Uncovering Untapped Carboxylic Acid Reductases (CARs) for One-Step Biosynthesis and Diversification of Bioactive Nitrogen-Containing Heterocycles

Abstract

<p>Nitrogen-containing heterocycles (N-heterocycles) are essential in drug discovery, with a majority of best-selling small-molecule drugs containing at least one N-heterocycle. Various enzymes have been characterized or engineered for N-heterocycle biosynthesis, but these attempts are frequently restricted to specific N-heterocycle skeletons and substitutes. Multidomain carboxylic acid reductase (CAR) is gaining attention for its ability to reduce amino acids to amino aldehydes, which can be converted into a variety of N-heterocycle skeletons through nonenzymatic dimerization and rearrangement. This unique cyclization process could expand the biocatalytic potential of CARs beyond those of specific N-heterocycles. Here, we applied domain-based genome mining to identify 32,735 CAR enzymes, providing the most comprehensive landscape of CARs to date. Through <em>in vivo</em> and <em>in vitro</em> assays, we characterized an untapped CAR family that exhibited substrate tolerance on amino acids and efficiently catalyzed the formation of various N-heterocycles from amino acids. By harnessing the potential of promiscuous CARs for one-step biosynthesis and diversification of N-heterocycles, we created a library of N-heterocycles for bioassays, identifying multiple quorum sensing (QS) inhibitors and protease inhibitors. Our discovery of untapped promiscuous CARs offers a promising alternative for the biosynthesis and diversification of N-heterocycles, making them a valuable resource for chemical and synthetic biologists.<br></p>