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Journal of Bacteriology, October 2009, p. 6457-6464, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00497-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation in Thermoanaerobacterium saccharolyticum and Demonstration of Increased Ethanol Yield via Hydrogenase Knockout{triangledown} ,{dagger}

A. Joe Shaw,1,2 David A. Hogsett,2 and Lee R. Lynd1,2,3*

Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire,1 Mascoma Corporation, Lebanon, New Hampshire,2 Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire3

Received 9 April 2009/ Accepted 25 July 2009

Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum.

* Corresponding author. Mailing address: Dartmouth College, Thayer School of Engineering, 8000 Cummings Hall, Hanover, NH 03755. Phone: (603) 646-2231. Fax: (603) 646-2277. E-mail: lee.r.lynd{at}dartmouth.edu

{triangledown} Published ahead of print on 31 July 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, October 2009, p. 6457-6464, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00497-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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