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A barley diterpene synthase (HvKSL4) was found to produce (14S)-cleistantha-8,12-diene (1). Formation of the nearly planar cyclohexa-1,4-diene configuration leaves the ring poised for aromatization, but necessitates a deceptively complicated series of rearrangements steered through a complex energetic landscape, as elucidated here through quantum chemical calculations and labeling studies.

The multiproduct chimeric sesterterpene synthase AcAS from Aspergillus calidoustus yielded spirocyclic calidoustene, which exhibits a novel skeleton, besides five known sesterterpenes. The complex cyclisation mechanism to all six compounds was investigated by isotopic labelling experiments in combination with DFT calculations. Chemically synthesised 8-hydroxyfarnesyl diphosphate was converted with isopentenyl diphosphate and AcAS into four oxygenated sesterterpenoids that structurally resemble cytochrome P450 oxidation products of the sesterterpene hydrocarbons. Protein engineering of AcAS broadened the substrate scope and gave significantly improved enzyme yields.

A reinvestigation of the linalool synthase from Chryseobacterium polytrichastri uncovered its diterpene synthase activity, yielding polytrichastrene A and polytrichastrol A with new skeletons, besides known wanju-2,5-diene and thunbergol. The enzyme mechanism was investigated by isotopic labeling experiments and DFT calculations to explain an unusual ethyl group formation. Rationally designed exchanges of active site residues showed major functional switches, resulting for I66F in the production of five more new compounds, including polytrichastrene B and polytrichastrol B, while A87T, A192V and the double exchange A87T, A192V gave a product shift towards wanju-2,5-diene.

A reinvestigation of the linalool synthase from Chryseobacterium polytrichastri uncovered its diterpene synthase activity, yielding polytrichastrene A and polytrichastrol A with new skeletons, besides known wanju-2,5-diene and thunbergol. The enzyme mechanism was investigated by isotopic labeling experiments and DFT calculations to explain an unusual ethyl group formation. Rationally designed exchanges of active site residues showed major functional switches, resulting for I66F in the production of five more new compounds, including polytrichastrene B and polytrichastrol B, while A87T, A192V and the double exchange A87T, A192V gave a product shift towards wanju-2,5-diene.