Timecourse transcriptomics of S. ambofaciens ATCC 23877 pirin defective strain. Streptomyces ambofaciens ATCC 23877

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 in spiramycin-producing strain Streptomyces ambofaciens ATCC 23877 insertional inactivation of pirin-like gene SAM23877_RS18305 (pirA) by C31 Att/Int system-based vectors 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, metabolomic profiling, and genetic complementation we show that most of these effects could be traced to inability of the pirA-defective strain to modulate beta-oxidation pathway leading to unbalanced supplying 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 catalyzing the first committed step of the beta-oxidation pathway.

Pirin蛋白（Pirin）是一类进化保守的含铁蛋白，广泛存在于所有生命界，参与多种分子生物学过程，且多与细胞应激应答密切相关。本研究基于如下实验证据：在产螺旋霉素的链霉菌菌株Streptomyces ambofaciens ATCC 23877中，利用基于ΦC31 Att/Int位点特异性重组系统的载体对pirin样基因SAM23877_RS18305（命名为pirA）实施插入失活后，该菌株的中心碳代谢与能量代谢相关基因表达出现显著改变，对氧化损伤呈现高度敏感性，同时聚酮类抗生素的生物合成受到显著抑制。通过整合转录组学、蛋白质组学、代谢组学分析以及遗传互补实验，我们证实上述多数异常表型均可归因于pirA缺陷菌株无法正常调控β-氧化途径，进而造成聚酮类生物合成所需的前体单体供应失衡。进一步通过计算机模拟蛋白质相互作用建模与体外实验验证，我们证明PirA是一种新型氧化还原敏感型负调控因子，可靶向作用于催化β-氧化途径首个关键不可逆步骤的极长链酰基辅酶A脱氢酶（very long chain acyl-CoA dehydrogenase）。