Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

Abstract

Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at about 90% of the theoretical maximum yield (2 ethanol molecules per glucose equivalent) and a titer of 70 g/liter. 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 ϳ50%, whereas multiple gene deletions of all annotated alcohol dehydrogenase genes except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked alcohol dehydrogenase (ADH) activity (acetaldehyde-reducing direction) by 93%.IMPORTANCE In this study, we set out to identify the alcohol dehydrogenases necessary for high ethanol production in T. saccharolyticum. Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase gene. 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.

Expression of adhA from different organisms in Clostridium thermocellum

“…In general, most of the additional ethanol production came at the expense of acetate production (Table 2 ). This is consistent with other reports indicating that there appears to be an oversupply of NADPH in C. thermocellum [ 14 , 15 ], and that this can be used to divert C2 flux (i.e., acetyl-CoA) to ethanol in the presence of an NADPH-linked ADH enzyme [ 8 , 9 , 16 ]. Lactate and malate were minor fermentation products.…”

“…End-product measurements were taken on the 3rd transfer. Cultures were grown in Corning™ Falcon™ 15 ml Conical Centrifuge Tubes and incubated anaerobically without shaking at 55 °C for 72 h. Upon harvesting, cultures were prepared as previously described for HPLC (High-Pressure Liquid Chromatography) analysis [ 8 ]. Ethanol yield was calculated as the percentage of theoretical yield based on the amount of ethanol produced and substrate consumed: .…”

“…Strategies to increase ethanol yield in C. thermocellum include deleting the pathways for acetic acid, lactic acid, and hydrogen production [ 3 – 6 ], and introducing heterologous genes from ethanol production pathways in other organisms [ 2 , 7 ], such as Thermoanaerobacterium saccharolyticum. Recently, it was shown that AdhA plays an important role in ethanol production in strains of T. saccharolyticum engineered for homoethanol production [ 8 ]. This enzyme was subsequently expressed in C. thermocellum and shown to increase ethanol yield and titer by 40% [ 3 ].…”

Enhanced ethanol production from lignocellulosic hydrolysates by inhibiting the hydrogen synthesis in Thermoanaerobacterium aotearoense SCUT27(Δldh)

“…In thermophilic anaerobic bacteria, ethanol production is almost exclusively mediated by two enzymes, AdhE and AdhA (Fig. ) . The proteins encoded by adhE and adhA can catalyze the conversion of acetaldehyde to ethanol using NADH and NADPH respectively as cofactors .…”

“…1). 28,29 The proteins encoded by adhE and adhA can catalyze the conversion of acetaldehyde to ethanol using NADH and NADPH respectively as cofactors. 30 Therefore we tested the activity of ADH (responsible for the last step of ethanol formation).…”