Voelker, F.; Kohlstedt, M.; Halmschlag, B.; Mueller, J.; Weinert, J.; Wittmann, C.; Blank, L. M.

Improving carbon yield and process time is a key requirement for the industrially viable production of biopolymers. During poly-{gamma}-glutamic acid production with Bacillus subtilis, a considerable fraction of the carbon source is diverted into by-products. These originate from accumulating pyruvate, constraining carbon flux towards glutamate synthesis. Disrupting by-product pathways reduces their secretion, but the underlying imbalance at the pyruvate node persists. Here, we rewired the central carbon metabolism to redirect glycolytic flux from pyruvate formation towards oxaloacetate and 2-oxoglutarate in an improved medium. Deletion of pyruvate kinase combined with heterologous expression of phosphoenolpyruvate carboxylase established an alternative anaplerotic entry route into the TCA cycle. Subsequent disruption of gluconeogenetic reactions (OF5) reduced by-product formation by 81% and increased {gamma}-PGA yield to 0.41 g g-1 glucose, but lowered the glucose uptake rate. Alternatively, disruption of the TCA cycle downstream of 2-oxoglutarate combined with enhanced glutamate synthesis (OF4.2) increased substrate uptake and improved the {gamma}-PGA production rate by 39% compared to the wild type, while product and by-product titers remained comparable. Both strategies thus revealed a trade-off between improved yield or production rate. Subsequent 13C metabolic flux analysis and intracellular metabolite profiling revealed distinct carbon routing phenotypes. While strain OF5 maintained carbon within the TCA cycle, OF4.2 cycled carbon between oxaloacetate, malate and pyruvate, thereby altering redox and energy balances. These results highlight the need for coordinated control of carbon flux, redox metabolism and energy supply for improving {gamma}-PGA synthesis, and identify the oxaloacetate-malate-pyruvate node as a promising strain engineering target.