CN117004635A - A high-throughput screening method for glycosaminoglycan backbone synthases - Google Patents

Abstract

The invention relates to a high-throughput screening method for glycosaminoglycan backbone synthase. The method includes steps: (1) constructing a glycosaminoglycan skeleton synthase library; (2) constructing a recombinant vector; (3) constructing recombinant bacteria for screening glycosaminoglycan skeleton synthases; (4) cultivating the recombinant bacteria; (5) converting After the bacterial fluid of the recombinant bacteria is analyzed by flow cytometry or flow cytometry sorting, the fluorescence is measured to screen out the target glycosaminoglycan skeleton synthase. The present invention uses Bacillus subtilis BS168ASS as the host bacterium, overexpresses the uridine diphosphate-glucose-6-dehydrogenase gene and the glycosaminoglycan skeleton synthase gene, and constructs a recombinant bacterium for screening glycosaminoglycan skeleton synthase. Then by measuring the fluorescence of the strains, high-throughput and accurate screening of glycosaminoglycan backbone synthase strains is achieved, which improves the screening efficiency and is useful for new sources of enzymes, especially new microorganisms that cannot be cultured in metagenomic libraries. The discovery of glycosaminoglycan backbone synthases provides a new starting point.

Description

A high-throughput screening method for glycosaminoglycan backbone synthases

Technical field

The present invention relates to a high-throughput screening method for glycosaminoglycan backbone synthase, specifically to the transformation of the azide-modified glycosaminoglycan backbone synthesis pathway of Bacillus subtilis and the glycosaminoglycan backbone synthesis pathway based on this strain. Fluorescence-activated cell sorting of glycoskeletal synthase belongs to the technical field of biotechnology.

Background technique

Glycosaminoglycans (GAGs) are long linear heteropolysaccharides widely present in the connective tissues of higher animals. They are composed of repeating disaccharide units of hexuronic acid or hexose and hexosamine. According to the monosaccharide residues, Based on the type of linkage between residues and the number and position of sulfate groups, glycosaminoglycans can be divided into: hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (Dermatan) sulfate (DS), keratin sulfate (KS), heparan sulfate (HS) and heparin (HP). Glycosaminoglycans can interact with growth factors, cytokines, chemokines and enzymes, and have an important impact on body growth, inflammatory response, coagulation, tumor metastasis and other processes.

Uridine diphosphate glucose-6-dehydrogenase (tuaD), which is present in the genomic DNA of Bacillus subtilis, can catalyze the conversion of uridine diphosphate glucose-6-dehydrogenase (UDP-Glucose) into urine. Glycoside diphosphate-glucuronic acid (UDP-GlcA), existing studies have proven that UDP-GlcA limits glycosaminoglycan skeleton biosynthesis, so tuaD is the key to high-level glycosaminoglycan skeleton biosynthesis. .

Glycosyltransferase (GTs) is an enzyme that catalyzes the transfer of monosaccharide units from sugar donors to polysaccharides in a regio- and stereospecific manner, providing a very efficient method for the synthesis of complex oligosaccharides. However, the limited number of available GTs, coupled with their instability and strict substrate specificity, severely hinders the widespread application of these enzymes and, therefore, the acquisition of glycosyltransferases that can efficiently synthesize specific oligosaccharides and glycoconjugates. Enzymes have profound meaning.

Bacillus subtilis is a species of the genus Bacillus and is a Gram-positive bacterium. It is considered GRAS (Generally recognized as safe) and has good tolerance to the environment. According to existing research, Bacillus subtilis has a relatively clean background on the glycosaminoglycan synthesis pathway.

In past studies, directed evolution methods through protein engineering have achieved limited success in broadening the substrate scope, changing substrate specificity, and improving the activity of GTs, partly due to the lack of effective high-throughput screening methods. Measuring the activity of GTs is extremely challenging because there are no significant changes in fluorescence or absorbance associated with glycosidic bond formation. The selection of ideal phenotypes is a random process in most cases. Therefore, it is of great significance to develop high-throughput, low-cost screening methods that can be applied to large GTs libraries.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a high-throughput screening method for glycosaminoglycan backbone synthase. Specifically, a recombinant Bacillus subtilis strain that efficiently synthesizes azide-modified glycosaminoglycan backbone was constructed, and then high-throughput screening of glycosaminoglycan backbone synthase was performed using this strain.

