Nonstationary 13C metabolic flux analysis reveals cryptic phenylacetic acid catabolism in glucose-limited Penicillium chrysogenum chemostat cultures.

Penicillium chrysogenum is the key industrial microorganism for penicillin production, and understanding its metabolic regulation is crucial for optimizing yields. While different chemostat strategies influence fermentation outcomes, the underlying metabolic mechanisms remain poorly understood. To address this, non-stationary 13C metabolic flux analysis (13C-MFA) was employed combined with quantitative metabolite profiling to investigate the physiological response of P. chrysogenum Wisconsin 54-1255 under both overflow- and feedback-controlled chemostats. Under overflow control, extracellular glucose concentrations were reduced to one-fifth of feedback levels, while the intracellular amount of glucose doubled. Concomitantly, the specific uptake rate of phenylacetic acid (PAA) increased twofold, while penicillin productivity decreased by 25%. Importantly, 13C-MFA revealed a shift towards a benzoic acid degradation pathway for PAA, sustaining the TCA cycle flux comparable to those under feedback control. These findings underscore the uncharacterized role of PAA catabolism in maintaining central carbon metabolism under overflow control, offering insights and strategies for further enhancing industrial penicillin production.