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Title: Ferredoxin:NAD + oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation [Identification of a ferredoxin:NAD + oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation]

Ferredoxin:NAD + oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD +. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum. In wild-type T. saccharolyticum, deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. As a result, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol.
Authors:
 [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Dartmouth College, Hanover, NH (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Report Number(s):
NREL/JA-2700-67275
Journal ID: ISSN 0099-2240
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 82; Journal Issue: 24; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; metabolic engineering; ferredoxins; ethanol production
OSTI Identifier:
1339505