초록
<P><B>ABSTRACT</B><P> Gpd1 and Gpd2 are the two isoforms of glycerol 3-phosphate dehydrogenase (GPDH), which is the rate-controlling enzyme of glycerol formation in Saccharomyces cerevisiae. The two isoenzymes play crucial roles in osmoregulation and redox balancing. Past approaches to increase ethanol yield at the cost of reduced glycerol yield have most often been based on deletion of either one or two isogenes ( <I>GPD1</I> and <I>GPD2</I> ). While single deletions of <I>GPD1</I> or <I>GPD2</I> reduced glycerol formation only slightly, the <I>gpd1</I> Δ <I>gpd2</I> Δ double deletion strain produced zero glycerol but showed an osmosensitive phenotype and abolished anaerobic growth. Our current approach has sought to generate “intermediate” phenotypes by reducing both isoenzyme activities without abolishing them. To this end, the <I>GPD1</I> promoter was replaced in a <I>gpd2</I> Δ background by two lower-strength <I>TEF1</I> promoter mutants. In the same manner, the activity of the <I>GPD2</I> promoter was reduced in a <I>gpd1Δ</I> background. The resulting strains were crossed to obtain different combinations of residual <I>GPD1</I> and <I>GPD2</I> expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to a <I>gpd1</I> Δ <I>gpd2</I> Δ double-deletion strain, these strains were able to completely ferment the sugars under quasi-anaerobic conditions in both minimal medium and during simultaneous saccharification and fermentation (SSF) of liquefied wheat mash (wheat liquefact). This result implies that our strains can tolerate the ethanol concentration at the end of the wheat liquefact SSF (up to 90 g liter <SUP>−1</SUP> ). Moreover, a few of these strains showed no significant reduction in osmotic stress tolerance compared to the wild type. </P></P>