Cloning, expression and characterization of the Ste6 gene encoding a UDP-glucose dehydrogenase in Streptomyces

Streptomyces sp. 139 was identified to produce a new exopolysaccharide Ebosin (139A) with antirheumatic arthritis activity in vivo. The Ebosin biosynthesis gene cluster(31.3 kb; GenBank Accession Number: AY131229) containing 22 ORFs (ste1-ste22) of Streptomyces sp. 139 had been reported previously. In this paper,we present experimental evidence for the identity of the ste6 gene product as a UDP-glucose dehydrogenase (UDPGDH). With pET-30a as vector,the gene was cloned and expressed in Escherichia coli BL21 (DE3). The expressed protein was purified to homogeneity by His-Bind resin affinity chromatograpy and it was able to catalyze UDP-glucose to UDP-glucuronic acid. To evaluate the function of ste6, the gene was disrupted by a single-crossover homologous recombination event and the result showed that ste6 is required in Ebosin biosynthesis.