STRATEGY DESIGN FOR THERAPEUTICAL PROTEIN PRODUCTION by Pichia pastoris

Introduction. Yeasts are widely used in production of recombinant proteins (r-proteins) of medical or industrial interest. For the production of a biomolecule in a host a creative metabolic engineering design is achieved, and then optimized both on the molecular genetic and fermentation level, by taking into account the properties of the product, the host organism and the expression cassette. Amongst, the methylotropic yeast Pichia pastoris has become a frequently used expression system for rprotein production owing to its strong and tightly regulated promoter alcohol oxidase I (AOX1). Since methanol is used not only as the carbon and energy source, but also as an inducer of the expression of r-proteins, despite at high concentrations inhibits the growth, fed-batch feeding strategies are employed to increase the product yield [1]. In this work, to improve r-protein productions under strong methanol inducible alcohol oxidase I (AOX1) promoter, feeding strategies for semi-batch operations were developed in pilot scale bioreactors, for the: i) nonglycosylated protein recombinant human growth hormone (rhGH) production by the constructed P. pastoris M13 strain (pPICZαA::hGH-Mut) [2]; ii) the glycoprotein recombinant human erythropoietin (rHuEPO) production by the constructed P. pastoris E17 strain (pPICZαA::epoMut+) [3]. Further, the influences of the co-substrate feeding strategies on the intracellular reaction network of P.pastoris will be discussed through metabolic flux analysis.