芳香聚酮类化合物产生菌的分子筛选及其代谢产物的研究

Study on molecular screening of actinomycetes producing aromatic polyketides and its metabolic products

目的建立一种芳香聚酮类化合物产生菌的基因筛选方法并获得相应结构类型的化合物。方法依据芳香聚酮类化合物生物合成途径所用的II型酮基合酶的同源性设计简并引物,通过PCR方法扩增出约0.6kb目的基因片段,同时通过同源进化和抗菌活性分析对海洋放线菌是否产生芳香聚酮类化合物进行早期评估,并最终从阳性菌中分离得到相应结构类型的化合物。结果利用建立的芳香聚酮类化合物产生菌基因筛选体系对100株海洋放线菌进行筛选,获得了18株携带II型酮基合酶基因的阳性菌株,通过同源进化和抗菌活性分析后从中筛选到1株与多环氧杂蒽酮类化合物lysolipin和xantholipin的酮基合酶基因同源的野野村菌(Nonomuraea sp.)FIM02-765,并最终从该菌中分离得到多环氧杂蒽酮类化合物simaomicinα。结论本研究通过聚酮合酶编码基因的同源进化分析以及建立起来的II型酮基合酶基因与化合物之间的联系,验证了芳香聚酮类化合物产生菌的基因筛选方法的可行性,同时本文还首次从野野村菌(Nonomuraea sp.)中分离得到多环氧杂蒽酮类化合物simaomicinα,这些都为高效利用海洋放线菌资源和基因资源奠定了一定的基础。

Objective To establish type II ketosythase gene screening system and identify the metabolic products correspondence to the results of gene screening. Methods A pair of degenerate PCR primers, targeting ketosynthase gene of aromatic polyketides, was designed and synthesized. The DNA fragment with the length of 0.6kb was amplified by PCR reaction. The phylogenetic analysis and antimicrobial activity was applied to predict the potential of marine-derived actinomycete to produce aromatic polyketide. Finally, the metabolic product, corresponding to the results of gene screening, was isolated by chromatography procedure. Results By PKS- II gene screening, 18 strains containing type II ketosythase gene were picked out from 100 marine actinomycete strains. The phylogenetic analysis based on the protein sequence of type II polyketide synthase and antimicrobial activity demonstrated that strain FIM02-765 belonging to Nonomuraea genus had a potential to synthesize aromatic polyketide compounds structurally related to lysolipins and xantholipin. As a result, a polycyclic xanthone compound, simaomicin a, was isolated from the fermentation broth of strain FIM02-765. Conclusion In our study, phylogenetic and chemical analyses showed a strong correlation between the sequence of type II polyketide synthase and the structure of encoded aromatic polyketides, which demonstrated the feasibility of using this PKS-II gene screening strategy. Meantime, this study firstly reported that the strain affiliated with the genus of Nonomuraea can produce simaomicin α, a polycyclic xanthone-type antibiotic. The above results laid foundation for further exploitation of the resource of marine actinomycetes and genes.