Streptomyces sahachiroi ATCC 33158中孢子色素合成基因簇(sah)的鉴定

摘要

[目的]Streptomyces sahachiroi ATCC 33158全基因组测序后,通过生物信息学分析找到一个Ⅱ型聚酮合酶( polyketide synthase,PKS)基因簇sah.我们通过基因敲除和异源表达的方法对sah的生物学功能进行了研究.[方法和结果]对sah基因簇ORF( open reading frame)进行分析后发现,除了一个额外的氧甲基转移酶基因sahI以外,该基因簇与天蓝色链霉菌中负责孢子色素合成的基因簇whiE具有很高的相似性.将sah中负责后修饰的3个基因sahG、sahH和sahI分别敲除后,发现孢子色素的颜色随之发生明显的变化.将sah-minimal PKS基因和whiE-minimal PKS基因分别导入变铅青链霉菌ZX1中进行异源表达,高效液相色谱及液质联用分析证实它们产生相同的水溶性红色色素物质.[结论]sah与whiE基因簇具有相似的生物学功能,负责链霉菌孢子色素的合成.这两个基因簇所合成的孢子色素具有相同的母核,差别在于sah基因簇中多了一个编码氧甲基转移酶的后修饰基因,这可能是导致两种链霉菌孢子在颜色上有细微差别的原因.%[Objective ] A putative type II polyketide synthase ( PKS ) gene cluster ( sah ) was found in the genome sequence of Streptomyces sahachiroi ATCC 33158 by bioinformatics. In order to discover its biological function, we cloned the sah cluster and made functional analyses by gene knockout and heterologous expression. [Methods & Results] Annotation of the predicted open reading frames (ORFs) by protein-protein blast revealed that the sah cluster is highly homologous with the spore-pigment biosynthetic cluster ( whiE) in S. coelicolor A3 ( 2 ) , except an additional 0-methyltransferase gene (sahl). Three genes of the sah cluster, sahG, sahH and sahl, which are putatively involved in post-PKS modifications, were respectively in-frame deleted by double crossover. We observed the color of the mutant strains spores apparently changed. HPLC and LC-MS analyses demonstrated that heterologous expression of the sah-minimal PKS gene and the whiE-minimal PKS gene in S. lividans ZX1 produced the same hydrophilic red pigments. [ Conclusion] The sah cluster is responsible for the spore-pigment biosynthesis of S. sahachiroi ATCC 33158. It catalyzes the formation of polyketides with the same core structures as that of the whiE cluster.