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Background  Glycerol is the major by-product accounting for up to 5% of the carbon in  Saccharomyces cerevisiae  ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process.    Results  The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native  GPD1  promoter in the yeast genome by previously generated well-characterized  TEF  promoter mutant versions in a  gpd2 Δ background. Two  TEF  promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as  TEFmut7  and  TEFmut2 . The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in  TEFmut7  and 88% in  TEFmut2 . The overall ethanol production yield on glucose was improved from 0.43 g g -1  in the wild type to 0.44 g g -1  measured in  TEFmut7  and 0.45 g g -1  in  TEFmut2 . Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for  TEFmut7  and  TEFmut2  respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 ± 4 g L -1 ), growth-inhibiting ethanol concentration (87 ± 3 g L -1 ) and volumetric ethanol productivity (2.1 ± 0.15 g l -1  h -1 ) measured in wild-type remained virtually unchanged in the engineered strains.    Conclusions  This work demonstrates the power of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Under the conditions used in this study (VHEP fed-batch), the two strains with "fine-tuned"  GPD1  expression in a  gpd2 Δ background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains  gpd1 Δ or  gpd2 Δ. Although glycerol reduction is known to be even higher in a  gpd1 Δ  gpd2 Δ double deletion strain, our strains could much better cope with process stress as reflected by better growth and viability.

Quantitative evaluation of yeast's requirement for glycerol formation in very high ethanol performance fed-batch process