Enhanced pyruvate production in <i>Candida glabrata</i> by engineering ATP futile cycle system.

Energy metabolism plays an important role in the growth and central metabolic pathways of cells. Manipulating energy metabolism is an efficient strategy to improve the formation of target products and to understand the effects of altering intracellular energy levels on global metabolic networks. </i>Candida glabrata<i>, as a dominant yeast strain for producing pyruvate, principally converts glucose to pyruvate through the glycolytic pathway. However, this process can be severely inhibited by a high intracellular ATP content. Here, in combination with the physiological characteristics of </i>C. glabrata<i>, efforts have been made to construct an ATP futile cycle system (ATP-FCS) in </i>C. glabrata<i> to decrease the intracellular ATP level without destroying F0F1-ATPase function. ATP-FCS was capable of decreasing the intracellular ATP level by 51.0% in </i>C. glabrata<i>. The decrease in the ATP level directly led to an increased pyruvate production and glycolysis efficiency. Moreover, we further optimized different aspects of the ATP-FCS to maximize pyruvate accumulation. Combining ATP-FCS with further genetic optimization strategies, we achieved a final pyruvate titer of 40.2 g/L, with 4.35 g pyruvate/g dry cell weight and a 0.44 g/g substrate conversion rate in 500-mL flasks, which represented increases of 98.5%, 322.3% and 160%, respectively, compared with the original strain. Thus, these strategies hold great potential for increasing the synthesis of other organic acids in microbes.