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
18.104.22.168) purified from different mould species. It is a
reversible enzyme active with dihydroxyacetone or
glycerol as substrates. The GLD1 (EC 22.214.171.124) catalyses
the conversion of D-glyceraldehyde and L glyceraldehyde
to glycerol, however there is tiny activity in reverse
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.
|Conference||International Specialised Symposium on Yeasts, ISSY 25 |
|Abbreviated title||ISSY 25|
|Period||18/06/06 → 21/06/06|