Abstract
A gene-shuffling technique was identified, optimized and used to generate diverse libraries of recombinant [FeFe]-hydrogenases. Six native [FeFe]-hydrogenase genes from species of Clostridia were first cloned and separately expressed in Escherichia coli concomitantly with the assembly proteins required for [FeFe]-hydrogenase maturation. All enzymes, with the exception of C. thermocellum HydA, exhibited significant activity when expressed. Single-stranded DNA fragments from genes encoding the two most active [FeFe]-hydrogenases were used to optimize a gene-shuffling protocol and generate recombinant enzyme libraries. Random sampling demonstrates that several shuffled products are active. This represents the first successful application of gene-shuffling using hydrogenases. Moreover, we demonstrate that a single set of [FeFe]-hydrogenase maturation proteins is sufficient for the heterologous assembly of the bioinorganic active site of several native and shuffled [FeFe]-hydrogenases.
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Acknowledgements
We would like to thank the entire NREL and CSM biohydrogen teams for insightful discussions regarding this research. Moreover, we acknowledge the insights and technical suggestions provided by the Air Force Office of Scientific Research BioSolar H2 research team led by G. Charles Dismukes at Princeton University. We would also like to thank John Spear (CSM) for previewing this manuscript and offering editorial suggestions.
This work was supported by U.S. Air Force Office of Scientific Research MURI award FA9550-05-1-0365 (MCP), the National Science Foundation award 0328187 (MCP, DA) and the Division of Energy Biosciences, Office of Science, U.S. Department of Energy (MLG, MS).
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Nagy, L.E., Meuser, J.E., Plummer, S. et al. Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries. Biotechnol Lett 29, 421–430 (2007). https://doi.org/10.1007/s10529-006-9254-9
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DOI: https://doi.org/10.1007/s10529-006-9254-9