Abstract
Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.
Highlights
Terpenoids are the most abundant and largest class (>75000) of natural products
Geranyl pyrophosphate (GPP), the substrate used by monoterpene synthases is formed by coupling one molecule of dimethylallyl pyrophosphate (DMAPP) with isopentenyl pyrophosphate (IPP), while farnesyl pyrophosphate (FPP), the substrate for sesquiterpenes, is synthesized by coupling three individual isoprene precursors.[9]
To investigate the suitability of both enzymes for monoterpenoid production in engineered E. coli strains, bLinS and bCinS were inserted in an E. coli “plug-andplay” monoterpenoid production platform, which consists of two gene modules.[3]
Summary
Terpenoids are the most abundant and largest class (>75000) of natural products. Most are commonly found in plants, and their biological roles range from interspecies communication to intracellular signaling and defense against predatory species.[1]. The protonation states of titratable residues were estimated using PropKA3.1,41,42 and the enzyme was solvated using a box of TIP3P43 water molecules (with a minimum buffer or 13 Å around the protein) using the solvate plugin of the VMD package.[44] Counterions were added to neutralize the system using autoionize plugin of VMD.[44] The CHARMM27 forcefield[39] was used to describe the protein with parameters for GPP and NPP that were adapted from those used for FPP in the work of van der Kamp et al.[45] The position of the GPP or NPP substrate was based on the position of the fluorinated analogue resolved in the crystal structure.
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