1. Studies Of Functions Of Genes Ste22, Ste23 And Characterization Of Ste 19 In Streptomyces Sp.139 2. Construction Of Genomic Library Of Streptomyces Verticillus Var. Pingyangensis N.SP.

Streptomyces, a gram-positive bacterium, is well known as an important industrial microorganism for its production of antibiotics. However, exopolysaccharide (EPS) production in Streptomyces was not reported before despite the fast progress at molecular level in other bacterial species (e.g. in lactic acid bacteria). A screening model for IL-1R antagonist was constructed in our lab, through which Streptomyces sp. 139 was obtained. Ebosin produced by S. sp. 139 is a new heteroexopolysaccharide with repeating unit consisting of galactose, mannose, glucose, arabinose, fucose, xylose, rhamnose and galacturonic acid. Ebosin has obvious anti-rheumatic arthritis activity in vivo and very low toxity. Now Ebosin is being applied for clinic study and may be a promising new drug.Ebosin biosynthesis gene cluster (ste) with the length of 34kb containing 24 ORFs was first cloned from 5. sp. 139 by Dr. Wang Lingyan. Functions of these ORFs should be characterized and identified profoundly to elucidate Ebosin biosynthesis pathway and to lay a foundation for obtaining new derivatives of Ebosin through modify the biosynthesis paths consciously by recombinative biology. We selected ste\9, ste22 and ste23 as research objects in this paper.The stel9 gene in ste gene cluster was identified to encode a new UDP-Glucose-4-epimerase by data base comparison and experimental validation. The enzyme catalyzes the interconversion of UDP-Glucose and UDP-galactose. The gene had a length of 1026 bp and encoded for a protein with 341 amino acids with deduced molecular weight of 36.5kD. To identify the function of ste 19, ste19 was cloned into E. coli expression plasmid pET-30a. SDS-PAGE of expressed product showed there was a new band in site of 37KD as expected. Recombiant protein was purified by His-Bind Resin with the His-tag and the purity was 92.9%. Activity analysis showed the expressed product had the activity of UDP-Glucose 4-epimerase. The characterizations of the enzyme such as Km, opium temperature and opium pH were investigated.The gene ste22 had a length of 948bp and encoded for a protein with 315 amino acids with deduced molecular weight of 35.5kD. The product encoded by ste22...