High-level extracellular production of glucose oxidase by recombinant Pichia pastoris using a combined strategy.

In this work, a combined strategy was developed to improve the production of glucose oxidase (GOD) (EC 1.1.3.4) in Pichia pastoris. One of the main challenges facing protein production by the high-density fermentation of P. pastoris is the high demand for oxygen. Another challenge is how to balance a reduction in oxygen consumption and its effects on protein production. Herein, a combined strategy involving mannitol co-feeding, two-stage methanol induction, and the co-expression of the transcriptional activator general control non-derepressible 4 (GCN4) from P. pastoris was used. A two-stage, co-feeding strategy, based on a mannitol/methanol mixture in a 3-L fermentor was used to enhance cell viability and protein production. This resulted in an increased GOD yield of 1208.2 U/mL compared with a control strain (427.6 U/mL). An increase in the copy number of the GCN4 gene enhanced the GOD yield (1634.7 U/mL) by 2.8-fold and the protein concentration (19.55 g/L) by 1.58-fold compared with the control (7.59 g/L). This strategy illustrates a way to overcome the high oxygen requirement during high-density fermentation of P. pastoris and balances the reduction of oxygen consumption and protein production. Moreover, the series of strategies presented in this work provide valuable and novel information for the industrial production of GOD.