title: Identification and functional analysis of the gene ste9 involving in Ebosin biosynthesis from Streptomyces sp. 139.
abstract: Ebosin is a novel exopolysaccharide produced by Streptomyces sp. 139 with remarkable antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste) consisting of 27 ORFs has been identified. For functional analysis, one of the ste genes, ste9, was disrupted and then the gene complementation was performed. The resultant mutant Streptomyces sp. 139 (ste9(-)) produced polysaccharides with molecular weights of about 4.153 × 10(5) which is much smaller than that of Ebosin (9.03 × 10(5)). The complemented strain Streptomyces sp. 139 (pKC9c) showed recovery in the molecular weights of EPS produced (8.004 × 10(5)). As the theoretical protein product of ste9 is a chain length determinant (Wzz) homologue by sequence similarity, ste9 was cloned and expressed in E. coli 086:H2 (wzz(-)) for a complementation test. SDS-PAGE analysis showed that E. coli 086:H2 (wzz(-)) (pET30a-ste9) produced a modal chain length lipid polysaccharide (LPS) similar to that of the wild-type E. coli 086:H2. In addition, the expression of ste9 was able to restore the serum resistance of E. coli 086:H2 (wzz(-)) to almost the level of the wild-type strain. These results indicate that the ste9 gene is coding for a chain length determinant which plays an important role in Ebosin biosynthesis.
authors.affiliation: Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Li
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Qi
authors.forename: Rong
authors.initials: R
authors.lastname: Jiang
authors.forename: Lianhong
authors.initials: L
authors.lastname: Guo
authors.forename: Reng
authors.initials: R
authors.lastname: Zhang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011135:Polysaccharides, Bacterial / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins
keywords: Ebosin biosynthesis; Streptomyces; chain length determinant; exopolysaccharide; gene ste9