Andriy Luzhetskyy

1 Introduction Despite the obvious progress in medicinal chemistry, the natural products and their derivatives still remain the leading players on the pharmaceutical market, especially among approved anticancer and antibacterial drugs (Newman and Cragg 2020).

Thioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing.