Production of copper nanoparticles using genetically engineered Komagataella phaffii.

With the increasing application of copper nanoparticles (CuNPs) as antibacterial agents, numerous studies have emerged in recent years focusing on their preparation and utilization. However, the existing physical and chemical processes for CuNPs synthesis are complex and environmentally hazardous, creating a demand for greener alternatives. Komagataella phaffii has been recognized as a cost-effective system for metal biosorption. Nevertheless, high concentrations of heavy metal particles inhibit cell growth and result in low biosorption efficiency of metal-based nanoparticles (NPs). To address this issue, we engineered the K. phaffii strain X-33-Cyb5R by expressing the cytochrome b-5 reductase (Cyb5R) enzyme, enhancing its tolerance to elevated heavy metal concentrations and promoting CuNPs biosorption. Through further optimization of biosorption conditions, CuNPs production reached 14.27 mg/g dry cell weight (DCW) after 36 h, utilizing 12 mmol/L CuSO₄ at 30 °C and pH 4. The adsorbed particles on the surface of the modified strain K. phaffii X-33-Cyb5R were confirmed to be CuNPs with diameters ranging from 40 to 80 nm. Notably, the CuNPs synthesized in this study exhibited potent antibacterial activity. This research not only provides a novel approach for the construction of highly metal-tolerant strains and efficient CuNPs production but also offers new insights for the development and utilization of environmentally friendly antibacterial agents.