Construction of high-producing strains of kirromycin

Abstract:

[Background] Kirromycin is a complex linear polyketide-nonribosomal peptide compound produced by Streptomyces collinus Tü 365, which interacts with the bacterial elongation factor EF-Tu, thereby inhibiting protein synthesis and exhibiting narrow-spectrum antibacterial activity. Currently, kirromycin is widely used as an experimental reagent in studying ribosomal structure and function. Recent studies have reported that kirromycin and its analogs exhibit superior activity against Wolbachia compared to the registered drug doxycycline, making them potential lead compounds for treating filarial worm infections. [Objective] Combinatorial metabolic engineering methods were used to construct high-production strains of kirromycin to enhance kirromycin production and lay the foundation for further applications. [Methods] Based on the kirromycin biosynthetic gene cluster and biosynthetic pathways analysis, overexpression or co-overexpression of the genes encoding crotonyl CoA reductase (kirN), aspartate-1-decarboxylase (kirD), phosphopantetheinyl transferase (kirP), efflux pump proteins of the major facilitator superfamily (kirTI and kirTII), and acetyl-CoA carboxylase (acc) from Streptomyces coelicolor A3(2) under the control of constitutive promoter kasOp* were performed to increase kirromycin production in S. collinus. [Results] Overexpression or co-overexpression of the aforementioned genes had a certain effect on increasing kirromycin production. Better results were observed from the overexpression of the acc gene and co-overexpression of kirD, kirN, kirP, kirTI, and kirTII genes. Compared to the wild-type strain, kirromycin production increased by 57.8% and 65.6%, reaching 198.3 mg/L and 208.1 mg/L, respectively. A systematic search for homologous proteins-coding genes of kirromycin biosynthetic enzymes in publicly available bacterial genome databases revealed 31 biosynthetic gene clusters potentially synthesizing kirromycin-like natural products. [Conclusion] This study effectively increased kirromycin production in S. collinus using metabolic engineering methods, laying a solid foundation for further applications and development of kirromycin.