Identification And Characterization Of A New Exopolysaccharide 139A Biosynthesis Gene Cluster From Streptomyces

Streptomyces 139 has been identified to produce a new exopolysaccharide designated EPS 139A that shows anti-rheumatic arthritis activity remarkable in vivo. Qualitative and quantitative sugar analysis showed that the EPS 139A consisted 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. It is possible that 139 A could be developed to be a new drug in the future. For increasing yield and modification the structure of 139 A with the combinatorial biosynthetic pathway, study of biosynthesis gene cluster for 139 A has been carried out.In recent years, several EPS gene clusters have been identified in Gram-positive and Gram-negative bacteria. Genes involved in regulation, nucleotide sugar precursor synthesis, glycosyl transfer, polymerization and export are found in EPS gene clusters. These clusters constitute complex machinery with the biosynthesis of LPSs or CPSs. The repeating polysaccharide units are assembled at a phosphorylated lipid carrier by glycosyltransferases, subsequently polymerized to be a high molecular weight EPS and exported to the cell surface.The strategy of studying EPS 139A biosynthesis is to clone the key gene in the EPS biosynthesis pathway, i.e. the priming glycosyltransferase gene catalyzing the first step of nucleotide sugar transfer. According to the homologous regions of several identified priming glycosyltransferases and taking into account information on Streptomyces codon usage degenerate primers were designed. A distinctive PCR product with the expected size of 0.3 kb was amplified from Streptomyces 139 total genomic DNA. Sequence analysis showed that it is a part of the putative priming glycosyltransferase gene and contains the predicted conserved domain A, B and C. To isolate the EPS 139A gene cluster, a Streptomyces 139 genomic library was constructed with the E. coli-Streptomyces shuttle vector pOJ446. A Streptomyces 139 genomic library was screened by colony hybridization with the PCR-amplified 0.3 kb fragment as a probe, 17 positive colonies were isolated, and the the ste gene cluster for exopolysaccharide biosynthesis in approximately 65 kb chromosomal region of Streptomyces 139 were localized.