Method for do novo biosynthesis of chondroitin sulfate

Abstract

The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCl buffer assisted with 3′-phosphoadenosine 5′-phophosulfate (PAPS). C4ST and C46ST came from bioengineered Escherichia coli or Pichia pastoris. Chondroitin came from bioengineered Bacillus subtilis 168.

Claims

1. A method for biosynthesis of Chondroitin sulfate (CS), comprising: expressing in Escherichia coli or Pichia pastoris a heterogeneous gene encoding chondroitin 4-sulfotransferase (C4ST) and a heterogeneous gene encoding chondroitin 6-sulfotransferase (C6ST); wherein a sequence of the heterogeneous gene encoding C4ST is set forth in SEQ ID NO:15; and wherein a sequence of the heterogeneous gene encoding C6ST is set forth in SEQ ID NO:16; collecting the C4ST and C6ST; and incubating the C4ST and C6ST with a 3′-phosphoadenosine-5′-phosphosulfate (PAPS) regeneration system for 20-50 hours; and wherein the recombinant Escherichia coli or the Pichia pastoris comprise a recombinant expression plasmid comprising the heterogeneous gene encoding C4ST and the heterogeneous gene encoding C6ST.

2. The method according to claim 1, wherein the plasmid is pET or pPIC.

3. The method according to claim 1, wherein the PAPS regeneration system catalyzes p-nitrobenzenesulphonic acid (PNPS) to PAPS, and wherein the PAPS regeneration system comprises: 0.1-100 μg aryl sulfotransferase IV (ASST IV), 0.1-50 mM PNPS, 1-200 μM PAP (3′5′-adenosine diphosphate), and 1-200 mM Tris-HCl, pH 5-9.

4. The method according to claim 3, further comprising expressing ASST IV in Escherichia coli encoded on a pET plasmid; wherein a sequence of a gene encoding ASST IV is set forth in SEQ ID NO:17.

5. The method according to claim 1, comprising adding 0.1-100 μg C4ST and C6ST to the PAPS regeneration system for CS synthesis.

6. The method according to claim 3, wherein a specific activity of ASST IV is 0.1-100 nmol/min.Math.mg.Math.protein.

7. The method according to claim 1, wherein a specific activity of C4ST is 0.1-100 pmol/min.Math.mg.Math.protein, and wherein a specific activity of C6ST is 0.1-100 pmol/min.Math.mg.Math.protein.

8. The method according to claim 1, comprising sulfating chondroitin using C4ST and C6ST assisted with the PAPS regeneration system under 25-50° C. within 1-50 hours.

9. The method according to claim 8, comprising producing the chondroitin by a recombinant Bacillus subtilis 168 (B. subtilis 168); wherein the recombinant B. subtilis 168 is constructed by expressing KfoC and KfoA in a genome and co-expressing genes of a synthetic pathway of chondroitin, comprising genes of the synthetic pathway of UDP-glucuronic acid (UDP-GlcUA) or UDP-N-Acetylglucosamine (UDP-GlcNAc).

10. The method according to claim 9, wherein genes of the synthetic pathway of UDP-GlcUA comprise pgcA, gtaB, and tuaD, and wherein genes of the synthetic pathway of UDP-GlcNAc comprise glmS, glmM, and glmU.

11. The method according to claim 9, further comprising co-expressing genes tuaD and glmU, or co-expressing genes tuaD, glmU, gtaB, glmM and glmS.

12. The method according to claim 9, comprising using a plasmid pP43NMK to express genes of the synthetic pathway of chondroitin.

13. The method according to claim 9, comprising culturing the recombinant B. subtilis 168 at 37° C. for 24-60 hours to produce the chondroitin, and collecting the chondroitin from a supernatant of the culture; wherein a fermentation medium of the culture comprises 20 g/L yeast extract, 15 g/L or 50 g/L sucrose, 3.9 g/L K.sub.2SO.sub.4, 1.5 g/L MgSO.sub.4 and 50 mM phosphate buffer, and has a pH of 6.5-7.5.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 SDS-PAGE analysis of recombinant E. coli which expressed ASST IV. M, marker; 1, SDS-PAGE analysis of whole cellular of E. coli BL21-pET20b-ASST IV; 2, SDS-PAGE analysis of whole cellular of E. coli BL21-pUC19-ASST IV; 3, SDS-PAGE analysis of the culture supernatant of E. coli BL21-pET20b-ASST IV; 4, SDS-PAGE analysis of the culture supernatant of E. coli BL21-pUC19-ASST IV.

