Rerouting and Improving Dauc‐8‐en‐11‐ol Synthase from Streptomyces venezuelae to a High Yielding Biocatalyst

Abstract

The dauc-8-en-11-ol synthase from Streptomyces venezuelae was investigated for its catalytic activity towards alternative terpene precursors, specifically designed to enable new cyclisation pathways. Exchange of aromatic amino acid residues at the enzyme surface by site-directed mutagenesis led to a 4-fold increase of the yield in preparative scale incubations, which likely results from an increased enzyme stability instead of improved enzyme kinetics.

Using Terpene Synthase Plasticity in Catalysis: On the Enzymatic Conversion of Synthetic Farnesyl Diphosphate Analogues

“…Finally, with 13 new reaction paths may be observed with BbS, while with SmTS6 a 1,10‐cyclisation could still be possible, and for HcS the result may depend on the question of initial 1,6‐ or 1,11‐cyclisation, potentially leading to new reaction paths. The FPP analogues 10 – 12 were synthesised as reported previously,[ 19 , 30 ] and 13 was synthesised through Corey‐Seebach umpolung from 1,3‐dithiane 14 and iodide 15 to yield 16 (Scheme 3 B). Deprotection via 17 to 18 followed by bromination and phosphorylation yielded 13 .…”

“…The main compound was isolated and identified as 22 ([α] D 25 =−16.2, c 0.21, CH 2 Cl 2 ), the enantiomer of ent ‐ 22 ([α] D 20 =+51.7, c 0.12, CH 2 Cl 2 ) that we had previously obtained from 12 with dauc‐8‐en‐11‐ol synthase (DcS) from Streptomyces venezuelae (Scheme 5 ). [19] The formation of 22 requires isomerisation to ( R )‐ A6 and subsequent 1,6‐cyclisation by anti ‐S N 2’ reaction, resulting in ( S )‐ B 6 . Final attack of water yields 22 .…”

“…These natural systems raise the question whether also canonical TPSs have the potential to convert substrates other than the regular oligoprenyl diphosphates. Recent research has demonstrated that this is indeed the case, [14] revealing that e. g. halogenated substrates,[ 15 , 16 ] substrates with additional or missing Me groups or altered methylation pattern,[ 16 , 17 , 18 , 19 ] functional groups attached to[ 20 , 21 ] or heteroatoms inserted into the chain, [22] with hydrogenated double bonds,[ 23 , 24 ] or stereoisomers with Z ‐configured olefins such as (2 Z ,6 E )‐FPP can be converted. [ 25 , 26 ] The substrate modifications can block reactivity, as for compounds with hydrogenated olefins, or open new reaction pathways as for compounds with altered methylation patterns in which cations can be stabilised at different carbons of the isoprenoid chain.…”

Isotopic labelling experiments and enzymatic preparation of iso-casbenes with casbene synthase from Ricinus communis

“…See DOI: 10.1039/d1qo01707a inserted into the carbon chain, 25,26 epoxidised 27 or hydrogenated double bonds, 24,28,29 and altered methylation patterns. [30][31][32] Here we report on isotopic labelling experiments to study the cyclisation mechanism of CS and the EI-MS fragmentation mechanisms of its product 1. A synthesis of two new oligoprenyl diphosphate analogs and their enzymatic conversion with CS into iso-casbenes was performed.…”

Mechanistic Investigations on Microbial Type I Terpene Synthases through Site-Directed Mutagenesis

“…Interestingly, the H197F/Y241F double mutant exhibited a 4-fold higher enzyme yield that turned out to be preparatively useful (46% isolated yield of 41 from 80 mg FPP, in comparison to 11% obtained with the wildtype). 61 It is possible that these exchanges cause some structural rearrangement in the mutant, because the nonpolar Phe residues may turn from the surface towards the inside of the enzyme. Structural work on DcS and the double mutant will be required for a detailed understanding of the observed effects.…”