High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

Kuchenreuther, Jon M.; Grady-Smith, Celestine S.; Bingham, Alyssa S.; George, Simon J.; Cramer, Stephen P.; Swartz, James R.

doi:10.1371/journal.pone.0015491

High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

Journal Article · Wed Nov 24 00:00:00 EST 2010 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0015491· OSTI ID:1627440

Kuchenreuther, Jon M. ^[1]; Grady-Smith, Celestine S. ^[2]; Bingham, Alyssa S. ^[3]; George, Simon J. ^[2]; Cramer, Stephen P. ^[2]; Swartz, James R. ^[4]

Stanford University, CA (United States); DOE/OSTI
University of California, Davis, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Stanford University, CA (United States)
Stanford University, CA (United States); Stanford University, CA (United States)

The realization of hydrogenase-based technologies for renewable H2 production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. In this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H₂ evolution with rates comparable to those of enzymes isolated from their respective native organisms. The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H₂-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1627440

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 11 Vol. 5; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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