A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study
Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. How...
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Elsevier
2024-12-01
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| Series: | Metabolic Engineering Communications |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214030124000142 |
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| author | Priti Regmi Melanie Knesebeck Eckhard Boles Dirk Weuster-Botz Mislav Oreb |
| author_facet | Priti Regmi Melanie Knesebeck Eckhard Boles Dirk Weuster-Botz Mislav Oreb |
| author_sort | Priti Regmi |
| collection | DOAJ |
| description | Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L−1 xylitol from 20 g L−1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH. |
| format | Article |
| id | doaj-art-f715c287cc824cf6869ecc547fa08f78 |
| institution | DOAJ |
| issn | 2214-0301 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj-art-f715c287cc824cf6869ecc547fa08f782025-08-20T02:49:27ZengElsevierMetabolic Engineering Communications2214-03012024-12-0119e0024510.1016/j.mec.2024.e00245A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case studyPriti Regmi0Melanie Knesebeck1Eckhard Boles2Dirk Weuster-Botz3Mislav Oreb4Goethe University Frankfurt, Faculty of Biological Sciences, Institute of Molecular Biosciences, Max-von-Laue Straße 9, 60438, Frankfurt am Main, GermanyTechnical University of Munich, Chair of Biochemical Engineering, Boltzmannstr. 15, 85748, Garching, GermanyGoethe University Frankfurt, Faculty of Biological Sciences, Institute of Molecular Biosciences, Max-von-Laue Straße 9, 60438, Frankfurt am Main, GermanyTechnical University of Munich, Chair of Biochemical Engineering, Boltzmannstr. 15, 85748, Garching, GermanyGoethe University Frankfurt, Faculty of Biological Sciences, Institute of Molecular Biosciences, Max-von-Laue Straße 9, 60438, Frankfurt am Main, Germany; Corresponding author. Institute of Molecular Biosciences, Max-von-Laue Straße 9, 60438, Frankfurt, Germany.Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L−1 xylitol from 20 g L−1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.http://www.sciencedirect.com/science/article/pii/S2214030124000142NADPH supplyD-xylitolGlucose-6-phosphate dehydrogenasel-galactonateSaccharomyces cerevisiae |
| spellingShingle | Priti Regmi Melanie Knesebeck Eckhard Boles Dirk Weuster-Botz Mislav Oreb A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study Metabolic Engineering Communications NADPH supply D-xylitol Glucose-6-phosphate dehydrogenase l-galactonate Saccharomyces cerevisiae |
| title | A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study |
| title_full | A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study |
| title_fullStr | A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study |
| title_full_unstemmed | A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study |
| title_short | A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study |
| title_sort | comparative analysis of nadph supply strategies in saccharomyces cerevisiae production of d xylitol from d xylose as a case study |
| topic | NADPH supply D-xylitol Glucose-6-phosphate dehydrogenase l-galactonate Saccharomyces cerevisiae |
| url | http://www.sciencedirect.com/science/article/pii/S2214030124000142 |
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