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Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae.
Appl Environ Microbiol. 2011 Sep; 77(17):5857-67.AE

Abstract

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 (GPD1 and GPD2). While single deletions of GPD1 or GPD2 reduced glycerol formation only slightly, the gpd1Δ gpd2Δ 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 GPD1 promoter was replaced in a gpd2Δ background by two lower-strength TEF1 promoter mutants. In the same manner, the activity of the GPD2 promoter was reduced in a gpd1Δ background. The resulting strains were crossed to obtain different combinations of residual GPD1 and GPD2 expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to a gpd1Δ gpd2Δ 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.

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Authors+Show Affiliations

Hubmann G
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31–Bus 2438, B-3001 Heverlee, Flanders, Belgium.
Guillouet S
No affiliation info available
Nevoigt E
No affiliation info available

MeSH

BiotechnologyCrosses, GeneticDown-RegulationEthanolGene DeletionGene Expression Regulation, FungalGenetic EngineeringGlycerolGlycerol-3-Phosphate Dehydrogenase (NAD+)Promoter Regions, GeneticSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsTriticum

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

21724879

Citation

Hubmann, Georg, et al. "Gpd1 and Gpd2 Fine-tuning for Sustainable Reduction of Glycerol Formation in Saccharomyces Cerevisiae." Applied and Environmental Microbiology, vol. 77, no. 17, 2011, pp. 5857-67.
Hubmann G, Guillouet S, Nevoigt E. Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae. Appl Environ Microbiol. 2011;77(17):5857-67.
Hubmann, G., Guillouet, S., & Nevoigt, E. (2011). Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 77(17), 5857-67. https://doi.org/10.1128/AEM.05338-11
Hubmann G, Guillouet S, Nevoigt E. Gpd1 and Gpd2 Fine-tuning for Sustainable Reduction of Glycerol Formation in Saccharomyces Cerevisiae. Appl Environ Microbiol. 2011;77(17):5857-67. PubMed PMID: 21724879.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae. AU - Hubmann,Georg, AU - Guillouet,Stephane, AU - Nevoigt,Elke, Y1 - 2011/07/01/ PY - 2011/7/5/entrez PY - 2011/7/5/pubmed PY - 2011/12/14/medline SP - 5857 EP - 67 JF - Applied and environmental microbiology JO - Appl Environ Microbiol VL - 77 IS - 17 N2 - 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 (GPD1 and GPD2). While single deletions of GPD1 or GPD2 reduced glycerol formation only slightly, the gpd1Δ gpd2Δ 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 GPD1 promoter was replaced in a gpd2Δ background by two lower-strength TEF1 promoter mutants. In the same manner, the activity of the GPD2 promoter was reduced in a gpd1Δ background. The resulting strains were crossed to obtain different combinations of residual GPD1 and GPD2 expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to a gpd1Δ gpd2Δ 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. SN - 1098-5336 UR - https://www.unboundmedicine.com/medline/citation/21724879/Gpd1_and_Gpd2_fine_tuning_for_sustainable_reduction_of_glycerol_formation_in_Saccharomyces_cerevisiae_ L2 - https://journals.asm.org/doi/10.1128/AEM.05338-11?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -