

[0019] Starting with Bacillus subtilis subsp. subtilis str.168, the gene glmS encoding the key enzyme molecule in the UDP-GlcNAc pathway was knocked out. Then, the gene NahK encoding N-acetylglucosamine-1-kinase and the gene AGX1 encoding N-acetylglucosamine-1-phosphate-uracil transferase were inserted, resulting in Bacillus subtilis BS168ASS. The enzyme encoded by glmS catalyzes the conversion of fructose-6-phosphate to glucose-6-phosphate. The enzyme encoded by NahK can catalyze the conversion of GlcNAc-1-P using ATP and GlcNAc. The enzyme encoded by AGX1 can catalyze the conversion of GlcNAc-1-P to UDP-GlcNAc. Bacillus subtilis BS168ASS can utilize N-azidoacetylated glucosamine GlcNAz as a substrate to generate UDP-GlcNAz.

[0020] Further preferably, the NCBI database ID of the gene glmS is 938736, the N-acetylglucosamine-1-kinase gene NahK is from Bifidobacterium longum and has an NCBI database ID of 69578838, and the N-acetylglucosamine-1-phosphate-uracil transferase gene AGX1 is from human and has an NCBI database ID of 6675.

[0021] According to a preferred embodiment of the present invention, in steps (4) and (5), the LB liquid culture medium containing the special substrate has the following components: 10 g / L yeast extract, 20 g / L peptone, 20 g / L sodium chloride, 5 μg / mL chloramphenicol and 100 μg / mL glycosaminoglycan backbone synthase natural substrate GlcNAc.

[0022] According to a preferred embodiment of the present invention, in step (5), the fluorescein is Cyanine5 DBCO, the final concentration of the fluorescein is 0.5mM, and the reaction time in the dark is 1h.

[0023] According to a preferred embodiment of the present invention, in step (5), the specific parameters for measuring fluorescence intensity and absorbance are: emission wavelength of 646 nm, excitation wavelength of 662 nm, and absorbance of 600 nm.

[0024] Technical features of the present invention:

[0025] This invention utilizes GlcNAz to culture recombinant strain BS168ASSGD containing glycosaminoglycan backbone synthase, resulting in an azide-modified glycosaminoglycan backbone on the strain surface. This polysaccharide backbone undergoes a click chemical reaction with the fluorescent dye Cyanine5 DBCO, causing the strain surface to fluoresce. The ratio of fluorescence intensity to absorbance of strains containing glycosaminoglycan backbone synthase is more than twice that of strains without glycosaminoglycan backbone synthase. Combined with fluorescence-activated cell sorting, high-throughput screening of active glycosaminoglycan backbone synthase can be achieved.

[0026] The reaction equation for the click chemistry reaction between the fluorescent dye Cyanine5 DBCO and GlcNAz is as follows:

[0027]

[0028] Beneficial effects:

[0029] This invention uses *Bacillus subtilis* BS168ASS, which blocks the endogenous GlcNAc synthesis pathway and expresses the GlcNAc (GlcNAz) salvage synthesis pathway, as the host bacterium. It overexpresses the uridine diphosphate-glucose-6-dehydrogenase gene *tuaD* and the glycosaminoglycan backbone synthase gene to construct a recombinant bacterium for screening glycosaminoglycan backbone synthases. Then, using this strain as a baseline, high-throughput and accurate screening of active glycosaminoglycan backbone synthase strains is achieved by measuring fluorescence intensity or the ratio of fluorescence intensity to absorbance, greatly improving screening efficiency. Furthermore, this invention provides a new starting point for the discovery of enzymes from novel sources, especially novel glycosaminoglycan backbone synthases from microbial communities that cannot be cultured in metagenomic libraries. Attached Figure Description

[0030] Figure 1 : Polyacrylamide gel electrophoresis image of the PCR amplification product of uridine diphosphate-glucose-6-dehydrogenase gene tuaD.

[0031] In the figure: M is the marker; 1 is the PCR amplification product of the tuaD gene;

[0032] Figure 2 Polyacrylamide gel electrophoresis image of PCR amplification products of chondroitin sulfate backbone synthase KfoC gene, hyaluronic acid backbone synthase PmHAS gene, and heparin backbone synthase PmHS2 gene.

[0033] In the figure: M is the marker; 1 is the PCR amplification product of the KfoC gene; 2 is the PCR amplification product of the PmHAS gene; 3 is the PCR amplification product of the PmHS2 gene.

[0034] Figure 3Electrophoresis images of PCR amplification products of CcCS, CvCS, NdCS, MsCS, RhCS, MeCS, HfCS, LeCS, PsCS and HfCS genes identified by the BLASTp algorithm.

