Genetic
engineering of a yeast strain for hyperproduction of glycerol.
Hélène Cordier,
Jean M. François
Centre Bioing. Gilbert Durand, UMR CNRS 5504/INRA 792/INSA, 135 av. de
Rangueil, Toulouse, 31077, France (helene.cordier@insa-tlse.fr)
In order to redirect part of the carbon flux through glycerol formation in S. cerevisiae, we carried out four successive genetic modifications in the yeast strain CENPK-2. GPD1 encoding the main glycerol phosphate dehydrogenase isoform and ALD3 encoding one of the acetaldehyde dehydrogenases were overexpressed, while TPI1 encoding triose phosphate isomerase and ADH1 encoding alcohol dehydrogenase were disrupted. As expected, GPD1 overexpression leads to a 10-fold increase in glycerol production, with a yield of 0.2g glycerol/g glucose. TPI1 disruption severely impairs growth, which is partially restored by overexpression of GPD1 in a tpi1 delta mutant, likely by consuming excess of DHAP. Interestingly, ADH1 disruption results in an extracellular glycerol production similar to that observed in a GPD1 overexpressing strain. Strains bearing TPI1 disruption together with GPD1 overexpression and ADH1 deletion produce up to 0.45g glycerol/g glucose, which almost reaches the maximum theoretical yield (0.51g/g). While TPI1 disruption proves to be efficient in redirecting the carbon flux through glycerol synthesis, it results in poor growth, and hence in low glycerol productivity. However, TPI1 disruption does not entirely account for the slow growth phenotype, and two additional genetic modifications that are targeted to reduce intracellular levels of glycerol and DHA are currently in progress.