Enforced ATP wasting as a means for bioprocess intensification

Bio-based production processes are key for shifting the chemical industry from fossil-based towards sustainable and CO₂-neutral manufacturing. However, to achieve this goal, bioprocesses must become economically viable and able to compete with conventional petrochemical production. This requires a constant improvement of the three major performance measures of bioprocesses: yield (product per substrate), titer (product per volume) and productivity (product per volume and time).

In our group, we have been developing the concept of enforced ATP wasting as a generic metabolic engineering strategy to improve the productivity of microbial cell factories. The rationale behind this approach is that artificially enforcing a high turnover (“wasting”) of ATP can increase product synthesis rates and yields, if production of the target product is coupled with ATP formation.

I will show experimental results demonstrating the remarkable potential of this approach. We first found that E. coli and the yeast S. cerevisiae, two major production hosts in biotech industry, increase the synthesis rates of natural fermentation products (ethanol, formate, lactate, acetate and succinate in E. coli and ethanol in S. cerevisiae) if the cytosolic F₁-portion of the ATPase from E. coli, which hydrolyzes (wastes) ATP, is introduced in the cells [1,2]. As a realistic application example, we used then enforced ATP wasting to enhance the conversion of glucose to the platform chemical 2,3-butanediol (2,3-BDO) by an engineered E. coli strain [3]. For various cultivation conditions, we compared the 2,3-BDO production performance of the existing production strain (control strain) against the production strain additionally expressing the F₁-ATPase encoding genes (ATPase strain). Under all cultivation conditions tested, the specific glucose uptake and 2,3-BDO synthesis rates in the ATPase strain improved markedly and were up to sixfold and tenfold higher, respectively. Likewise, the 2,3-BDO yield was increased by up to 45%.

To further shed light on the physiological response of E. coli under (extreme) ATP-demanding conditions, we analyzed systematically the impact of different levels of uncoupled ATPase activity in wild type E. coli under aerobic and anaerobic conditions, both with cell growth and growth arrest [4]. As one key result, we found that the glucose uptake rate shows always a biphasic response curve with respect to increasing ATPase activity, reaching a maximum at a medium ATPase level, but dropping markedly when this level is exceeded. This finding suggests that there is an optimal level of ATP wasting to achieve maximal metabolic activity. Using metabolomic and proteomic data in combination with a kinetic model of E. coli’s central metabolism we revealed the underlying mechanism of the biphasic behavior. This study provided valuable new insights on the functioning of glycolysis under extreme conditions and guides rational engineering of cell factories with high productivity.