Studies Of Characterization And Function Of Ste16 Involved In Ebosin Biosynthesis, Double Disruption Of Ste15-ste22 And Heterologous Expression Of Ste Gene Cluster

Streptomyces sp. 139 was identified to produce a novel exopolysaccharide (EPS) designated Ebosin. It has obvious anti-rheumatic arthritis activity in vivo and may be developed to be a new drug in the future.The biosynthesis gene cluster consisting of 27 ORFs for Ebosin was confirmed in our laboratory. The individual roles of the putative genes in the cluster have been characterized for studying of the relationship between structure and activity of Ebosin and obtaining new derivatives. In this research, the objective of present investigation was to functionally assess ste16.ste16 (1,236bp) was predicted to specify a protein with homology to known NDP-hexose methyltransferases from different sources of microbes, which has the ability catalyzing transfer of the methyl group from S-adenosylmethionine (AdoMet) to dTDP-4-keto-6-deoxy-D-glucose.For characterization of the protein encoded by ste16, the gene was expressed in E. coli BL21 and the recombinant protein was purified to ~ 90% with a Mw 47kD in agreement with the expected size. Enzymatic reaction showed that Ste16 protein possesses the methyltransferase activity which catalyzes transfer of the methyl group from S-adenosylmethionine (AdoMet) to dTDP-4-keto-6-deoxy-D-glucose and then production of S-adenosylhomocysteine (AdoHcy). So that Ste16 was identified as a NDP-hexose methyltransferase with Km 0.43±0.02mM.For understanding the function of ste16 in the biosynthesis of Ebosin, the gene was disrupted with a double crossover via homologous recombination. The mutant strain Streptomyces sp.139 (ste16~-) was confirmed with Southern blot. The monosaccharide composition of EPS-16m produced by the mutant strain was found varied from that of Ebosin. The monosaccharide analysis of EPS-16c produced by the ste16 complemented mutant strain showed no remarkable changes compared with EPS-16m. The Mw for both of EPS-16m and EPS-16c were remarkably smaller than Ebosin. The antagonist activity of EPS-16m for IL-1R was significantly lower than Ebosin, but the activity of EPS-16c recovered partially compared with that of EPS-16m. According to these results, we assume Ste16 transfers methyl groups to rhamnose or fucose residues (6DOHs) of Ebosin in vivo, although the direct evidence has yet to be provided. Ste16 may be as a modificator gene during the biosynthesis of Ebosin. The products encoded by ste15 and ste22 were identified as the glucosyltranferase and the rhamnose-glycosyltranferase respectively. The ste22 was disrupted with a double crossover via homologous recombination in Streptomyces sp.139 (ste15~-). Southern blot demonstrated the mutant strain Streptomyces sp.139 (ste15~- ste22~-). The Mw of EPS-15m22m produced by the mutant strain was smaller more than 50% comparing with Ebosin. The antagonist activity of EPS-15m22m for IL-1R was significantly lower than the original levels of Ebosin. The monosaccharide analysis of EPS-15m22m has been carried out. From these research data, it can be concluded that both of stel5 and ste22 played essential roles in relationship between structure and activities in Ebosin and affected tremendously the polymerization of the repeating units of Ebosin also.The biosynthesis gene cluster (ste) of Ebosin was cloned into Streptomyces lividans TK24 with cosmid pOJ446 as vector. The monosaccharide analysis of EPS-TK, EPS-pOJ and EPS-ste produced by Streptomyces lividans TK24, Streptomyces lividans TK24 (pOJ446) and Streptomyces lividans TK24 (ste) separately showed that them were similar for the EPSs. The Mw of EPSs were remarkably lower than Ebosin and all of them had not the antagonist activities for IL-1R. From the results mentioned above, the biosynthesis gene cluster (ste) of Ebosin has not been expressed in Streptomyces lividans TK24. More detail research works should be performed further.In a word, the presenting studies identified the function of ste16 in the biosynthesis of Ebosin as a modificator gene and the essential roles of glucose, rhamnose in the bioactivity of Ebosin. These studies have laid the better groundwork for studying the relationship between structure and bioactivity of EPSs and obtaining more novel derivatives of Ebosin with new activities.