The technical solution of the present invention is as follows:

A high-throughput screening method for glycosaminoglycan backbone synthase, including the following steps:

(1) Use the BLASTp method to randomly obtain glycosaminoglycan backbone synthase genes and construct a glycosaminoglycan backbone synthase library;

(2) Connect the glycosaminoglycan backbone synthase gene and uridine diphosphate-glucose-6-dehydrogenase gene tuaD expression cassette in the library to the pHCMC04 plasmid respectively to obtain the recombinant vector pHCMC04-GTs-tuaD;

(3) Transform the recombinant vector pHCMC04-GTs-tuaD into Bacillus subtilis BS168ASS, select positive recombinants, and obtain the recombinant strain GD for screening glycosaminoglycan backbone synthase;

(4) Inoculate the recombinant strain GD for screening glycosaminoglycan skeleton synthase into LB liquid culture medium containing a special substrate at a volume ratio of 0.05 to 0.15%, and culture overnight at 35 to 40°C and 200 to 250 rpm. Obtain the bacterial liquid of the recombinant strain GD;

(5) Inoculate the bacterial liquid of the recombinant strain GD into the LB liquid medium containing a special substrate at a volume ratio of 0.5 to 1.5%, culture it until the OD ₆₀₀ is 0.6 to 0.8, and then transfer it to M9 low-salt medium without glucose. In the culture medium, culture for 0.5 to 1.5 hours, add xylose inducer with a final concentration of 15 to 25 g/L and N-azidoacetylglucosamine with a final concentration of 80 to 120 μg/mL, induce and culture for 4 to 8 hours, and then The bacterial cells react with luciferin in the dark for 0.5 to 1.5 hours. When the number of genes in the glycosaminoglycan skeleton synthase library is less than 20, the bacterial cells after the fluorescence reaction are analyzed by flow cytometry, and then the fluorescence intensity or fluorescence is measured. The ratio of intensity to absorbance is used to screen out the target glycosaminoglycan skeleton synthase; when the number of genes in the glycosaminoglycan skeleton synthase library exceeds 20, the bacteria after the fluorescence reaction are flow sorted, and then the fluorescence is measured. Intensity, collect the bacteria with the top 0.5-2% fluorescence intensity, and screen out the target glycosaminoglycan skeleton synthase.

Preferably according to the present invention, in step (1), the glycosaminoglycan skeleton synthase is chondroitin sulfate skeleton synthase, heparin skeleton synthase or hyaluronic acid synthase.

Preferably according to the present invention, in step (2), the ID of the NCBI database of the uridine diphosphate-glucose-6-dehydrogenase gene tuaD is 936766.

According to the preferred embodiment of the present invention, in step (3), the Bacillus subtilis BS168ASS blocks the endogenous uridine diphosphate-N-acetylglucoside (GlcNAc) synthesis pathway and expresses heterologous uridine diphosphate-N-acetylglucoside. Glucoside salvage synthesis pathway in Bacillus subtilis.

According to the preferred method of the present invention, the construction method of the Bacillus subtilis BS168ASS is as follows:

Using Bacillus subtilis subsp.subtilis str.168 as the starting strain, the gene glmS encoding the key enzyme molecule for the synthesis of UDP-GlcNAc pathway was deleted, and then the gene NahK encoding N-acetylhexosamine-1-kinase and the encoding The gene AGX1 of N-acetylglucosamine-1-phosphate-uracil transferase was obtained from Bacillus subtilis BS168ASS. The enzyme encoded by gene glmS is responsible for catalyzing fructose-6-phosphate to glucose-6-phosphate. The enzyme encoded by gene NahK can catalyze GlcNAc-1-P using ATP and GlcNAc. The enzyme encoded by gene AGX1 can catalyze GlcNAc-1-P to UDP-GlcNAc, Bacillus subtilis BS168ASS can use N-azide acetylated glucosamine GlcNAz as a substrate to generate UDP-GlcNAz.

Further preferably, the NCBI database ID of the gene glmS is 938736, the N-acetylhexosamine-1-kinase gene NahK comes from Bifidobacterium longum, the NCBI database ID is 69578838, and the N-acetylhexosamine-1-kinase gene NahK The gene AGX1 of glucosamine-1-phosphate-uracil transferase comes from human, and the ID of the NCBI database is 6675.

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

According to the preferred method of the present invention, in step (5), the fluorescein is Cyanine5 DBCO, the final concentration of fluorescein is 0.5mM, and the reaction time in the dark is 1 hour.

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

Technical features of the invention:

The present invention uses GlcNAz to cultivate the recombinant strain BS168ASSGD containing glycosaminoglycan skeleton synthase, so that an azide-modified glycosaminoglycan skeleton is produced on the surface of the strain, and the polysaccharide skeleton reacts with the fluorescent dye Cyanine5 DBCO to cause a click chemical reaction on the surface of the strain. It is fluorescent. The ratio of fluorescence intensity to absorbance of strains containing glycosaminoglycan backbone synthase is more than 1 times that of strains without glycosaminoglycan backbone synthase. Combined with fluorescence-activated cell sorting, active glycosamines can be detected High-throughput screening of glycan backbone synthases.

The reaction formula for the click chemical reaction between the fluorescent dye Cyanine5 DBCO and GlcNAz is as follows:

Beneficial effects:

The present invention uses Bacillus subtilis BS168ASS, which blocks the endogenous GlcNAc synthesis pathway and expresses the GlcNAc (GlcNAz) salvage synthesis pathway, as the host bacterium, and overexpresses the uridine diphosphate-glucose-6-dehydrogenase gene tuaD and glycosaminoglycans. Backbone synthase gene, and a recombinant strain for screening glycosaminoglycan backbone synthase was constructed. Then, based on this strain, high-throughput and accurate screening of active glycosaminoglycan backbone synthase strains can be achieved by measuring the fluorescence intensity or the ratio of fluorescence intensity to absorbance, which greatly improves the screening efficiency. It also provides a new starting point for the discovery of new sources of enzymes, especially new glycosaminoglycan backbone synthases from microbiota that cannot be cultured in metagenomic libraries.

