Application of Gene-Shuffling for the Rapid Generation of Novel [FeFe]-Hydrogenase Libraries

Lauren Nagy, Jonathan Meuser, Scott Plummer, Michael Seibert, Maria Ghirardi, Paul King, Dianne Ahmann, Matthew Posewitz

Research output: Contribution to journalArticlepeer-review


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 languageAmerican English
Pages (from-to)421-430
Number of pages10
JournalBiotechnology Letters
Issue number3
StatePublished - 2007

NREL Publication Number

  • NREL/JA-270-41455


  • gene shuffling
  • heterologous expression
  • hydrogen
  • hydrogenase


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