Both adhE and a separate NADPH-dependent alcohol dehydrogenase (adhA) are necessary for high ethanol production in Thermoanaerobacterium saccharolyticum.

Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at ∼90% theoretical yield and titer of 70 g/L. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ∼50%, whereas multiple gene deletions of all annotated alcohol dehydrogenases except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked ADH activity (acetaldehyde-reducing) by 93%. IMPORTANCE In this study we set out to identify the necessary alcohol dehydrogenases for high ethanol production in T. saccharolyticum Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase. Here we show that both adhA and adhE are needed for high ethanol yield in the engineered strain LL1049. This is the first report showing adhA is important for ethanol production in a native adhA host, which has important implications for achieving higher ethanol yields in other microorganisms.