In ethanol fermentation of

Saccharomyces cerevisiae

(

S. cerevisiae

)

glycerol is one of the main by-products. The purpose of this investigation was to increase ethanol yield through minimizing glycerol yield by using mutants in which

FPS1

encoding a channel protein that mediates glycerol export and

GPD2

encoding one of glycerol-3-phosphate dehydrogenase were knocked-out using one-step gene replacement.

GLT1

and

GLN1

that encode glutamate synthase and glutamine synthetase

respectively

were overexpressed using two-step gene replacement in

fps1Δ

gpd2Δ

mutant. The fermentation properties of ZAL69(

fps1Δ

::

LEU2

gpd2Δ

::

URA3

) and ZAL808 (

fps1Δ

::

LEU2

gpd2Δ

::

URA3

PPGK1

-

GLT1

PPGK1

-

GLN1

) under microaerobic conditions were investigated and compared with those of wild type(DC124). Consumption of glucose

yield of ethanol

yield of glycerol

acetic acid

and pyruvic acid were monitored. Compared with wild type

the ethanol yield of ZAL69 and ZAL808 were improved by 13.17% and 6.66%

respectively

whereas glycerol yield decreased by 37.4% and 41.7%. Meanwhile

acetic acid yield and pyruvic acid yield decreased dramatically compared to wild type. Our results indicate that

FPS1

and

GPD2

deletion of

S. cerevisiae

resulted in reduced glycerol yield and increased ethanol yield

but simultaneous over-expression of

GLT1

and

GLN1

in

fps1Δ

gpd2Δ

mutant did not have a higher ethanol yield than

fps1Δ

gpd2Δ

mutant.