Description of the drawings

Figure 1: Polyacrylamide gel electrophoresis pattern of the PCR amplification product of uridine diphosphate-glucose-6-dehydrogenase gene tuaD;

In the figure: M is marker; 1 is tuaD gene PCR amplification product;

Figure 2: Polyacrylamide gel electrophoresis pattern of the PCR amplification products of the chondroitin sulfate skeleton synthase KfoC gene, hyaluronic acid skeleton synthase PmHAS gene, and heparin skeleton synthase PmHS2 gene;

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

Figure 3: Electropherogram of PCR amplification products of CcCS gene, CvCS gene, NdCS gene, MsCS gene, RhCS gene, MeCS gene, HfCS gene, LeCS gene, PsCS gene and HfCS gene searched by BLASTp algorithm;

In the figure: M is marker; 1 is the PCR amplification product of CcCS gene; 2 is the PCR amplification product of CvCS gene; 3 is the PCR amplification product of NdCS gene; 4 is the PCR amplification product of MsCS gene; 5 is the PCR amplification product of RhCS gene. Products; 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;

Figure 4: Electrophoresis pattern of the pHCMC04 plasmid backbone after double digestion with SpeI and BamHI;

In the figure: M is the marker; 1 is the double-digestion fragment of plasmid pHCMC04;

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

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;

Figure 6: Recombinant plasmid construction map containing CcCS gene, CvCS gene, NdCS gene, MsCS gene, RhCS gene, MeCS gene, HfCS gene, LeCS gene, PsCS gene and TmCS gene found by BLASTp algorithm;

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

Figure 7: Fluorescence measured by a microplate reader after the click chemical reaction of the recombinant bacteria BS168SSAED, BS168SSACD, BS168SSAHS2D and BS168SSAHASD;

In the figure: the abscissa represents the recombinant strain type, and the ordinate represents the ratio of fluorescence intensity to absorbance;

Figure 8: Fluorescence measured by flow cytometry after the click chemical reaction of the recombinant bacteria BS168SSAED, BS168SSACD, BS168SSAHS2D and BS168SSAHASD;

In the figure: A is the fluorescence intensity comparison between BS168SSACD and BS168SSAED; B is the fluorescence intensity comparison between BS168SSAHS2D and BS168SSAED; C is the fluorescence intensity comparison between BS168SSAHASD and BS168SSAED;

Figure 9: Fluorescence measured by a microplate reader after click chemical reaction of recombinant Bacillus subtilis containing CcCS gene, CvCS gene, NdCS gene, MsCS gene, RhCS gene, MeCS gene, HfCS gene, LeCS gene, PsCS gene and TmCS gene Condition;

In the figure: the abscissa is the type of recombinant strain; the ordinate is the ratio of fluorescence intensity to absorbance;

Figure 10: Construction map of recombinant plasmid containing CvCS gene, HfCS gene, and MeCS gene;

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;

Figure 11: Polyacrylamide gel electrophoresis pattern of CvCs protein, HfCS protein and MeCS protein;

In the figure: M is marker; 1 is CvC protein; 2 is HfCS protein; 3 is MeCS protein;

Figure 12: UDP-GalNAc/UDP-GalNAc transferase activity diagram of CvCs protein, HfCS protein and MeCS protein;

In the figure: the abscissa is the protein type; the ordinate is the conversion rate.

Detailed ways

The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. The following examples are intended to further illustrate the present invention but not to limit the scope of the present invention.

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; the plasmid extraction kit and gel recovery kit were all purchased from Vazyme. Purchased from OMEGA bio-tek (USA); restriction endonuclease was purchased from Thermo Scientific; fluorescein Cyanine5 DBCO was purchased from Lumiprobe. Experimental procedures not otherwise specified were performed in accordance with the product instructions.

Example 1: Construction of Bacillus subtilis engineering strain BS168SSAGD for efficient synthesis of azide-modified glycosaminoglycan skeleton

1. Construction of Bacillus subtilis BS168ASS

The Bacillus subtilis BS168ASS blocks the endogenous uridine diphosphate-N-acetylglucoside (GlcNAc) synthesis pathway and expresses heterologous uridine diphosphate-N-acetylglucoside (N-azidoacetylglucosamine, GlcNAz ) rescue synthesis pathway of Bacillus subtilis, the specific construction method is as follows:

(1) Blocking of the endogenous synthesis pathway of UDP-GlcNAc

The pUC57-neo plasmid containing the neomycin resistance gene glmS gene homology arm fragment synthesized by the company (GenScript) was digested with BamHI to obtain a 1800bp fragment;

The enzyme digestion system is as follows:

Enzyme digestion reaction conditions: 37°C water bath for 1 hour. And recover the desalting liquid of the enzyme-digested system.

