CN117004635A

CN117004635A - High-throughput screening method of glycosaminoglycan skeleton synthase

Info

Publication number: CN117004635A
Application number: CN202210468072.3A
Authority: CN
Inventors: 生举正; 胡宏羽; 侯进; 王钰佳
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-11-07

Abstract

The invention relates to a high-throughput screening method of glycosaminoglycan skeleton synthase. The method comprises the following steps: (1) constructing a glycosaminoglycan backbone synthase library; (2) constructing a recombinant vector; (3) constructing recombinant bacteria for screening glycosaminoglycan skeleton synthase; (4) culturing recombinant bacteria; (5) And (3) after the bacterial liquid of the recombinant bacteria is subjected to flow analysis or flow sorting, measuring fluorescence, and screening out the target glycosaminoglycan skeleton synthase. The invention uses bacillus subtilis BS168ASS as host bacteria, and expresses uridine diphosphate-glucose-6-dehydrogenase gene and glycosaminoglycan skeleton synthase gene to construct recombinant bacteria for screening glycosaminoglycan skeleton synthase. And then, by measuring the fluorescence of the strain, the high-throughput and accurate screening of the glycosaminoglycan skeleton synthase strain is realized, the screening efficiency is improved, and a new starting point is provided for the discovery of new glycosaminoglycan skeleton synthases in enzymes of new sources, particularly in microbiota which cannot be cultured in a metagenomic library.

Description

High-throughput screening method of glycosaminoglycan skeleton synthase

Technical Field

The invention relates to a high-throughput screening method of glycosaminoglycan skeleton synthase, in particular to transformation of an azide modified glycosaminoglycan skeleton synthesis path of bacillus subtilis and fluorescence activated cell sorting for the glycosaminoglycan skeleton synthase based on the strain, and belongs to the technical field of biology.

Background

Glycosaminoglycans (GAGs), a long linear heteropolysaccharide widely present in connective tissue of higher animals, are composed of repeating disaccharide units of hexuronic acid or hexose, hexosamine, and can be classified into: hyaluronic Acid (HA), chondroitin sulfate (chondroitin sulfate, CS), dermatan Sulfate (DS), keratan Sulfate (KS), heparan Sulfate (HS) and Heparin (HP). Glycosaminoglycans interact with growth factors, cytokines, chemokines and enzymes, and have important effects on processes such as body growth, inflammatory reactions, coagulation, tumor metastasis, etc.

Uridine diphosphate-Glucose-6-dehydrogenase (Uridine diphosphate Glucose-6-dehydrogenase, tuaD), which is present in Bacillus subtilis genomic DNA, catalyzes the conversion of uridine diphosphate-Glucose (UDP-Glucose) to uridine diphosphate-glucuronic acid (UDP-GlcA), has been demonstrated in prior studies to limit glycosaminoglycan backbone biosynthesis, and tuaD is therefore critical for high level glycosaminoglycan backbone biosynthesis.

Glycosaminoglycan backbone Synthases (GTs) are enzymes that catalyze the transfer of monosaccharide units from a sugar donor to a polysaccharide in a regio-and stereotactic manner, providing a very efficient method for the synthesis of complex oligosaccharides. However, the limited number of available GTs, together with their instability and stringent substrate specificity, severely hamper the wide range of applications of these enzymes, and thus the availability of glycosyltransferases that can efficiently synthesize specific oligosaccharides and glycoconjugates is of profound interest.

Bacillus subtilis (Bacillus subtilis), a species of Bacillus belonging to the genus gram-positive, is considered GRAS (Generally recognized as safe), is well tolerated by the environment and, according to existing studies, has a cleaner background with respect to the glycosaminoglycan synthesis pathway.

