Efficient in vitro refactoring and biosynthetic gene cluster amplification for the overproduction and accelerated discovery of anticancer thioamitides

Thioamitides, a class of highly modified bacterial ribosomally synthesised and post-translationally modified peptides (RiPPs), have potent activities against multiple cancer cell lines. Among these compounds, the structurally divergent thioalbamide combines promising in vivo antiproliferative activity with a superior chemical stability respect to its counterparts. However, thioalbamide is produced in low yields by its genetically intractable native producer and its biosynthetic pathway was initially not productive when transferred into the heterologous host Streptomyces coelicolor M1146. These circumstances substantially hamper to increase the production of this promising compound. Here, we show how in vitro Gibson-like assemblies can be employed for the quick and efficient refactoring of the thioalbamide biosynthetic gene cluster (BGC), leading to substantially increased levels of production in S. coelicolor M1146 through a prioritised selection of promoters. Via this work, PtsrA and PgroEL2 were identified as beneficial additions to the Streptomyces synthetic biology toolbox. We then assessed bacterial genomes for biosynthetic gene clusters (BGCs) predicted to produce thioalbamide-like compounds with improved hydrophilicity. This rational discovery campaign led to the identification a silent thioamitide BGC encoding a thioalbamide-like core peptide but clustered with additional tailoring enzymes, including a previously unknown cupin-fold protein. Applying the refactoring strategy together with the simultaneous expression of multiple BGC copies, we characterised the product of this pathway, thiocupinamide, a polyhydroxylated thioamitide closely related to thioalbamide. We show that thiocupinamide has potent anticancer and antibacterial activities.