Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
J Microbiol Biotechnol. 2013 Nov 28;23(11):1500-8. doi: 10.4014/jmb.1305.05035.
Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has obvious antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste), consisting of 27 open reading frames, has been identified. This paper reports our study of the gene functionality of ste8, the predicted protein product of which is homologous to some bacterial chain length determinant Wzz proteins. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain E. coli 086:H2 (Δwzz). The functional complementation of wzz by ste8 was demonstrated by the restoration of wild-type lipopolysaccharide biosynthesis and increased levels of serum resistance of E. coli 086:H2 (Δwzz) (pET30a-ste8). To examine the function of ste8 in ebosin biosynthesis, the gene was knocked out with a double crossover via homologous recombination. The molecular weight of the ebosin derivative EPS-8m produced by the mutant Streptomyces sp. 139 (ste8(-)) was much lower than that of ebosin, and the binding activity of EPS-8m for IL-1R decreased significantly compared with ebosin. These results demonstrate that ste8 encodes a chain length determinant (Wzz) that functions in ebosin biosynthesis.
埃博霉素是由链霉菌 sp. 139 产生的一种新型胞外多糖，具有明显的体内抗风湿性关节炎活性，其生物合成基因簇（ste）已被鉴定，由 27 个开放阅读框组成。本文报道了我们对 ste8 基因功能的研究，其预测的蛋白产物与一些细菌链长决定因子 Wzz 蛋白同源。为了研究 Ste8 的功能，将 ste8 克隆并在突变株大肠杆菌 086:H2（Δwzz）中表达。通过 ste8 对 wzz 的功能互补，恢复了野生型脂多糖的生物合成，并提高了大肠杆菌 086:H2（Δwzz）（pET30a-ste8）的血清抗性水平。为了研究 ste8 在埃博霉素生物合成中的功能，通过同源重组进行了双交换敲除。突变株链霉菌 sp. 139（ste8(-)）产生的埃博霉素衍生物 EPS-8m 的分子量明显低于埃博霉素，并且 EPS-8m 与 IL-1R 的结合活性与埃博霉素相比显著降低。这些结果表明 ste8 编码一个在埃博霉素生物合成中起作用的链长决定因子（Wzz）。
