초록
<B>ABSTRACT</B><P> NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP <SUP>+</SUP> . 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 <I>nfnAB</I> gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum , deletion of <I>nfnAB</I> 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 <I>nfnAB</I> 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 Δ <I>nfnAB</I> strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the Δ <I>nfnAB</I> strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of <I>nfnAB</I> 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 <I>nfnAB</I> . This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from <I>nfnAB</I> ) 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. </P><P>IMPORTANCE Thermophilic 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) <SUP>+</SUP> . The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP <SUP>+</SUP> and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion of <I>nfnAB</I> 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>