In the past studies, methods directed evolution by protein engineering have met with limited success in broadening the range of substrates, altering the substrate specificity, and increasing GTs activity, in part because of the lack of efficient high throughput screening methods. Determination of GTs activity is very challenging because there is no significant change in fluorescence or absorbance associated with glycosidic bond formation. While screening for the desired phenotype is in most cases a random process. Therefore, it would be of great interest to develop high throughput, low cost screening methods that can be applied to large GTs libraries.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a high-throughput screening method of glycosaminoglycan skeleton synthase. Specifically, a recombinant bacillus subtilis for efficiently synthesizing an azide-modified glycosaminoglycan skeleton is constructed, and then high-throughput screening is carried out on glycosaminoglycan skeleton synthase by the strain.

The technical scheme of the invention is as follows:

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) The bacterial liquid of recombinant bacterial GD is mixed with 0.5 to 1.5 percent of bacterial liquidInoculating the product ratio into LB liquid culture medium containing special substrate, culturing to OD ₆₀₀ Transferring to an M9 low-salt culture medium without glucose for 0.6-0.8 h, culturing for 0.5-1.5 h, adding a xylose inducer with the final concentration of 15-25 g/L and N-azidoacetyl glucosamine with the final concentration of 80-120 mu g/mL, inducing and culturing for 4-8 h, then carrying out light-shielding reaction on thalli and fluorescein for 0.5-1.5 h, carrying out flow analysis on the thalli after fluorescence reaction when genes in a glycosaminoglycan skeleton synthase library are below 20, and then measuring the fluorescence intensity or the ratio of the fluorescence intensity to the 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.

According to a preferred embodiment of the present invention, in the step (1), the glycosaminoglycan skeleton synthase is chondroitin sulfate skeleton synthase, heparin skeleton synthase or hyaluronate synthase.

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

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

According to the invention, the construction method of the bacillus subtilis BS168ASS comprises the following steps:

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. The enzyme coded by the gene glmS is responsible for catalyzing fructose-6-phosphate to glucose-6-phosphate, the enzyme coded by the gene NahK can be catalyzed by ATP and GlcNAc to obtain GlcNAc-1-P, the enzyme coded by the gene AGX1 can be catalyzed by GlcNAc-1-P to UDP-GlcNAc, and the bacillus subtilis BS168ASS can be used for generating UDP-GlcNAz by using N-azido acetylated glucosamine GlcNAz as a substrate.

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

According to the present invention, preferably, in the steps (4) and (5), the composition of the LB liquid medium containing the special 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.

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

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

The invention has the technical characteristics that:

according to the invention, the recombinant strain BS168 ASSDG containing the glycosaminoglycan skeleton synthase is cultivated by utilizing GlcNAz, so that the azide-modified glycosaminoglycan skeleton is generated on the surface of the strain, the surface of the strain is provided with fluorescence after click chemical reaction of the polysaccharide skeleton and fluorescent dye Cyanine5 DBCO, the ratio of the fluorescence intensity to absorbance of the strain containing the glycosaminoglycan skeleton synthase is more than 1 time of that of the strain without the glycosaminoglycan skeleton synthase, and the high-throughput screening of the glycosaminoglycan skeleton synthase with activity can be realized by combining fluorescence activated cell sorting.

The click chemistry reaction of the fluorescent dye Cyanine5 DBCO and GlcNAz is as follows:

the beneficial effects are that:

according to the invention, bacillus subtilis BS168ASS for blocking an endogenous GlcNAc synthesis pathway and expressing GlcNAc (GlcNAz) salvage synthesis pathway is used as a host bacterium, and uridine diphosphate-glucose-6-dehydrogenase genes tuaD and glycosaminoglycan skeleton synthase genes are expressed, so that recombinant bacteria for screening glycosaminoglycan skeleton synthase are constructed. And then, by taking the strain as a reference, the high-throughput and accurate screening of the active glycosaminoglycan skeleton synthase strain is realized by measuring the fluorescence intensity or the ratio of the fluorescence intensity to the absorbance, the screening efficiency is greatly improved, and a new starting point is provided for the discovery of new glycosaminoglycan skeleton synthases in enzymes of new sources, particularly in microbiota which cannot be cultured in a metagenomic library.

