title: In VitroBioconversion of Pyruvate ton-Butanol with Minimized Cofactor Utilization.
abstract: Due to enhanced energy content and reduced hygroscopicity compared with ethanol,n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems,n-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-freen-butanol biosynthesis from the central metabolic intermediate pyruvate involves cofactors [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-freen-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 ton-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate ton-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-freen-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as cofactor. Our proof-of-concept demonstrates thatn-butanol was synthesized at neutral pH and 50°C. This integrated reaction concept allowed GC detection of all reaction intermediates andn-butanol production of 148 mg L-1(2 mM), which compares well with other cell-freen-butanol production processes.
authors: Department of Chemistry, Technical University of Munich, Garching, Germany; B&B Sustainable Innovations GmbH, Köln, Germany.
authors: Steven
authors: S
authors: Reiße
authors: Department of Chemistry, Technical University of Munich , Garching , Germany.
authors: Martina
authors: M
authors: Haack
authors: Department of Chemistry, Technical University of Munich , Garching , Germany.
authors: Daniel
authors: D
authors: Garbe
authors: Department of Chemistry, Technical University of Munich , Garching , Germany.
authors: Bettina
authors: B
authors: Sommer
authors: Straubing Center of Science, Technical University of Munich , Straubing , Germany.
authors: Fabian
authors: F
authors: Steffler
authors: Straubing Center of Science, Technical University of Munich , Straubing , Germany.
authors: Jörg
authors: J
authors: Carsten
authors: B&B Sustainable Innovations GmbH , Köln , Germany.
authors: Frank
authors: F
authors: Bohnen
authors: Straubing Center of Science, Technical University of Munich , Straubing , Germany.
authors: Volker
authors: V
authors: Sieber
authors: Department of Chemistry, Technical University of Munich, Garching, Germany; B&B Sustainable Innovations GmbH, Köln, Germany.
authors: Thomas
authors: T
authors: Brück
keywords: biocatalysis; butanol; cell-free; chemoenzymatic; enzyme cascade; synthetic biotechnology