Metabolic Engineering of Plasmid-Free Escherichia coli for the Highly Efficient Biosynthesis of cis,cis-Muconic Acid From Glucose

Cis , cis -muconic acid (ccMA) is a promising platform chemical for the production of polymers and fine chemicals. However, conventional synthesis suffers from poor selectivity and environmental concerns, while plasmid-dependent biosynthesis requires expensive antibiotics and inducers for large-scale fermentation. Here, we constructed a plasmid-free and inducer-free Escherichia coli strain for the efficient production of from glucose. The biosynthetic pathway was established via the endogenous shikimate pathway, through 3-dehydroshikimate, which is sequentially converted to protocatechuate by a 3-dehydroshikimate dehydratase ( Ap AroZ R363A ), to catechol by a protocatechuate decarboxylase ( Kp AroY), and finally to ccMA by a catechol 1,2-dioxygenase ( Pp CatA). To increase the supply of ccMA, the competitive pathway genes ( aroE , pykF , and ptsG ) were disrupted, and the essential pathway genes ( galP , glK , tktA , talB , aroG , aroB , and aroD ) were overexpressed. To address the carbon flux imbalance caused by aroE and pykF knockouts, their expression was dynamically regulated using P fliA -ATG- aroE and P flgB -TTG- pykF designs. To eliminate the plasmid burden and the need for antibiotics or inducers, the ccMA pathway genes were chromosomally integrated, and the final engineered strain WMA101 produced 9.54 g/L of ccMA with a yield of 40.3% (mol/mol) from glucose in shake flasks. These studies demonstrate a sustainable, scalable platform for ccMA biomanufacturing.