Cell-free directed evolution of [iron iron] hydrogenases

Hydrogenase enzymes catalyze the formation or consumption of hydrogen gas, which is regarded as a potential pollution-free energy carrier. Hydrogenases have the potential to enable large-scale biological hydrogen production schemes or to replace the expensive metal catalysts that activate hydrogen in fuel cells. However, the evolutionary forces that produced these catalytic marvels have optimized them for use in their natural hosts, leaving them lacking in certain industrially important traits. [FeFe] hydrogenases, which produce hydrogen at high turnover rates with a unique iron-sulfur center called the H-cluster, are extremely sensitive to oxygen, making them difficult to apply in industrial situations. The development of an [FeFe] hydrogenase variant resistant to deactivation by oxygen could represent a turning point for biological energy conversion.;Directed evolution is an established method for identifying beneficial protein mutations that could not have been predicted rationally. The procedure mimics natural evolution by creating a library of mutants and selecting the fittest among them. At the heart of a directed evolution effort is a high-throughput method for evaluating each mutant for the desired phenotype.;We have established an E. coli-based cell-free protein synthesis (CFPS) system that allows in vitro transcription, translation, and activation of [FeFe] hydrogenases. Three maturase proteins are required for H-cluster assembly and insertion into the hydrogenase scaffold. These proteins are not naturally present in the E. coli extract source strain. Heterologous expression of the three maturases in an anaerobic E. coli fermentation followed by cell lysis yielded a cell extract capable of [FeFe] hydrogenase maturation. We then used this technology as the basis to develop two separate high-throughput screens capable of identifying oxygen-tolerant [FeFe] hydrogenase mutants.;In one screening method, mutant genes are isolated in the wells of microtiter plates, amplified by single-molecule polymerase chain reaction (smPCR), expressed by CFPS, and assayed for activity before and after exposure to oxygen. This screen is also capable of identifying mutants with increased specific activity, and we have characterized one such mutant.;To enable screening of larger libraries, a second method was developed in which mutant hydrogenase genes are isolated and expressed using in vitro compartmentalization. The CFPS mixture is emulsified into a continuous oil phase, creating billions of independent picoliter-scale reactors. Within each droplet a different mutant gene is transcribed and translated, and the gene and the protein product are attached to the surface of a microbead. We have linked hydrogenase activity to the generation of a fluorescent signal that allows sorting of beads displaying positive mutants by fluorescence-activated cell sorting (FACS).