Bacillus subtilis subsp.subtilis str.168 was inoculated into LB liquid medium at a volume ratio of 0.1%, and incubated overnight at 37°C and a 225rpm shaker. Take 2.6 mL of the overnight culture and inoculate it into 40 mL of culture medium (LB + 0.5 M sorbitol), and culture at 37°C and 200 rpm until OD ₆₀₀ = 0.8 to 0.9. Bath the bacterial solution in ice water for 10 minutes, then centrifuge at 5000g and 4°C for 5 minutes to collect the bacteria. Use 50 mL of pre-cooled electroporation culture medium (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol), resuspend the cells, centrifuge at 5000g for 5 minutes at 4°C to remove the supernatant, and rinse 4 times. The washed bacterial cells were suspended in 1 mL of electroporation culture medium, and 60 μL was dispensed into each EP tube to obtain BS168 electroporation competent cells.

Add 6 μL of pUC57-neo plasmid enzymatic digestion liquid to 60 μL of electroporated competent cells to recover the product, incubate on ice for 2 minutes, add to a pre-cooled electroporation cup (1 mm), and electrocute once. Electrodemeter settings: 2kV, 1mm, 1 electric shock. After electroshock, take out the cup and immediately add 1mL of RM medium (LB+0.5M sorbitol+0.38M mannitol) at 37°C, 200rpm. After recovery for 3 hours, culture on LB solid medium containing 50μg/mL neomycin. Overnight, the positive transformants were picked and recorded as BS168△glmS strain.

(2) Introduction of UDP-GlcNAc (UDP-GlcNAz) rescue synthesis pathway that can utilize GlcNAc (GlcNAz)

Design the neomycin resistance gene homology arm fragment P _veg -AGX1-P _veg -NahK with kanamycin resistance gene to knock out the neomycin resistance gene on the genome, pUC57-P _veg -AGX1 -P _veg -NahK plasmid was synthesized in the company (Sangon). Use Acc65I and SmaI to digest pUC57-P _veg -AGX1-P _veg -NahK plasmid;

The enzyme digestion system is as follows:

Enzyme digestion reaction conditions: 37°C water bath for 1 hour. And recover the desalting liquid of the enzyme-digested system.

Prepare the BS168△glmS electroporation competence according to the method described in (1), and electroporate the homologous arm fragment P _veg -AGX1-P _veg -NahK into the BS168△glmS electroporation competence, in the presence of 50 μg/mL kanamycin Cultivation was carried out overnight on LB solid medium containing sterol, and positive transformants were selected to obtain Bacillus subtilis BS168ASS.

The ID of the NCBI database of the gene glmS is 938736, the gene NahK of the N-acetylhexosamine-1-kinase comes from Bifidobacterium longum, the ID of the NCBI database is 69578838, and the N-acetylglucosamine-1 -The gene AGX1 of phospho-uracil transferase comes from the human NCBI database with ID 6675.

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

2. Acquisition of uridine diphosphate-glucose-6-dehydrogenase gene tuaD

Extract the genomic DNA of Bacillus subtilis, use the genomic DNA as the template, tuaD F and tuaDR as the primers to perform PCR, the primer sequence is:

tuaD F:5’-AGGTACCAAGAGAGGAATGTACACATGAAAAAAATAGCTGTCATTGG-3’,

tuaD R:5’-GACGTCGACTCTAGAGGATCCTTATAAATTGACGCTTCCCAAGTC-3’;

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

PCR reaction conditions: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 72°C for 15 seconds, extension at 58°C for 2 minutes, a total of 30 cycles, extension at 72°C for 5 minutes, and storage at 4°C. 1% agarose gel electrophoresis for 30 minutes, gel recovery and purification of PCR products. The obtained PCR product was analyzed and detected by 1% agarose gel electrophoresis. The results are shown in Figure 1. An electrophoresis band with a size of approximately 1390 bp was obtained, which is the tuaD fragment.

3. Construction of glycosaminoglycan backbone synthase library

Obtained the chondroitin sulfate skeleton synthase KfoC derived from Escherichia coli, the ID of the NCBI database is CAD5992240.1; the heparin scaffold synthase PmHS2 derived from Pasteurella multocida, the ID of the NCBI database is AY292200.1; the hyaline derived from Pasteurella multocida Acid backbone synthase pmHAS, the ID of the NCBI database is AAC38318.2. These three enzymes are all highly active glycosaminoglycan backbone synthases.

