Cell-Free Reaction System for ATP Regeneration from d-Fructose
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| Titel: | Cell-Free Reaction System for ATP Regeneration from d-Fructose |
|---|---|
| Autoren: | Kraußer, Franziska, Rabe, Kenny, Topham, Christopher, Kundoch, Jan-Ole, Ohde, Daniel, Liese, Andreas, Walther, Thomas |
| Quelle: | ACS synthetic biology. 14(4):1250-1263 |
| Verlagsinformationen: | 2025. |
| Publikationsjahr: | 2025 |
| Schlagwörter: | acetyl phosphate synthesis | ATP cofactor regeneration | in vitro biocatalysis | molecular modeling | phosphoketolase | semirational enzyme engineering |
| Beschreibung: | Adenosine triphosphate (ATP)-dependent in vitro bioprocesses, such as cell-free protein synthesis and the production of phosphorylated fine chemicals, are of considerable industrial significance. However, their implementation is mainly hindered by the high cost of ATP. We propose and demonstrate the feasibility of a cell-free ATP regeneration system based on the in situ generation of the high-energy compound acetyl phosphate from low-cost d-fructose and inorganic phosphate substrates. The enzyme cascade chains d-fructose phosphoketolase, d-erythrose isomerase, d-erythrulose phosphoketolase, and glycolaldehyde phosphoketolase activities theoretically enabling production of 3 mol ATP per mol of d-fructose. Through a semirational engineering approach and the screening of nine single-mutation libraries, we optimized the phosphoketolase (PKT) from Bifidobacterium adolescentis, identifying the improved variant Bad.F6Pkt H548N. This mutant exhibited a 5.6-fold increase in d-fructose activity, a 2.2-fold increase in d-erythrulose activity, and a 1.3-fold increase in glycolaldehyde activity compared to the wild-type enzyme. The Bad.F6Pkt H548N mutant was initially implemented in a cell-free reaction system together with an acetate kinase from Geobacillus stearothermophilus and a glycerol kinase from Cellulomonas sp. for the production of glycerol-3 phosphate from ADP and glycerol. We demonstrated the feasibility of ATP regeneration from 25 mM d-fructose with a stoichiometry of 1 mol of ATP per mol of C6 ketose. Subsequently, the reaction system was enhanced by incorporating d-erythrose isomerase activity provided by a l-rhamnose isomerase from Pseudomonas stutzeri. In the complete system, the ATP yield increased to 2.53 mol molfructose-1 with a maximum productivity of 7.2 mM h-1 |
| Publikationsart: | Article |
| Sprache: | English |
| ISSN: | 2161-5063 |
| DOI: | 10.15480/882.15055 |
| Rights: | CC BY |
| Dokumentencode: | edsair.dris...01170..32459a24b02cc989652f979f82c7c9ef |
| Datenbank: | OpenAIRE |
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| Items | – Name: Title Label: Title Group: Ti Data: Cell-Free Reaction System for ATP Regeneration from d-Fructose – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Kraußer%2C+Franziska%22">Kraußer, Franziska</searchLink><br /><searchLink fieldCode="AR" term="%22Rabe%2C+Kenny%22">Rabe, Kenny</searchLink><br /><searchLink fieldCode="AR" term="%22Topham%2C+Christopher%22">Topham, Christopher</searchLink><br /><searchLink fieldCode="AR" term="%22Kundoch%2C+Jan-Ole%22">Kundoch, Jan-Ole</searchLink><br /><searchLink fieldCode="AR" term="%22Ohde%2C+Daniel%22">Ohde, Daniel</searchLink><br /><searchLink fieldCode="AR" term="%22Liese%2C+Andreas%22">Liese, Andreas</searchLink><br /><searchLink fieldCode="AR" term="%22Walther%2C+Thomas%22">Walther, Thomas</searchLink> – Name: TitleSource Label: Source Group: Src Data: <i>ACS synthetic biology</i>. 