Drawings

Fig. 1: polyacrylamide gel electrophoresis of PCR amplified product of uridine diphosphate-glucose-6-dehydrogenase gene tuaD;

in the figure: m is marker;1 is tuaD gene PCR amplified product;

fig. 2: a polyacrylamide gel electrophoresis diagram of PCR amplified products of 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 a KfoC gene PCR amplification product; 2 is a PCR amplification product of the PmHAS gene; 3 is a PCR amplification product of the PmHS2 gene;

fig. 3: PCR amplified product electrophoretogram of CcCS gene, cvCS gene, ndCS gene, msCS gene, rhCS gene, meCS gene, hfCS gene, leCS gene, psCS gene and HfCS gene found by BLASTP algorithm;

in the figure: m is marker;1 is a CcCS gene PCR amplification product; 2 is a CvCS gene PCR amplification product; 3 is the NdCS gene PCR amplification product; 4 is the PCR amplification product of the MsCS gene; 5 is a PCR amplification product of the RhCS gene; 6 is a MeCS gene PCR amplification product; 7 is a PCR amplification product of the HfCS gene; 8 is a PCR amplification product of the LeCS gene; 9 is a PCR amplification product of the PsCS gene; 10 is a TmCS gene PCR amplification product;

fig. 4: electrophoretogram of pHCMC04 plasmid skeleton after SpeI and BamHI double enzyme cutting;

in the figure: m is marker;1 is plasmid pHCMC04 double enzyme cutting fragment;

fig. 5: contains chondroitin sulfate skeleton synthase KfoC gene; the hyaluronan backbone synthase PmHAS gene; constructing a recombinant plasmid construction map of the heparin skeleton synthase PmHS2 gene;

in the figure: a is pHCMC04-KfoC-tuaD recombinant plasmid; b is pHCMC04-pmHAS-tuaD recombinant plasmid; c is pHCMC04-PmHS2-tuaD recombinant plasmid;

fig. 6: constructing a map of recombinant plasmids containing the CcCS gene, the CvCS gene, the NdCS gene, the MsCS gene, the RhCS gene, the MeCS gene, the HfCS gene, the LeCS gene, the PsCS gene and the TmCS gene which are searched by BLASTP algorithm;

in the figure: a is pHCMC04-CcCS-tuaD recombinant plasmid; b is pHCMC04-CvCS-tuaD recombinant plasmid; c is pHCMC04-NdCS-tuaD recombinant plasmid; d is pHCMC04-MsCS-tuaD recombinant plasmid; 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;

fig. 7: fluorescence conditions of recombinant bacteria BS168SSAED, BS168SSACD, BS168SSAHS2D and BS168SSAHASD measured by an enzyme-labeled instrument after click chemical reaction;

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

fig. 8: fluorescence conditions determined by flow analysis after click chemistry reactions of recombinant bacteria BS168 ssad, BS168SSACD, BS168SSAHS2D and BS168SSAHS asd;

in the figure: a is the comparison of the fluorescence intensity of BS168SSACD and BS168 SSAED; b is the comparison of the fluorescence intensity of BS168SSAHS2D and BS168 SSAED; c is the comparison of the fluorescence intensity of BS168SSAHASD and BS168 SSAED;

fig. 9: the fluorescence 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, which is measured by an enzyme-labeled instrument after click chemical reaction;

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

FIG. 10 is a diagram showing construction of recombinant plasmid containing CvCS gene, hfCS gene and MeCS gene;

in the figure: a is a Pet28a (+) -His-CvCS recombinant plasmid; b is a Pet28a (+) -His-HfCS recombinant plasmid; a is a Pet28a (+) -His-MeCS recombinant plasmid;

fig. 11: polyacrylamide gel electrophoresis of CvCs protein, hfCS protein and MeCS protein;

in the figure: m is marker;1 is a CvC protein; 2 is HfCS protein; 3 is MeCS protein;

fig. 12: a map of UDP-GalNAc/UDP-GalNAc transferase activity of Cvcs protein, hfCS protein and MeCS protein;

in the figure: the abscissa is the protein type; the ordinate is the conversion.

