Strategies to enhance the hydrolytic activity of Escherichia coli BL21 penicillin G acylase based on heterologous expression and targeted mutagenesis

Abstract

Penicillin G acylase (PGA) serves as a critical biocatalyst for the hydrolysis of penicillin G, yielding 6-aminopenicillanic acid, a vital precursor for β-lactam semi-synthetic antibiotics. The catalytic efficiency of PGA, however, remains suboptimal in native Escherichia coli strains. To improve this, E. coli BL21 was engineered as a microbial cell factory via heterologous expression and site-directed mutagenesis to enhance PGA activity. The heterologous pga gene from Providencia rettgeri was integrated into E. coli BL21 (DE3) for the biosynthesis of PGA, achieving a PGA activity of 253 ± 2 U/mL after 16 hours of fermentation. The N167 site underwent mutation, producing the sites N167A and N167I. Plasmids carrying these mutations were introduced into E. coli BL21(DE3), and the enzymatic activities were recorded as 293 ± 3 U/mL for the N167A mutant and 238 ± 2 U/mL for the N167I mutant. This study not only introduces a novel approach to enhancing PGA activity but also illustrates the potential for catalytic optimization through targeted modifications of the enzyme's active site.