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Talks and Poster Presentations (with Proceedings-Entry):

F. Rudroff:
"Synthesis of fragrance aldehydes by the combination of chemo- and biocatalysis in microbial cell factories";
Talk: Bioflavour 2018, Frankfurt am Main; 09-18-2018 - 09-21-2018; in: "Bioflavour 2018", (2018), 1 - 162.



English abstract:
In this study, we present the synthesis of chiral fragrance aldehydes, which was tackled by a combination of chemo-catalysis and a multi-enzymatic in vivo cascade reaction and the development of a highly versatile high-throughput assay for the enzymatic reduction of carboxylic acids.
We investigated a biocompatible metal-catalyzed synthesis for the preparation of α or β substituted cinnamic acid derivatives which are fed directly into the biocatalytic system. Subsequently, the target molecules were synthesized by an enzymatic cascade consisting of a carboxylate reduction, followed by the selective C-C double bond reduction catalyzed by appropriate enoate reductases (Figure 1).
A key step of this study was the selective reduction of the carboxylic acid towards the corresponding aldehydes. Nature provides carboxylic acid reductases (CARs), as elegant and efficient tools for the direct ATP and NADPH dependent reduction of carboxylic acids to aldehydes. Within this study, we developed a simple HTA that allows the substrate independent and chemoselective quantification of aldehydes (irrespective of their chemical structure), is sensitive to the nM range, and works in the presence of microbial cells. The HTA was validated by re-examination of the substrate scope of the well-known CAR from Nocardia iowensis (CARNi). A mutagenesis study with 6,000 clones was conducted to improve the activity of CARNi for the sterically demanding 2-methoxycarboxylic acid. This identified a hot spot in the active site of CARNi that increased the affinity of 2-methoxybenzoic acid 4-fold upon mutation. Within this study, we present the first HT-screening assay for aldehydes, which works in the presence of living cells and is completely structural independent with respect to the aldehyde residue.

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