Using the amino acid sequences of these three enzymes as sequence comparison templates, we used the BLASTp algorithm to perform sequence comparisons in the bioinformatics database (NCBI database) and found ten amino acid sequences that may be glycosaminoglycan backbone synthases (GTs). They are respectively abbreviated as CcCS, the ID of the NCBI database is WP_150081707.1; CvCS, the ID of the NCBI database is WP_168227469.1; HfCS, the ID of the NCBI database is NBI12747.1; LeCS, the ID of the NCBI database is WP_1661815; MsCS, the ID of the NCBI database is WP_1661815. The ID is WP_024460064.1; MeCS, the ID of the NCBI database is WP_167432605.1; NdCS, the ID of the NCBI database is WP_085359788.1; PsCS, the ID of the NCBI database is WP_144187813.1; RhCS, the ID of the NCBI database is WP_077587478.1; TmCS, the ID of the NCBI database is WP_136735112.1

The above 13 gene fragments were double-codon optimized in E. coli and Bacillus subtilis and synthesized by the company (GenScript) into the corresponding plasmid pET28a(+)-His-GT, which is present in E. coli TOP10. Design primers respectively and perform PCR on the plasmid to obtain the corresponding gene fragments, collectively called GT fragments. The primer sequences are:

KfoC F:5’-TGACAAATGGTCCAAACTAGTATGAGTATTCTTAATCAAGCAATA-3’;

KfoC R:5’-GTACATTCCTCTCTTGGTACCTTATAAATCATTCTCTATTTTTT-3’;

PmHAS F:5’-TGACAAATGGTCCAAACTAGTATGAACACACTGAGCCAAGCAA-3’;

PmHAS R:5’-GTACATTCCTCTCTTGGTACCTTACAGTGTAATTGAATTAATAA-3’;

PmHS2 F:5’-TGACAAATGGTCCAAACTAGTATGAAAGGCAAAAAAGAAATGA-3’;

PmHS2 R:5’-GTACATTCCTCTCTTGGTACCTTAAAGAAAATAAAACGGCAGGC-3’;

CcCS F:5’-TGACAAATGGTCCAAACTAGTATGCAACAATCTAGCAAATCATTT-3’;

CcCS R:5’-GTACATTCCTCTCTTGGTACCTTATATATTTTTGTGAAAAGAGGT-3’;

CvCS F:5’-TGACAAATGGTCCAAACTAGTATGACAATTTTGAATCAAGCGATT-3’;

CvCS R:5’-GTACATTCCTCTCTTGGTACCTTATATAAACTGCTGAATACACAG-3’;

HfCS F:5’-TGACAAATGGTCCAAACTAGTATGAATATACTGTCCAAGGCAATT-3’;

HfCS R:5’-GTACATTCCTCTCTTGGTACCTTAAGCAAAGTTAAGCCGATGGGT-3’;

LeCS F:5’-TGACAAATGGTCCAAACTAGTATGTCGATTTTTAACGAAGCAATT-3’;

LeCS R:5’-GTACATTCCTCTCTTGGTACCTTATATAAATTTGAAACCGATTTT-3’;

MeCS F:5’-TGACAAATGGTCCAAACTAGTATGGCCAGCTTTGTAGAAGCTAAC-3;’

MeCS R:5’-GTACATTCCTCTCTTGGTACCTTATTTTTTGAAAAAAACTACCTG-3’;

MsCS F:5’-TGACAAATGGTCCAAACTAGTATGGGGGCAGGTCAAAATATACTG-3’;

MsCS R:5’-GTACATTCCTCTCTTGGTACCTTAAAGAAAATAAAACGGCAGGC-3’;

NdCS F:5’-TGACAAATGGTCCAAACTAGTATGGAAAAGATCCTGAGCCGCGCA-3’;

NdCS R:5’-GTACATTCCTCTCTTGGTACCTTATATGTTTTTAAACTGAATCAG-3’;

PsCS F:5’-TGACAAATGGTCCAAACTAGTATGAAGATCCTGAGCAATGCGATT-3’;

PsCS R:5’-GTACATTCCTCTCTTGGTACCTTACGTTATAAAATTACCAATGGC-3’;

RhCS F:5’-TGACAAATGGTCCAAACTAGTATGAATATTCTGTCAAAAGCCGTT-3’;

RhCS R:5’-GTACATTCCTCTCTTGGTACCTTAATAATAGGTAATGTAAAAGTT-3’;

TmCS F:5’-TGACAAATGGTCCAAACTAGTATGAATAAAAATATATTCGATCAA-3’;

TmCS R: 5’-GTACATTCCTCTCTTGGTACCTTAAAGCAAAGTGCTGAACTGCTC-3’.

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

PCR reaction conditions: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 72°C for 15 seconds, extension at 60°C for 3 minutes, a total of 30 cycles, extension at 72°C for 5 minutes, and storage at 4°C. 1% agarose gel electrophoresis for 30 minutes, gel recovery and purification of PCR products. The obtained PCR products were analyzed and detected by 1% agarose gel electrophoresis, and the results are shown in Figures 2 and 3.

4. Construction of recombinant expression plasmid

(1) Double digest the vector plasmid pHCMC04 with SpeI and BamHI to obtain an 8000bp fragment. The electrophoresis pattern of this fragment is shown in Figure 4;

The enzyme digestion system is as follows:

Enzyme digestion reaction conditions: 37°C water bath for 1 hour.