Detailed Description

The invention is described below by means of specific embodiments. The technical means employed in the present invention are methods well known to those skilled in the art unless specifically stated. The following examples are intended to further illustrate the present invention and are not intended to limit the scope of the invention.

PCR Taq enzyme, a seamless cloning kit, E.coli DH 5. Alpha. Competent cells, E.coli BL21 (de 3) competent cells, all purchased from Vazyme, used in the following examples; plasmid extraction and gel recovery kits, both purchased from OMEGA bio-tek (USA); restriction endonucleases were purchased from Thermo Scientific; fluorescein Cyanine5 DBCO was purchased from Lumiprobe. Experimental procedures not specifically described were all performed according to the product specifications.

Example 1: construction of bacillus subtilis engineering strain BS168SSAGD for efficiently synthesizing azide modified glycosaminoglycan skeleton

1. Construction of Bacillus subtilis BS168ASS

The bacillus subtilis BS168ASS is used for blocking the synthesis path of endogenous uridine diphosphate-N-acetylglucoside (GlcNAc), expressing heterologous uridine diphosphate-N-acetylglucoside (N-azidoacetylglucosamine, glcNAz) and remedying the synthesis path, and specifically comprises the following construction steps of:

(1) Blocking of UDP-GlcNAc endogenous synthesis pathway

pUC57-neo plasmid synthesized by the company (Kirsrui) and carrying neomycin resistance gene glmS gene homologous arm fragment is digested with BamHI, so as to obtain 1800bp fragment;

the enzyme digestion system is as follows:

cleavage reaction conditions: water bath at 37 ℃ for 1h. And recovering the desalted liquid of the system after enzyme digestion.

Bacillus subtilis Bacillus subtilis subsp.subsstr.168 was inoculated in 0.1% by volume in LB liquid medium and overgrown overnight at 37℃in a 225rpm shaker. 2.6mL of the overnight culture was inoculated into 40mL of medium (LB+0.5M sorbitol), and cultured at 37℃and 200rpm to OD ₆₀₀ =0.8 to 0.9. The bacterial liquid was subjected to ice-water bath for 10 minutes, and then 5000g was centrifuged at 4℃for 5 minutes to collect bacterial cells. The cells were resuspended in 50mL of pre-chilled electrotransfer medium (0.5M sorbitol, 0.5M mannitol, 10% glycerol), centrifuged at 5000g for 5min at 4℃and the supernatant removed by rinsing 4 times. The washed thalli are blown and suspended in 1mL electrotransformation culture medium, and 60 mu L of each EP tube is split charging to obtain BS168 electrotransformation competence.

mu.L of pUC57-neo plasmid was added to 60. Mu.L of electrocompetent cells, and the product was recovered after digestion with water, incubated on ice for 2 minutes, and added to a pre-chilled electrocuvette (1 mm) for one shock. And (3) setting an electric converter: 2kV,1mm, 1 shock. After completion of the electric shock, the cup was removed and 1mL of RM medium (LB+0.5M sorbitol+0.38M mannitol) was immediately added thereto, and after resuscitating for 3 hours at 37℃and 200rpm, positive transformants were picked up and designated as BS 168. DELTA. GlmS strain, which were cultured overnight on LB solid medium containing 50. Mu.g/mL neomycin.