[0035] In the figure: M is the marker; 1 is the PCR amplification product of the CcCS gene; 2 is the PCR amplification product of the CvCS gene; 3 is the PCR amplification product of the NdCS gene; 4 is the PCR amplification product of the MsCS gene; 5 is the PCR amplification product of the RhCS gene; 6 is the PCR amplification product of the MeCS gene; 7 is the PCR amplification product of the HfCS gene; 8 is the PCR amplification product of the LeCS gene; 9 is the PCR amplification product of the PsCS gene; 10 is the PCR amplification product of the TmCS gene.

[0036] Figure 4 Electrophoresis image of the pHCMC04 plasmid backbone after double digestion with SpeI and BamHI;

[0037] In the figure: M is the marker; 1 is the double digestion fragment of plasmid pHCMC04;

[0038] Figure 5 Recombinant plasmid construction map containing chondroitin sulfate backbone synthase KfoC gene; hyaluronic acid backbone synthase PmHAS gene; and heparin backbone synthase PmHS2 gene;

[0039] In the figure: A is the pHCMC04-KfoC-tuaD recombinant plasmid; B is the pHCMC04-pmHAS-tuaD recombinant plasmid; C is the pHCMC04-PmHS2-tuaD recombinant plasmid.

[0040] Figure 6 : Contains recombinant plasmid construction maps for CcCS, CvCS, NdCS, MsCS, RhCS, MeCS, HfCS, LeCS, PsCS and TmCS genes identified by the BLASTp algorithm;

[0041] In the figure: A is the pHCMC04-CcCS-tuaD recombinant plasmid; B is the pHCMC04-CvCS-tuaD recombinant plasmid; C is the pHCMC04-NdCS-tuaD recombinant plasmid; D is the pHCMC04-MsCS-tuaD recombinant plasmid; E is the pHCMC04-RhCS-tuaD recombinant plasmid; F is the pHCMC04-MeCS-tuaD recombinant plasmid; G is the pHCMC04-HfCS-tuaD recombinant plasmid; H is the pHCMC04-LeCS-tuaD recombinant plasmid; I is the pHCMC04-LeCS-tuaD recombinant plasmid; J is the pHCMC04-TmCS-tuaD recombinant plasmid.

[0042] Figure 7 The fluorescence of recombinant bacteria BS168SSAED, BS168SSACD, BS168SSAHS2D and BS168SSAHASD after a click chemical reaction was measured by an ELISA reader.

[0043] In the figure: the horizontal axis represents the type of recombinant strain, and the vertical axis represents the ratio of fluorescence intensity to absorbance;

[0044] Figure 8 Fluorescence characteristics of recombinant bacteria BS168SSAED, BS168SSACD, BS168SSAHS2D, and BS168SSAHASD after a click chemistry reaction, as determined by flow cytometry.

[0045] In the figure: A shows the comparison of fluorescence intensity between BS168SSACD and BS168SSAED; B shows the comparison of fluorescence intensity between BS168SSAHS2D and BS168SSAED; C shows the comparison of fluorescence intensity between BS168SSAHASD and BS168SSAED.

[0046] Figure 9 Fluorescence measured by an ELISA reader after recombinant Bacillus subtilis containing CcCS, CvCS, NdCS, MsCS, RhCS, MeCS, HfCS, LeCS, PsCS and TmCS genes undergoes a click chemical reaction.

[0047] In the figure: the horizontal axis represents the type of recombinant strain; the vertical axis represents the ratio of fluorescence intensity to absorbance.

[0048] Figure 10 : Construction map of recombinant plasmids containing CvCS, HfCS and MeCS genes;

[0049] In the figure: A is the Pet28a(+)-His-CvCS recombinant plasmid; B is the Pet28a(+)-His-HfCS recombinant plasmid; A is the Pet28a(+)-His-MeCS recombinant plasmid.

[0050] Figure 11 : Polyacrylamide gel electrophoresis images of CvCs protein, HfCS protein and MeCS protein;

[0051] In the diagram: M stands for marker; 1 represents CvC protein; 2 represents HfCS protein; 3 represents MeCS protein;

[0052] Figure 12 : UDP-GalNAc / UDP-GalNAc transferase activity of CvCs, HfCS and MeCS proteins;

[0053] In the figure: the horizontal axis represents protein type; the vertical axis represents conversion rate. Detailed Implementation

[0054] The present invention will now be described through specific implementation schemes. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. The following embodiments are intended to further illustrate the content of the present invention, but not to limit the scope of protection of the present invention.

[0055] The PCR Taq enzyme, seamless cloning kit, *E. coli* DH5α competent cells, and *E. coli* BL21(de3) competent cells used in the following examples were all purchased from Vazyme; plasmid extraction kits and gel extraction kits were purchased from OMEGA bio-tek (USA); restriction endonucleases were purchased from Thermo Scientific; and Cyanine5 DBCO was purchased from Lumiprobe. Unless otherwise specified, all experimental procedures were performed according to the product instructions.

