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The quest for a cell factory for the production of recombinant proteins: Pichia pastoris vs Yarrowia lipolytica
Vandermies, Marie; Theron, Chrispian; Fickers, Patrick
2019ECAB5
 

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Keywords :
Pichia pastoris; Yarrowia lipolytica; recombinant protein production; secretion; lipase; EGFP; ERAD
Abstract :
[en] 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) largely outperformed those of P. pastoris (µ = 0.07 and final DCW = 5 g/L, respectively). Moreover, 5-fold higher CalB production levels were observed with Y. lipolytica as a host than with P. pastoris. Maximal specific lipase activity was reached earlier in the culture with Y. lipolytica, i.e. 5700 U/gDCW after 28h, versus 1200 U/gDCW after 56h for P. pastoris. In contrast, a 7-fold higher CalB expression level was observed in P. pastoris than in Y. lipolytica. Additional experiments with CalB, EGFP, and fusion protein EGFP-CalB in P. pastoris ruled out the hypotheses of processing or secretion issues, as well as CalB inactivation in P. pastoris supernatant. In fact, CalB suffers from degradation within P. pastoris cells due to the activation of unfolded protein response, endoplasmic-reticulum associated degradation, and proteasomal degradation in response to high intracellular levels of recombinant protein. 4. Conclusions In the present study, Y. lipolytica outperformed P. pastoris in terms of growth performances, recombinant enzyme activity and promoter induction easiness (erythritol being much more convenient to handle than methanol). Such elements tend to point out Y. lipolytica as an advantageous host for recombinant protein production compared to P. pastoris. Further studies focusing on other recombinant proteins shall refine these conclusions.
Disciplines :
Biotechnology
Author, co-author :
Vandermies, Marie ;  Université de Liège - ULiège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Microbial, food and biobased technologies
Theron, Chrispian ;  Université de Liège - ULiège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Microbial, food and biobased technologies
Fickers, Patrick ;  Université de Liège - ULiège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Microbial, food and biobased technologies
Language :
English
Title :
The quest for a cell factory for the production of recombinant proteins: Pichia pastoris vs Yarrowia lipolytica
Publication date :
17 September 2019
Event name :
ECAB5
Event organizer :
ESBES
Event place :
Firenze, Italy
Event date :
15/09/2019 au 19/09/2019
Audience :
International
Funders :
FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture [BE]
Available on ORBi :
since 01 September 2019

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