Robert Kourist

Overcoming the Incompatibility Challenge in Chemoenzymatic and Multi‐Catalytic Cascade Reactions

Multi-catalytic cascade reactions bear a great potential to minimize downstream and purification steps, leading to a drastic reduction of the produced waste. In many examples, the compatibility of chemo- and biocatalytic steps could be easily achieved. Problems associated with the incompatibility of the catalysts and their reactions, however, are very frequent. Cascade-like reactions can hardly occur in this way. One possible solution to combine, in principle, incompatible chemo- and biocatalytic reactions is the defined control of the microenvironment by compartmentalization or scaffolding. Current methods for the control of the microenvironment of biocatalysts go far beyond classical enzyme immobilization and are thus believed to be very promising tools to overcome incompatibility issues and to facilitate the synthetic application of cascade reactions. In this Minireview, we will summarize recent synthetic examples of (chemo)enzymatic cascade reactions and outline promising methods for their spatial control either by using bio-derived or synthetic systems.

Bio‐based α,ω‐Functionalized Hydrocarbons from Multi‐step Reaction Sequences with Bio‐ and Metallo‐catalysts Based on the Fatty Acid Decarboxylase OleTJE

OleT from Jeotgalicoccus sp. ATCC 8456 catalyzes the decarboxylation of ω‐functionalized fatty acids to the corresponding alkenols, which can themselves serve as starting material for the synthesis of polymers and fine chemicals. To show the versatility of possible reactions, a series of in vitro reaction cascades was developed where an alkenol produced by the decarboxylation of ω‐hydroxy fatty acids can be further converted into alkenylamines and diols. By coupling OleT with an alcohol dehydrogenase or alcohol oxidase as well as an amino‐transaminase, an oxidative decarboxylation followed by the oxidation of the terminal alcohol and a subsequent reductive transamination could be carried out. By using different cofactors or electron sources, the reactions could be performed sequentially or simultaneously. The combination of enzymatic decarboxylation with a ruthenium catalyst in a chemo‐enzymatic cascade provides a novel way to synthesize long‐chain diols.

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells

Abstract Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label‐free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC‐MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.

Practical Considerations Regarding the Choice of the Best High-Throughput Assay

All protein engineering studies include the stage of identifying and characterizing variants within a mutant library by employing a suitable assay or selection method. A large variety of different assay approaches for different enzymes have been developed in the last few decades, and the throughput performance of these assays vary considerably. Thus, the concept of a protein engineering study must be adapted to the available assay methods. This introductory review chapter describes different assay concepts on selected examples, including selection and screening approaches, detection of pH and cosubstrate changes, coupled enzyme assays, methods using surrogate substrates and selective derivatization. The given examples should guide and inspire the reader when choosing and developing own high-throughput screening approaches.

Enzymatic Decarboxylation as a Tool for the Enzymatic Defunctionalization of Hydrophobic Bio-based Organic Acids

Abstract The decarboxylation of organic acids is an emerging tool for the synthesis of bio-based olefins. Although terminal alkenes are the key intermediates for the chemical industry, they play only a marginal role in nature. Nature relies on the activation of carboxylic acids via thioesters or phosphate esters. Formation of terminal alkenes coupled to their selective functionalization has been rarely observed in metabolism. Enzymatic systems for the conversion of organic acids to alkenes are therefore scarce. Interestingly, several systems were identified in the last few years that catalyze the direct decarboxylation of organic acids to terminal olefins ( D’Espaux etal., 2015 , Herman and Zhang, 2016 ; Kang and Nielsen,2017 ; Lennen and Pfleger,2013 ; Schwartz etal.,2014 ; Zhou etal.,2014 ). This decarboxylation often proceeds under mild reaction conditions and produces terminal olefins without formation of unwanted internal alkenes. Nature offers different, highly promising catalytic systems with substrate spectra ranging from mid- to long-chain olefins. Most decarboxylases involved in lipid modification have been discovered in the last 10 years, and the recent elucidation of structure and mechanisms has laid the basis for an optimization by molecular engineering, leading to variants with improved catalytic activity and extended substrate spectrum.

Cloning and characterization of a new delta-specific l-leucine dioxygenase from Anabaena variabilis

Optically pure hydroxy amino acids show several bioactivities and are valuable building blocks for the pharmaceutical industry. Fe(II)/α-ketoglutarate dependent dioxygenases catalyze the hydroxylation or sulfoxidation of l-amino acids with high regio- and stereoselectivity. While several β- and γ-specific enzymes have been described, only one δ-specific hydroxylase has been reported so far. Based on its similarity to the known l-leucine 5-hydroxylase from Nostoc punctiforme, an open reading frame from the cyanobacterium Anabaena variabilis was identified as putative l-leucine dioxygenase (AvLDO). Here we report the cloning and characterization of this dioxygenase. The enzyme showed a high preference for acidic conditions and moderate reaction temperatures. AvLDO catalyzed the regio- and stereoselective hydroxylation of several aliphatic amino acids in δ-position. In case of the sulfoxidation of l-methionine, AvLDO produced the opposite diastereomer than isoleucine dioxygenase. AvLDO is thus an interesting addition to the toolbox of Fe(II)/α-ketoglutarate dependent dioxygenases. On the genomic DNA of Anabaena variabilis ATCC 29413, the avldo gene is located on a gene cluster involved (2S,4S)-4-methylproline biosynthesis, which is contained in bioactive peptides often found from cyanobacteria. This fact suggests the metabolic functional role of this amino acid dioxygenase in cyanobacteria.

Editorial: Applied Microbiology for Chemical Syntheses.

1 Lehrstuhl für Bioprozesstechnik, Technische Universität Dortmund, Dortmund, Germany, Department of Technichal Chemistry, Process Engineering and Biotechnology, Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria, 3 EntreChem SL, Edificio Científico Tecnológico, Oviedo, Spain, Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Centre for Water and Environmental Research, University of Duisburg-Essen, Essen, Germany

Lichtgetriebene Ganzzellbiotransformation mit rekombinanten Cyanobakterien

Photosynthetic microorganisms have received considerable attention as production organisms for chemicals. An investigation of the photosynthetic NADPH supply for enantioselective biotransformations with recombinant cyanobacteria showed that the specific activity of the cells is comparable to heterotrophic organisms. Light-dispersion of the cells, however, limits the approach. A wide use as production organisms requires an improvement of several magnitudes.