PDF(Pubmed)
Abstract：
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 (PPi) 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 (pHi) 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.
摘要：
在酿酒酵母中，由于使用质子-易位ATP酶(H+-ATP酶),pH稳态依赖于ATP,其构成细胞ATP供应中的主要消耗。这里,来自拟南芥的外源质子易位焦磷酸酶（H-PPase），使用无机焦磷酸盐(PPi)而不是ATP，评估了其降低ATP负担的效果。H+-Ppase定位于液泡膜或细胞膜，并在低pH下的乙酸盐胁迫下研究了它们的影响。使用生物传感器（pHluorin和mQueen-2m）观察葡萄糖或木糖生长过程中细胞内pH（pHi）和ATP水平的变化。与亲本菌株相比，液泡膜H-PPase菌株在pH为3.7和6g·L-1乙酸胁迫下的生长速率显着提高了35％。在厌氧葡萄糖和木糖发酵期间，同一菌株中的ATP水平升高。在厌氧木糖发酵过程中，pHluorin和液泡膜H-PPase的共表达通过提高的生长速率（11.4％）和延长的对数生长持续时间来改善生长特性。我们的研究确定了在工业中存在的苛刻条件下提高酿酒酵母作为细胞工厂的生产率的潜在方法。
