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

Abstract

BackgroundAs 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.ResultsHuman interferon α2b (huIFN α2b) 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 huIFNα2b 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 α2b degradation by an aspartic protease secreted by Y. lipolytica was prevented by adding pepstatin (10 μM), leading to produce a 19-fold more active huIFN α2b (26.2 × 107 IU mg-1).ConclusionY. 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.

Carbon source dependent promoters in yeasts

“…In the meantime P POX2 has been frequently used, especially, when hydrophobic substrate conditions were required. The performance of P POX2 was further improved, testing human interferone alpha 2b expression, by co-feeding glucose at a limited rate during induction with oleate [139]. …”

Control of specific growth rate for the enhanced production of human interferon α2b in glycoengineered Pichia pastoris: process analytical technology guided approach

“…Specific huIFNα2b secretion rate, q huIFNα2b for μ sp ≤ 0.04 h −1 was found to be greater than 0.4 mg g −1 h ‐1 , which emerges as the highest among different recombinant proteins expressed in glycoengineered Pichia pastoris under AOX promoter . The huIFNα2b titer (1483 mg L −1 ) obtained at μ sp = 0.04 h −1 is the highest reported value for Pichia pastoris and also for any other yeast expression platforms (Table S4 Supporting Information) reported in the literature . Hence, this present study delineates that the specific growth rate is a paramount factor establishing superior specific protein productivity.…”

“…53,55,56 The huIFN 2b titer (1483 mg L −1 ) obtained at sp = 0.04 h −1 is the highest reported value for Pichia pastoris and wileyonlinelibrary.com/jctb also for any other yeast expression platforms (Table S4 Supporting Information) reported in the literature. [57][58][59][60][61][62] Hence, this present study delineates that the specific growth rate is a paramount factor establishing superior specific protein productivity. Feedback control from a PID output is a relatively simpler methodology and robustly controlled the inducer feeding in response to the real-time signal (ΔC) than a pre-set feed-rate.…”

Lipid Accumulation by Fungi Grown on Palm Oil Mill Effluent

“…There was slight differences in concentration of lipid species among fungi evaluated (Wynn 2001;Khot 2012;Chan 2010). The variability in fatty acid composition of oleaginous organism could be due to culture technique (Chi 2011) and species dependence (Gasmi . 2011).…”