Optimization of Liquid Fermentation Conditions for N-Glycosylated Ganoderma lucidum Immunomodulatory Protein Production by an Engineered Pichia pastoris

The Ganoderma lucidum immunomodulatory protein (FIP-glu) holds promise as a potential anti-tumor therapeutic agent. Enhancing the expression and stability of the engineered yeast strain is crucial for its industrial viability. This study focused on optimizing the fermentation parameters to produce an N-glycosylated protein, designated T36N, using the engineered Pichia pastoris GS115 strain with a mutant gene FIP-glu_T36N, a FIP-glu variant with an N-linked glycan modification. The response surface methodology (RSM) was employed to investigate the fermentation parameters that influence the yield of the N-glycosylated protein. Initially, the impact of fermentation duration on the optical density (OD600) and protein yield of P. pastoris GS115 were assessed, leading to the determination of a 96-hour fermentation period as optimal. Subsequently, the interplay among fermentation temperature, initial pH, and methanol concentration on protein yield was explored. The RSM analysis revealed the most favorable conditions for fermentation: a temperature of 26 degrees C, an initial pH of 6.5, and a methanol concentration of 1.00%. Verification tests confirmed that under these optimized conditions, the T36N yield peaked at 346.64 +/- 3.47 mg.L-1, marking a 32.9% increase. The results underscored the capability of achieving high yields of T36N through the optimization of P. pastoris GS115 fermentation conditions. This research laid the groundwork for the industrial-scale production of N-glycosylated immunomodulatory proteins, offering a platform for the development of novel therapeutic agents.