[0056] Example 1: Construction of an engineered Bacillus subtilis strain BS168SSAGD with an efficient azide-modified glycosaminoglycan backbone

[0057] 1. Construction of Bacillus subtilis BS168ASS

[0058] The Bacillus subtilis BS168ASS strain is a Bacillus subtilis strain that blocks the endogenous uridine diphosphate-N-acetylglucosinolate (GlcNAc) synthesis pathway and expresses the heterologous uridine diphosphate-N-acetylglucosinolate (N-azidoacetylglucosamine, GlcNAz) salvage synthesis pathway. The specific construction method is as follows:

[0059] (1) Blocking the UDP-GlcNAc endogenous synthesis pathway

[0060] The pUC57-neo plasmid containing the homologous arm fragment of the neomycin resistance gene glmS, synthesized by the company (Genscript), was digested with BamHI to obtain an 1800bp fragment.

[0061] The enzyme digestion system is as follows:

[0062]

[0063] Enzyme digestion reaction conditions: 37℃ water bath for 1 hour. The desalted liquid from the digested system was then recovered.

[0064] Bacillus subtilis subsp. subtilis str. 168 was inoculated at a volume ratio of 0.1% into LB liquid medium and incubated overnight at 37°C and 225 rpm in a shaker. 2.6 mL of the overnight culture was inoculated into 40 mL of medium (LB + 0.5 M sorbitol) and incubated at 37°C and 200 rpm until OD reached. 600 =0.8~0.9. Incubate the bacterial culture in an ice-water bath for 10 minutes, then centrifuge at 5000g, 4℃ for 5 minutes to collect the bacterial cells. Resuspend the bacterial cells in 50mL of pre-chilled electroporation medium (0.5M sorbitol, 0.5M mannitol, 10% glycerol), centrifuge at 5000g, 4℃ for 5 minutes, and discard the supernatant. Repeat this washing process 4 times. Resuspend the washed bacterial cells in 1mL of electroporation medium, aliquoting 60μL into each EP tube to obtain BS168 electroporation competent cells.

[0065] Add 6 μL of the pUC57-neo plasmid digestion product to 60 μL of electroporation competent cells, incubate on ice for 2 minutes, then transfer to a pre-chilled electroporation cuvette (1 mm) and electroporate once. Electroporator settings: 2 kV, 1 mm, 1 electroporation. After electroporation, remove the cuvette and immediately add 1 mL of RM medium (LB + 0.5 M sorbitol + 0.38 M mannitol), incubate at 37°C, 200 rpm for 3 hours to recover, then culture overnight on LB solid medium containing 50 μg / mL neomycin. Positive transformants are picked and designated as strain BS168△glmS.

[0066] (2) The introduction of the UDP-GlcNAc (UDP-GlcNAz) salvage synthesis pathway of GlcNAc (GlcNAz) can be utilized.

[0067] Design a neomycin resistance gene homologous arm fragment P carrying the kanamycin resistance gene. veg -AGX1-P veg -NahK, to knock out the neomycin resistance gene in the genome, pUC57-P veg -AGX1-P vegThe NahK plasmid was synthesized at the company (Sangon Biotech). pUC57-P was digested using Acc65I and SmaI enzymes. veg -AGX1-P veg -NahK plasmid;

[0068] The enzyme digestion system is as follows:

[0069]

[0070] Enzyme digestion reaction conditions: 37℃ water bath for 1 hour. The desalted liquid from the digested system was then recovered.

[0071] The BS168ΔglmS electrotransfer competent state was prepared according to the method described in (1), and the homologous arm fragment P was... veg -AGX1-P veg -NahK was electroporated into BS168△glmS electrocompetent cells, cultured overnight on LB solid medium containing 50 μg / mL kanamycin, and positive transformants were picked to obtain Bacillus subtilis BS168ASS.

[0072] The NCBI database ID for the gene glmS is 938736, the NCBI database ID for the N-acetylglucosamine-1-kinase gene NahK is derived from Bifidobacterium longum and is 69578838, and the NCBI database ID for the N-acetylglucosamine-1-phosphate-uracil transferase gene AGX1 is derived from the human NCBI database and is 6675.

[0073] This specific method has been disclosed in Chinese patent document 2021100967185.

