Modeling of Metabolic Overflow in Recombinant Pichia pastoris

Batch and fed-batch cultivations of Pichia pastoris KM71 were carried out to study the metabolic overflow when cultured in high concentration of glycerol. Pyruvate found in the culture medium was identified as a by-product of the metabolic overflow. Exponential feed profile was used in the fed-batch cultivations. Cell concentration of 96.23 g⋅lP-1P and 191.87 g⋅lP-1P were obtained from two fed-batch experiments. A dynamic metabolic overflow model of Pichia pastoris KM71 was proposed to describe the glycerol consumption, pyruvate formation, and cell growth under high glycerol concentration in an aerobic cultivation process. The model is based on the capability of Pichia pastoris KM71 to uptake glycerol, which is a bit higher than the limited substrate utilization rate for growth of cells. This excess glycerol uptake rate is converted into an overflow by-product, pyruvate. The modeling results agreed well with the experimental results of both batch and fed-batch experiments.

The critical glycerol concentration, defined as a glycerol concentration above which the metabolic overflow could occur, was calculated to be approximately 0.7-0.9 g⋅lP-1P. The value of critical glycerol concentration is corresponding to the limited substrate utilization for growth of Pichia pastoris KM71. Changes in the overflow behavior were observed at high pyruvate concentration and high cell mass concentration. The result obtained from this study is useful for the high cell density cultivation of Pichia pastoris using fed-batch technique where the minimization of by-product formation is needed.