Heterologous production of thiostrepton A and biosynthetic engineering of thiostrepton analogs

Abstract

Thiostrepton A 1, produced by Streptomyces laurentii ATCC 31255 (S. laurentii), is one of the more well-recognized thiopeptide metabolites. Thiostrepton A 1 and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens. Although numerous lines of evidence have established that the thiopeptides arise from the post-translational modification of ribosomally-synthesized peptides, few details have been revealed concerning this elaborate process. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton post-translational machinery. Due to the difficulties in the genetic manipulation of S. laurentii, the heterologous production of thiostrepton A 1 from an alternate streptomycete host was sought to facilitate the biosynthetic investigations of the peptide metabolite. The production of thiostrepton A 1 from the non-cognate hosts did not lend itself to be as robust as S. laurentii-based production, therefore an alternate strategy was pursued for the production of thiostrepton variants. The introduction of a fosmid used in the heterologous production of thiostrepton A 1, harboring the entire thiostrepton biosynthetic gene cluster, into the tsrA deletion mutant permitted restoration of thiostrepton A 1 production near to that of the wild-type level. The fosmid was then engineered to enable the replacement of wild-type tsrA. Introduction of expression fosmids encoding alternate TsrA sequences into the S. laurentii tsrA deletion mutant led to the production of thiostrepton variants retaining antibacterial activity, demonstrating the utility of this expression platform toward thiopeptide engineering.