Production de protéines recombinantes chez la levure Yarrowia lipolytica

Les protéines recombinantes (rProt) revêtent une importance capitale (comme produit fini et comme catalyseur) dans les domaines de la pharmaceutique, de la chimie, des cosmétiques, de l'alimentation humaine et animale, et du traitement des déchets. Pour des raisons de qualité, une partie de ces protéines est produite par des systèmes-hôtes levuriens, tels Saccharomyces cerevisiae et Pichia pastoris (syn. Komagataella sp.). Outre ces hôtes établis, la levure non-conventionnelle Yarrowia lipolytica représente une usine cellulaire prometteuse pour la production de rProt. En effet, Y. lipolytica synthétise et sécrète des protéines de manière substantielle, jusqu’à 1–2 g·L-1. De plus, certains des inconvénients inhérents à ses prédécesseurs (hyperglycosylation élevée, effet Crabtree, dépendance au méthanol pour induire l'expression hétérologue) sont absents chez Y. lipolytica. Néanmoins, un manque de vue d'ensemble et d'outils spécifiques pour répondre aux exigences de cette levure l’empêchent toujours d'atteindre son plein potentiel de production de rProt. Dans le cadre de cette thèse, deux problèmes majeurs de la production de rProt chez Y. lipolytica ont été sélectionnés et traités : l’absence de promoteurs inductibles pratiques et la difficulté à maîtriser le dimorphisme au cours des processus en bioréacteur. Ces deux problèmes d’ordre pratique ont été résolus suite à la découverte et au détournement d’un gène de sa fonction primaire. Dans un premier temps, l'identification et la caractérisation du gène EYK1 dans le cadre du métabolisme de l'érythritol et de l'érythrulose chez Y. lipolytica ont conduit au développement de promoteurs régulés par ces polyols. Leur facilité d'utilisation et leur polyvalence se révèlent correspondre aux critères de production d'une large gamme de rProt. En utilisant une souche-hôte et des plasmides adaptés à l'expression inductible par l'érythritol et l'érythrulose, environ 45 000 U·mL-1 de lipase CalB sécrétée – le rendement le plus élevé jamais atteint chez la levure – ont été obtenus en 24 h dans un bioréacteur planctonique en mode batch. Deuxièmement, la découverte du gène YlHsl1 a permis d’obtenir des cellules mutantes bloquées dans une morphologie pseudohyphale (ne pouvant plus changer de conformation en réponse à des stimuli environnementaux). Ces cellules s’adaptent parfaitement à la croissance à l’état immobilisé au sein du bioréacteur, ce qui constitue un atout supplémentaire pour les bioprocédés continus et simplifie le traitement en aval des protéines sécrétées. Enfin, le système d'expression induit par l'érythritol développé pour Y. lipolytica au cours de cette thèse a été directement comparé pour la première fois à celui de P. pastoris, pour leur capacité à produire des rProt (ici la lipase CalB). La comparaison a révélé un titre de lipase CalB sécrétée 5 fois plus élevé dans les cultures en bioréacteur de Y. lipolytica que dans celles de P. pastoris, confirmant l'efficacité de Y. lipolytica pour la production de rProt

La levure comme usine cellulaire

Présentation des travaux du laboratoire levures du MiPI lors de l'inauguration du bâtiment TERR

Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast <i>Yarrowia lipolytica</i>

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and the possibility of post-translational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at a high level, and to secrete protein in large amounts. In recent years, several reviews have been dedicated to genetic tools developed for this purpose. Though the construction of efficient cell factories for recombinant protein synthesis is important, the development of an efficient process for recombinant protein production in a bioreactor constitutes an equally vital aspect. Indeed, a sports car cannot drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering

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

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

The quest for the best cell factory for recombinant protein production: Yarrowia lipolytica vs Pichia pastoris

In the present study, the performances of the emerging cell factories Y. lipolytica and P. pastoris were compared for their ability to synthetize and secrete recombinant proteins in bioreactors. As a case study, the lipase CalB from Candida antarctica was cloned under the control of the strong inducible promoters pEYK300A3B and pAOX1 and expressed in Y. lipolytica EYK1ko and P. pastoris MutS recipient strains, respectively. Surprisingly, Y. lipolytica performances were far superior in terms of cell growth, extracellular lipase activity, although P. pastoris showed a significantly higher level of CalB gene expression. According to our results, neither of codon usage bias, protein processing and secretion, or CalB lipase inactivation could be incriminated. It is therefore hypothesized that the observed difference lies in post-translational mechanisms activated by the overexpression of recombinant proteins, namely the unfolded protein response (UPR) and the endoplasmic reticulum (ER) associated degradation (ERAD). Here indeed, the proteasome was shown activated in P. pastoris following recombinant protein expression. In conclusion, keeping specific process constraints in mind, the selection of the adequate cell factory can dramatically improve the production of a given recombinant protein

Useful tools for genome editing in the non-conventional yeast Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica is increasingly used for the production of recombinant proteins or biomolecules with biotechnological or pharmaceutical applications. The development of such a cell factory requires steps of genome editing that rely on selectable markers. The recently identified EYK1, encoding erythrulose kinase, can serve as an efficient catabolic selectable marker for genome editing in Y. lipolytica. Besides, the cloning-free strategy developed here simplifies the construction of disruption cassettes for genome editing in Y. lipolytica

Enhancing the sweetener erythritol productivity in Yarrowia lipolytica using metabolic engineering

Erythritol (1,2,3,4-butanetetrol) is a four-carbon sugar alcohol used as sweeteners in food and beverage industry. It is most commonly generated from glucose via fermentation using osmophilic yeasts. Yarrowia lipolytica is highly proficient at producing erythritol and can use raw glycerol instead of glucose as its main carbon source. Raw glycerol is a byproduct of the biodiesel industry, and it is a cheaper and more efficient carbon source than glucose when it comes to producing erythritol. To date, most studies seeking to improve erythritol production have used wild type strains or randomly generated mutants and have focused on optimizing the culture medium or culturing conditions. In this study, we demonstrated that metabolic engineering can be used to enhance the production of erythritol from glycerol. To this end, we constructed a set of eleven strains that overexpressed genes encoding key enzymes of either glycerol catabolism and erythritol synthesis pathway. For the best of our engineered strains, erythritol productivity in bioreactor was increased 78% relative to the wild type strain and maximum concentrations were obtained in 40% less time

Catabolic selectable marker eases genome editing in Yarrowia lipolytica

Selectable markers are a central component of genome edition technologies. In the yeast Yarrowia lipolytica, these markers are traditionally based on antibiotic resistance (hygromycin B) or auxotrophy (e.g., leucine, uracil). However, the use of the former is impaired by a high level of spontaneous resistance, and the use of the latter by continuous complementation of the culture medium or restoration of prototrophy to the strains. As an alternative, genes related to the catabolism of carbon sources, or “catabolic selectable markers”, present the advantage of not being involved in essential metabolic pathways. The recently identified EYK1, encoding erythrulose kinase, can serve as an efficient catabolic selectable marker for genome editing in Y. lipolytica. Compared to auxotrophic markers such as URA3 and LEU2, EYK1 increases the growth rate of transformants on selective medium and the efficiency of genome edition. The utility of the marker EYK1 in a replicative vector was also demonstrated. Besides, the cloning-free strategy developed here simplifies the construction of disruption cassettes for genome editing in Y. lipolytica