(2) FIG. 2 SDS-PAGE analysis of recombinant E. coli which expressed C4ST or C6ST. M, marker; 1, SDS-PAGE analysis of culture supernatant of E. coli BL21-pET20b-C4ST; 2, SDS-PAGE analysis of culture supernatant of E. coli BL21-pUC19-C4ST; 3, SDS-PAGE analysis of culture supernatant of E. coli BL21-pET20b-C6ST; 4, SDS-PAGE analysis of culture supernatant of E. coli BL21-pUC19-C6ST.

(3) FIG. 3 Western blot analysis of recombinant P. pastoris which expressed C4ST or C6ST; M, marker; 1, Western blot analysis of culture supernatant of recombinant P. pastoris expressing C4ST based on pPIC; 2, Western blot analysis of culture supernatant of recombinant P. pastoris expressing C4ST based on pGAPZ; 3, Western blot analysis of culture supernatant of recombinant P. pastoris expressing C6ST based on pPIC; 4, Western blot analysis of culture supernatant of recombinant P. pastoris expressing C6ST based on pGAPZ.

(4) FIG. 4 Activity analysis of C4ST and C6ST based on chondroitin, commercial CSA and CSC from animal.

(5) FIG. 5 CS disaccharide fractionation analysis. LC-MS spectra of chondroitin disaccharides (a); LC-MS spectra of CSC disaccharides (b); LC-MS spectra of CSA disaccharides (c).

(6) FIG. 6 Schematic of plasmid construction for co-expression of pathway genes in B. subtilis 168.

(7) FIG. 7 Electrophoresis analysis of recombinant strains: M, marker; 1, B. subtilis E168C/pP43-DBA; 2, B. subtilis E168C/pP43-UMS; 3, B. subtilis E168C/pP43-DU; 4, B. subtilis E168C/pP43-DU-PBMS.

(8) FIG. 8 Production of chondroitin by the recombinant B. subtilis 168.

(9) FIG. 9 Time course of chondroitin production and cell growth by B. subtilis E168C/pP43-DU-PBMS and B. subtilis E168C.

(10) FIG. 10 Time course of chondroitin production with feed-back culture in 3 L fermenter by B. subtilis E168C/pP43-DU-PBMS.

DETAILED DESCRIPTION

(11) Analysis of C4ST and C6ST activity: The activity of C4ST and C6ST was analysis based on chondroitin. The reaction liquor were 20 mM Tris-HCl (pH7.0), 3 mM PNPS, 20 μM PAP, 10 mg ASST IV, 5 mg/mL chondroitin, and 20 μg C4ST or C6ST. The reaction was happened at 37° C. for 20 h, and terminated by heating at 100° C. for 5 min. At last, the absorbance was determined at 400 nm. The blank was reaction liquor components with no C4ST and C6ST. Product rate was calculation by the formula Y=10.sup.−3*(18.83*(AC-AASST IV)+0.38; Ac: the absorbance of C4ST or C6ST; ASST IV: the absorbance of blank.

(12) Chondroitin molecular weight analysis: The molecular weight of chondroitin was analysed by combination of Multi-Angle Laser Light Scattering Instrument and Size Exclusion Chromatography with Ultrahydrogel Linea on refractive index detector. It was performed with 0.5 mL/min phase (0.1 M NaNO.sub.3) at 50° C. for 20 min and injection volume was 20 μL.

Example 1: The Production of ASST IV, C4ST, and C6ST by Bioengineered E. coli

(13) The genes encoding Rat ASST IV C4ST, and C6ST were separately inserted between the Nde I and Not I sites of pET26b after the T7 promoter and fusing with His-tag in the N-terminal to obtain the engineering plasmids pET26b-C4ST, pET26b-C6ST, and pET26b-ASST IV (The primers used were listed in Tab. 2). Finally, those plasmids were transformed into E. coli BL21 (DE3) and plated on LB plate with 50 μg/mL ampicillin for screening.

(14) The E. coli BL21 separately containing pET26b-C4ST, pET26b-C6ST, and pET26b-ASST IV was cultivated in Luria-Bertani (LB) medium with 50 μg/mL ampicillin at 37° C. in a rotary shake at 220 rpm. Two milliliter of culture was inoculated into 50 mL of LB medium with 50 μg/mL ampicillin and cultivated at 37° C. Then, after 2 h, OD.sub.600 of the broth reached 0.6-0.8, 30 μL of 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was add into the broth. After it was induced at 16° C. for 48 h, cells were harvested by centrifugation at 8000 rpm for 5 min at 4° C.

