Cloning, Expression And Functional Studies Of Gene Ste5 And Ste22 Involved In Ebosin Biosynthesis

It is well known that members of the genus Streptomyces produce a great many antibiotics and other classes of biologically active secondary metabolites,but little is known of the EPSs produced by these valuable microbes.Recently,a novel EPS named Ebosin was isolated and identified in our laboratory from the culture of Streptomyces sp. 139,which has antagonist activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo.The application of clinical trial for Ebosin has been made. The biosynthesis gene cluster(ste) of Ebosin consisting of 27 ORFs was identified in our lab.We have endeavored to elucidate the functions of the ste genes present in this cluster.Here we presented our study on ste5 and ste22.Based on the database analysis,the deduced product of ste5 showed high homology with glycosyltransferases,which may take responsibility in Ebosin biosynthesis at the first step.For understanding the function of ste5 in the biosynthesis of Ebosin,the gene was disrupted with a double crossover via homologous recombination.The proportions of monosaccharides in EPS-5m produced by the gene deficient strain Streptomyces sp. 139(ste5) were changeable especially in reducing galactose compared with Ebosin, while its antagonist activity for IL-1R was lost and Mw was apparently lower than Ebosin.The gene ste5 was cloned and expressed in E.coli.The recombinant protein with Mw～54 KD was purified,which was identified as a galactosyltransferase catalyzing the transfer of galactose-1-phosphate from UDP-galactose to the lipid carrier in the cytoplasmic membrane of Streptomyces sp.139.As a priming glycosyltransferases,Ste5 is functional at the first step during biosynthesis of Ebosin.Gene ste22 was also cloned and expressed in E.coli with pBV220 as vector.The recombinant protein Ste22 with Mw～35 KD was purified.The result of enzymatic study showed that the protein of ste22 was rhamnosyltransferase catalyzing the transference of rhamnose from TDP-rhamnose to a rhamnose-acceptor,a lipophilic carrier in the cytoplasmic membrane of Streptomyces sp.139.Gene ste22 plays essential role undoubtedly in biosynthesis of Ebosin.In short,the functions of ste5 and ste22 were identified during biosynthesis of Ebosin.The foundation has been laid on explaining the relationship between structure and biological activities in Ebosin and producing the novel derivates of Ebosin with activities.