New filamentous mutant of Yarrowia lipolytica and its use in biofilm bioreactors

The non-conventional yeast Yarrowia lipolytica is regarded as a promising cell factory for the production of recombinant proteins as well as added value chemicals. This organism possesses interesting metabolic properties, such as high capacity of biomolecule synthesis and secretion, substrate flexibility and adequacy with culture at high-cell density. Another characteristic trait of Y. lipolytica lies in its ability to adopt an ovoid or filamentous morphology (hyphae and pseudohyphae) according to environmental conditions. To date, several effectors of the dimorphic transition have been identified and characterized at the molecular level. However, the dimorphic transition remains difficult to master during cultures in bioreactor, thus negatively affecting the process reproducibility. Yet, filamentous organisms are particularly well suited for the development of immobilized-cell processes. In the present study, we have isolated and characterized a Y. lipolytica morphological mutant growing exclusively in the pseudohyphal morphology by means of insertion mutagenesis and high throughput screening methods. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the stainless steel structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica.