三烷基取代芳香聚酮gombapyrones生物合成基因簇的确认及其生物合成推导

Investigation of the biosynthetic gene cluster of trialkyl-substituted aromatic polyketide gombapyrones from Streptomyces rubellomurinus

摘要:

【目的】本研究旨在确认链霉菌Streptomyces rubellomurinus ATCC 31215来源芳香聚酮化合物(gombapyrones, GOMs)的生物合成基因簇(biosynthetic gene cluster, BGC)，并对其生物合成途径进行推导。【方法】对链霉菌S. rubellomurinus ATCC 31215进行大规模发酵及提取分离，得到GOM-B和GOM-D；以三烷基取代芳香聚酮生物合成途径保守存在的P450单氧化酶的蛋白序列作为探针，在GOMs产生菌S. rubellomurinus基因组中进行BLAST搜索获得潜在的GOMs生物合成基因簇(gom BGC)；通过对gom BGC中的聚酮合成酶(polyketide synthase, PKS)结构基因进行同框缺失突变，对突变株发酵产物进行高效液相色谱-质谱(high performance liquid chromatography-mass spectrometry, HPLC-MS)分析以确认gom BGC与GOMs的产生相关；基于生物信息学分析，推导GOM-B的生物合成途径。【结果】从S. rubellomurinus发酵产物中分离得到了化合物GOM-B和GOM-D，并通过一维核磁数据分析确认其结构。从S. rubellomurinus基因组中获得了潜在的GOMs生物合成基因簇gom BGC，相关基因序列和功能注释已递交PubMed数据库(GenBank编号：OQ831859)；基因缺失突变PKS基因gomB后导致S. rubellomurinus发酵产物中GOM-B和GOM-D组分消失；基于生物信息学分析对GOM-B的I型PKS生物合成途径进行了推导。【结论】首次确认了三烷基取代芳香聚酮GOM-B和GOM-D的生物合成基因簇gom BGC；该生物合成途径属于I型PKS，推测其中P450单氧化酶GomJ具有独特的多烯聚酮链芳构化功能；与最近报道的GOM-G生物合成基因簇gbn BGC相比较，gom BGC编码的PKS装配线缺少一个碳链延伸功能模块，这与其产物GOM-B的碳骨架结构一致；本工作所报道的gom BGC展现了细菌I型聚酮生物合成基因演变导致其产物结构多样性的范例；gom BGC编码的P450氧化酶GomJ与已报道同源蛋白(78.3%序列相同) GbnP相比，作用底物分子骨架少了2个碳，因此是探究细菌三烷基取代芳香聚酮芳构化酶反应底物耐受性的理想材料。

Abstract:

[Objective] To identify the biosynthetic gene cluster (BGC) of gombapyrones (GOMs), the trialkyl-substituted aromatic polyketides derived from Streptomyces rubellomurinus ATCC 31215, and deduce the biosynthetic pathway. [Methods] GOM-B and GOM-D were extracted for the large-scale fermentation broth of S. rubellomurinus ATCC 31215. The P450 monooxygenase catalyzing the polyene chain aromatization is conserved in the biosynthetic pathways of trialkyl-substituted aromatic polyketides in bacteria. Thus, BLAST searching was carried out with the P450 monooxygenase as a probe to identify the candidate BGC for GOMs (gom BGC) from the genome of S. rubellomurinus. Through deletion of the polyketide synthase (PKS) gene in gom BGC and high performance liquid chromatography-mass spectrometry (HPLC-MS) identification of the fermentation products of the mutant strain, the gom BGC can be confirmed to direct the production of GOMs. Furthermore, bioinformatics tools were used to deduce the biosynthetic pathway of GOM-B. [Results] GOM-B and GOM-D were extracted from the fermentation broth of S. rubellomurinus and their structures were identified by NMR. The gom BGC identified in the work was submitted to the PubMed, with the GenBank accession number: OQ831859. The deletion of a PKS gene gomB resulted in the disappearance of GOM-B and GOM-D in the fermentation broth. The biosynthetic pathway of GOM-B was deduced as a type I PKS based on bioinformatics analysis. [Conclusion] This work identified a new BGC which directs the biosynthesis of trialkyl-substituted aromatic polyketides, including GOM-B and GOM-D. The type I PKS involves a P450 monooxygenase GomJ putatively catalyzing the unique polyene chain aromatization. Compared with the gbn BGC recently reported to direct the GOM-G biosynthesis, the PKS assembly line encoded by gom BGC lacks one elongation module, which is consistent with the carbon skeleton of GOM-B. The gom BGC could serve as an example that the gene evolution of bacterial type I PKS leads to the structural diversity of polyketides. Moreover, GomJ shows 78.3% sequence identity to the P450 monooxygenase GbnP functionally identified from gbn BGC, whereas the putative substrate of GomJ has two carbons less than the substrate of GbnP. Thus, the discovery of this work helps to decipher the substrate-specificity mechanism of the unusual P450 monooxygenase which catalyzes polyene chain aromatization in the biosynthetic pathways of trialkyl-substituted aromatic polyketides in bacteria.