(2) The introduction of the synthetic pathway can be remedied using UDP-GlcNAc (UDP-GlcNAz) of GlcNAc (GlcNAz)

Design of neomycin resistance Gene homology arm fragment P with kanamycin resistance Gene _veg -AGX1-P _veg NahK to knock out neomycin resistant groups on the genomepUC57-P, therefore _veg -AGX1-P _veg NahK plasmid was synthesized at the company (Industry). pUC57-P was digested with Acc65I and SmaI _veg -AGX1-P _veg -NahK plasmid;

the enzyme digestion system is as follows:

Preparation of BS168 ΔglmS electrotransport competence according to the method described in (1), and homology arm fragment P _veg -AGX1-P _veg NahK electrotransformation into BS168 delta glmS electrotransformation competence, culturing overnight on LB solid medium containing 50 mug/mL kanamycin, and picking positive transformant to obtain bacillus subtilis BS168ASS.

The NCBI database of the gene glmS has an ID of 938736, the gene NahK of the N-acetylhexosamine-1-kinase is derived from bifidobacterium longum, the NCBI database has an ID of 69578838, and the gene AGX1 of the N-acetylglucosamine-1-phosphate-uracil transferase is derived from the human NCBI database has an ID of 6675.

This particular method has been disclosed in chinese patent document 2021100967185.

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

Extracting bacillus subtilis genome DNA, and carrying out PCR by taking the genome DNA as a template and tuaD F and tuaD R as primers, wherein the sequences of the primers are as follows:

tuaD F:5’-AGGTACCAAGAGAGGAATGTACACATGAAAAAAATAGCTGTCATTGG-3’，

tuaD R:5’-GACGTCGACTCTAGAGGATCCTTATAAATTGACGCTTCCCAAGTC-3’；

the PCR reaction system is as follows: (primer concentration was 10. Mu.M)

PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15s, annealing at 72℃for 15s, extension at 58℃for 2min for 30 cycles; extending at 72 ℃ for 5min, and preserving at 4 ℃.1% agarose gel electrophoresis for 30min, and gel recovery and purification of PCR products. The PCR product obtained was analyzed and detected by 1% agarose gel electrophoresis, and the result is shown in FIG. 1, and an electrophoresis band with a size of about 1390bp was obtained, namely a tuaD fragment.

3. Construction of glycosaminoglycan backbone synthase library

Obtaining chondroitin sulfate skeleton synthase KfoC from Escherichia coli, wherein the ID of NCBI database is CAD5992240.1; the ID of the NCBI database of the heparin skeleton synthase PmHS2 from Pasteurella multocida is AY292200.1; the ID of NCBI database is AAC38318.2, the hyaluronan backbone synthase pmHAS from Pasteurella multocida, and all three enzymes are highly active glycosaminoglycan backbone synthases.

The amino acid sequences of the three enzymes are used as sequence alignment templates, the sequence alignment of a bioinformatics database (NCBI database) is carried out through BLASTP algorithm, ten amino acid sequences which are possibly glycosaminoglycan skeleton Synthases (GTs) are searched, and the amino acid sequences are respectively abbreviated as CcCS, and the ID of the NCBI database is WP_150081707.1; the ID of the NCBI database of the CvCS is WP_168227469.1; the ID of the NCBI database of HfCS is NBI12747.1; the ID of the NCBI database is WP_1661815; the ID of the NCBI database of the MsCS is WP_024460064.1; the ID of the NCBI database of MeCS is WP_167432605.1; the ID of the NCBI database of NdCS is WP_085359788.1; the ID of the NCBI database is WP_144187813.1; the ID of the NCBI database of RhCS is WP_077587478.1; tmCS, NCBI database with ID WP_136735112.1

The 13 gene fragments are synthesized into corresponding plasmids pET28a (+) -His-GT by company (gold Style) through double codon optimization of escherichia coli and bacillus subtilis, and exist in E.coli TOP 10. Primers are respectively designed, and PCR is carried out on plasmids to obtain corresponding gene fragments, which are collectively called GT fragments. Wherein the primer sequences are respectively as follows:

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 was 10. Mu.M)

PCR reaction conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15s, annealing at 72℃for 15s, extension at 60℃for 3min for 30 cycles; extending at 72 ℃ for 5min, and preserving at 4 ℃.1% agarose gel electrophoresis for 30min, and gel recovery and purification of PCR products. The obtained PCR product was analyzed and detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 2 and FIG. 3.

