Gpd1 and Gpd2 Fine-Tuning for Sustainable Reduction of Glycerol Formation in Saccharomyces cerevisiae

Gpd1 and Gpd2 are the two isoforms of glycerol 3-phosphate dehydrogenase (GPDH), which is the rate-controlling enzyme of glycerol formation inSaccharomyces 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 (GPD1 andGPD2 ). While single deletions ofGPD1 orGPD2 reduced glycerol formation only slightly, thegpd1 Δgpd2 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, theGPD1 promoter was replaced in agpd2 Δ background by two lower-strengthTEF1 promoter mutants. In the same manner, the activity of theGPD2 promoter was reduced in agpd1Δ background. The resulting strains were crossed to obtain different combinations of residualGPD1 andGPD2 expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to agpd1 Δgpd2 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−1 ). Moreover, a few of these strains showed no significant reduction in osmotic stress tolerance compared to the wild type.