[0074] 2. Obtaining the uridine diphosphate-glucose-6-dehydrogenase gene tuaD

[0075] Genomic DNA was extracted from Bacillus subtilis. Using the genomic DNA as a template, PCR was performed using tuaDF and tuaDR as primers. The primer sequences are as follows:

[0076] tuaD F:5'-AGGTACCAAGAGAGGAATGTACACATGAAAAAAATAGCTGTCATTGG-3',

[0077] tuaD R:5'-GACGTCGACTCTAGAGGATCCTTATAAATTGACGCTTCCCAAGTC-3';

[0078] The PCR reaction system is as follows: (primer concentration is 10 μM)

[0079]

[0080]

[0081] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 72℃ annealing for 15 s, 58℃ extension for 2 min, for a total of 30 cycles; 72℃ extension for 5 min, and storage at 4℃. PCR products were purified by 1% agarose gel electrophoresis for 30 min, and the results are as follows. Figure 1 As shown, an electrophoretic band of approximately 1390 bp was obtained, which is the tuaD fragment.

[0082] 3. Construction of a glycosaminoglycan backbone synthase library

[0083] The following enzymes were obtained: chondroitin sulfate backbone synthase KfoC from Escherichia coli (NCBI database ID: CAD5992240.1); heparin backbone synthase PmHS2 from Pasteurella multocida (NCBI database ID: AY292200.1); and hyaluronic acid backbone synthase pmHAS from Pasteurella multocida (NCBI database ID: AAC38318.2). All three enzymes are highly active glycosaminoglycan backbone synthases.

[0084] Using the amino acid sequences of these three enzymes as sequence alignment templates, the BLASTp algorithm was used to perform sequence alignment in bioinformatics databases (NCBI database). Ten amino acid sequences that may be glycosaminoglycan backbone synthases (GTs) were identified, abbreviated as CcCS (NCBI database ID: WP_150081707.1); CvCS (NCBI database ID: WP_168227469.1); HfCS (NCBI database ID: NBI12747.1); and LeCS (NCBI database ID: WP_168227469.1). _1661815; MsCS, NCBI database ID is WP_024460064.1; MeCS, NCBI database ID is WP_167432605.1; NdCS, NCBI database ID is WP_085359788.1; PsCS, NCBI database ID is WP_144187813.1; RhCS, NCBI database ID is WP_077587478.1; TmCS, NCBI database ID is WP_136735112.1

[0085] The above 13 gene fragments were optimized using double codons in *E. coli* and *Bacillus subtilis*, and the corresponding plasmids pET28a(+)-His-GT were synthesized by GenScript and found in *E. coli* TOP10. Primers were designed to perform PCR on the plasmids to obtain the corresponding gene fragments, collectively referred to as the GT fragments. The primer sequences are as follows:

[0086] KfoC F:5'-TGACAAATGGTCCAAACTAGTATGAGTATTCTTAATCAAGCAATA-3';

[0087] KfoC R:5'-GTACATTCCTCTCTTGGTACCTTATAAATCATTCTCTATTTTTT-3';

[0088] PmHAS F:5'-TGACAAATGGTCCAAACTAGTATGAACACACTGAGCCAAGCAA-3';

[0089] PmHAS R:5'-GTACATTCCTCTCTTGGTACCTTACAGTGTAATTGAATTAATAA-3';

[0090] PmHS2 F:5'-TGACAAATGGTCCAAACTAGTATGAAAGGCAAAAAAGAAATGA-3';

[0091] PmHS2 R:5'-GTACATTCCTCTCTTGGTACCTTAAAGAAAATAAAACGGCAGGC-3';

[0092] CcCS F:5'-TGACAAATGGTCCAAACTAGTATGCAACAATCTAGCAAATCATTT-3';

[0093] CcCS R:5'-GTACATTCCTCTCTTGGTACCTTATATATTTTTGTGAAAAGAGGT-3';

[0094] CvCS F:5'-TGACAAATGGTCCAAACTAGTATGACAATTTTGAATCAAGCGATT-3';

[0095] CvCS R:5'-GTACATTCCTCTCTTGGTACCTTATATAAACTGCTGAATACACAG-3';

[0096] HfCS F:5'-TGACAAATGGTCCAAACTAGTATGAATACTGTCCAAGGCAATT-3';

[0097] HfCS R:5'-GTACATTCCTCTCTTGGTACCTTAAGCAAAGTTAAGCCGATGGGT-3';

[0098] LeCS F:5'-TGACAAATGGTCCAAACTAGTATGTCGATTTTTAACGAAGCAATT-3';

[0099] LeCS R:5'-GTACATTCCTCTCTTGGTACCTTATAAAATTTGAAACCGATTTT-3';

[0100] MeCS F:5'-TGACAAATGGTCCAAACTAGTATGGCCAGCTTTGTAGAAGCTAAC-3;'

[0101] MeCS R:5'-GTACATTCCTCCTTGGTACCTTATTTTTTGAAAAAAACTACCTG-3';

