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

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.

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
944476
DOE Contract Number:  
AC36-99-GO10337
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biotechnology Letters; Journal Volume: 29; Journal Issue: 3, 2007
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; 14 SOLAR ENERGY; 59 BASIC BIOLOGICAL SCIENCES; DNA; ENZYMES; ESCHERICHIA COLI; GENES; HYDROGENASES; PROTEINS; SAMPLING; Basic Sciences

Citation Formats

Nagy, L. E., Meuser, J. E., Plummer, S., Seibert, M., Ghirardi, M. L., King, P. W., Ahmann, D., and Posewitz, M. C. Application of Gene-Shuffling for the Rapid Generation of Novel [FeFe]-Hydrogenase Libraries. United States: N. p., 2007. Web. doi:10.1007/s10529-006-9254-9.
Nagy, L. E., Meuser, J. E., Plummer, S., Seibert, M., Ghirardi, M. L., King, P. W., Ahmann, D., & Posewitz, M. C. Application of Gene-Shuffling for the Rapid Generation of Novel [FeFe]-Hydrogenase Libraries. United States. doi:10.1007/s10529-006-9254-9.
Nagy, L. E., Meuser, J. E., Plummer, S., Seibert, M., Ghirardi, M. L., King, P. W., Ahmann, D., and Posewitz, M. C. Mon . "Application of Gene-Shuffling for the Rapid Generation of Novel [FeFe]-Hydrogenase Libraries". United States. doi:10.1007/s10529-006-9254-9.
@article{osti_944476,
title = {Application of Gene-Shuffling for the Rapid Generation of Novel [FeFe]-Hydrogenase Libraries},
author = {Nagy, L. E. and Meuser, J. E. and Plummer, S. and Seibert, M. and Ghirardi, M. L. and King, P. W. and Ahmann, D. and Posewitz, M. C.},
abstractNote = {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.},
doi = {10.1007/s10529-006-9254-9},
journal = {Biotechnology Letters},
number = 3, 2007,
volume = 29,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}