(15) The recombinant E. coli BL21 were washed by 20 mM Tris-HCl (pH7.0), and diluted in 20 mM Tris-HCl (pH7.0) to have an absorbance of 10 at 600 nm. The suspended cells were lysed by sonication at 4° C. The cleared lysate was mixed were separated by 8-12% SDS-PAGE and identified by size comparison to Pre-stained standard (ThermoFisher, Germany) (FIG. 1, 2) and MALDI-TOF-MASS (Shimadzu, Japan).

Example 2: Construction of Bioengineered P. pastoris

(16) Genes encoding Rat C4ST, C6ST were amplified by PCR using the PrimeSTAR HS (Premix) with the primes containing the sequence of pPIC3.5K as overlapping overhangs in the 5′-terminal, followed by Gibson isothermal assembly cloning to circularize to obtain the engineering pPIC3.5K-C4ST, and pPIC3.5K-C6ST with alpha signal peptide (The primers used were listed in Table 2). Then, the transformation and recombinant screening were proposed according to the instructions of A Pichia Vector for Multicopy Integration and Secreted Expression (Invitrogen, Germany).

(17) pPIC3.5K-C4ST and pPIC3.5K-C6ST were separately transformed into P. pastoris GS115 to get recombinant P. pastoris GS115. Recombinant P. pastoris GS115 was cultivated in 50 mL BMMY medium containing 0.5 g/L methanol at 20° C., 200 rpm, for 5d. The culture supernatants were collected for C4ST and C6ST purification. The supernatants were filtered through a 0.22 μm membrane and concentrated with Millipore ultrafiltration system according to the manufacturer's instructions with a membrane of 3 kDa cut off, the resulted samples were analysis and identified by SDS and MALDI-FOR-MASS (FIG. 3).

(18) C4ST and C6ST activity was also assayed by changes of absorbance at 400 nm due to the formation of free 4-nitrophenol as described with some modification (FIG. 4).

Example 3: Synthesis of CSA, CSC

(19) The conversion of chondroitin to CSA or CSC involved two steps, including PAPS regeneration and sulfotransferase modification. In brief, the standard reaction mixture containing 3 mM 3′-phosphoadenosine 5′-phosphate (PNPS), 10 mg ASST IV (0.1-100 nmol/min.Math.mg.Math.protein) and 5 mg/mL chondroitin, and 20 μg C4ST (0.1-100 pmol/min.Math.mg.Math.protein) or C6ST (0.1-100 pmol/min.Math.mg.Math.protein), and 20 mM Tris-HCl (pH7.0) buffer. The mixture was incubated at 37° C. for 20 h for CSA or CSC.

Example 4 CS Disaccharide Fractionation Analysis

(20) CS disaccharide fraction was analysed by LCMS after it was lysed by chondroitinase ABC at 37° C. for 5-20 h and terminated at 100° C. for 5 min. The LC were performed at C18 Reverse phase column, 0.3 mm*250 mm, with phase A (8 mM CH.sub.3COOH), phase B (8 mM CH.sub.3COOH 70% methanol/H.sub.2O). The elution condition was in Table 1. The MS were performed with nitrogen as the desolation gas and as a nebulizer in negative-ion modes. The nebulizer flow was 0.75 L/min and nozzle temperature was 140° C. The N.sub.2 was also the drying gas with the flow 1.2 mL/min. The negative ion spectra were generated by scanning the range of 40-2000 m/z, with special ion peaks M/Z 397 and 458 (FIG. 5).

(21) TABLE-US-00001 TABLE 1 Time (min) PhaseA(%) PhaseB(%) 0 100 0 3 100 0 13 98 2 28 86 14 41 75 25 57 61 39 74 45 55 84 10 90 104 100 0

(22) TABLE-US-00002 Time (min) Phase A(%) Phase B(%) 13 98 2 28 86 14 41 75 25 57 61 39 74 45 55 84 10 90 104 100 0

Example 5: Construction of Recombinant B. subtilis with Coexpression Pathway Gene

