High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.
BACKGROUND
The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.


RESULTS
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 H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms.


CONCLUSIONS
The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-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.
Department of Chemical Engineering, Stanford University, Stanford, California, USA.
Jon M
JM
Kuchenreuther
Celestine S
CS
Grady-Smith
Alyssa S
AS
Bingham
Simon J
SJ
George
Stephen P
SP
Cramer
James R
JR
Swartz
D000693:Anaerobiosis; D055162:Biocatalysis / Q000187:drug effects; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D003013:Clostridium / Q000201:enzymology; D003545:Cysteine / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D015971:Gene Expression Regulation, Enzymologic; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007501:Iron / Q000378:metabolism / Q000494:pharmacology; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D006859:Hydrogen; D007501:Iron; C118830:iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine