The influence of carbon sources on recombinant-human- growth-hormone production by Pichia pastoris is dependent on phenotype: a comparison of Muts and Mut+ strains

The influence of carbon sources on rhGH (recombinant human growth hormone) production by two Pichia pastoris strains having different methanol utilization phenotypes (P. pastoris‐hGH‐Mut+ and P. pastoris‐hGH‐Muts) was investigated using batch bioreactors. The effect of methanol concentration (CMeOH) in defined and complex media, and further glycerol/methanol mixed defined media, was analysed systematically over a wide range. With methanol as the sole carbon source, strain Muts grew only slightly, whereas with Mut+, a cell concentration (CX) of 6.0 g of dry cells/dm3 was obtained and an rhGH concentration (CrhGH) of 0.032 g/dm3 was produced. In complex medium without glycerol at a CMeOH of 2% (v/v), a CrhGH of 0.16 g of rhGH/dm3 was produced by Muts, a value 3‐fold higher than that produced by Mut+, despite the fact that the CX of Mut+ (6.1 g/dm3) was 2‐fold higher than that of Muts (3.0 g/dm3). In a glycerol/methanol mixed defined medium, methanol consumption began when glycerol was totally depleted, indicating that glycerol is a repressor of the AOX1 (alcohol oxidase‐1 gene) promoter. With strain Muts at a glycerol concentration (CGly) of 30 g/dm3 and a CMeOH of 1% (v/v), the CrhGH produced was 0.11 g/dm3, whereas, with the Mut+ strain, a CrhGH of 0.06 g/dm3 was obtained at a CGly of 30 g/dm3 and a CMeOH of 4%. As methanol is not consumed by Muts strain effectively and the presence of methanol in the fermentation broth triggers induction of the AOX1 promoter, our results encourage the use of the Muts strain for rhGH production. In addition to rhGH production, the specific cell growth rates, specific methanol and/or glycerol utilization rates and maintenance coefficients in methanol‐ and glycerol‐based defined media were determined. With a methanol‐based defined medium and using the Mut+ strain, a higher specific growth rate (μ) of approx. 0.14 h−1 was observed during the exponential cell growth phase at a CMeOH of ≤2.0%. When glycerol was used as a sole carbon source, both phenotypes showed similar cell‐growth and glycerol‐utilization rates. The results of the present study should enable one to optimize the expression of other therapeutic proteins by P. pastoris.