Reprogramming Corynebacterium glutamicum metabolism to efficiently synthesis protocatechuic acid from flask to pilot...

Protocatechuic acid (PCA), a crucial active component in Chinese herbal medicine with diverse biological functions, encounters challenges in effective extraction yield and preservation of its activity. In this investigation, a precise metabolic network model of C. glutamicum was designed for PCA production. Flux Balance Analysis (FBA) is systematically employed to compute metabolic fluxes and thereby direct metabolic engineering strategies, including increasing the flow of pentose phosphate pathway (PPP) and shikimic acid pathway, eliminating the endogenous degradation pathway of PCA and implementing the heterologous PCA biosynthesis pathway, the highest titer of PCA in C. glutamicum was increased to 22.11 g per liter (g/L) in shake flask. The transcriptome analysis revealed that the engineered C. glutamicum exhibited improved redistribution of carbon flow and enhanced energy metabolism. Subsequently, by optimizing the ratio of C/N and feeding strategy, the titers of PCA achieved 63.22 g/L and yield of 0.411 mol/mol in a 5-liter (L) bioreactor and 52.77 g/L yield of 0.405 mol/mol in a 100-L bioreactor which is maximum yield documented in the existing literature on pilot scale. Moreover, the effective antibacterial activity and antioxidant activity of mass-produced PCA can be used as feed additives for livestock. In summary, these metabolic engineering strategies open up the possibility of constructing C. glutamicum efficiently to produce high-value chemicals.