The physiology of an engineered Saccharomyces cerevisiae strain that carries both an improved glycerol-3-phosphate and the synthetic dihydroxyacetone pathway for glycerol utilization.

Our laboratory previously established variants of the Saccharomyces cerevisiae strain CEN.PK113-1A able to grow in synthetic glycerol medium. One approach focused on improving the endogenous l-glycerol-3-phosphate (G3P) pathway, while a second approach aimed to replace the endogenous pathway with the dihydroxyacetone (DHA) pathway. The latter approach led to a significantly higher maximum specific growth rate (µmax) of 0.26 h-1 compared to 0.14 h-1. The current study focused on combining all genetic modifications in one strain. Apart from the so-called "TWO pathway strain" (CEN TWOPW), two isogenic control strains, CEN G3PPW and CEN DHAPW, were constructed. The µmax of CEN TWOPW (∼0.24 h-1) was virtually identical to that of CEN DHAPW. Remarkable characteristics of the strain CEN TWOPW compared to CEN DHAPW include a higher specific glycerol consumption rate, the capacity to deplete glycerol completely, and a much higher ethanol and lower biomass formation during oxygen-limited shake flask cultivations. The results obtained with different alleles of the GUT1 gene, encoding for glycerol kinase, suggest that the phenotype of the strain CEN TWOPW is at least partly attributed to the particular point mutation in the GUT1 allele used from the strain JL1, which was previously generated through adaptive laboratory evolution.