[0102] MsCS F:5'-TGACAAATGGTCCAAACTAGTATGGGGGCAGGTCAAAATACTG-3';

[0103] MsCS R:5'-GTACATTCCTCTCTTGGTACCTTAAAGAAAATAAAACGGCAGGC-3';

[0104] NdCS F:5'-TGACAAATGGTCCAAACTAGTATGGAAAAGATCCTGAGCCGCGCA-3';

[0105] NdCS R:5'-GTACATTCCTCCTTGGTACCTTATATGTTTTTAAACTGAATCAG-3';

[0106] PsCS F:5'-TGACAAATGGTCCAAACTAGTATGAAGATCCTGAGCAATGCGATT-3';

[0107] PsCS R:5'-GTACATTCCTCTCTTGGTACCTTACGTTATAAAATTACCAATGGC-3';

[0108] RhCS F:5'-TGACAAATGGTCCAAACTAGTATGAATATTCTGTCAAAAGCCGTT-3';

[0109] RhCS R:5'-GTACATTCCTCTCTTGGTACCTTAATAATAGGTAATGTAAAAGTT-3';

[0110] TmCS F:5'-TGACAAATGGTCCAAACTAGTATGAATAAAAATATATTCGATCAA-3';

[0111] TmCS R:5'-GTACATTCCTCTCTTGGTACCTTAAAGCAAAGTGCTGAACTGCTC-3'.

[0112] The PCR reaction system is as follows: (primer concentration is 10 μM)

[0113]

[0114] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 72℃ annealing for 15 s, 60℃ extension for 3 min, for a total of 30 cycles; 72℃ extension for 5 min, and storage at 4℃. PCR products were purified by 1% agarose gel electrophoresis for 30 min. The obtained PCR products were analyzed by 1% agarose gel electrophoresis, and the results are as follows. Figure 2 and Figure 3 As shown.

[0115] 4. Construction of recombinant expression plasmids

[0116] (1) The vector plasmid pHCMC04 was digested with SpeI and BamHI to obtain an 8000bp fragment. The electrophoresis image of this fragment is shown below. Figure 4 As shown;

[0117] The enzyme digestion system is as follows:

[0118]

[0119]

[0120] Enzyme digestion reaction conditions: 37℃ water bath for 1 hour.

[0121] (2) The enzyme-digested vector plasmids were ligated with the tuaD and GT fragments using a seamless DNA cloning method, and then chemically transformed into *E. coli* DH5α competent cells. After sequencing verification, a total of 13 recombinant expression vectors were obtained, namely pHCMC04-CcCS-tuaD recombinant plasmid, pHCMC04-CvCS-tuaD recombinant plasmid, pHCMC04-NdCS-tuaD recombinant plasmid, pHCMC04-MsCS-tuaD recombinant plasmid, pHCMC04-RhCS-tuaD recombinant plasmid, pHC... The recombinant plasmids MC04-MeCS-tuaD, pHCMC04-HfCS-tuaD, pHCMC04-LeCS-tuaD, pHCMC04-LeCS-tuaD, pHCMC04-TmCS-tuaD, pHCMC04-KfoC-tuaD, pHCMC04-PmHAS-tuaD, and pHCMC04-PmHS2-tuaD are collectively referred to as GD expression vectors. The specific construction diagrams of these recombinant plasmids are shown below. Figure 5 and Figure 6 As shown.

[0122] The seamless cloning reaction system is as follows:

[0123]

[0124] Seamless cloning reaction conditions: react in a water bath at 50°C for 30–50 min.

[0125] 5. Constructing recombinant Bacillus subtilis containing the GD expression vector

[0126] BS168ASS electrotransformation competent cells were prepared according to the method described in (1). The GD expression vector was transferred into Bacillus subtilis BS168ASS by electrotransformation. The cells were cultured overnight on LB solid medium containing 5 μg / mL chloramphenicol. Transformants were grown, which were the recombinant bacteria BS168SSAGD that efficiently synthesizes the azide-modified glycosaminoglycan backbone.

[0127] Example 2: Application of recombinant strain BS168SSAGD in high-throughput screening of glycosaminoglycan backbone synthases

[0128] 1. Validation of high-throughput screening systems based on known activities of glycosaminoglycan backbone synthases KfoC, PmHS2, and PmHAS.

[0129] The recombinant bacteria containing the KfoC gene and tuaD gene constructed in Example 1 were named BS168SSACD; the recombinant bacteria containing the PmHS2 gene and tuaD gene were named BS168SSAHS2D; the recombinant bacteria containing the PmHAS gene and tuaD gene were named BS168SSAHASD; and the Bacillus subtilis containing the pHCMC04 empty plasmid was named BS168SSAE, which served as a control.

