The quest for a cell factory for the production of recombinant proteins: Pichia pastoris vs Yarrowia lipolytica

• Main Authors: Vandermies, Marie, Theron, Chrispian, Fickers, Patrick
• Format: Conference Object
• Language: English
• Published: 2019
• Subjects: Pichia pastoris; Yarrowia lipolytica; recombinant protein production; secretion; lipase; EGFP; ERAD; Life sciences; Biotechnology; Sciences du vivant; Biotechnologie; antartic*
• Online Access: https://orbi.uliege.be/handle/2268/239060

1. Introduction Mastering microbial recombinant protein production is critical for application fields ranging from low-value agribusiness processes to high-value pharmaceutical products. Hyperglycosylation and alcoholic fermentation issues of model yeast Saccharomyces cerevisiae stimulated the development of non-conventional yeasts as alternative hosts for recombinant expression. In this regard, Pichia pastoris has been preferentially employed, despite a lack of comparative basis with other non-conventional yeasts. Here we report on the direct comparison of P. pastoris with another non-conventional yeast, Yarrowia lipolytica, for the production of industrial lipase B from Candida antartica (CalB) in small-scale bioreactors. 2. Methods In Y. lipolytica, CalB gene was expressed under the control of the erythritol-inducible promoter pEYK1-3AB, in a strain deleted for the gene EYK1 (allowing to use erythritol as a free inducer, no longer metabolized by the cells). Glycerol was employed as main carbon source. In P. pastoris, CalB gene was expressed under the control of the methanol-inducible promoter pAOX1, in a MutS strain (metabolizing methanol slower due to main alcohol oxidase deletion). Sorbitol was employed as main carbon source. The same CalB sequence was cloned in both strains, since no rare codon was detected and no significant difference in codon usage was observed between Y. lipolytica and P. pastoris. Batch cultures were realized in duplicate in Eppendorf DASbox® Mini Bioreactor System (120 mL working volume) over 72h. Temperature, pH, agitation rate, and aeration were adapted to suit both yeasts. Culture samples were analyzed for CalB gene expression, lipase activity and carbon source concentration (using HPLC). 3. Results and discussion For the present comparison, Y. lipolytica and P. pastoris were cultivated under conditions considered as the most efficient for each species. Under the specified conditions, Y. lipolytica growth rate and final biomass (µ = 0.26 and final DCW = 9 g/L, respectively) ...