Efficient secretory expression of human milk Osteopontin in Komagataella phaffii
Zhihang Zhang,
Yangyang Li,
Zhenmin Liu,
Shixiu Cui,
Xianhao Xu,
Yanfeng Liu,
Jianghua Li,
Guocheng Du,
Xueqin Lv,
Long Liu
Affiliations
Zhihang Zhang
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China; Food Laboratory of Zhongyuan, Jiangnan University, Wuxi 214122, PR China
Yangyang Li
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China
Zhenmin Liu
State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd, Shanghai 200436, PR China
Shixiu Cui
Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China; Jiaxing Institute of Future Food, Jiaxing 314015, PR China
Xianhao Xu
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China
Yanfeng Liu
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China
Jianghua Li
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China; Corresponding author.
Guocheng Du
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China
Xueqin Lv
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China
Long Liu
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China; Science Center for Future Foods, Jiangnan University, Wuxi 214122, PR China
Osteopontin (OPN), a bioactive milk protein in breast milk, has diverse applications in food and medicine. OPN is predominantly extracted from fresh bovine milk; however, the concentration of OPN in bovine milk is exceedingly low. To address these issues, in the present study, we used Komagataella phaffii for the recombinant expression of human OPN (hOPN). The results of host cell screening revealed that X33 cells were more suitable than other K. phaffii hosts for hOPN expression, yielding a titer of 340.5 μg/L. Subsequently, it was observed that the extracellular production of hOPN significantly increased to 8.02 mg/L from strain XPSA01 using the PAOX1 promoter and the hybrid signal peptide PROSCW10-α. Meanwhile, the co-expression of transcription factors, translation factors, and molecular chaperones resulted in a 2.94-fold enhancement in extracellular hOPN production compared with that in XPSA01, resulting in a titer of 23.6 mg/L. Finally, the above strategies were combined to obtain the recombinant strain XPSA01-CP, which achieved titers of 35.6 and 128.5 mg/L hOPN in shake-flask fermentation and 3-L bioreactor, respectively. To the best of our knowledge, this is the highest extracellular yield of hOPN ever reported.