4. Construction of recombinant expression plasmids

(1) The vector plasmid pHCMC04 was digested with SpeI and BamHI to give a 8000bp fragment, the electrophoresed pattern of which is shown in FIG. 4;

the enzyme digestion system is as follows:

cleavage reaction conditions: water bath at 37 ℃ for 1h.

(2) The digested vector plasmid is connected with the tuaD fragment and the GT fragment by a DNA seamless cloning method, and then is chemically transformed into escherichia coli DH5 alpha competent cells, and after sequencing verification, 13 recombinant expression vectors are obtained, wherein the 13 recombinant expression vectors are 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-TmCS-tuaD recombinant plasmid, pHCMC04-KfoC-tuaD recombinant plasmid, pHCMC04-Pm recombinant plasmid and pHCMC04-PmHS2-tuaD recombinant plasmid, and the specific expression map is constructed as shown in figure 5 and figure 6.

The seamless cloning reaction system is as follows:

seamless cloning reaction conditions: and carrying out water bath reaction for 30-50 min at 50 ℃.

5. Construction of recombinant Bacillus subtilis containing GD expression vector

Preparing BS168ASS electrotransformation competence according to the method in (1), transferring a GD expression vector into bacillus subtilis BS168ASS by an electrotransformation method, culturing overnight on LB solid medium containing 5 mug/mL chloramphenicol, and growing a transformant, namely the recombinant bacterium BS168SSAGD for efficiently synthesizing the azide-modified glycosaminoglycan skeleton.

Example 2: application of recombinant bacterium BS168SSAGD in high-throughput screening of glycosaminoglycan skeleton synthase

1. Verification of high throughput screening systems based on the known active glycosaminoglycan backbone synthases KfoC, pmHS2 and PmHAS.

The recombinant bacterium constructed in example 1 and containing the KfoC gene and the tuaD gene was referred to as BS168SSACD; the recombinant bacterium containing the PmHS2 gene and the tuaD gene is called BS168SSAHS2D; the recombinant bacterium containing the PmHAS gene and the tuaD gene is called BS168SSAHASD; bacillus subtilis containing the pHCMC04 null plasmid was designated as BS168SSAE as a control.

Inoculating into LB liquid culture medium with special substrate at a volume ratio of 0.1%, and culturing at 37deg.CCulturing overnight in a 225rpm shaker. Expanding the overnight cultured strain into LB liquid medium with special substrate at 1% volume ratio, and keeping the strain grow to OD ₆₀₀ At 0.6-0.8, the strain is washed 3 times by 1 XPBS buffer solution, transferred to a low-salt M9 culture medium without glucose for culture, and after consuming GlcNAc contained in the bacterial cells per se, xylose inducer with the final concentration of 20g/L and glycosyltransferase unnatural substrate GlcNAz with the final concentration of 100 mu g/mL are added after 1h. The culture was induced in a shaker at 37℃and 225rpm for 6h.

The components of the LB liquid medium with the special substrate are 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 glycosyltransferase.

After the strain after induced culture is washed 3 times by a 1 XPBS buffer solution through resuspension centrifugation, the strain is resuspended in 200 mu L of the 1 XPBS buffer solution, fluorescein Cyanine5 DBCO with the final concentration of 0.5mM is added into a light-shielding 1.5mL centrifuge tube, the strain is subjected to standing reaction for 1h at 37 ℃ and then is washed 3 times by the 1 XPBS buffer solution, an enzyme-labeled instrument is adopted to detect the fluorescence intensity at the emission wavelength of 646nm and the absorbance at the excitation wavelength of 662nm and 600nm, and the fluorescence intensity and OD are calculated ₆₀₀ The results are shown in FIG. 7.

