An efficient lysate-based approach for biosynthesis of the pyrrolobenzodiazepine natural product tilimycin

Many bacteria use nonribosomal peptide synthetases (NRPSs), a family of multidomain enzymes that produce peptide natural products using an assembly line strategy. One class of such compounds are pyrrolobenzodiazepines, which have DNA alkylating activity. One example is tilimycin, a compound produced by the human gut microbiota that plays a role in epithelial damage during antibiotic-associated dysbiosis. The production of analogs of these natural products may facilitate the discovery of novel bioactive molecules. However, the synthesis of these natural products typically requires significant resources and time to produce in sufficient amounts for microbial and biochemical testing. Biocatalysis offers an environmentally friendly approach, but enzyme yield and stability, particularly with large multidomain enzymes that are often used in natural product pathways, can limit biochemical reactions with purified protein. Here, we demonstrate a cell lysate-based method to synthesize the nonribosomal peptide natural product tilimycin directly from the substrates 3-hydroxyanthranilic acid and L-proline with lysates from E. coli cell lines that express the tilimycin biosynthetic proteins. We present our protocol optimization and scale-up to produce tilimycin in a fast, efficient manner.