Studies Of Function, Characterization For Ste11 Involving In Ebosin Biosynthesis And The Replacement Of Ste7, Ste22 With The Glycosyltransferase Originated From Streptococcus Thermophilus

Ebosin produced by Streptomyces sp.139 is a novel exopolysaccharide(EPS) with remarkable anti-rheumatic arthritis activity in vivo. It may be developed to a new drug in the future.A biosynthesis gene cluster consisting of 27 ORFs (ste1-ste27) for Ebosin was identified in our lab. Among them, ste 6, ste7, ste10 ste15 and ste22 were confirmed to play important roles in the biosynthesis of Ebosin before.Ste11 (2100bp) was predicted to specify a protein with homology to known threonine dehydrogenase from different sources of microbes. For characterization of the protein encoding by ste11, the gene was cloned and expressed in E. coli BL21. The recombinant Ste11 was purified with the affinity chromatography and found capable of catalyzing NAD~+ and L-threonine to NADH and 2-amino-3-ketobutyrate, hence identified as a threonine dehydrogenase with optimum temperature 37℃, pH 7.5 and Km 0.2mmol/L.To investigate its function in the biosynthesis of Ebosin , the ste11 gene was knocked out with a double crossover via homologous recombination to produce Streptomyces sp. 139 (ste11~-),which was identified by Southern Blot. Gene complementation of the knock-out mutant was achieved producing the ste11 -complemented mutant strain Streptomyces sp. 139 (pKC11). GC analysis of monosacharride composition in EPS-m produced by Streptomyces sp. 139 (ste11~-) showed that it was composed of the same monosaccharides found in Ebosin but some proportional changes occurred in comparison with Ebosin. Analysis of EPS-c produced by the ste11-complemented mutant strain with GC showed no significant changes compared with EPS-m for most of the monosaccharides. The antagonist activities of EPS-m for IL-1R were much lower than those of Ebosin but the activities of EPS-c recovered partly compared from EPS-m. The Mw of EPS-m and EPS-c are remarkably smaller than Ebosin. With its function verified, we assume that threonine presents in the molecule of Ebosin, although the direct evidence has yet to be provided. Ste11 affects not only the bioactivity significantly but also the polymerization of the repeating units consisting of monosaccharides in Ebosin. The gene may function as a modificator gene of Ebosin during its biosynthesis.Glycosyltransferases (GTs) that sequentially link sugars from intracellular nucleotide sugar to a lipid carrier are key enzymes in EPSs biosynthesis. Replacing the GTs gene in the biosynthesis of Ebosin with the heterologous GTs gene will be benefit to produce Ebosin derivatives and study the relationship between structure and activity of Ebosin. The gene encoding glucosyltransferase originating from Streptococcus thermophilus was amplified with PCR and cloned into Streptomyces sp.139(ste 7~-) and Streptomyces sp.139 (ste22~-) respectively using a double crossover via homologous recombination. The mutants Streptomyces sp.139 (ste22HC) and Streptomyces sp.139( ste7 HC) were identified by Southern Blot.GC analysis of monosaccharide composition in EPS-HC22 and EPS-HC7 produced by Streptomyces sp. 139 (ste22 HC) and Streptomyces sp. 139 (ste7 HC) separately showed that glucose increased remarkably , at meant wile rhamnose and fucose decreased tremendously in two Ebosin derivatives in comparison with Ebosin. The antagonist activities of IL-1R for two Ebosin derivatives lost and their Mw were significantly lower than Ebosin. Such results demonstrated that rhamnose, fucose and gluocse are essential in biosynthesis of Ebosin.In briefly, these studies have laid the ground works for producing more new derivatives of Ebosin with various bioactivities. As our knowledge, the threonine dehydrogenase was reported in Streptomyces at first time.