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.

溶葡萄球菌素是一种抗菌剂，可特异性裂解金黄色葡萄球菌的细胞壁，因此具有预防金黄色葡萄球菌引起的食物腐败的潜力。然而，其在医疗和食品行业的应用仍然受到其高成本的限制。在这项研究中，溶葡萄球菌素编码基因经过密码子优化（Opt - lys），然后连接到pET-22b（+）载体中，实现在大肠杆菌BL21（DE3）中的异源表达。令人惊讶的是，溶葡萄球菌素也可以由大肠杆菌细胞外分泌. 基于这一特点，建立了一种利用双层板筛选具有分泌型溶葡萄球菌素突变体的高通量方法。结合常温和室温等离子诱变，获得了具有最高细胞外活性的菌株BL21(DE3)/p Opt - lys 4-18。此外，经过优化发酵条件，确定最大胞外活性为155.3 U/mL，胞外蛋白浓度达到442.26 mg/L。因此，本研究提供了一种高效生产溶葡萄球菌素的重组菌株和更好的突变体筛选方法，为溶葡萄球菌素的应用和研究奠定了基础。