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Pagliardini et al. Microbial Cell Factories 2010, 9:36 http://www.microbialcellfactories.com/content/9/1/36

RESEARCH

Open Access

Quantitative evaluation of yeast’s requirement for glycerol formation in very high ethanol performance fed-batch process
Julien Pagliardini1, Georg Hubmann2,3, Carine Bideaux1, Sandrine Alfenore1, Elke Nevoigt2,3,4, Stéphane E Guillouet1*

AbstractBackground: 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 glycerolproduction 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 apreviously developed Very High Ethanol Performance (VHEP) fed-batch process. Results: The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The finetuned 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 gpd2Δ background. Two TEFpromoter 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 wildtype was 61% in TEFmut7 and 88% in TEFmut2. The overall ethanolproduction 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), growthinhibiting ethanolconcentration (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 otherhand. Under the conditions used in this study (VHEP fed-batch), the two strains with “fine-tuned” GPD1 expression in a gpd2Δ background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains gpd1Δ or gpd2Δ. Although glycerol reduction is known to be even higher in a gpd1Δ gpd2Δ double deletion strain, our strains could much better cope with processstress as reflected by better growth and viability.

* Correspondence: stephane.guillouet@insa-toulouse.fr 1 Université de Toulouse, INSA, UPS, INP, LISBP, 135 Av de Rangueil, F-31077 Toulouse France INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France CNRS, UMR5504, F-31400 Toulouse, France
© 2010 Pagliardini et al; licensee BioMed Central Ltd. This is anOpen Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pagliardini et al. Microbial Cell Factories 2010, 9:36 http://www.microbialcellfactories.com/content/9/1/36

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