Multilevel regulation of gene expression in brasilicardin biosynthetic gene cluster

Brasilicardin A, BraA, is a secondary metabolite produced by the bacterium Nocardia terpenica, and a promising drug due to potent immunosuppressive activity and low cytotoxicity. Currently, a semisynthetic approach confers production of a complete compound but suffers from insufficient biosynthesis of BraA intermediates used in the chemical synthesis steps leading to only lab scale quantities of the compound. A better understanding of gene expression regulatory pathways within the brasilicardin biosynthetic gene cluster, Bra-BGC, is a prerequisite to further improvements in the biosynthesis titers. However, a transcriptional regulation in Bra-BGC has only been superficially analyzed, till now. In this study, we comprehensively analyze the functions of several unstudied transcriptional regulators, KstR, SdpR and OmpR, encoded within the close vicinity of the Bra-BGC, and delve the role of the previously described cluster-situated activator Bra12. We present, that the Bra12 and the novel regulator SdpR, bind several DNA sequences located in the promoter regions of the genes essential for BraA biosynthesis. Subsequently, we demonstrate complex regulatory network through which both regulators are capable of controlling activity of those gene promoters and thus gene expression in Bra-BGC. Furthermore, using heterologous producer strain Amycolatopsis japonicum, we present, that Bra12 and SdpR regulators play opposite roles in brasilicardin congeners biosynthesis in. Finally, we propose a comprehensive model of multilevel gene expression regulation in Bra-BGC and propose the roles of locally encoded transcriptional regulators Overall design: Differential whole transcriptome shotgun sequencing of Nocardia terpenica IFM 0406 was performed in duplicates at two time points of growth.