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

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 in vivo and in vitro 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.