Using directed evolution to improve hydrogen production in chimeric hydrogenases from algal species.

Algae generate hydrogen from sunlight and water utilizing high-energy electrons generated during photosynthesis. The amount of hydrogen produced in heterologous expression of the wild-type hydrogenase is currently insufficient for industrial applications. One approach to improve hydrogen yields is through directed evolution of the DNA of the native hydrogenase. Here, we created 113 chimeric algal hydrogenase gene variants derived from combining segments of three parent hydrogenases, two from Chlamydomonas reinhardtii (CrHydA1 and CrHydA2) and one from Scenedesmus obliquus (HydA1). To generate chimeras, there were seven segments into which each of the parent hydrogenase genes was divided and recombined in a variety of combinations. The chimeric and parental hydrogenase sequences were cloned for heterologous expression in Escherichia coli, and 40 of the resultant enzymes expressed were assayed for H2 production. Chimeric clones that resulted in equal or greater production obtained with the cloned CrHydA1 parent hydrogenase were those comprised of CrHydA1 sequence in segments #1, 2, 3, and/or 4. These best-performing chimeras all contained one common region, segment #2, the part of the sequence known to contain important amino acids involved in proton transfer or hydrogen cluster coordination. The amino acid sequence distances among all chimeric clones to that of the CrHydA1 parent were determined, and the relationship between sequence distances and experimentally-derived H2 production was evaluated. An additional model determined the correlation between electrostatic potential energy surface area ratios and H2 production. The model yielded several algal mutants with predicted hydrogen productions in a range of two to three times that of the wild-type hydrogenase. The mutant data and the model can now be used to predict which specific mutant sequences may result in even higher hydrogen yields. Overall, results provide more precise details in planning future directed evolution to functionally improve algal hydrogenases.

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production.

Modeling the folding and hydrogen production of Clostridium acetobutylicum and Clostridium saccharobutylium mutants using electrostatic potential surfaces and molecular dynamics.

Electrostatic potential surfaces (EPS) were used with molecular dynamics to model the folding mechanisms and kinetics of hydrogenase mutants from wild types Clostridium acetobutylicum and Clostridium saccharobutylium. The purpose of the EPS approach was to incorporate long range electrostatic forces between widely separated regions of the mutants which contain 575 amino acids. Also, it was demonstrated that the ratio of positive to negative EPS of unfolded mutants could be used to predict the production of molecular hydrogen from the folded mutants. Using the prediction model, mutant compositions were determined that should yield hydrogen of up to 40 times that obtainable from the wild type C. acetobutylicum. It is expected that the developed EPS techniques can be used to study the folding of other proteins and to predict the reactivity of the folded protein structures.