tRNA-deacylase-directed discovery of biosynthetic pathways

Amino acids are one of nature's most privileged building blocks for generating molecular diversity on length scales ranging from small molecules to proteins. While amino acid products arising from certain class-defined biosynthetic pathways can be found with established bioinformatic strategies, those that fall outside of these types remain difficult to identify. Here, to address this challenge, we have developed an approach to find biosynthetic gene clusters (BGCs) that utilize and modify amino acid monomers while remaining agnostic to biosynthetic class. We demonstrate that tRNA deacylases specific for host-synthesized non-canonical amino acids (ncAAs) serve as a common genomic marker of ncAA metabolism. Using this approach, we show that thousands of cryptic BGCs can be identified and demonstrate the discovery of BGCs for several distinct ncAAs as well as a hydrazide-containing tripeptide. We anticipate this approach will have broad applications for discovering natural products with ncAAs and beyond.