Biocatalytic Synthesis of 7-Aminodesacetoxycephalosporanic Acid with Engineered E. coli Dual Cells and Immobilized Enzymes

7-Aminodesacetoxycephalosporanic acid (7-ADCA) is a crucial β-lactam antibiotic core. Traditional chemical synthesis of 7-ADCA is plagued by high energy consumption and environmental pollution. In contrast, whole-cell biosynthesis offers a more ecofriendly and cost-effective alternative. In this study, we developed a three-enzyme cascade reaction using a deacetoxycephalosporin C synthase, an l -glutamate oxidase, and a catalase, achieving cofactor recycling and converting low-cost penicillin G (PenG) to phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA) within recombinant Escherichia coli strains. An innovative dual-cell catalytic system (SCell-A and SCell-B) was developed to overcome the challenges of heterologous expression and low production yields. After metabolic optimization and adjustment of the SCell-A-to-SCell-B ratio, the combination of strains SCA07 and SCB03 was ultimately determined. Eventually, 50 g/L PenG was transformed to 38.30 g/L G-7-ADCA in a scaled-up 15 L reactor within 10 h, with a conversion as high as 95.32%. Further hydrolysis of 133 g/L G-7-ADCA by acylase produced 81.32 g/L 7-ADCA, with a conversion of 95.62% and a space-time yield of 40.7 g/L/h. The innovative dual-cell catalytic strategy employed in this study provides an economical, environmentally friendly, and efficient alternative for the industrial production of G-7-ADCA and 7-ADCA.