(2) The digested vector plasmid, tuaD fragment and GT fragment were connected through DNA seamless cloning method, and then chemically transformed into E. coli DH5α competent cells. After sequencing verification, a total of 13 recombinant expression vectors were obtained, respectively. It is 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, pHCMC04-MeCS-tuaD recombinant plasmid, pHCMC04 -HfCS-tuaD recombinant plasmid, pHCMC04-LeCS-tuaD recombinant plasmid, pHCMC04-LeCS-tuaD recombinant plasmid, pHCMC04-TmCS-tuaD recombinant plasmid, pHCMC04-KfoC-tuaD recombinant plasmid, pHCMC04-PmHAS-tuaD recombinant plasmid and pHCMC04-PmHS2 -tuaD recombinant plasmids are collectively called GD expression vectors. The specific recombinant plasmid construction maps are shown in Figure 5 and Figure 6.

The seamless cloning reaction system is as follows:

Seamless cloning reaction conditions: react in a water bath at 50°C for 30 to 50 minutes.

5. Construction of recombinant Bacillus subtilis containing GD expression vector

Prepare BS168ASS electroporation competent cells according to the method described in (1), transfer the GD expression vector into Bacillus subtilis BS168ASS through electroporation, and culture it overnight on LB solid medium containing 5 μg/mL chloramphenicol. The transformant is the recombinant bacterium BS168SSAGD that efficiently synthesizes azide-modified glycosaminoglycan skeleton.

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

1. Validation of high-throughput screening system based on glycosaminoglycan backbone synthases KfoC, PmHS2 and PmHAS with known activity.

The recombinant bacterium containing the KfoC gene and tuaD gene constructed in Example 1 is called BS168SSACD; the recombinant bacterium containing the PmHS2 gene and tuaD gene is called BS168SSAHS2D; the recombinant bacterium containing the PmHAS gene and tuaD gene is called BS168SSAHASD; the recombinant bacterium containing the pHCMC04 empty plasmid is called subtilis, called BS168SSAE, served as a control.

Then it was inoculated into the special substrate LB liquid medium at a volume ratio of 0.1%, and cultured overnight in a shaker at 37°C and 225 rpm. Expand the strain cultured overnight into the special substrate LB liquid medium at a volume ratio of 1%. When the strain grows to an OD ₆₀₀ of 0.6-0.8, wash the strain three times with 1×PBS buffer and transfer the strain to another Cultivate in low-salt M9 medium containing glucose to consume the GlcNAc contained in the bacteria themselves. After 1 hour, add xylose inducer with a final concentration of 20g/L and glycosyltransferase non-natural substrate GlcNAz with a final concentration of 100μg/mL. . Induced and cultured for 6 hours at 37°C, 225rpm shaker.

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

The induced cultured strain was washed three times with 1×PBS buffer by resuspension and centrifugation, and then resuspended in 200 μL of 1×PBS buffer. Add a final concentration of 0.5mM fluorescein Cyanine5 DBCO into a light-proof 1.5mL centrifuge tube. , let stand for 1 hour at 37°C and then wash 3 times with 1×PBS buffer. Use a microplate reader to detect the fluorescence intensity at the emission wavelength of 646nm, the excitation wavelength of 662nm and the absorbance at 600nm, and calculate the ratio of the fluorescence intensity to OD ₆₀₀ . , the results are shown in Figure 7.

It can be seen from Figure 7 that the fluorescence intensity and absorbance ratio of the recombinant strains BS168SSACD, BS168SSAHS2D, and BS168SSAHASD containing glycosaminoglycan backbone synthase are significantly higher than those of the strain BS168SSAE that does not contain glycosaminoglycan backbone synthase, and the fluorescence is higher than that of the strain BS168SSAE that does not contain glycosaminoglycan backbone synthase. The fluorescence of sugar skeleton synthase strain BS168SSAE is more than doubled.

The bacterial solution that participated in the fluorescence reaction was diluted with 1×PBS buffer until the OD ₆₀₀ was between 0.3 and 0.5, and a single-color flow cytometry analysis was performed at an emission wavelength of 646 nm and an excitation wavelength of 662 nm. The results are shown in Figure 8.

As can be seen from Figure 8, the fluorescence intensity of the recombinant strains BS168SSACD, BS168SSAHS2D, and BS168SSAHASD containing glycosaminoglycan backbone synthase is significantly higher than that of the strain BS168SSAE that does not contain glycosaminoglycan backbone synthase. This result is consistent with the results of the microplate reader.

2. Screening of glycosaminoglycan backbone synthase libraries with unknown activity

(1) Screening of glycosaminoglycan backbone synthase library based on the ratio of fluorescence intensity to absorbance

The ten recombinant bacteria containing tuaD gene and GT fragment constructed in Example 1 are respectively abbreviated as: CcCS, CvCS, HfCS, LeCS, MsCS, MeCS, NdCS, PsCS, RhCS, and TmCS. Cultivate and induce in a 96-well deep well plate according to the method described in 1, and perform a click chemical reaction with Cyanine5 DBCO. Use a microplate reader to detect its fluorescence intensity at the emission wavelength of 646nm, excitation wavelength of 662nm and absorbance at 600nm. The ratio of its fluorescence intensity to OD ₆₀₀ was calculated, and the results are shown in Figure 9.

