Secondary metabolites overproduction through transcriptional gene cluster refactoring

We present a random rational approach enabling the construction of overproducing strains in two steps. The approach first involves creating a library of clusters of interest, in which native promoters are substituted with randomly generated constitutive synthetic promoters, and then expressing this library in an appropriate host strain. This strategy is fast, easy to use, accounts for the architecture of a cluster and completely decouples the expression of a gene cluster from complex native regulatory networks. The strategy was applied to improve the production of a macrocyclic peptide, bottromycin, which possesses antibacterial activity against multidrug-resistant bacteria and is a blueprint for a new class of antibacterials. We successfully optimized the expression of genes in operons and created several variants of the bottromycin gene cluster that provide 5-50 fold higher titres of bottromycin than the natural one, thus resulting in the identification of several new bottromycin derivatives not previously described. Moreover, due to the higher bottromycin yield, bottromycin derivatization was performed via the biosynthetic engineering of the gene cluster. The abovementioned features make this generic strategy a promising tool for the overproduction of known secondary metabolites and the activation of silent secondary metabolites in Actinobacteria.