14(4):1250-1263 – Name: Publisher Label: Publisher Information Group: PubInfo Data: 2025. – Name: DatePubCY Label: Publication Year Group: Date Data: 2025 – Name: Subject Label: Subject Terms Group: Su Data: <searchLink fieldCode="DE" term="%22acetyl+phosphate+synthesis+|+ATP+cofactor+regeneration+|+in+vitro+biocatalysis+|+molecular+modeling+|+phosphoketolase+|+semirational+enzyme+engineering%22">acetyl phosphate synthesis | ATP cofactor regeneration | in vitro biocatalysis | molecular modeling | phosphoketolase | semirational enzyme engineering</searchLink> – Name: Abstract Label: Description Group: Ab Data: Adenosine triphosphate (ATP)-dependent in vitro bioprocesses, such as cell-free protein synthesis and the production of phosphorylated fine chemicals, are of considerable industrial significance. However, their implementation is mainly hindered by the high cost of ATP. We propose and demonstrate the feasibility of a cell-free ATP regeneration system based on the in situ generation of the high-energy compound acetyl phosphate from low-cost d-fructose and inorganic phosphate substrates. The enzyme cascade chains d-fructose phosphoketolase, d-erythrose isomerase, d-erythrulose phosphoketolase, and glycolaldehyde phosphoketolase activities theoretically enabling production of 3 mol ATP per mol of d-fructose. Through a semirational engineering approach and the screening of nine single-mutation libraries, we optimized the phosphoketolase (PKT) from Bifidobacterium adolescentis, identifying the improved variant Bad.F6Pkt H548N. This mutant exhibited a 5.6-fold increase in d-fructose activity, a 2.2-fold increase in d-erythrulose activity, and a 1.3-fold increase in glycolaldehyde activity compared to the wild-type enzyme. The Bad.F6Pkt H548N mutant was initially implemented in a cell-free reaction system together with an acetate kinase from Geobacillus stearothermophilus and a glycerol kinase from Cellulomonas sp. for the production of glycerol-3 phosphate from ADP and glycerol. We demonstrated the feasibility of ATP regeneration from 25 mM d-fructose with a stoichiometry of 1 mol of ATP per mol of C6 ketose. Subsequently, the reaction system was enhanced by incorporating d-erythrose isomerase activity provided by a l-rhamnose isomerase from Pseudomonas stutzeri. In the complete system, the ATP yield increased to 2.53 mol molfructose-1 with a maximum productivity of 7.2 mM h-1 – Name: TypeDocument Label: Document Type Group: TypDoc Data: Article – Name: Language Label: Language Group: Lang Data: English – Name: ISSN Label: ISSN Group: ISSN Data: 2161-5063 – Name: DOI Label: DOI Group: ID Data: 10.15480/882.15055 – Name: Copyright Label: Rights Group: Cpyrght Data: CC BY – Name: AN Label: Accession Number Group: ID Data: edsair.dris...01170..32459a24b02cc989652f979f82c7c9ef |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.15480/882.15055 Languages: – Text: English PhysicalDescription: Pagination: PageCount: 14 StartPage: 1250 Subjects: – SubjectFull: acetyl phosphate synthesis | ATP cofactor regeneration | in vitro biocatalysis | molecular modeling | phosphoketolase | semirational enzyme engineering Type: general Titles: – TitleFull: Cell-Free Reaction System for ATP Regeneration from d-Fructose Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Kraußer, Franziska – PersonEntity: Name: NameFull: Rabe, Kenny – PersonEntity: Name: NameFull: Topham, Christopher – PersonEntity: Name: NameFull: Kundoch, Jan-Ole – PersonEntity: Name: NameFull: Ohde, Daniel – PersonEntity: Name: NameFull: Liese, Andreas – PersonEntity: Name: NameFull: Walther, Thomas IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 01 Type: published Y: 2025 Identifiers: – Type: issn-print Value: 21615063 – Type: issn-locals Value: edsair Numbering: – Type: volume Value: 14 – Type: issue Value: 4 Titles: – TitleFull: ACS synthetic biology Type: main |
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