(23) At first, B. subtilis E168C components were prepared. The inorganic ion components were (g/L): K.sub.2HPO.sub.4, 140; KH.sub.2PO.sub.4, 60; (NH.sub.4).sub.2SO.sub.4, 20; (Na.sub.3C.sub.6H.sub.5O.sub.7.2H.sub.2O),10; MgSO.sub.4.7H.sub.2O, 2. GMI solutions used for component preparation containing 9.7 mL inorganic ions, 2.5 mL 20% glucose, 0.4 mL 5% casein, 1 mL 10% yeast extract per 100 mL. GMII solutions used for component preparation containing 9.7 mL inorganic ions, 2.5 mL 20% glucose, 0.08 mL 5% casein, 0.04 mL 10% yeast extract, 0.25 mL 1M MgCl.sub.2, 0.05 mL 1M CaCl.sub.2 per 100 mL. In detail, the colony of B. subtilis E168C was inoculated in 5 mL GMI, and cultivated at 30° C., 125 rpm for 16 h. Then, it was inoculated into 18 mL GMI with 10% (V/V), and cultivated at 37° C., 200 rpm for 3.5 h. Following, it was inoculated into 90 mL GMII with 10% (V/V), and cultivated at 37° C., 200 rpm for 1.5 h. Then, the cells were collected by centrifugation at 4° C., 5000 g for 10 min, and resuspended in 10 mL GMII, packaged it in 500 μl for each one.

(24) The recombinant plasmids pP43-DBA, pP43-UMS, pP43-DU, and pP43-DU-PBMS used for co-expression pathway genes were construction based on the parent expression plasmid pP43NMK (Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168, Metabolic Engineering, 2016, Jinpeng). Then, they were transformed into B. subtilis E168C components by chemical transformation, and cultured in LB plates with 50 μg/mL kanamycin for transformant selection. All the transformant were confirmed by colony PCR with the primers gtaB-F/pgcA-R, glmM-F/glmS-R, tuaD-F/glmU-R, and glmU-F/gtaB-R. The results of electrophoresis were in FIG. 7. All the recombinant strains were named as B. subtilis E168C/pP43-DBA, B. subtilis E168C/pP43-UMS, B. subtilis E168C/pP43-DU, B. subtilis E168C/pP43-DU-PBMS.

(25) TABLE-US-00003 Primers sequences: 5′-3′: gtaB-F: ATGAAAAAAGTACGTAAAGCCATAA (SEQ ID NO: 7) pgcA-R: TTATTTTGCTGTTGACTCAACAA (SEQ ID NO: 8) glmM-F: ATGGGCAAGTATTTTGGAACAGACG (SEQ ID NO: 9) glmS-R: TTACTCCACAGTAACACTCTTCGCA (SEQ ID NO: 10) tuaD-F: GTGAAAAAAATAGCTGTCATTGGAAC (SEQ ID NO: 11) glmU-R: TTATTTTTTATGAATATTTTTCACATAATC (SEQ ID NO: 12) glmU-F: ATGGATAAGCGGTTTGCAGTTG (SEQ ID NO: 13) gtaB-R: TTAGATTTCTTCTTTGTTTAGTAAAC (SEQ ID NO: 14)

Example 6: Production of Chondroitin by Recombinant Strains in Flask

(26) All the four recombinant strains were inoculated into LB with 50 μg/mL kanamycin, cultured at 37° C., 200 rpm for 16 h. Then, they were inoculated with 10% (V/V) into fermentation medium (20 g/L yeast extract, 50 g/L sucrose, 3.9 g/L K.sub.2SO.sub.4, 1.5 g/L MgSO.sub.4, 50 mM phosphate buffer, pH7.0 50 μg/mL kanamycin), cultured in 37° C., 200 rpm for 54 h. And then, it was induced at the second hours by 20 g/L xylose. All the medium for B. subtilis E168C culture have no kanamycin. After fermentation, the culture was centrifugated at 10000 g for 5 min, and the supernatants were collected. Then 3V ethanol were added and mixed at 4° C. for 1 h for the precipitation, after centrifugated at 10000 g for 5 min, the sediment was collected and resuspended in H.sub.2O. Then, the centrifugation and resuspend steps were repeats for three times for chondroitin purification. The chondroitin concentration was assayed by Bitter-Muir carbazole assay. 200 μl samples and 1 mL Na.sub.2B.sub.4O.sub.2.10H.sub.2O—H.sub.2SO.sub.4 were mixed in tubes and boiled for 15 min. After they were cooled down, 50 μl carbazole was added and mixed, then boiled for 15 min. The optical density was determined after they were cooled down, and the chondroitin yield was calculated.

