Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy.
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Palermo, Italy; Advanced Technologies Network (ATeN) Center, University of Palermo, Palermo, Italy.
Metab Eng. 2018 Jul;48:254-268. doi: 10.1016/j.ymben.2018.06.008. Epub 2018 Jun 23.
Pirins are evolutionarily conserved iron-containing proteins that are found in all kingdoms of life, and have been implicated in diverse molecular processes, mostly associated with cellular stress. In the present study, we started from the evidence that the insertional inactivation of pirin-like gene SAM23877_RS18305 (pirA) by ΦC31 Att/Int system-based vectors in spiramycin-producing strain Streptomyces ambofaciens ATCC 23877 resulted in marked effects on central carbon and energy metabolism gene expression, high sensitivity to oxidative injury and repression of polyketide antibiotic production. By using integrated transcriptomic, proteomic and metabolite profiling, together with genetic complementation, we here show that most of these effects could be traced to the inability of the pirA-defective strain to modulate beta-oxidation pathway, leading to an unbalanced supply of precursor monomers for polyketide biosynthesis. Indeed, in silico protein-protein interaction modeling and in vitro experimental validation allowed us to demonstrate that PirA is a novel redox-sensitive negative modulator of very long-chain acyl-CoA dehydrogenase, which catalyzes the first committed step of the beta-oxidation pathway.
吡嗪是一种进化上保守的含铁蛋白，存在于所有生命领域，与多种分子过程有关，主要与细胞应激有关。在本研究中，我们首先发现，通过 ΦC31 Att/Int 系统载体对螺旋霉素产生菌变铅青链霉菌 ATCC 23877 中的吡嗪样基因 SAM23877_RS18305（pirA）进行插入失活，会对中心碳和能量代谢基因表达产生显著影响，对氧化损伤高度敏感，并抑制聚酮类抗生素的产生。通过整合转录组学、蛋白质组学和代谢物分析，以及遗传互补，我们在这里表明，这些影响大多数可以追溯到 pirA 缺陷菌株无法调节β-氧化途径，导致聚酮类生物合成的前体单体供应不平衡。事实上，通过计算机蛋白质-蛋白质相互作用模型和体外实验验证，我们证明了 PirA 是一种新型的、对氧化还原敏感的非常长链酰基辅酶 A 脱氢酶的负调节剂，该酶催化β-氧化途径的第一个关键步骤。
