Ferredoxin:NAD ⁺ Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

Abstract

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. Finally, 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. IMPORTANCERedox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD ؉ , a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms. Ferredoxins are iron-sulfur proteins found in many anaerobic bacteria and archaea and mediate electron transfer in various metabolic processes, including photosynthesis (1, 2), alcohol production (3, 4), nitrogen fixation (5, 6), and hydrogen production (7). Lack of knowledge about these ferredoxin-dependent pathways currently limits our ability to incorporate them into metabolic engineering strategies. The ferredoxin:NAD ϩ /NADP ϩ oxidoreductase (FNOR) enzyme (EC 1.18.1.2/EC 1.18.1.3) forms a key bridge in metabolism between the nicotinamide cofactor (i.e., NAD ϩ , NADH, NADP ϩ , and NADPH)-dependent pathways and ferredoxin-dependent pathways (Fig. 1, equation a) (8-11). Recently, the thioredoxin reductase-like (TrxR-type) FNORs were found, and they are widely distributed among the bacteria and archaea (12)(13)(14). Even these types of FNORs are more homologous to bacterial NADPH-TrxRs, but they have catalytic properties similar to those of FNOR (14). FNOR enzymes are widely believed to be of central importance for the bioenergetics of anaerobic bacteria due to their ability to couple electron transport with ion or Na ϩ gradient generation (15) or transhydrogenation. Furthermore, they are the key enzymes for many biochemical and biofuel pathways, including isopropanol, ethanol, and n-butanol (3,4,16). Since ferredoxin has a lower standard reduction potential than...

CO2-Induced Transcriptional Reorganization: Molecular Basis of Capnophillic Lactic Fermentation in Thermotoga neapolitana

“…In this view, synthesis of LA could be another mechanism to get rid of the excess of reducing equivalents. A similar cycle has been postulated between RNF and ferredoxin-dependent NADH oxidoreductase (FNOR) in T. saccharolyticum in order to operate a net transfer of protons across the cell membrane and eliminate the proton gradient that could be lethal for the cells (Tian et al, 2016).…”

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase

“…Protein purification for the purposes of demonstrating in vitro conversion of sedoheptulose 7-phosphate to sedoheptuoloase 1,7-bisphosphate by the purified PP i -PFK in the presence of PP i was performed as previously described (39). To obtain purified PP i -PFK protein, the induced E. coli cells were resuspended in 100 mM Tris-HCl buffer (pH 7 at 55°C).…”

“…Mass spectrometry analyses of assay samples-Assay samples were analyzed as previously described (39), using an LC-MS/ MS system a Thermo Scientific Vanquish UHPLC coupled by heated electrospray ionization (HESI) to a hybrid quadrupole-high resolution mass spectrometer (Q Exactive Orbitrap, Thermo Scientific). Liquid chromatography separation was performed using an ACQUITY UPLC BEH C18 (2.1 ϫ 100 mm column, 1.7-m particle size), with a flow rate of 0.2 ml/min.…”

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

“…Previous work suggests that C. thermocellum ferredoxins are compatible with T. saccharolyticum ferredoxin-utilizing enzymes [13]. However, given that they play an important role in electron transfer, and that it is possible that some ferredoxins work better than others with certain enzymes, we decided to include the introduction of T. saccharolyticum ferredoxin into C. thermocellum in this study.…”