Adaptive laboratory evolution of Thermoanaerobacterium aotearoense for enhanced ethanol production from raw cassava starch

Thermophiles have the potential to utilize starch for bioethanol production in consolidated bioprocessing procedure, but they are usually limited by the intolerance of high ethanol titer. In this study, we performed adaptative laboratory evolution (ALE) on Thermoanaerobacterium aotearoense strain PRH-B3 (Delta ldh Delta rex Delta hfsB::BCD), which produced ethanol as the only product. After hundreds of generations of domestication with the increasing ethanol concentration, two lineages of ethanol-tolerant mutants were gained and the effects of alcohol dehydrogenase (ADH) domain mutations in aldehyde/alcohol dehydrogenase AdhE were analyzed. The assay of ADH activities in vivo showed that the mutants exhibited reduced NADH-dependent ADH activities and increased NADPH-dependent ADH activities, compared to that from PRH-B3. AdhA was subsequently demonstrated to contribute to the increased NADPH-linked ADH activities in the mutants. This was accompanied by an increased expression level of NADH-dependent ferredoxin:NADP(+) oxidoreductase and improved contents of NADP(H), indicating that the ethanol-tolerant strains preferred to use NADPH for ethanol synthesis with a potential regulatory mechanism. The maximum ethanol production (similar to 39 g/L) of the mutants had increased by 26 % after ALE from untreated cassava starch. The enhanced comprehension of the mechanisms underlying ethanol tolerance elucidated herein paves the way for the development of other ethanologenic strains with elevated ethanol titers.