title: Identification of the [FeFe]-hydrogenase responsible for hydrogen generation in Thermoanaerobacterium saccharolyticum and demonstration of increased ethanol yield via hydrogenase knockout.
abstract: 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.
authors: Dartmouth College, Thayer School of Engineering, 8000 Cummings Hall, Hanover, NH 03755, USA.
authors: A Joe
authors: AJ
authors: Shaw
authors: David A
authors: DA
authors: Hogsett
authors: Lee R
authors: LR
authors: Lynd
mesh_terms: D019342:Acetic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D050260:Carbohydrate Metabolism; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism*; D005810:Multigene Family; D046950:Thermoanaerobacterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000431:Ethanol; D006859:Hydrogen; D006864:Hydrogenase; D019342:Acetic Acid