As can be seen from FIG. 7, the ratio of fluorescence intensity to absorbance of recombinant bacteria BS168SSACD and BS168SSAHS2D, BS SSAHASD containing glycosaminoglycan skeleton synthase is significantly higher than that of strain BS168SSAE without glycosaminoglycan skeleton synthase, and the fluorescence is more than 1 time that of strain BS168SSAE without glycosaminoglycan skeleton synthase.

Diluting the bacterial solution after the fluorescence reaction to OD by using 1 XPBS buffer solution ₆₀₀ The results of the single-color flow analysis at the emission wavelength of 646nm and the excitation wavelength of 662nm, which were between 0.3 and 0.5, are shown in FIG. 8.

As can be seen from FIG. 8, the fluorescence intensities of recombinant bacteria BS168SSACD and BS168SSAHS2D, BS SSAHASD containing glycosaminoglycan skeleton synthase are obviously higher than those of the strain BS168SSAE without glycosaminoglycan skeleton synthase, and the results are consistent with those of an enzyme labelling instrument.

2. Screening of glycosaminoglycan backbone synthase libraries based on unknown Activity

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

Ten recombinant bacteria containing tuaD gene and GT fragment constructed in example 1 were abbreviated as: ccCS, cvCS, hfCS, leCS, msCS, meCS, ndCS, psCS, rhCS, tmCS. Culturing and inducing in 96-well deep well plate according to the method described in 1, performing click chemistry reaction with Cyanine5 DBCO, detecting its fluorescence intensity at 646nm emission wavelength, excitation wavelength 662nm and absorbance at 600nm by using enzyme-labeling instrument, and calculating its fluorescence intensity and OD ₆₀₀ The results are shown in FIG. 9.

As can be seen from fig. 9, the CvCs gene, hfCS gene, and MeCS gene exhibited fluorescence to an extent comparable to or even stronger than that of the KfoC gene.

(2) Verification of in vitro glycosaminoglycan backbone synthase Activity

The construction patterns of the CvCs gene, hfCS gene and MeCS gene which show stronger fluorescence in (1) and original plasmids pET28a (+) -His-CvCS, pET28a (+) -His-HfCS and pET28a (+) -His-MeCS synthesized by corresponding plasmid companies synthesized by the company (Kirsrui) are shown in FIG. 10. Transformants were grown in LB solid medium of 50. Mu.g/mL kanamycin by chemical transformation into E.coli BL21 (DE 3), single colonies were picked up, and plasmids were extracted and verified by restriction enzyme digestion to obtain BL21 (DE 3) expression strains containing the CvCs gene, the HfCS gene and the MeCS gene.

The enzyme digestion system is as follows:

cleavage reaction conditions: water bath at 37 ℃ for 20min.

BL21 (DE 3) expression strain containing CvCs, hfCS, meCS gene is respectively expanded to 1L of LB liquid medium in a volume ratio of 1%, and the strain grows to OD ₆₀₀ When the ratio is 0.6 to 0.8IPTG with a final concentration of 0.2mM was added at 22℃and 225rpm to induce 12-18 h.

