Enzymes for Synthetic Biology of Ambroxide-Related Diterpenoid Fragrance Compounds

Abstract

Ambrox and related ambroxides are highly priced in the fragrance industry, and valued for their delicate odor and fixative properties. Historically, ambrox was obtained from ambergris, a waxy excretion produced by sperm whales, now an endangered species. Synthetic ambroxides have replaced ambergris in perfume manufacture. Plant labdane diterpenoids can serve as starting material for ambroxide synthesis. