BE-7585A is an angucycline class natural product produced by Amycolatopsis orientalis subsp. vinearia BA-07585, which possesses an unusual thiosugar moiety. While sulfur is an essential element in biological systems, few thiosugars exist in nature. The primary focus of this research is to elucidate the biosynthetic pathway for sulfur incorporation into the thiosugar product. If successful, this will be the first established pathway of thiosugar biosynthesis. To accomplish this goal, a cosmid library was constructed from A. orientalis genomic DNA and was screened by designed primers to locate the BE-7585A biosynthetic gene cluster. We have successfully identified a putative gene cluster containing the angucycline core and deoxysugar biosynthetic genes. Interestingly, ORF34, which is similar to the established thiazole biosynthesis protein (ThiG) in thiamin biosynthesis, may play an important role in thiosugar formation. Using the ThiG-mediated reaction as a guide, we have proposed a sulfur incorporation mechanism for the thiosugar moiety of BE-7585A. ORF34 was expressed, purified and incubated with putative substrates. While 1-deoxy-D-xylulose-5-phosphate (the substrate of ThiG) did not react with ORF34, D-glucose-6-phosphate formed a covalent adduct with the Lys110 residue of ORF34. This observation supports our proposed thiosugar biosynthesis and suggests difference between ORF34- and ThiG- mediated catalysis. A proposed biosynthetic pathway for BE-7585A, including angucycline core formation, rhodinose biosynthesis, and thiosugar biosynthesis, will be discussed.