After induction, the bacterial liquid was centrifuged at 8000g at 4℃for 20min, the supernatant was discarded, the bacterial pellet was collected and resuspended in loading buffer (20 mM Tris-HCl, pH= 7.5,0.5M NaCl,5mM imidazole); crushing thalli for 30min by using an ultrasonic crusher, wherein the working conditions are as follows: working for 15s, intermittent for 45s, amplitude of 35%, energy 1500KJ,4 ℃; the crushed cells were centrifuged at 12000g for 30min at 4℃using an ultra-low temperature centrifuge, and the supernatant was collected and filtered with a 0.22 μm filter membrane. The filtered supernatant was purified using Ni-charged MagBeads, followed by ddH ₂ O, completely suspending the magnetic beads in the test tube, placing the test tube on a magnetic bead separation frame to collect the magnetic beads, and discarding the magnetic bead preservation solution which is cleaned and washed to remove the original 20% ethanol; the beads were resuspended twice with loading buffer (20 Mm Tris-HCl, ph= 7.5,0.5M NaCl,35mM imidazole) and the system was equilibrated; re-suspending the filtered supernatant with magnetic beads, and incubating in a shaker at 4 ℃ and 225rpm for 30min; washing off the hybrid protein with equilibration buffer (20 Mm Tris-HCl, ph= 7.5,0.5M NaCl,35mM imidazole); the protein of interest was eluted with elution buffer (20 Mm Tris-HCl, ph= 7.5,0.5M NaCl,200mM imidazole) and collected.

The expression and purification of the induced proteins were identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the results are shown in FIG. 11, which shows successful separation of CvCs, hfCS and MeCS proteins.

The glycosaminoglycan backbone synthase activities of the Cvcs, hfCS and MeCS proteins were measured with GlcA-pNP as acceptor substrate and UDP-GalNAc/UDP-GlcNAc as donor substrate, respectively.

The reaction system is as follows:

reaction conditions: the reaction was carried out overnight at 37 ℃.

The reacted system was filtered through a 0.22 μm filter and analyzed by PAMN-HPLC. The specific absorbance peak of the pNP group was detected at 310 nm. The chromatographic column used in HPLC analysis was YMC-Pack PolymerAn ine II chromatographic column with mobile phase of water and 1mol/L KH ₂ PO ₄ The solution flow rate was 0.5mL/min, and the UV absorption of each component at 310/254nm after each protein catalytic product was separated by chromatography column was measured, and the results are shown in FIG. 12.

The HPLC analysis procedure was as follows:

as can be seen from FIG. 12, the Cvcs protein, the HfCS protein and the MeCS protein all exhibit strong UDP-GalNAc transferase activity, i.e., the Cvcs protein, the HfCS protein and the MeCS protein are glycosaminoglycan skeleton synthases having high activity. Consistent with the screening results of the glycosaminoglycan backbone synthase library based on the ratio of fluorescence intensity to absorbance in this example 2.

3. High throughput screening of glycosaminoglycan backbone synthases based on Fluorescence Activated Cell Sorting (FACS)

The method for measuring fluorescence based on the enzyme label instrument can only screen a small amount of glycosaminoglycan skeleton synthase, and the high-throughput screening needs to rely on a fluorescence activated cell sorting technology by referring to a strain BS168SSACD containing KfoC genes and tuaD gene expression cassettes as KfoC+; only the pHCMC04 null plasmid strain BS168SSAE, designated KfoC-.

KfoC+ and KfoC-were mixed and cultured in such a manner that KfoC+/total bacteria were 10: 1. 1: 1. the enrichment efficiency of FACS on positive groups was further analyzed at a volume ratio of 1:10, 1:100.

The mixed cultured strain was induced to culture and fluorescent reaction as described in this example 1, washed and analyzed by flow cytometry. The cells with fluorescence intensities of the first 2% were collected to minimize possible false positives. The bacteria after sorting were cultured overnight in LB solid medium containing 5. Mu.g/mL chloramphenicol, and after single colony was picked, the positive proportion after enrichment was identified and compared with that before sorting, and the results are shown in Table 1.

Table 1 effect of efficient synthesis of recombinant bacillus subtilis with azide-modified glycosaminoglycan backbone in FACS system, enrichment of recombinant strain containing high activity glycosaminoglycan backbone synthase from a large number of background bacteria.

As can be seen from Table 1, the recombinant strain KfoC+ containing highly active glycosaminoglycan backbone synthase was enriched with up to 97% probability in the past under single round of high stringency flow separation conditions at 1:100 mixed culture conditions.

Claims

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

(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.