high-yield expression of heterologous [fefe] hydrogenases in escherichia coli高收益的表达不同的(象皮病)氢化酶在大肠杆菌.pdfVIP

High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli 1 2,3 1 2,3 Jon M. Kuchenreuther , Celestine S. Grady-Smith , Alyssa S. Bingham , Simon J. George , Stephen P. Cramer2,3, James R. Swartz1,4* 1 Department of Chemical Engineering, Stanford University, Stanford, California, United States of America, 2 Department of Applied Science, University of California Davis, Davis, California, United States of America, 3 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 4 Department of Bioengineering, Stanford University, Stanford, California, United States of America Abstract Background: 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. Principal Findings: 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,