As can be seen from Figure 9, the CvCs gene, HfCS gene and MeCS gene exhibit fluorescence that is comparable to or even stronger than that of the KfoC gene.

(2) Verification of glycosaminoglycan backbone synthase activity in vitro

The CvCs gene, HfCS gene and MeCS gene that showed strong fluorescence in (1) were synthesized by the company (GenScript) into corresponding plasmids. The company synthesized the original plasmid pET28a(+)-His-CvCS, pET28a(+)- His-HfCS, pET28a(+)-His-MeCS, and the specific recombinant plasmid construction map is shown in Figure 10. They were chemically transformed into E. coli BL21 (DE3), and the transformants were grown in LB solid medium with 50 μg/mL kanamycin. Single colonies were picked, and the plasmid was extracted and verified by enzyme digestion to obtain the CvCs gene containing, BL21(DE3) expression strain of HfCS gene and MeCS gene.

The enzyme digestion system is as follows:

Enzyme digestion reaction conditions: 37°C water bath for 20 minutes.

Expand the BL21 (DE3) expression strain containing CvCs, HfCS, and MeCS genes into 1 L of LB liquid culture medium at a volume ratio of 1%. When the strain grows to an OD ₆₀₀ of 0.6 to 0.8, add a final concentration of 0.2 mM IPTG, 22°C, 225rpm, induction for 12 to 18 hours.

After induction, centrifuge the bacterial solution at 4°C and 8000g for 20 minutes, discard the supernatant, collect the bacterial pellet and resuspend it in loading buffer (20Mm Tris-HCl, pH=7.5, 0.5M NaCl, 5mM imidazole) ;Use an ultrasonic crusher to crush the bacterial cells for 30 minutes. The working conditions are: working for 15 seconds, interval of 45 seconds, amplitude 35%, energy 1500KJ, 4℃; use ultra-low temperature centrifuge to centrifuge the broken bacterial cells for 30min at 4℃, 12000g, and collect the The clear liquid was filtered with a 0.22 μm filter membrane. Use Ni-charged MagBeads to purify the filtered supernatant. First use ddH ₂ O to completely resuspend the magnetic beads in the test tube. Place the test tube on the magnetic bead separation rack to collect the magnetic beads. Discard the supernatant and wash away the original 20% ethanol. Magnetic bead storage solution; resuspend the magnetic beads twice in loading buffer (20MmTris-HCl, pH=7.5, 0.5M NaCl, 35mM imidazole) to balance the system; resuspend the filtered supernatant and add Incubate for 30 minutes in a shaker at 225 rpm at 4°C; wash away impurity proteins with equilibrium buffer (20Mm Tris-HCl, pH=7.5, 0.5M NaCl, 35mM imidazole); use elution buffer (20Mm Tris-HCl, pH=7.5, 0.5M NaCl, 200mM imidazole) to elute the target protein and collect.

The expression and purification of the protein after induction were identified by polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown in Figure 11, indicating that CvCs protein, HfCS protein and MeCS protein were successfully separated.

GlcA-pNP was used as the acceptor substrate, and UDP-GalNAc/UDP-GlcNAc was used as the donor substrate respectively to measure the glycosaminoglycan backbone synthase activities of CvCs protein, HfCS protein and MeCS protein.

The reaction system is as follows:

Reaction conditions: overnight reaction at 37°C.

The reaction system was filtered with a 0.22 μm filter membrane and analyzed by PAMN-HPLC. The specific absorption peak of the pNP group was detected at 310 nm. The chromatographic column used in HPLC analysis is a YMC-Pack Polyamine II column. The mobile phase is water and 1mol/L KH ₂ PO ₄ solution. The flow rate is 0.5mL/min. Each protein catalytic product is detected after separation by the chromatographic column. The UV absorption of each component at 310/254nm is shown in Figure 12.

The HPLC analysis procedure is as follows:

It can be seen from Figure 12 that CvCs protein, HfCS protein and MeCS protein all exhibit strong UDP-GalNAc transferase activity, that is, CvCs protein, HfCS protein and MeCS protein are all highly active glycosaminoglycan skeleton synthases. It is consistent with the screening results of the glycosaminoglycan backbone synthase library based on the ratio of fluorescence intensity to absorbance in Example 2.

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

The method based on measuring fluorescence using a microplate reader can only screen a small number of glycosaminoglycan backbone synthases. High-throughput screening requires fluorescence-activated cell sorting technology, by dividing strains containing KfoC gene and tuaD gene expression cassettes. BS168SSACD is called KfoC+; strain BS168SSAE, which only contains the pHCMC04 empty plasmid, is called KfoC-.

KfoC+ and KfoC- were mixed and cultured, and the FACS enrichment efficiency of 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 respectively.

The mixed cultured strains were cultured and fluorescently reacted according to the method described in Example 1, and analyzed by flow cytometry after washing. Collect bacterial cells with fluorescence intensity in the top 2% to minimize possible false positives. The sorted bacteria were cultured overnight in LB solid medium containing 5 μg/mL chloramphenicol. After picking single colonies, the positive ratio after enrichment was identified and compared with before sorting. The results are shown in Table 1 Show.

