Metabolic engineering of Corynebacterium glutamicum for increased cis, cis-muconate production from plant-derived p-hydroxycinnamates via deregulated pathway flux and increased CoA intermediate availability

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biochemical production, with p-hydroxycinnamates emerging as key aromatic building blocks derived from agricultural residues and grassy plants. C. glutamicum has recently been engineered to produce cis, cis-muconate (MA), a high-value platform chemical used in biobased plastics, resins, and specialty chemicals. However, unlike other aromatics, the metabolism of the p-hydroxycinnamates p-coumarate, ferulate, and caffeate in MA-producing C. glutamicum is inefficient, limiting MA production performance. Here, we discovered that p-hydroxycinnamate metabolism, encoded by the phd operon, is repressed by the local repressor PhdR under glucose-rich conditions, while the global regulator GlxR activates the pathway in the absence of glucose. The deregulated C. glutamicum MA-10 lacking phdR exhibited an up to 98-fold increase in the conversion of p-coumarate, ferulate, and aromatic mixtures derived from plant waste into MA. Transcriptomic and metabolomic analyses revealed strong induction of the phd operon in strain MA-10 and a marked increase in intracellular aromatic CoA-esters and acetyl-CoA, indicating enhanced flux through the p-hydroxycinnamate degradation pathway. ¹³C-tracer studies demonstrated a substantial contribution of aromatic side-chain carbon to central metabolic pathways, supporting biomass formation and enabling MA production even in the absence of sugars. Additionally, MA-10 showed broadened substrate flexibility, degrading cinnamate into MA and methoxylated cinnamates into valuable benzoate derivatives. The strain also successfully converted aromatics from real straw lignin hydrolysates into MA. Our findings reveal the potential of targeted regulatory engineering to optimize C. glutamicum for lignin valorization. The newly developed strain MA-10 provides a robust platform for the biobased production of MA from lignocellulosic feedstocks, paving the way for sustainable and economically viable biorefinery processes.