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。
[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 LC-MS for their capability of producing BS. [Results] We defined the boundary of the essential BS biosynthetic gene cluster that contains ten genes blsD to blsM. Disruption of blsK leads to the accumulation of demethylblasticidin S （DBS） and BS. [Conclusion] Ten genes blsD to blsM are sufficient for BS biosynthesis. BlsK was responsible for adding leucine to the β-amino group of arginine-derived side chain of DBS to form leucyldemthylblasticidin S （LDBS）, There are two biosynthetic pathway for BS： DBS is directly methylated to BS; the alternative is that DBS is first converted to LDBS that is then methylated and finally hydrolyzed into BS.
出处
《微生物学通报》
CAS
CSCD
北大核心
2014年第7期1318-1325,共8页
Microbiology China
基金
国家973计划项目(No.2012CB721004)
