In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization
Due to enhanced energy content and reduced hygroscopicity compared to ethanol, n-butanol is flagged as a next generation biofuel and platform chemical. In addition to conventional cellular systems, butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In con...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-10-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fbioe.2016.00074/full |
| _version_ | 1811305576426110976 |
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| author | Thomas Bartholomäus Brück |
| author_facet | Thomas Bartholomäus Brück |
| author_sort | Thomas Bartholomäus Brück |
| collection | DOAJ |
| description | Due to enhanced energy content and reduced hygroscopicity compared to ethanol, n-butanol is flagged as a next generation biofuel and platform chemical. In addition to conventional cellular systems, butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free butanol biosynthesis from the central metabolic intermediate pyruvate involves co-factors (NAD(P)H, CoA) and acetyl-CoA dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine – aldol – condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as co-factor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50 °C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L-1 (2 mM), which compares well with other cell-free butanol production processes. |
| first_indexed | 2024-04-13T08:27:57Z |
| format | Article |
| id | doaj.art-6f7ca46e668844bf96684bfe95df927b |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-13T08:27:57Z |
| publishDate | 2016-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-6f7ca46e668844bf96684bfe95df927b2022-12-22T02:54:21ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852016-10-01410.3389/fbioe.2016.00074205442In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilizationThomas Bartholomäus Brück0Technische Universität MünchenDue to enhanced energy content and reduced hygroscopicity compared to ethanol, n-butanol is flagged as a next generation biofuel and platform chemical. In addition to conventional cellular systems, butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free butanol biosynthesis from the central metabolic intermediate pyruvate involves co-factors (NAD(P)H, CoA) and acetyl-CoA dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine – aldol – condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as co-factor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50 °C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L-1 (2 mM), which compares well with other cell-free butanol production processes.http://journal.frontiersin.org/Journal/10.3389/fbioe.2016.00074/fullBiocatalysisbutanolCell-freeSynthetic biotechnologychemo-enzymaticEnzyme cascade |
| spellingShingle | Thomas Bartholomäus Brück In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization Frontiers in Bioengineering and Biotechnology Biocatalysis butanol Cell-free Synthetic biotechnology chemo-enzymatic Enzyme cascade |
| title | In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization |
| title_full | In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization |
| title_fullStr | In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization |
| title_full_unstemmed | In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization |
| title_short | In vitro bioconversion of pyruvate to n-butanol with minimized cofactor utilization |
| title_sort | in vitro bioconversion of pyruvate to n butanol with minimized cofactor utilization |
| topic | Biocatalysis butanol Cell-free Synthetic biotechnology chemo-enzymatic Enzyme cascade |
| url | http://journal.frontiersin.org/Journal/10.3389/fbioe.2016.00074/full |
| work_keys_str_mv | AT thomasbartholomausbruck invitrobioconversionofpyruvatetonbutanolwithminimizedcofactorutilization |