| Streptomyces sp.139 was identified to produce a new exopolysaccharide designatedEbosin in our laboratory. Qualitative and quantitative sugar analysis showed that Ebosinconsisted of galactose, arabinose, mannose, fucose, xylose, rhamnose, galacturonic acid andglucose with a molar ratio of 19:16:5.0:5.0:4.0:3.0:3.0:2.0. The structure of the repeating unitsof sugars for Ebosin has been identified. The pharmacology studies shows that it hasremarkable anti-rheumatic arthritis activity in vivo and the application for the clinical trial hasbeen made. It is possible that Ebosin will be developed to be a new drug treating rheumaticarthritis.The biosynthesis gene cluster of Ebosin in Streptomyces sp.139 was confirmed by DrWang Lingyan in our laboratory. The cluster consists of 22 ORFs designated stel to ste22,including the functions related to regulation(stel-ste4), nucleotide sugar precursorsynthesis(ste6, ste10, ste11, ste17 and ste19), glycosyltransfer(ste5, ste7, ste15 and ste22),polymerization and export (ste8, ste9, ste13, ste14 and ste21), and modification (ste12, ste16,ste18, ste20). On these bases, the further studies have been carried out and some of results arepresented in this paper.Genes ste15, ste17 and ste19 were disrupted respectively by the double crossover viahomologous recombination. EPS-m produced by these mutants of Streptomyces sp. 139 wereanalyzed individually monosaccharide composition, molecular mass and bioactivity. Theseresults demonstrated that the ste15, ste17 and ste19 are functionally involved in Ebosinbiosynthesis with different ways. 1. The ste15 is deduced to encode a glycosyltransferase (GTF) appeared to be involved insequential transfer of glucose from nucleotide glucose to the repeating units on a lipid carrierfor subsequent polymerization in biosynthesis pathway of Ebosin.2. The ste17 is putatively to encode a glucose-1-phosphate cytidylyltransferase catalyzingD-glucose-1-phosphate and CTP to form CDP-glucose. As one of the nucleotide sugarprecursors, CDP -glucose is involved in the biosynthesis of Ebosin. The ste17 was cloned andexpression in E.coli, and the recombinant protein of the ste17 was purified.3. The ste19 is identified to encode a UDP-glucose 4-epimerase, which mainly convertsUDP-galactose to UDP-glucose. As the nucleotide sugar precursors, both UDP-glucose andUDP-galactose are involved in the biosynthesis pathway of Ebosin.Using the 0.9 kb DNA fragment (containing sequences of ste22) as probe, the genelaborary of Streptomyces sp. 139 was screened. With the chromosomal walking, the biosynthesisgene cluster of Ebosin was identified to be ending at ste27. |
