Identification of metabolic engineering targets for improving glycerol assimilation ability of Saccharomyces...

Glycerol, a by-product of biodiesel production, has been utilized as a raw material for bioproduction. Saccharomyces cerevisiae, which has been used as a host microorganism for bioproduction, possesses the metabolic pathways for glycerol assimilation, but it cannot grow on glycerol as a carbon source. In this study, we identified metabolic engineering targets to improve the glycerol assimilation ability of S. cerevisiae based on adaptive laboratory evolution experiments using serial transfer of culture on glycerol and transcriptome analysis of the evolved cells using RNA-sequencing. The transcriptome data revealed that the upregulation of genes related to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation contributed to the increased specific growth rate on glycerol during adaptive evolution. Furthermore, genes related to the pentose phosphate pathway were downregulated. Based on these observations, we identified metabolic engineering targets for improving glycerol assimilation. Overexpression of HAP4, which encodes one of the subunits of the Hap2p/3p/4p/5p transcription factor complex involved in the upregulation of the TCA cycle genes, or disruption of RIM15, which encodes a protein kinase related to the transcription regulator Gis1p, as well as overexpression of STL1, which encodes the glycerol/H⁺ symporter, improved the growth of S. cerevisiae on glycerol as the main carbon source. Our results indicate that the engineering targets can be identified based on adaptive laboratory evolution and transcriptome analysis of the evolved cells, and that the glycerol assimilation ability of S. cerevisiae is indeed improved by engineering the identified targets.