Engineering in vivo and in vitro systems for expression and activation of [FeFe] hydrogenases [electronic resource]
Keywords: Chemical Engineering

Hydrogenases reversibly catalyze the reduction of protons into molecular hydrogen using unique iron-based cofactors. Compared to [NiFe] and [Fe]-only hydrogenases, [FeFe] hydrogenases preferentially and more rapidly evolve hydrogen. These enzymes also have turnover rates similar to platinum catalysts, and are thus being considered as the central catalysts for novel renewable energy technologies. Researchers have developed solid-state devices and engineered microbes that use hydrogenases to produce hydrogen, yet, several constraints must be addressed before such biotechnologies can be deployed at industrial scales. For example, the [FeFe] hydrogenase iron-sulfur clusters and catalytic cofactor, termed the H-cluster, can be damaged by molecular oxygen. This susceptibility to oxygen not only limits the use of these enzymes, but it also makes their production quite challenging. Furthermore, additional proteins called the HydE, HydF, and HydG maturases are necessary for assembling the H-cluster and activating the [FeFe] hydrogenases, and these biosynthetic pathways remain poorly understood. Although microorganisms have been engineered to overexpress [FeFe] hydrogenases, no reported systems can provide sufficient yields to make the production of these enzymes both scalable and cost-effective for the application of hydrogenases at the industrial level. We report the development of multiple Escherichia coli based approaches for the production of active [FeFe] hydrogenases. Not only do we create an anaerobic cell-free hydrogenase expression platform, which has been instrumental in our efforts to evolve an oxygen-tolerant enzyme, but we also detail the first example of post-translational hydrogenase activation that is dependent on several extrinsic small molecules. By using tools such as design of experimentation, we elucidate that S-adenosyl methionine, cysteine, and tyrosine are required for reconstructing the H-cluster biosynthetic pathway in a cell-free environment. We also describe an improved in vivo expression system in which, relative to other published methods, hydrogenase yields are substantially higher. Furthermore, the specific activities of purified [FeFe] hydrogenases are comparable to those reported for enzyme isolated from the native organism. To develop such a system, we combined media optimizations with using an E. coli strain engineered for improved maturation of proteins containing iron-sulfur clusters. These advancements increased the production of active hydrogenase, resulting in 30 milligrams of isolated and fully matured enzyme per liter of culture, which exceeds the highest reported yields by more than 20-fold. The same in vivo methods were used for individual expression of the HydE, HydF, and HydG maturases, facilitating the development of an improved in vitro maturation platform. The efficacy and scalability of this enhanced in vitro system enables the routine preparation of isolated and fully active hydrogenase at the milligram scale. We are therefore able to utilize isotopic labeling approaches along with Fourier transform infrared spectroscopy to investigate the H-cluster biosynthetic pathway. In doing so, we conclusively prove that tyrosine is the substrate for the carbon monoxide and cyanide ligands associated with the H-cluster [2Fe] sub-cluster that is required to activate the [FeFe] hydrogenases. Certainly, the interest in sustainable energy technologies has never been higher, and this work provides substantial advances toward the understanding of [FeFe] hydrogenase activation as well as new methods for further investigating how to effectively synthesize these excellent hydrogen-producing catalysts. The capability to readily produce large quantities of hydrogenases will also facilitate the development of new technologies that use these enzymes. Moreover, the approaches described for anaerobic expression of oxygen-sensitive proteins can be extended for studying other enzymes such as [NiFe] hydrogenases and nitrogenases.