Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage-sensing domains, we engineered flavin mononucleotide-based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.

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.

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The combination of a heterogeneously catalyzed reaction with a biotransformation as a one-pot cascade process is an important strategy to reduce costs, time, and labor efforts in the production of chemicals from biogenic resources. Although one-pot cascade-type approaches generally result in more efficient chemical processes by reducing the number of workup operations needed and time consumed, the combination of different types of catalysts, both chemical and enzymatic, into a single reaction vessel often remains challenging. During our study, aimed at the direct synthesis of 2-keto-3-deoxy sugar acids as one intermediate toward biobased building blocks starting from the corresponding sugars by combining heterogeneous inorganic catalysis with enzyme catalysis, we encountered several incompatibility problems. These were overcome by a chemoenzymatic method in different compartments, which involves the gold-catalyzed direct oxidation by molecular oxygen and the subsequent conversion of the sugar acids through an enzymatic dehydration step. The described procedure represents an efficient synthesis route toward four different 2-keto-3-deoxy sugar acids and serves as a proof of concept for the combination of one-pot-incompatible catalysts under continuous flow.

The application of unusual high pH-values within enzymatic cyanohydrin synthesis has been investigated. Usually enzymatic cyanohydrin synthesis in two-phase systems requires low pH-values within the aqueous phase to suppress the non-enzymatic side reaction. In contrast, we investigated the usage of pH-values above pH 6 by using the highly enantioselective (S)-selective hydroxynitrile lyase from Manihot esculenta. With these unusual reaction conditions also the unfavorable substrate 3-phenoxy-benzaldehyde can be converted by the wild type enzyme with excellent conversion and enantiomeric excess yielding pure (S)-3-phenoxy-benzaldehyde cyanohydrin with an enantiomeric excess of 97%. Although the variant MeHNL-W128A shows a higher activity with respect to this reaction, the enantioselectivity was reduced (85% e.e.(S)). Additionally, a new continuous spectroscopic cyanohydrin assay monitoring the formation of 3-phenoxy-benzaldehyde cyanohydrin was developed.

The production of chemicals from biogenic resources is widely performed using microorganisms. These often do not meet the requirements of technical processes due to the narrow limits of microbial physiology. As a consequence, alternatives are sought. Cell‐free enzymatic reaction cascades were recently proposed as next‐generation bioproduction systems. Here, we address problems of microbial systems, show potential advantages of cell‐free concepts and give an overview over the state of the technology. Furthermore, we address current challenges of the cell‐free strategy and give an outlook on future developments.

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A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.