Rewiring central carbon metabolism reveals a trade-off between yield and production rate in poly-γ-glutamic acid synthesis by Bacillus subtilis

Achieving high carbon yield and process time is crucial for commercially viable production of biopolymers like poly-γ-glutamic acid by *Bacillus subtilis*. However, a significant portion of the carbon source gets diverted into by-products when pyruvate accumulates, limiting the carbon flow towards glutamate synthesis. Disrupting these by-product pathways reduces their secretion, yet the imbalance at the pyruvate node remains.

To address this, researchers rewired central carbon metabolism, redirecting glycolytic flux from pyruvate formation towards oxaloacetate and 2-oxoglutarate in an improved medium. Deleting pyruvate kinase and introducing heterologous phosphoenolpyruvate carboxylase created an alternative anaplerotic entry into the TCA cycle. Subsequently, disrupting gluconeogenic reactions (OF5) resulted in an 81% reduction in by-product formation and a 0.41 g g-1 glucose yield of {gamma}-PGA, but reduced the glucose uptake rate.

Alternatively, targeting the TCA cycle downstream of 2-oxoglutarate with enhanced glutamate synthesis (OF4.2) boosted substrate uptake and improved the {gamma}-PGA production rate by 39%, maintaining comparable product and by-product titers. Metabolic flux analysis and intracellular metabolite profiling revealed distinct carbon routing phenotypes.

OF5 strain maintained carbon within the TCA cycle, whereas OF4.2 cycled carbon between oxaloacetate, malate, and pyruvate, altering redox and energy balances. These findings underscore the importance of coordinated control of carbon flux, redox metabolism, and energy supply for optimizing {gamma}-PGA synthesis, identifying the oxaloacetate-malate-pyruvate node as a promising target for strain engineering.