[0130] Then, the strain was inoculated at a volume ratio of 0.1% into LB liquid medium containing a special substrate and cultured overnight at 37°C and 225 rpm in a shaker. The overnight cultured strain was then expanded into LB liquid medium containing a volume ratio of 1% until the strain reached OD. 600 When the pH was 0.6–0.8, the strain was washed three times with 1×PBS buffer and transferred to low-salt M9 medium without glucose for culture, consuming the GlcNAc naturally present in the cells. After 1 hour, xylose inducer was added to a final concentration of 20 g / L and non-natural glycosyltransferase substrate GlcNAz to a final concentration of 100 μg / mL. The culture was then induced and cultured at 37°C and 225 rpm for 6 hours.

[0131] The components of the special substrate LB liquid medium are as follows: 10 g / L yeast extract, 20 g / L peptone, 20 g / L sodium chloride, 5 μg / mL chloramphenicol and 100 μg / mL natural substrate of glycosyltransferase GlcNac.

[0132] After the induced culture was completed, the strain was resuspended in 200 μL of 1×PBS buffer by resuspending and centrifugation three times. A final concentration of 0.5 mM fluorescein Cyanine5 DBCO was added to a 1.5 mL centrifuge tube protected from light. The mixture was incubated at 37°C for 1 h, then washed three times with 1×PBS buffer. The fluorescence intensity at emission wavelength 646 nm and excitation wavelength 662 nm, and the absorbance at 600 nm were measured using a microplate reader. The fluorescence intensity and OD were calculated. 600 The ratio, the result is as follows Figure 7 As shown.

[0133] Depend on Figure 7 It can be seen that the fluorescence intensity to absorbance ratio of the recombinant strains BS168SSACD, BS168SSAHS2D, and BS168SSAHASD containing glycosaminoglycan backbone synthase is significantly higher than that of the strain BS168SSAE without glycosaminoglycan backbone synthase, and the fluorescence is more than 1 times that of the strain BS168SSAE without glycosaminoglycan backbone synthase.

[0134] The bacterial culture that participated in the fluorescence reaction was diluted to OD using 1×PBS buffer. 600Monochromatic flow cytometry analysis was performed with a wavelength between 0.3 and 0.5, at an emission wavelength of 646 nm and an excitation wavelength of 662 nm. The results are as follows: Figure 8 As shown.

[0135] Depend on Figure 8 It can be seen that the fluorescence intensity of recombinant bacteria BS168SSACD, BS168SSAHS2D, and BS168SSAHASD containing glycosaminoglycan backbone synthase is significantly higher than that of strain BS168SSAE which does not contain glycosaminoglycan backbone synthase. This result is consistent with the results of the microplate reader.

[0136] 2. Screening of glycosaminoglycan backbone synthase libraries with unknown activities

[0137] (1) Screening of glycosaminoglycan backbone synthase libraries based on the ratio of fluorescence intensity to absorbance

[0138] The ten recombinant bacteria containing the tuaD gene and GT fragment constructed in Example 1 were abbreviated as: CcCS, CvCS, HfCS, LeCS, MsCS, MeCS, NdCS, PsCS, RhCS, and TmCS. They were cultured and induced in 96-well deep-well plates according to the method described in Example 1, and underwent a click chemistry reaction with Cyanine5 DBCO. The fluorescence intensity at emission wavelength of 646 nm and excitation wavelength of 662 nm, and the absorbance at 600 nm were detected using a microplate reader. The fluorescence intensity and OD were calculated. 600 The ratio, the result is as follows Figure 9 As shown.

[0139] Depend on Figure 9 It can be seen that the CvCs gene, HfCS gene, and MeCS gene exhibit fluorescence to a degree comparable to or even stronger than that of the KfoC gene.

[0140] (2) Validation of in vitro glycosaminoglycan backbone synthase activity

[0141] The CvCs, HfCS, and MeCS genes, which exhibited strong fluorescence in (1), were used by GenScript to synthesize corresponding plasmids. The original plasmids synthesized by GenScript were pET28a(+)-His-CvCS, pET28a(+)-His-HfCS, and pET28a(+)-His-MeCS. The specific recombinant plasmid construction map is shown below. Figure 10 As shown. Transformants were chemically transformed into Escherichia coli BL21(DE3), and transformed into LB solid medium containing 50 μg / mL kanamycin. Single colonies were picked, and plasmids were extracted and verified by enzyme digestion to obtain BL21(DE3) expression strains containing CvCs, HfCS and MeCS genes.

[0142] The enzyme digestion system is as follows:

[0143]

[0144]

[0145] Enzyme digestion reaction conditions: 37℃ water bath for 20 min.

