Studies Of Gene Cloning, Expression And Function Of Ste7 And Ste10 Involved In Ebosin Biosynthesis And Construction Of The Strain With Ste7-ste15 Double Disruption

Streptomyces,a gram-positive bacterium,is well known as an important industrial microorganism producing antibiotics.A screening model for IL-1R antagonists was constructed in our lab;Ebosin produced by Streptomyces sp.139 was obtained with the model.Ebosin is a new heteroexopolysaccharide with repeating unit consisting of galactose,arabinose,mannose,fucose,xylose,rhamnose,galacturonic acid and glucose with a molar ratio of 19:16:5.0:5.0:4.0:3.0:3.0:2.0.The structure of the repeating units for Ebosin has been identified.The pharmacology studies show that Ebosin has obvious anti-rheumatic arthritis activity in vivo with lower toxicity.It has been applied for clinic trial and may be developed to a new drug treating rheumatic arthritis.Ebosin biosynthesis gene cluster(ste) containing of 24 ORFs was identified by Dr.Wang.The cluster include the functions related to regulation(ste1-ste4), glycosyltransferation(ste5,ste7,ste15 and ste22),precursor synthesis of nucleotide sugar(ste6,ste10,ste11,ste17and ste19),polymerization and export(ste8,ste9,ste13, ste14 and ste21),and modification(ste12,ste16,ste18 and ste20).For elucidating the biosynthesis pathway of Ebosin,studying the relationship between structure and bioactivity and obtaining new derivatives,the functions of the ORFs involving in the gene cluster will be studied in more detail.Both ste7 and ste10 were selected for functional study in this thesis.According to the data base analysis,the protein encoded by ste10 showed high homology with asparagine synthetase originated from various microorganisms.It is well known that the asparagine synthetase can catalyze the synthesis of asparagines from aspartate,using either glutamine or ammonia as a nitrogen source.The asparagine synthetase distributes widely in prokaryotes and eukaryotes.As our knowledge,the enzyme has not been reported in Streptomyces before us.To identify the function of ste10,it was expressed in E.coli with plasmid pET-30a as vector.After inducing with IPTG and cultivating at 37℃,SDS-PAGE analysis of and cell lysates showed that there was a new band in site of 70KD as expected and the recombinant Ste10 was expressed in the inclusion body form.After renaturation,the protein was purified with Ni²⁺-NTA affinity chromatogram to 93% purity analyzing with HPLC.Ste 10 was shown to be able of catalyzing the transfer of amide nitrogen of glutamine to the side chain of aspartate to produce asparagine.Its Kin,optimum temperature and pH were determined to be 0.9 mM,37℃and 7.38, respectively.For understanding the function of ste10 in the biosynthesis of Ebosin,the gene was disrupted with a double crossover via homologous recombination.Compared with Ebosin,the monosacchafide composition of EPS-10m produced by the gene deficient strain Streptomyces sp.139(ste10^-) was changeable.EPS-10c produced by the gene complement strain Streptomyces sp.139(ste10c) was similar to EPS-10m. ELISA assay showed that the antagonist activity of EPS-m for IL-1R was much lower than that of Ebosin and this lost in the bioactivity was recovered in EPS-c after the gene complementation.We deduced that asparagine might be in the side chain of Ebosin.Ste10 is probably involved in the biosynthesis of Ebosin as a modificator gene.Base on data base comparison,the protein encoded by ste7 showed high homology with glycosyltransferases originated from different microorganisms.The gene was disrupted with a double crossover via homologous recombination and the mutant Streptomyces sp.139(ste7^-) verified by Southern blot and PCR analyses. Compared with Ebosin,the monosaccharide composition of exopolysaccharide (EPS-7m) produced by the mutant Streptomyces sp.139(ste7^-) was found quite different.Six out of eight sugar components were reduced in proportion but most noticeable were the drops(about 80%) in glucose and fucose contents.The other two, galactose and rhamnose were increased in proportion.Gene complementation largely reversed the composition changes including the decrease of fucose but had little effect on glucose content.The antagonist activity of EPS-m for IL-1R in vitro was remarkably lower than Ebosin at both tested concentrations.Compared with EPS-m this bioactivity of EPS-c produced by the complemented strain was significantly higher and it was even higher than Ebosin at the lower concentration(at 4.5μg/ml). These results have demonstrated the functional involvement of ste7 gene in the biosynthesis of Ebosin mostly probably by encoding a glycosyltransferse for fucose transfer. According to the data base comparison,proteins encoded by ste7 and ste15 showed high homology with that of the glycosyltransferase groupâ… (Pfam00534). Previous studies showed that ste15 gene encodes a glycosyltransferse for glucose transfer.In this study,we disrupted ste7 with the same strategy mentioned above in the mutant Streptomyces sp.139(ste15^-).The mutant strain Streptomyces sp.139 (ste7^-ste15^-) was identified by Southern blot and PCR.The studies of gene complementation,monosaccharide composition analysis and the IL-1R antagonist bioactivities for mutants have been carried out in the laboratory.In conclusion,the functions of ste7 and ste 10 were identified in the biosynthesis of Ebosin with gene disruptions.Such results demonstrated the relationship of structure and bioactivity of Ebosin and laid on the foundation of studying new derivatives of Ebosin.Studying characterazation and function of asparagine synthetase in streptomyces has not been reported before us.