Skip to main content
Article
Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase
Journal of Biological Chemistry
  • S. K. Chanrayan
  • P. M. McTernan
  • R. C. Hopkins
  • J. Sun
  • Francis E. Jenney, Jr., Philadelphia College of Osteopathic Medicine
  • M. W. Adams
Document Type
Article
Publication Date
1-1-2012
Disciplines
Abstract

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H 2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Comments

This article was published in Journal of Biological Chemistry, Volume 287, Issue 5, Pages 3257-3264.

The published version is available at http://dx.doi.org/10.1074/jbc.M111.290916.

Copyright © 2012 ASBMB.

Citation Information
S. K. Chanrayan, P. M. McTernan, R. C. Hopkins, J. Sun, et al.. "Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase" Journal of Biological Chemistry Vol. 287 Iss. 5 (2012) p. 3257 - 3264
Available at: http://works.bepress.com/francis_jenney/65/