[0146] BL21(DE3) expression strains containing CvCs, HfCS, and MeCS genes were amplified at a 1% volume ratio to 1L of LB liquid medium. The strains were allowed to grow to OD200. 600 When the concentration is 0.6–0.8, add IPTG to a final concentration of 0.2 mM, and induce at 22°C and 225 rpm for 12–18 h.

[0147] After induction, the bacterial culture was centrifuged at 8000g for 20 min at 4℃, the supernatant was discarded, the bacterial pellet was collected and resuspended in loading buffer (20Mm Tris-HCl, pH=7.5, 0.5M NaCl, 5mM imidazole); the bacterial cells were disrupted by ultrasonic disruption for 30 min under the following conditions: 15s operation, 45s interval, 35% amplitude, 1500KJ energy, 4℃; the disrupted bacterial cells were centrifuged at 12000g for 30 min at 4℃ using an ultra-low temperature centrifuge, the supernatant was collected and filtered through a 0.22μm filter membrane. The supernatant after filtration was purified using Ni-charged MagBeads. The magnetic beads were first completely resuspended in the test tube with ddH2O. The test tube was then placed on a magnetic bead separator to collect the beads. The supernatant was discarded, and the original 20% ethanol preservative solution was washed away. The magnetic beads were resuspended twice with loading buffer (20 Mm Tris-HCl, pH 7.5, 0.5 M NaCl, 35 mM imidazole) to equilibrate the system. The filtered supernatant was then resuspended and incubated at 4 °C and 225 rpm for 30 min on a shaker. Impurities were washed away with equilibration buffer (20 Mm Tris-HCl, pH 7.5, 0.5 M NaCl, 35 mM imidazole). The target protein was eluted with elution buffer (20 Mm Tris-HCl, pH 7.5, 0.5 M NaCl, 200 mM imidazole) and collected.

[0148] The expression and purification of the induced protein were identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 11 As shown, CvCs protein, HfCS protein, and MeCS protein were successfully isolated.

[0149] Using GlcA-pNP as the acceptor substrate and UDP-GalNAc / UDP-GlcNAc as the donor substrate, the glycosaminoglycan backbone synthase activities of CvCs, HfCS, and MeCS proteins were measured.

[0150] The reaction system is as follows:

[0151]

[0152] Reaction conditions: Reaction overnight at 37°C.

[0153] The reaction mixture was filtered through a 0.22 μm filter membrane and then analyzed by PAMN-HPLC. A specific absorption peak of the pNP group was detected at 310 nm. The HPLC analysis used a YMC-Pack Polyamine II column, with water and 1 mol / L KH₂PO₄ solution as the mobile phase, and a flow rate of 0.5 mL / min. The UV absorption at 310 / 254 nm was measured for each protein catalytic product after separation by the column. The results are as follows: Figure 12 As shown.

[0154] The HPLC analysis procedure is as follows:

[0155]

[0156]

[0157] Depend on Figure 12 It can be seen that CvCs, HfCS, and MeCS proteins all exhibit strong UDP-GalNAc transferase activity, meaning that CvCs, HfCS, and MeCS proteins are all highly active glycosaminoglycan backbone synthases. This is consistent with the screening results of the glycosaminoglycan backbone synthase library based on the ratio of fluorescence intensity to absorbance in Example 2 of this paper.

[0158] 3. High-throughput screening of glycosaminoglycan backbone synthases based on fluorescence-activated cell sorting (FACS) technology

[0159] The method based on ELISA reader to measure fluorescence can only screen a small number of glycosaminoglycan backbone synthases. High-throughput screening requires fluorescence-activated cell sorting technology. The strain BS168SSACD containing the KfoC gene and tuaD gene expression cassette is called KfoC+; the strain BS168SSAE containing only the pHCMC04 empty plasmid is called KfoC-.

[0160] KfoC+ and KfoC- were cultured together, and the enrichment efficiency of FACS on the positive group was further analyzed according to the volume ratio of KfoC+ / total bacteria of 10:1, 1:1, 1:10, and 1:100.

[0161] The mixed-cultured bacterial strains were induced and subjected to fluorescence reaction as described in Example 1. After washing, the samples were analyzed by flow cytometry. The top 2% of cells with the highest fluorescence intensity were collected to minimize possible false positives. The sorted bacteria were cultured overnight in LB solid medium containing 5 μg / mL chloramphenicol. After single-cell picking, the positive rate after enrichment was identified and compared with that before sorting. The results are shown in Table 1.

[0162] Table 1. The effect of using recombinant Bacillus subtilis with azide-modified glycosaminoglycan backbone, which efficiently synthesizes glycosaminoglycan backbone, in the FACS system to enrich recombinant strains containing highly active glycosaminoglycan backbone synthase from a large number of background bacteria.

