海洋芽孢杆菌Bacillus methylotrophicus B-9987中difficidins生物合成基因簇的分析鉴定

目的 对海洋芽孢杆菌B-9987中difficidins生物合成基因簇及其关键基因进行分析、鉴定及功能研究。 方法 通过生物信息学手段对difficidins基因簇的基因组成和功能结构域进行了分析；结合其结构特征及聚酮化合物的生物合成原理,初步推导其生物合成途径；构建了烯酰辅酶A水合酶基因difO双交换阻断突变株,对其功能进行初步验证并对基因簇进行确认。 结果 从B-9987发酵液中分离纯化得到化合物oxydifficidin,鉴定了B-9987中该化合物的生物合成基因簇,其是由位于同一个操纵子的15个基因difA-O所编码的反式酰基转移酶聚酮合酶体系组装而成；difO基因阻断后,oxydifficidin不再产生。 结论 difficidins生物合成基因簇的鉴定为后续研究其生物合成途径及相关基因的功能奠定了基础。

Characterization of the biosynthetic gene cluster of difficidin from marine-derived Bacillus methylotrophicus B-9987

Objective To identify and characterize the difficidin biosynthetic gene cluster from Bacillus methylotrophicus B-9987. Method By bioinformatics analysis, the gene organization and functional domain composition of the difficidin gene cluster were predicted. The biosynthetic pathway was deduced by combining the structural characteristics of difficidins and the biosynthesis principle of polyketides. To identify the gene cluster, the difO double crossover mutant was constructed followed by HPLC analysis of fermentation and to probe its function. Result Oxydifficidin was obtained from B-9987 fermentation broth. The difficidin gene cluster of B-9987 was identified, which was encoded by 15 genes, difA-O, located in an operon and assembled via a trans-AT polyketide synthase system. Difficidins were no longer produced in the difO gene deletion mutant. Conclusion TheScharacterization of difficidin gene cluster from Bacillus methylotrophicus B-9987 set the stage for elucidation of theSbiosynthetic molecularSmechanism and further combinatorial biosynthesis of difficidins.