H2-driven enzymatic cascades for the biosynthesis of N-heterocycles

• Applicants: Professor Dr. Lars Lauterbach; Dr. Bettina Nestl
• Subject Area: Biochemistry ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2016 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 284111627

This collaborative research project aims at the design of an H2-based whole cell cofactor regeneration system of NAD(P)H dependent pathways to supply artificial, nicotinamide-dependent enzyme cascades with the corresponding reducing equivalents. Saturated N-heterocycles are prevalent in biologically active molecules and are increasingly attractive scaffolds in the development of new pharmaceuticals. Unlike their aromatic counterparts, there are limited strategies for facile and selective syntheses of substituted saturated N-heterocycles by chemical and biological methods. Herein we plan the establishement of a multi-enzymatic cascade for the preparation of substituted N-heterocycles: the transaminase-catalyzed regio- and stereoselective monoamination of diketones and the diamine oxidase-catalyzed oxidation of diamines result in the formation of the amino-ketone intermediate, which spontaneously forms the corresponding cyclic imine followed by the asymmetric reduction to the N-heterocyclic product by the application of the imine reductase. This is featured by the coupling of the imine reductase with either the transaminase/alanine dehydrogenase module or the diamine oxidase, wherein the regeneration of the reduced cofactors required is performed by a particular hydrogenase enzyme. For this purpose the principal characteristics of the individual reactions of the enzyme cascades will be first investigated. The basic interaction between biocatalysts will be investigated in vitro for a three-enzyme model system (cofactor regeneration by a hydrogenase, substrate conversion by the coupling of an imine reductase with either transaminase/alanine dehyrogenase system or amine oxidase). Based on results and the improvement of individual enzymes through enzyme engineering approaches, the complexity of the redox reaction cascade will be progressively increased. The ultimate goal is the creation of an optimized whole cell cascade in the host organism (Pseudomonas putida).

• DFG Programme: Research Grants
• Co-Investigator: Dr. Oliver Lenz