NADP dependent glycerol dehydrogenases in the mould Hypocrea jecorina and their application in cofactor engineering

Fungal microorganisms have the enzymes for a glycerol cycle consisting of the following enzymes: glycerol dehydrogenase (NADP+), dihydroxyacetone kinase, glycerol-3-phosphate dehydrogenase (NAD+) glycerol-3-phosphate phosphatase. In each cycle NADPH and NAD is formed from NADP and NADH at the expense of ATP. However an active glycerol cycle has never been reported. If active, such a cycle could be used in cofactor engineering. An application could be pentose fermentation with the yeast S. cerevisiae where the NADPH/NADP imbalance is a rate limiting factor. Our approach was to express an NADP dependent glycerol dehydrogenase and the endogenous DAK1 (dihydroxy acetone kinase) from a constitutive promoter as a strategy to introduce this glycerol cycle in yeast. NADP glycerol dehydrogenases can convert glycerol to glyceraldehyde or dihydroxy acetone. So far it was not possible to predict whether a glycerol dehydrogenase was glyceraldehyde or dihydroxy acetone forming. The genes gld1 and gld2 from mould Hypocrea jecorina (Trichoderma reesei) coding for enzymes with high similarity to the NADP-dependent glycerol dehydrogenases were cloned and expressed in a heterologous host. The encoded proteins were purified and their kinetic properties characterized. The GLD2 characteristics are similar to the previously described NADP-dependent glycerol-2-dehydrogenases (EC 1.1.1.156) purified from different mould species. It is a reversible enzyme active with dihydroxyacetone or glycerol as substrates. The GLD1 (EC 1.1.1.72) catalyses the conversion of D-glyceraldehyde and L glyceraldehyde to glycerol, however there is tiny activity in reverse reaction. The GLD2 was chosen for overexpression together with DAK1 to facilitate the glycerol cycle in S. cerevisiae. Preliminary studies on xylose fermenting S. cerevisiae will be presented.