Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

Abstract

Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut(+) strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed.

Keywords: Fusarium graminearum; Pichia pastoris; galactose oxidase; methanol feeding; optimization.

Figure 1

Volumetric GalOx activity in the fermentation broth at 25 °C (▪) and 30 °C (♦). Time corresponds to the total time of cultivation. Methanol feeding was initiated at 30 h. The detectable production of active and correctly folded GalOx sets off earlier at 30 °C, apparently due to the higher biomass concentration (data not shown). It stabilizes after 85 h of culture. At 25 °C, the volumetric GalOx activity increases continuously to 600 kU L⁻¹.

Figure 2

Volumetric GalOx activity as a function of time in culture using (A) high methanol-feeding rates, following the function F(t) = e^0.28t (♦) and F(t) = e^{0.28EXP(0.28t)} (▪), and (B) low methanol-feeding rates following the function F(t) = 6e^0.1t (•) and F(t) = 4.76e^0.1t (▴). Under mild conditions, the volumetric GalOx activity in the fermentation broth was almost 10 times higher than in the former case. Time refers to the total time in culture. The exponential phase started at 40 h.

Figure 3

Volumetric GalOx activity in the fermentation broth under different fed-batch conditions; (I) 4-h glycerol-feeding phase and constant methanol-feeding rate (3 mL h⁻¹ L_{initial culture volume}⁻¹) (▴), (J) 8-h glycerol feeding phase with constant methanol feeding rate (3 mL h⁻¹ L_{initial culture volume}⁻¹) (x), (K) 8-h glycerol-feeding phase and exponential methanol-feeding rate [F(t) = 3.6e^0.0213t] (♦), (L) 8-h glycerol feeding phase with constant methanol feeding rate to wet cell weight ratio (▪). Time corresponds to the total time of the cultivation. Methanol adaptation started at 30 h and lasted for 2 h.