Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

Abstract

NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP؉ . The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H 2 formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ⌬nfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ⌬nfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity. IMPORTANCEThermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P)؉ . The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP ؉ and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion of nfnAB and demonstrate a role for NfnAB in metabolism and ethanol formation in Thermoanaerobacterium saccharolyticum and show that this may be an important feature among other thermophilic ethanologenic anaerobes. P lant biomass is a sustainable and low-carbon source for biofuels; however, its recalcitrance makes conversion into fuels difficult (1). One of the leading strategies for low-cost conversion of plant biomass into fuels is consolidated bioprocessing (CBP), wherein one or more microbes solubilize plant cell walls and ferment the resulting sugars into fuel without exogenously added enzymes. The thermophilic anaerobe Thermoanaerobacterium saccharolyticum solubilizes components of plant biomass and produces ethano...

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

“…Another possibility is the presence of a set of enzymes whose net activity is equivalent to FNOR activity. As described in a previous study (19), there are two ethanol production pathways in T. saccharolyticum, one NADH based and one NADPH based (Fig. 4).…”

“…The deletion of these two genes led to the total loss of NADPH-FNOR activity (10, 19); however, NADH-FNOR activity remained and was sufficient for high-yield ethanol production (83% of the theoretical yield) under certain conditions. This suggested the possibility of an ethanol production pathway that used NADH (i.e., not NADPH) for all redox steps; however, the gene responsible for the NADH-FNOR activity was not known (19).…”

“…Engineered strains of T. saccharolyticum that can produce over 70 g/liter ethanol at neartheoretical yield have been developed (21), and this has inspired us to study the pathway this organism uses to produce ethanol. A key step in the pathway is the transfer of electrons from ferredoxin to NAD ϩ and/or NADP ϩ , which T. saccharolyticum performs readily (19). One candidate for this activity is the Nfn complex (Tsac_2085 NfnA and Tsac_2086 NfnB).…”

“…Another coupling reaction is the transhydrogenation reaction, resulting in the NADH-dependent reduced FNOR reaction (Fig. 1, equation c) (10,19,20).…”

“…Another coupling reaction is the transhydrogenation reaction, resulting in the NADH-dependent reduced FNOR reaction (Fig. 1, equation c) (10,19,20).Thermoanaerobacterium saccharolyticum is a thermophilic bacterium with the ability to ferment many components of the hemicellulose fraction of lignocellulosic biomass, including xylan, and can produce ethanol at high yields and titers. Engineered strains of T. saccharolyticum that can produce over 70 g/liter ethanol at neartheoretical yield have been developed (21), and this has inspired us to study the pathway this organism uses to produce ethanol.…”