Biosynthesis of Isoprenoids: Mechanistic Investigations, Enzyme Engineering and Enzymatic Synthesis of Novel Terpene Analogs

• Applicant: Professor Dr. Jeroen Dickschat
• Subject Area: Biological and Biomimetic Chemistry ; Organic Molecular Chemistry - Synthesis and Characterisation
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 584969299

This project will address the enzyme mechanisms of three terpene synthases (TSs) for which the structures of the products and biosynthetic proposals have recently been published. A deep analysis of the proposed mechanisms revealed in one case open stereochemical questions, and in two cases alternative possibilities. Experimental work to deeply clarify the mechanisms will include extensive isotopic labelling experiments accompanied by DFT calculations within a collaboration with Prof. Dr. Bernd Goldfuss (University of Cologne). A large library of labelled terpene precursors is available from our previous work, and newly designed precursors with special isotope substitutions will be synthesised within this project. The mechanisms of the three published enzymes will be further studied through site-directed mutagenesis of active site residues. These can initially be identified from Alphafold 3 protein models, but for deeper insights enzyme crystal structures, ideally liganded with metal cofactors and synthetic non-reactive substrate surrogates, will be obtained within a collaboration with Prof. Dr. Michael Groll (TU Munich). Productive variants of VniA together with the CYP450 from the same biosynthetic gene cluster involved in vinigrol biosynthesis will be heterologously expressed in A. oryzae, which may potentially lead to new bioactive derivatives. Substrate analogs with a blocked (double bond saturations) or buffered (Me group removals) reactivity will also be used to investigate the mechanisms of the three enzymes. It is expected that such substrate analogs lead to the formation of derailment products representing cationic intermediates along the cyclisation cascade, thus pointing to their existence. All newly obtained products from enzyme variants or substrate analogs with blocked/buffered reactivity will be isolated and structurally characterized up to the level of absolute configuration determination. Novel substrate analogs with functional groups that may result in changed reactivities will be synthesised and converted with all investigated TSs. This includes substrate analogs with alkyne function and cyclopropane rings. In cases where e. g. the steric bulk of the additional carbon of the cyclopropane ring is not tolerated, structure-based enzyme variants with widened cavity will be created. If the enzymes in the main focus of this project show a poor performance with substrate analogs, the synthetic compounds will be tested with other TSs made available in our previous work. All products will be isolated and their structures will be fully assigned.

• DFG Programme: Research Grants