Novel evolved Yarrowia lipolytica strains for enhanced growth and lipid content under high concentrations of crude glycerol

Yarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance.Y. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9-20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation.ALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable "chassis" for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.