Physiology and metabolism of Yarrowia lipolytica for the utilization of alternative carbon substrates

Efforts to valorize alternative carbon feedstocks from lignocellulosic hydrolysis or the biodiesel by-product glycerol have motivated investigations into new cell-factory hosts. The non-conventional yeast species Yarrowia lipolytica has attracted attention in recent years as a promising candidate for these novel and sustainable biotechnological applications. In this Ph.D. thesis, we analyzed the physiology, genetics and metabolism of Y. lipolytica for the usage of alternative carbon sources by methods of quantitative physiology and genomics.

We benchmarked the cellular performance of the three Y. lipolytica strains IBT 446, W29 and H222 on glucose, xylose, arabinose and glycerol using single and mixed substrate fermentations in controlled bioreactors. Glycerol was found to be the preferred carbon source for all three strains, leading to the highest growth rates and the production of sugar alcohols. Inter-strain variations were detected and, in particular, IBT 446 was found to differ in several characteristics from the commonly used strains W29 and H222. IBT 446, originally isolated from Danish feta cheese, possessed beneficial characteristics like the absence of hyphal growth, which usually causes problems in industrial fermentations. Since physiological differences were observed, we sequenced the genomes of the three Y. lipolytica strains.

All strains showed a characteristic sequential substrate utilization in mixed carbon fermentations. Interestingly, it was observed that the presence of glycerol can prevent the consumption of glucose and that this suppression is further strain dependent: IBT 446 exhibited a strong sequential utilization of glycerol and glucose, whereas W29 co-consumed the two substrates. This indicated so far unknown carbon regulation mechanisms, which are converse to well-described carbon repression systems (e.g. in S. cerevisiae or E. coli) ensuring the prioritized use of glucose. RNAseq analysis was performed in order to investigate the influence of glycerol on the gene expression. We could show that genes encoding several transporters and metabolic enzymes were expressed significantly higher in W29. Further, we found strain-specific carbon responses and that several differentially expressed genes encode proteins related to signal transduction and transcriptional regulation, e.g. S. cerevisiae orthologs RME1, STE4, STE6, SST2, GPA2 and AZF1.