杀稻瘟菌素生物合成基因簇的边界确定.docVIP

————————————————————————————————————————————————————— 杀稻瘟菌素生物合成基因簇的边界确定 Microbiology China tongbao@ 微生物学通报 Jul. 20, 2014, 41(7): 1318?1325 ? 2014 by Institute of Microbiology, CAS DOI: 10.13344/j.microbiol.china.130557 杜爱芹 吴俊 邓子新 贺新义* (上海交通大学 生命科学技术学院 微生物学代谢国家重点实验室 上海 200030) 摘 要：【目的】杀稻瘟菌素(BS)是六元糖环肽核苷类抗生素，在农业和基因工程方面有重要应用。由于在其原始产生菌中很难进行基因操作，且许多合成步骤未知，为了增加对此基因簇的认识，为后续生物合成途径的推导和改造提供更多依据，在变铅青链霉菌中确定该基因簇的边界，并对blsK基因功能进行初步研究。【方法】利用PCR-targeting法对已测序基因簇中的基因进行基因置换得到突变株，LC-MS检测突变株发酵液的产物从而确定基因置换是否影响杀稻瘟菌素的产生。【结果】明确了杀稻瘟菌素基因簇包含blsD–blsM共10个基因；在blsK的基因置换突变株中检测到去甲基杀稻瘟菌素(DBS)和少量杀稻瘟菌素的积累。【结论】blsD–blsM这10个基因负责杀稻瘟菌素的生成；从blsK的突变株产物积累结果推测，BlsK蛋白负责加载亮氨酸到DBS的精氨酸衍生侧链的β-氨基上，生成N-亮氨酰化去甲基杀稻瘟菌素(LDBS)；BS生物合成途径有两条，一条是由DBS直接甲基化生成BS；另一条是由DBS转化成LDBS继而由LDBS甲基化和水解生成BS。 关键词：杀稻瘟菌素，基因簇，PCR-targeting，去甲基杀稻瘟菌素，生物合成途径 Determination of the boundary of blasticidin S biosynthetic gene cluster DU Ai-Qin WU Jun DENG Zi-Xin HE Xin-Yi* (State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China) Abstract: [Objective] Blasticidin S (BS) is a hexose-containing peptidyl-nucleoside antibiotic widely used as fungicidal in agriculture and as an effective selection reagent in eukaryotic cells. Genetic manipulation in the native producer of BS was never successful, which hinder the studies of BS biosynthesis. We recently engrafted the BS biosynthetic gene cluster into the chromosome of Streptomyces lividans and achieved heterologous production of BS. In this study, we tried to localize the boundary of BS gene cluster to identify the essential genes, providing functional insights into its remaining unknown biosynthetic steps. [Methods] PCR-targeting was used to carry out gene replacements in S. lividans-derived BS producer. The mutants were fermented and assayed by 基金项目：国家973计划项目(No. 2012CB721004) *通讯作者：Tel：86-212131；：xyhe@ 收稿日期：2013-09-01；接受日期：2013-10-15；