Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, Yarrowia lipolytica

Background: As an oleaginous yeast, Yarrowia lipolytica is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Less is known about the behavior of Y. lipolytica in large bioreactors using these substrates. There is therefore a lack of established know-how concerning high cell density culture protocols of this yeast. Consequently, the establishment of suitable induction conditions is required, to maximize recombinant protein production under the control of these promoters. Results: Human interferon alpha 2b (huIFN alpha 2b) production in Yarrowia lipolytica was used as a model for the enhancement of recombinant protein production under the control of the oleic acid (OA)-inducible promoter POX2. Cell viability and heterologous protein production were enhanced by exponential glucose feeding, to generate biomass before OA induction. The optimal biomass level before induction was determined (73 g L(-1)), and glucose was added with oleic acid during the induction phase. Several oleic acid feeding strategies were assessed. Continuous feeding with OA at a ratio of 0.02 g OA per g dry cell weight increased huIFN alpha 2b production by a factor of 1.88 (425 mg L(-1)) and decreased the induction time (by a factor of 2.6, 21 h). huIFN alpha 2b degradation by an aspartic protease secreted by Y. lipolytica was prevented by adding pepstatin (10 mu M), leading to produce a 19-fold more active huIFN alpha 2b (26.2 x 10(7) IU mg(-1)). Conclusion: Y. lipolytica, a generally regarded as safe (GRAS) microorganism is one of the most promising non conventional yeasts for the production of biologically active therapeutic proteins under the control of hydrophobic substrate-inducible promoter.