Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae
Abstract Background Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been...
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| Format: | Article |
| Language: | English |
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BMC
2017-04-01
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| Series: | Biotechnology for Biofuels |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13068-017-0791-3 |
| _version_ | 1818232221610278912 |
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| author | Ioannis Papapetridis Marlous van Dijk Antonius J. A. van Maris Jack T. Pronk |
| author_facet | Ioannis Papapetridis Marlous van Dijk Antonius J. A. van Maris Jack T. Pronk |
| author_sort | Ioannis Papapetridis |
| collection | DOAJ |
| description | Abstract Background Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. Results GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L−1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Conclusions Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes. |
| first_indexed | 2024-12-12T11:02:50Z |
| format | Article |
| id | doaj.art-5d60f03c58ca419eb10b67602aa9f953 |
| institution | Directory Open Access Journal |
| issn | 1754-6834 |
| language | English |
| last_indexed | 2024-12-12T11:02:50Z |
| publishDate | 2017-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels |
| spelling | doaj.art-5d60f03c58ca419eb10b67602aa9f9532022-12-22T00:26:28ZengBMCBiotechnology for Biofuels1754-68342017-04-0110111410.1186/s13068-017-0791-3Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiaeIoannis Papapetridis0Marlous van Dijk1Antonius J. A. van Maris2Jack T. Pronk3Industrial Microbiology Section, Department of Biotechnology, Delft University of TechnologyIndustrial Microbiology Section, Department of Biotechnology, Delft University of TechnologyIndustrial Microbiology Section, Department of Biotechnology, Delft University of TechnologyIndustrial Microbiology Section, Department of Biotechnology, Delft University of TechnologyAbstract Background Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. Results GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L−1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Conclusions Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.http://link.springer.com/article/10.1186/s13068-017-0791-3YeastNADHNADPHRedox engineeringAcetic acidOsmotic stress |
| spellingShingle | Ioannis Papapetridis Marlous van Dijk Antonius J. A. van Maris Jack T. Pronk Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae Biotechnology for Biofuels Yeast NADH NADPH Redox engineering Acetic acid Osmotic stress |
| title | Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae |
| title_full | Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae |
| title_fullStr | Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae |
| title_full_unstemmed | Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae |
| title_short | Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae |
| title_sort | metabolic engineering strategies for optimizing acetate reduction ethanol yield and osmotolerance in saccharomyces cerevisiae |
| topic | Yeast NADH NADPH Redox engineering Acetic acid Osmotic stress |
| url | http://link.springer.com/article/10.1186/s13068-017-0791-3 |
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