Metabolic Engineering of Plasmid‐Free <i>Escherichia coli</i> for the Highly Efficient Biosynthesis of <i>cis,cis</i> ‐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 (ApAroZ^R363A), to catechol by a protocatechuate decarboxylase (KpAroY), and finally to ccMA by a catechol 1,2-dioxygenase (PpCatA). 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.