Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries.
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.
Nagy
Lauren E
LE
Environmental Science and Engineering Division, Colorado School of Mines, Golden, CO 80401, USA.
Meuser
Jonathan E
JE
Plummer
Scott
S
Seibert
Michael
M
Ghirardi
Maria L
ML
King
Paul W
PW
Ahmann
Dianne
D
Posewitz
Matthew C
MC
D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D032621:DNA Shuffling / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis
D007506:Iron-Sulfur Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; C118830:iron hydrogenase; D006864:Hydrogenase