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.
Original language | American English |
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Pages (from-to) | 421-430 |
Number of pages | 10 |
Journal | Biotechnology Letters |
Volume | 29 |
Issue number | 3 |
DOIs | |
State | Published - 2007 |
NREL Publication Number
- NREL/JA-270-41455
Keywords
- gene shuffling
- heterologous expression
- hydrogen
- hydrogenase