Abstract

Pichia pastoris and Pichia methanolica have been used as expression systems for the production of recombinant protein. The main problems of the production are the slow hierarchic consumption of ethanol and acetate which cause toxicity problems due to methanol accumulation when this surpasses 0.5 gl^-1. In some cases, the laboratory scale cultures does not show the methanol accumulation problems because the cells are usually washed before changing the depleted carbon source culture media, by a fresh one, supplemented with methanol; a strategy that is clearly inapplicable in the industrial productions. Other authors use to feed the methanol before the depletion of glycerol or D-glucose, but this practice does not guaranty the ethanol and acetate fast consumption; leading to methanol accumulation and toxicity problems. It was concluded that pre-induction stage strategy should be studied in detail and that it is very important to start the induction stage with a concentration of biomass as great as possible. On the other side, it is essential that there should be a monitoring of ethanol and acetate until reaching non-toxic methanol stable-concentration and these conditions should be maintained till the end of the process.

Key words: Carbon source, protein expression, Pichia pastoris, Pichia methanolica.

Abbreviation

SCP, Single cell protein; Mut pathway, methanol utilization pathway; AO, alcohol oxidase; MO, methanol oxidase, PTS, peroxisomal targeting signals; DHAS, dihydroxyacetone synthase, CAT, catalase; MOD2 (AUG2),alcohol utilizing genes; AOX, alcohol oxidase genes; Mut^S, slow methanol growth phenotype; Mut⁺, wild type; Mut^-, negative methanol utilization.