(23d) Evolutionary Engineering of Geobacillus Thermoglucosidans for Improved Ethanol Production

Thermophiles are organisms that grow at high temperatures (>50°C). Many researchers have proposed that thermophilic organisms are better suited for the production of cellulosic biofuels than mesophilic organisms as the overall processes are less susceptible to contamination and potentially more energy efficient. In this study, we explored the thermophile Geobacillus thermoglucosidans for the production of bio-ethanol. This bacterium is a facultative anaerobe, grows at an optimal temperature 60°C, and can ferment diverse carbohydrates including glucose, cellobiose, xylose, and arabinose. However, it natively performs mixed acid fermentation. To improve ethanol productivity in this thermophile, we engineered and evolved two strains of G. thermoglucosidasius BGSC 95A1 (95A1) and C56-YS93 (C56). We first deleted lactate dehydrogenase (Ldh) and pyruvate formate lyase (Pfl) in order to eliminate lactate and formation production and then heterologously expressed pyruvate decarboxylase (Pdc) from Sarcina ventriculi to directly produce ethanol from pyruvate. Elimination of Pfl, however, slowed growth significantly because the strain was unable to generate acetyl-CoA under anaerobic conditions. To restore growth, we added acetic acid to media to provide a source for acetyl-CoA. The strains were then evolved on glucose and cellobiose. We found that the evolved strains of 95A1 were able to efficiently produce ethanol during growth on glucose or cellobiose whereas the evolved C56 strains performed poorly. Genome sequencing of evolved 95A1 strains identified common loss-of-function mutations in adenine phosphoribosyltransferase (Aprt) and the stage III sporulation protein AA (SpoIIIAA). We disrupted the two genes encoding them and found that deletion of both enhanced the ethanol production, especially Aprt. In conclusion, we were able to engineer a strain of G. thermoglucosidans to efficiently produce ethanol from glucose and cellobiose using a combination of metabolic engineering and evolutionary strategies. This work helps establish this thermophile as a platform organism for biofuel and biochemical production.