Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris

Abstract

Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. Because this property of IBPs can increase the freeze-thaw survival of cells, IBPs has attracted the attention from industries for their potential use in biotechnoogical applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris sytem. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25°C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ~100 mg/L. The total amount of secreted proteins at these conditions (25°C, pH 6.0, and glycerol fed-batch phase) was ~443 mg/L, 60 % of which was rLeIBP, yielding ~272 mg/L. In the pilot scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/L. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for six days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42°C, which is almost the same to those reported previously. The avaotechnoogical applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris sytem. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25°C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ~100 mg/L. The total amount of secreted proteins at these conditions (25°C, pH 6.0, and glycerol fed-batch phase) was ~443 mg/L, 60 % of which was rLeIBP, yielding ~272 mg/L. In the pilot scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/L. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for six days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42°C, which is almost the same to those reported previously. The ava