(27) The chondroitin yield was increased by recombinant strains (B. subtilis E168C/pP43-DBA 2.17 g/L, B. subtilis E168C/pP43-UMS 2.37 g/L, B. subtilis E168C/pP43-DU 1.96 g/L, B. subtilis E168C/pP43-DU-PBMS 2.67 g/L), compared to parent (1.83 g/L) (FIG. 8). It was shown that the recombinant strains which co-expressed all the pathway genes had the highest chondroitin production. It meant that the balance between the concentration of UDP-GlcUA and UDP-GlcNAc contributed to chondroitin synthesis. For strains growth, it was stationary phase for B. subtilis E168C/pP43-DU-PBMS culture to the 12-24th hours (FIG. 9). The highest OD.sub.600 nm of B. subtilis E168C/pP43-DU-PBMS culture reached 21.5 while the parents' OD.sub.600 nm was 19.9. In conclusion, recombinant pP43-DU-PBMS have no effect on strains growth. Besides, the molecular weights of chondroitin produced by recombinant strains (84.36 kDa, 97.02 kDa, 72.78 kDa, and 119.20 kDa) were higher than the parents (83.51 kDa) according to MALLS-SEC (Table 2). The polydispersity Ip of chondroitin was between 1.08-1.51, and the value was closer to 1 when certain UDP-GlcUA and UDP-GlcNAc existed. That means, co-expressing genes related to the synthetic pathway of chondroitin, such as genes related to the synthetic pathway of UDP-glucuronic acid (UDP-GlcUA) or UDP-N-Acetylglucosamine (UDP-GlcNAc) was a successful method for uniformity chondroitin production.

(28) TABLE-US-00004 TABLE 2 Difference of chondroitin molecular weight produced by recombinant B. subtilis Strains .sup.aM.sub.n (kDa) .sup.bM.sub.w (kDa) .sup.cI.sub.p B. subtilis E168C 69.58 ± 0.62 83.51 ± 0.98 1.21 ± 0.03 B. subtilis E168C/ 59.83 ± 0.87 84.36 ± 1.02 1.41 ± 0.04 pP43-DBA B. subtilis E168C/ 89.83 ± 0.67 97.02 ± 1.88 1.08 ± 0.08 pP43-UMS B. subtilis E168C/ 48.20 ± 1.39 72.78 ± 1.60 1.51 ± 0.03 pP43-DU B. subtilis E168C/ 101.02 ± 1.08  119.20 ± 2.18  1.18 ± 0.02 pP43-DB-PBMS .sup.anumber-average molecular weight(M.sub.n); .sup.brelative molecular mass(M.sub.w); .sup.cpolydispersity(I.sub.p = M.sub.w/M.sub.n).

Example 7: Production of Chondroitin by B. subtilis E168C/pP43-DU-PBMS in 3 L Fermenter with Strategy of Feed-Batch

(29) B. subtilis E168C/pP43-DU-PBMS was inoculated into 150 mL LB broth, with 50 μg/mL kanamycin and cultivated at 37° C., 200 rpm for 16 h. Then, they were inoculated with 10% (V/V) into fermentation medium (20 g/L yeast extract, 50 g/L sucrose, 3.9 g/L K.sub.2SO.sub.4, 1.5 g/L MgSO.sub.4, 50 mM phosphate buffer, pH7.0 50 μg/mL kanamycin), and cultivated at 37° C., 2 vvm. 2h after the inoculation into fermentation medium, 20 g/L xylose was added. The pH was adjusted to 7.0 by 5 M NaOH. The stirred rate was adjusted to 600 rpm at the 6th hours, and 800 rpm at the 8th hours. 800 g/L sucrose solution was feed when the sucrose concentration of broth bellowed than 5 g/L to keep the sucrose concentration at 0-5 g/L. In detail, the feed strategy was 7.5, 7.5, 15, 10 g/L/h during 8-12 h. After the fermentation, the Mw, Mn, and 1p were assayed by HPSEC-MALLS.

(30) It showed that Time course of chondroitin production was in S style. Chondroitin mainly accumulated during later stationary phase (FIG. 10). And chondroitin accumulation was no couple with strains growth. The highest chondroitin concentration was 6.06 g/L at the 70th which was 2.27 times than chondroitin production in the flask. The Mw of chondroitin in the fermenter was 78.64 kDa which was smaller than that in the flask due to the shearing force caused by stirring. As a result, the 1p of chondroitin produced in fermenter was 1.89 higher than that in the flask. That means the chondroitin molecular weight was more dispersive when fermented in 3 L fermenter.