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

Journal Article · · Biotechnology Letters
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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.
Research Organization:
National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Sponsoring Organization:
USDOE
DOE Contract Number:
AC36-08GO28308
OSTI ID:
944476
Report Number(s):
NREL/JA-270-41455; MainId:63376; UUID:59bda6ba-f7d4-e411-b769-d89d67132a6d; MainAdminId:45450
Journal Information:
Biotechnology Letters, Journal Name: Biotechnology Letters Journal Issue: 3 Vol. 29; ISSN 0141-5492; ISSN BILED3
Country of Publication:
United States
Language:
English

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Related Subjects

BASIC BIOLOGICAL SCIENCES
HYDROGEN
SOLAR ENERGY
DNA
ENZYMES
ESCHERICHIA COLI
GENES
HYDROGENASES
PROTEINS
SAMPLING
gene shuffling
heterologous expression
hydrogen
hydrogenase