Towards the exploitation of glycerol's high reducing power in Saccharomyces cerevisiae-based bioprocesses.

One advantage of using glycerol as a carbon source for industrial bioprocesses is its higher degree of reduction compared to glucose. In order to exploit this reducing power for the production of reduced compounds thereby significantly increasing maximum theoretical yields, the electrons derived from glycerol oxidation must first be saved in the form of cytosolic NAD(P)H. However, the industrial platform organism Saccharomyces cerevisiae naturally uses an FAD-dependent pathway for glycerol catabolism transferring the electrons to the respiratory chain. Here, we developed a pathway replacement strategy forcing glycerol catabolism through a synthetic, NAD+-dependent route. The required expression cassettes were integrated via CRISPR-Cas9 targeting the endogenous GUT1 locus, thereby abolishing the native FAD-dependent pathway. Interestingly, this pathway replacement even established growth in synthetic glycerol medium of strains naturally unable to grow on glycerol and an engineered derivative of CEN.PK even showed the highest ever reported maximum specific growth rate on glycerol (0.26h-1).