Bifunctional small molecules for biosensing and continuous enzyme evolution

The National Institutes of Health Office of the Director awarded the University of California Santa Cruz $714,870 on September 4, 2026, under the Trans-NIH Research Support program (CFDA 93.310) to develop bifunctional small molecules for biosensing and continuous enzyme evolution.

The project addresses two bottlenecks in directed enzyme evolution: the low throughput of classical analytical methods, which explore only 10² variants per round, and the labor-intensive manual operations between rounds. The recipient will develop molecular tools—specifically bifunctional small molecules that engage two proteins and can be delivered into E. coli—to enable continuous phage-assisted evolution. This approach can explore approximately 10¹¹ variants with continuous selection over three days, and conduct hundreds of generations per day. The work includes a strategy for general small-molecule biosensing in E. coli and a platform to use bifunctional small molecules to control cellular processes, enabling high-throughput selection for enzymes that produce small molecules.

Work is performed in Santa Cruz, California. The period of performance runs from September 4, 2026, through June 30, 2031. The award is a Project Grant, classified under the assistance type of project grant, subject to standard NIH peer-review merit criteria, annual performance evaluation, and three-year record retention requirements.

Abstract

Enzymes are the world's most skilled organic chemists. At their best, enzymes outperform traditional chemical reagents in every consideration from cost, to selectivity and product purity, to environmental waste. However, finding an enzyme for a new reaction remains prohibitively difficult. There are two problems: first, low-throughput analytical chemistry (such as liquid chromatography-mass spectrometry) limits classical directed evolution to 102 variants per round; second, manual operations to generate mutant libraries take days between rounds. Continuous evolution is the solution. The rapid phage life cycle and ultra-high mutation rates in phage-assisted continuous evolution explore vastly more sequence space (~1011 variants with continuous selection over 3 days) than classical directed evolution. These numbers are sufficient to cover all double mutants and most triple mutants for a small evolving protein in a single generation of phage, with hundreds of generations per day. However, we cannot conduct selections for enzymes that make small molecules because we lack the molecular tools. We need biosensors to measure small-molecule concentration-for any small molecule-and translate that signal into gene expression in order to discriminate between low-producing and high-producing enzymes. My previous work revealed a technique to deliver large (CA. 1 KDA) small molecules into E. Coli, the host organism for phage-assisted continuous evolution. This discovery sets the stage to control cellular processes in E. Coli with bifunctional small molecules, molecules that engage two proteins with two ligands connected by a linker. Bifunctionals have revolutionized the study of (more permeable) mammalian cells in chemical biology. Armed with these tools and a means to deliver them, I propose: (1) a strategy for general small-molecule biosensing in E. Coli; (2) a platform to use these biosensors for continuous directed evolution of enzymes; (3) a fully enzymatic replacement of peptide synthesis reagents; and (4), a chemoenzymatic route to previously inaccessible glycopeptide antibiotics. The proposal constitutes a high-risk, high-reward, vision to completely reshape catalysis using continuous evolution, with broad impacts on natural and designed enzymes.