Highly efficient expression and secretion of human lysozyme using multiple strategies in Pichia pastoris

• Published in: Biotechnology journal 2023-11, Vol.18 (11), p.e2300259-n/a
• Main Authors: Wang, Yasen, Wang, Buqing, Gao, Yahui, Nakanishi, Hideki, Gao, Xiao‐Dong, Li, Zijie
• Format: Article
• Language: English
• Subjects: chaperones; human lysozyme; Pichia pastoris; transcription factors; vacuolar sorting receptor
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.202300259

Background Human lysozyme (hLYZ), an emerging antibacterial agent, has extensive application in the food and pharmaceutical industries. However, the source of hLYZ is particularly limited. Results To achieve highly efficient expression and secretion of hLYZ in Pichia pastoris, multiple strategies including G418 sulfate screening, signal sequence optimization, vacuolar sorting receptor VPS10 disruption, and chaperones/transcription factors co‐expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL−1, which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL−1 in a 5 L fermenter. Conclusion This research provides a very useful and cost‐effective approach for the hLYZ production in P. pastoris and can also be applied to the production of other recombinant proteins. Graphical and Lay Summary To achieve highly efficient expression and secretion of human lysozyme in Pichia pastoris, multiple strategies including gene copy numbers optimization, signal sequence optimization, Golgi to vacuole pathway engineering, and chaperones/transcription factors co‐expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL−1, which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL−1 in a 5‐L fermenter.