AI.zymes: A Modular Platform for Evolutionary Enzyme Design

Abstract

Abstract The ability to create new‐to‐nature enzymes would substantially advance bioengineering, medicine, and the chemical industry. Despite recent breakthroughs in protein design and structure prediction, designing novel biocatalysts remains challenging. Here, we present AI.zymes, a modular platform integrating cutting‐edge protein engineering algorithms within an evolutionary framework ( https://github.com/bunzela/AIzymes ). By combining bioengineering tools such as Rosetta, ESMFold, ProteinMPNN, and FieldTools in iterative rounds of design and selection, AI.zymes can optimize a broad range of catalytically relevant properties. In addition to enhancing transition state affinity and protein stability, AI.zymes can also improve properties that are not targeted by the employed design algorithms. For instance, AI.zymes can enhance electrostatic catalysis by iteratively selecting variants with stronger catalytic electric fields. Benchmarking AI.zymes on the promiscuous Kemp eliminase activity of ketosteroid isomerase led to a 7.7‐fold activity increase after experimentally testing just 7 variants. Due to its modularity, AI.zymes can readily incorporate emerging design algorithms, paving the way for a unifying framework for enzyme design.