In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H(+)-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H(+)-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PP(i)) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H(+)-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pH(i)) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g.L(-1) acetic acid stress was observed in the vacuolar membrane H(+)-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H(+)-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

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Sreenivas K, Eisentraut L, Brink DP, Persson VC, Carlquist M, Gorwa-Grauslund MF, van Niel EWJ

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