[0163]

[0164] As shown in Table 1, under single-round high-strict flow cytometry sorting conditions, the recombinant strain KfoC+ containing highly active glycosaminoglycan backbone synthase still has a 97% probability of enrichment even under 1:100 mixed culture conditions.

Claims

1. A high-throughput screening method for glycosaminoglycan backbone synthases, characterized in that, The steps include the following: (1) Using the BLASTp method, glycosaminoglycan backbone synthase genes were randomly obtained and glycosaminoglycan backbone synthase libraries were constructed. (2) The glycosaminoglycan backbone synthase gene and the uridine diphosphate-glucose-6-dehydrogenase gene tuaD expression cassette in the library were respectively ligated into the pHCMC04 plasmid to obtain the recombinant vector pHCMC04-GTs-tuaD; (3) The recombinant vector pHCMC04-GTs-tuaD was transformed into Bacillus subtilis BS168ASS, and positive recombinants were selected to obtain the recombinant strain GD for screening glycosaminoglycan backbone synthase; The Bacillus subtilis BS168ASS is a Bacillus subtilis strain that blocks the endogenous uridine diphosphate-N-acetylglucosinolate synthesis pathway and expresses the heterologous uridine diphosphate-N-acetylglucosinolate salvage synthesis pathway. The construction method is as follows: Bacillus subtilis B acillus subtilis subsp. subtilis str. 168 As the starting strain, the gene glmS encoding the key enzyme molecule for the synthesis of the UDP-GlcNAc pathway was knocked out, and then the gene NahK encoding N-acetylglucosamine-1-kinase and the gene AGX1 encoding N-acetylglucosamine-1-phosphate-uracil transferase were inserted to obtain Bacillus subtilis BS168ASS. The NCBI database ID for the gene glmS is 938736; the NCBI database ID for the N-acetylglucosamine-1-kinase gene NahK is from Bifidobacterium longum; and the NCBI database ID for the N-acetylglucosamine-1-phosphate-uracil transferase gene AGX1 is from human; the NCBI database ID for the N-acetylglucosamine-1-phosphate-uracil transferase gene AGX1 is from human. (4) The recombinant bacteria GD, which screened for glycosaminoglycan backbone synthase, was inoculated into LB liquid medium containing special substrate at a volume ratio of 0.05-0.15% and cultured overnight at 35-40℃ and 200-250 rpm to obtain the bacterial culture of recombinant bacteria GD. The LB liquid culture medium containing the special substrate has the following components: 10 g / L yeast extract, 20 g / L peptone, 20 g / L sodium chloride, 5 μg / mL chloramphenicol and 100 μg / mL glycosaminoglycan backbone synthase natural substrate GlcNAc. (5) Inoculate the recombinant bacterial GD culture into LB liquid medium containing a special substrate at a volume ratio of 0.5-1.5% and culture until OD. 600 The concentration of the glycosaminoglycan synthase was 0.6–0.8, and then the cells were transferred to M9 low-salt medium without glucose and cultured for 0.5–1.5 h. Xylose inducer at a final concentration of 15–25 g / L and N-azidoacetylglucosamine at a final concentration of 80–120 μg / mL were added, and the cells were induced for 4–8 h. The cells were then reacted with luciferin in the dark for 0.5–1.5 h. When the number of genes in the glycosaminoglycan backbone synthase library was less than 20, the cells after fluorescence reaction were analyzed by flow cytometry, and the fluorescence intensity or the ratio of fluorescence intensity to absorbance was measured to screen for the target glycosaminoglycan backbone synthase. When the number of genes in the glycosaminoglycan backbone synthase library exceeded 20, the cells after fluorescence reaction were sorted by flow cytometry, and the fluorescence intensity was measured. The cells with the highest fluorescence intensity (top 0.5–2%) were collected to screen for the target glycosaminoglycan backbone synthase.

2. The high-throughput screening method for glycosaminoglycan backbone synthases as described in claim 1, characterized in that, In step (1), the glycosaminoglycan backbone synthase is chondroitin sulfate backbone synthase, heparin backbone synthase or hyaluronic acid synthase.

3. The high-throughput screening method for glycosaminoglycan backbone synthases as described in claim 1, characterized in that, In step (2), the NCBI database ID of the uridine diphosphate-glucose-6-dehydrogenase gene tuaD is 936766.

4. The high-throughput screening method for glycosaminoglycan backbone synthases as described in claim 1, characterized in that, In step (5), the fluorescein is Cyanine5 DBCO, the final concentration of the fluorescein is 0.5 mM, and the reaction time in the dark is 1 h.

5. The high-throughput screening method for glycosaminoglycan backbone synthases as described in claim 1, characterized in that, In step (5), the specific parameters for measuring fluorescence intensity and absorbance are: emission wavelength of 646 nm, excitation wavelength of 662 nm, and absorbance of 600 nm.

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