The limited supply of fossil resources demands the development of renewable alternatives to petroleum-based products. Here, biobased higher alcohols such as isobutanol are versatile platform molecules for the synthesis of chemical commodities and fuels. Currently, their fermentation-based production is limited by the low tolerance of microbial production systems to the end products and also by the low substrate flux into cell metabolism. We developed an innovative cell-free approach, utilizing an artificial minimized glycolytic reaction cascade that only requires one single coenzyme. Using this toolbox the cell-free production of ethanol and isobutanol from glucose was achieved. We also confirmed that these streamlined cascades functioned under conditions at which microbial production would have ceased. Our system can be extended to an array of industrially-relevant molecules. Application of solvent-tolerant biocatalysts potentially allows for high product yields, which significantly simplifies downstream product recovery.

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-free n-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-free n-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 cofactor. 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 n-butanol production processes.

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Die Produktion biobasierter Hochleistungskunststoffe wie Polyamide nutzt Pflanzenöle als Rohstoffquelle. Fortschritte in chemischen und biotechnologischen Katalyse‐Verfahren ermöglichen heute die Konversion von Pflanzenölen in maßgeschneiderte Bausteine für die Polymerproduktion. Schon heute haben biobasierte Polymer‐Synthesebausteine äquivalente chemische und physikalische Eigenschaften als auch vergleichbare Kostenstrukturen wie petrochemische Synthesegrundstoffe. Dies ermöglicht die Integration Biomasse‐basierter Rohstoffströme in bestehende Prozesskonfigurationen im industriellen Maßstab. Jedoch wird erst die effiziente Nutzung von Synergien zwischen chemischen und biotechnologischen Verfahren ein nachhaltiges und umweltschonendes Prozessdesign in Zukunft erlauben. Um langfristig Bio‐Öle für die chemische Industrie bereitstellen zu können, ohne signifikant in die Nahrungsmittelproduktion einzugreifen, müssen neue Verfahren für die Produktion mikrobieller Öle auf Basis von Biomasse‐Reststoffströmen entwickelt werden.

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