Adaptive evolution of Yarrowia lipolytica

Abstract

We need renewable resources to allow sustainable production of fuels. By using lipid accu-mulating yeast, biodiesel can be produced in a sustainable way from resources that were previously not used, for example lignocellulose-based sources such as agricultural waste. However, for this process to be profitable, the tolerance of the yeast needs to be improved. This project aims to improve the tolerance of the oleaginous yeast Yarrowia lipolytica to-wards osmotic and saline stress by using the method adaptive laboratory evolution. This method has previously been shown to be efficient in constructing strains to tolerate new conditions without the need of prior knowledge. After evolving Y. lipolytica for 220 gen-eration in minimal medium containing 1.4 M NaCl, an improved performance in the same medium was observed, along with evolved cross-tolerance towards low pH. This indicates that this adaptive evolution of Y. lipolytica resulted in improved ionic tolerance rather than pure osmotic tolerance. The evolved strains were sent for whole genomic sequencing to find out which mutations that caused this phenotype. During this project, two previous CRISPR/Cas9 strategies were combined and adapted for efficient markerless reverse en-gineering. When genome data arrives, this strategy will be used for reconstruction of can-didate mutations to find out which mutations are important for the observed phenotype. The gained knowledge from this evolution experiment can later be used for constructing a robust industrial strain that efficiently converts lignocellulose-based material to biodiesel, allowing sustainable production of fuels.