Abstract
The oleaginous yeast species Rhodotorula toruloides is a promising candidate for applications in circular bioeconomy due to its ability to efficiently utilize diverse carbon sources being tolerant to cellular stress in bioprocessing. Previous studies including genome-wide analyses of the multi-stress tolerant strain IST536 MM15, derived through adaptive laboratory evolution from a promising IST536 strain for lipid production from sugar beet hydrolysates, suggested the occurrence of significant modifications in the cell wall. In this study, the cell wall integrity and carbohydrate composition of those strains was characterized to gain insights into the physicochemical changes associated to the remarkable multi-stress tolerance phenotype of the evolved strain. Compared to the original strain, the evolved strain exhibited a higher proportion of glucomannans, fucogalactomannans, and chitin relative to (1→4)-linked glucans, and an increased presence of glycoproteins with short glucosamine derived oligosaccharides, which have been found to be associated to ethanol stress tolerance and physical strength of the cell wall. Furthermore, the evolved strain cells were found to be significantly smaller than the original strain and more resistant to thermal and mechanical disruption, consistent with higher proportion of beta-linked polymers instead of glycogen, conferring a more rigid and robust cell wall. These findings provide further insights into the cell wall composition of this basidiomycetous red yeast species and into the alterations occurring in a multi-stress tolerant evolved strain. This new information can guide yeast genome engineering towards more robust strains of biotechnological relevance.