High-level secretory production of lysostaphin in Escherichia coli mutant by codon optimization and atmospheric and room temperature plasma mutagenesis

Abstract

Lysostaphin is an antimicrobial agent that can specifically lyse the cell walls of Staphylococcus aureus, thus having the potential in prevention of food corruption caused by S. aureus. Yet, its application in the medical and food industry is still limited by its high costs. In this study, the lysostaphin coding gene was codon-optimized (Opt-lys) and then ligated into the pET-22b(+) vector to achieve heterologous expression in Escherichia coli BL21(DE3). Surprisingly, lysostaphin could also be secreted extracellularly by Escherichia coli. Based on this characteristic, a high-throughput method for screening mutants with secretory lysostaphin using a double-layer plate was established. In combination with atmospheric and room temperature plasma mutagenesis, strain BL21(DE3)/pOpt-lys 4-18 with the highest extracellular activity was obtained. Furthermore, after optimization of fermentation conditions, the maximal extracellular activity was determined as 155.3 U/mL, and the extracellular protein concentration reached 442.26 mg/L. Therefore, this study provided a recombinant strain that could efficiently produce lysostaphin and a better mutant screening method, laying the foundation for the application and research of lysostaphin.