Fermentation Development of Recombinant Pichia pastoris Expressing the Heterologous Gene: Bovine Lysozyme

The methylotrophic yeast, Pichia pastoris, has been shown to be an outstanding host for high-level heterologous gene expression.'+ The alcohol oxidase promoter was isolated and cloned by Ellis et a/.' and transformation of Pichia pastoris was first reported in 1985.6 The success of the Pichia expression system is linked to the strong, tightly regulated alcohol oxidase (AOXl ) promoter. The strength of the promoter is demonstrated by the observation that AOX comprises up to 30% of the soluble protein in extracts of Pichia pastoris grown on methanol (MeOH) in a chemostat.' Another key feature of the system is that high cell densities can be achieved using a simple MeOH-salts medium.' The strong promoter coupled with the high-cell-density fermentations have allowed production of recombinant products a t high intercellular (e.g., 400 mg/L hepatitis surface antigen)' and extracellular (e.g., 2.5 g /L invertase and 250 mg/L bovine l y ~ o z y m e ) ~ ~ ~ concentrations. The recombinant Pichia fermentations referenced above'.234 employed an MeOH utilization mutant (Mut-) created by integration of the recombinant expression cassette into the host genome a t the AOXl locus, resulting in disruption of the AOXl structural gene. The Mutstrains were grown in a repressed, batch regime for 24 hours, followed by an MeOH-induced, fed-batch regime for 100-200 hours. The long induction period is a consequence of the slow growth rate of the Mutstrain on MeOH ( p = 0.01-0.04 h' ) . The Mutstrains are still able to utilize MeOH due to the presence of the AOX2 gene, which yields 10-20 times less AOX activity than the AOXI gene.' The original Mutprotocols offer several desirable features. They are extremely easy to scale up to large volumes and they consistently give maximal levels of expression.' Additionally, low growth rates may be desirable for production of certain recombinant product^.'*^^'^ Furthermore, the Mutstrains are not as sensitive to transient high residual MeOH concentrations, which can cause as much as a 99% loss of AOX activity and cell death following certain culture perturbations of wild-type met hylotrophic yeast. 'J I-'