Citrate as Cost-Efficient NADPH Regenerating Agent
The economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration....
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-12-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fbioe.2018.00196/full |
| _version_ | 1818909568317521920 |
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| author | Reinhard Oeggl Reinhard Oeggl Timo Neumann Jochem Gätgens Diego Romano Stephan Noack Dörte Rother Dörte Rother |
| author_facet | Reinhard Oeggl Reinhard Oeggl Timo Neumann Jochem Gätgens Diego Romano Stephan Noack Dörte Rother Dörte Rother |
| author_sort | Reinhard Oeggl |
| collection | DOAJ |
| description | The economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration. In this context we now present an easy-to-apply whole cell cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1-phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5-13C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolism pathways, the glyoxylate shunt, and the glutamate dehydrogenase. |
| first_indexed | 2024-12-19T22:28:59Z |
| format | Article |
| id | doaj.art-8f6fb604e257461cae376af25dc989e8 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-12-19T22:28:59Z |
| publishDate | 2018-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-8f6fb604e257461cae376af25dc989e82022-12-21T20:03:23ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852018-12-01610.3389/fbioe.2018.00196380120Citrate as Cost-Efficient NADPH Regenerating AgentReinhard Oeggl0Reinhard Oeggl1Timo Neumann2Jochem Gätgens3Diego Romano4Stephan Noack5Dörte Rother6Dörte Rother7Forschungszentrum Jülich GmbH, IBG-1: Biotechnology, Jülich, GermanyAachen Biology and Biotechnology, RWTH Aachen University, Aachen, GermanyForschungszentrum Jülich GmbH, IBG-1: Biotechnology, Jülich, GermanyForschungszentrum Jülich GmbH, IBG-1: Biotechnology, Jülich, GermanyDepartment of Food, Environmental and Nutritional Sciences, University of Milan, Milan, ItalyForschungszentrum Jülich GmbH, IBG-1: Biotechnology, Jülich, GermanyForschungszentrum Jülich GmbH, IBG-1: Biotechnology, Jülich, GermanyAachen Biology and Biotechnology, RWTH Aachen University, Aachen, GermanyThe economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration. In this context we now present an easy-to-apply whole cell cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1-phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5-13C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolism pathways, the glyoxylate shunt, and the glutamate dehydrogenase.https://www.frontiersin.org/article/10.3389/fbioe.2018.00196/fullcitrate oxidationoxidoreductase screeningnicotinamide cofactorreduction equivalent regenerationNADPH regenerationcofactor regeneration |
| spellingShingle | Reinhard Oeggl Reinhard Oeggl Timo Neumann Jochem Gätgens Diego Romano Stephan Noack Dörte Rother Dörte Rother Citrate as Cost-Efficient NADPH Regenerating Agent Frontiers in Bioengineering and Biotechnology citrate oxidation oxidoreductase screening nicotinamide cofactor reduction equivalent regeneration NADPH regeneration cofactor regeneration |
| title | Citrate as Cost-Efficient NADPH Regenerating Agent |
| title_full | Citrate as Cost-Efficient NADPH Regenerating Agent |
| title_fullStr | Citrate as Cost-Efficient NADPH Regenerating Agent |
| title_full_unstemmed | Citrate as Cost-Efficient NADPH Regenerating Agent |
| title_short | Citrate as Cost-Efficient NADPH Regenerating Agent |
| title_sort | citrate as cost efficient nadph regenerating agent |
| topic | citrate oxidation oxidoreductase screening nicotinamide cofactor reduction equivalent regeneration NADPH regeneration cofactor regeneration |
| url | https://www.frontiersin.org/article/10.3389/fbioe.2018.00196/full |
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