Glycerol is a polyol that can be produced either chemically from oils or propylene, or biologically by yeasts, mainly under osmotic stress. Currently, glycerol is an abundant byproduct generated during biodiesel manufacturing that can be used as substrate in fermentative processes. Its efficient assimilation varies widely among yeast species; therefore, understanding the regulation of both transport and catabolism is pivotal for optimizing biotechnological processes based on this carbon source. This review addresses current knowledge on the regulatory networks controlling glycerol metabolism in yeasts, compassing transport mechanisms, metabolic pathways, transcriptional control and enzyme regulation. We highlight the distinct roles of the Stl1p symporter and Fps1p aquaglyceroporin in mediating glycerol flux across the plasma membrane, as well as the species-specific reliance on either the glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA) pathway for glycerol assimilation. Classical biochemical studies have shown that glycerol catabolic enzymes are tightly regulated by carbon source availability, osmotic conditions, and feedback inhibition. Recently, transcriptomic and genetic analyses, particularly in Yarrowia lipolytica, show that the glycerol metabolism is governed by complex interactions between catabolite repression, nutrient signaling, and metabolic intermediates such as G3P, which acts as a key regulatory signal. Despite significant advances, regulatory mechanisms remain elusive in most non-conventional yeasts, underscoring the need for broader comparative studies. In addition, we review major metabolic engineering strategies aimed at enhancing glycerol utilization or redirecting carbon flux toward targeted bioproducts, emphasizing how mechanistic insights into glycerol metabolism and its regulation can guide the development of engineered strains with improved features for industrial applications. Lastly, we pointed out promising avenues for future research and biotechnological innovation.
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