Table 1 The effect of efficiently synthesizing recombinant Bacillus subtilis with azide-modified glycosaminoglycan backbone and applying it in the FACS system to enrich recombinant strains containing highly active glycosaminoglycan backbone synthase from a large number of background bacteria.

As can be seen from Table 1, under a single round of highly stringent flow sorting conditions, there is still a 97% probability of enriching the recombinant strain KfoC+ containing highly active glycosaminoglycan backbone synthase under 1:100 mixed culture conditions.

Claims (9)

1. A high throughput screening method of glycosaminoglycan backbone synthase, comprising the steps of:

(1) Randomly obtaining a glycosaminoglycan skeleton synthase gene by using a BLASTP method, and constructing a glycosaminoglycan skeleton synthase library;

(2) Respectively connecting a glycosaminoglycan skeleton synthase gene and a uridine diphosphate-glucose-6-dehydrogenase gene tuaD expression cassette in the library to pHCMC04 plasmid to obtain a recombinant vector pHCMC04-GTs-tuaD;

(3) Converting a recombinant vector pHCMC04-GTs-tuaD into bacillus subtilis BS168ASS, and selecting positive recombinants to obtain recombinant bacteria GD for screening glycosaminoglycan skeleton synthase;

(4) Inoculating recombinant bacteria GD for screening glycosaminoglycan skeleton synthase into LB liquid culture medium containing special substrate according to the volume ratio of 0.05-0.15%, and culturing overnight at 35-40 ℃ and 200-250 rpm to obtain bacterial liquid of the recombinant bacteria GD;

(5) Inoculating bacterial liquid of recombinant bacterial GD into LB liquid culture medium containing special substrate according to the volume ratio of 0.5-1.5%, culturing to OD ₆₀₀ Transferring to M9 low-salt culture medium without glucose for 0.6-0.8, culturing for 0.5-1.5 hr, adding xylose inducer with final concentration of 15-25 g/L and N-azidoacetyl glucosamine with final concentration of 80-120 μg/mL, inducing culture for 4-8 hr, and light-shielding thallus and fluoresceinReacting for 0.5-1.5 h, when the number of genes in the glycosaminoglycan skeleton synthase library is less than 20, performing flow analysis on the thalli after fluorescence reaction, measuring fluorescence intensity or measuring the ratio of the fluorescence intensity to absorbance, and screening out target glycosaminoglycan skeleton synthase; when the number of genes in the glycosaminoglycan skeleton synthase library exceeds 20, performing flow sorting on thalli after fluorescence reaction, measuring fluorescence intensity, collecting thalli with the fluorescence intensity of 0.5-2%, and screening out target glycosaminoglycan skeleton synthase.

2. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 1, wherein in step (1), the glycosaminoglycan backbone synthases are chondroitin sulfate backbone synthases, heparin backbone synthases or hyaluronan synthases.

3. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 1, wherein in step (2), the NCBI database of uridine diphosphate-glucose-6-dehydrogenase gene tuaD has an ID of 936766.

4. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 1, wherein in step (3), the bacillus subtilis BS168ASS is bacillus subtilis which blocks the endogenous uridine diphosphate-N-acetylglucoside synthesis pathway and expresses the heterologous uridine diphosphate-N-acetylglucoside salvage synthesis pathway.

5. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 4, wherein the construction method of bacillus subtilis BS168ASS is as follows:

taking bacillus subtilis Bacillus subtilis subsp.subtilis str.168 as an initial strain, knocking out a gene glmS encoding a key enzyme molecule for synthesizing UDP-GlcNAc pathway, and then inserting a gene NahK encoding N-acetylhexosamine-1-kinase and a gene AGX1 encoding N-acetylglucosamine-1-phosphate-uracil transferase to obtain bacillus subtilis BS168ASS.

6. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 5, wherein the gene glmS has an ID of 938736, the gene NahK of N-acetylhexosamine-1-kinase is derived from bifidobacterium longum, the gene AGX1 of N-acetylglucosamine-1-phosphate-uracil transferase is derived from human, and the gene NCBI database has an ID of 6675.

7. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 1, wherein in steps (4) and (5), the composition of the LB liquid medium containing the specific substrate is as follows: 10g/L yeast extract, 20g/L peptone, 20g/L sodium chloride, 5. Mu.g/mL chloramphenicol, and 100. Mu.g/mL GlcNAc, the natural substrate for glycosaminoglycan backbone synthase.

8. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 1, wherein in step (5), the fluorescein is Cyanine5 DBCO, the final concentration of the fluorescein is 0.5mM, and the light shielding reaction time is 1h.

9. The high throughput screening method of glycosaminoglycan backbone synthases according to claim 1, wherein in step (5), the specific parameters for determining fluorescence intensity and absorbance are: the emission wavelength was 646nm, the excitation wavelength was 662nm, and the absorbance was 600nm.