Enhancing the secretion pathway maximizes the effects of mixed feeding strategy for glucose oxidase production in the methylotrophic yeast Pichia pastoris

• Main Authors: Olufemi Emmanuel Bankefa, Meiyu Wang, Taicheng Zhu, Yin Li
• Format: Article
• Language: English
• Published: SpringerOpen 2018-05-01
• Series: Bioresources and Bioprocessing
• Subjects: Glucose oxidase; Hac1p; Mixed feeding; Pichia pastoris; Secretion pathway engineering
• Online Access: http://link.springer.com/article/10.1186/s40643-018-0211-y

Abstract Background A mixed feeding strategy (co-feeding of complex carbon sources with methanol) has become a common practice for process development in Pichia pastoris to increase cell biomass and enzyme production levels. However, in some cases mixed feeding did not have a significant impact or even had a negative effect on specific enzyme productivity. We hypothesized that this may be due to a bottleneck in the protein secretion pathway caused by too strong protein expression as a result of mixed feeding operation. Results Using glucose oxidase (Gox) as a model protein, the individual and synergistic effects of co-feeding of sorbitol or yeast extract (YE) with methanol and Hac1p overexpression on the secretory expression of Gox were investigated both in shake flasks and in a laboratory fermenter. The results showed that YE is superior to sorbitol in terms of stimulating protein expression and cell growth. Moreover, separate applications of the mixed feeding strategy and secretory pathway engineering only achieved limited success in enhancing Gox levels, while the combined use of the two strategies acted synergistically, leading to 297% increase of Gox production and the final enzyme titer reached 787.4 U/mL in GSgox-Pp on 1-L fermenter. Conclusions Co-feeding of YE combined with secretion pathway engineering significantly improved glucose oxidase secretion, which can be also applied to improve secretory expression of other foreign proteins in P. pastoris system.