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.
Highlights
As an oleaginous yeast, Yarrowia lipolytica is able to assimilate hydrophobic substrates
Effect of glucose feeding during the induction phase A three-stage fed-batch culture technique was adopted for the production of huIFN a2b, using Y. lipolytica JMY1852p
The ratio of oleic acid per gram of biomass required to trigger huIFN a2b gene expression was only one sixth that required when oleic acid was in excess. These results suggest that the specific amount of oleic acid added and the way in which oleic acid is used are the most important variables to be considered for efficient induction
Summary
Yarrowia lipolytica is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Yeasts have a great secretion potential including a strict quality control with the ability to perform complex post transcriptional modification [1,2]. In addition they are easier and less expensive to work with than insect or mammalian cells, and are adapted to fermentation processes. The most attractive yeast, especially in terms of performance reproducibility, was Y. lipolytica [3]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
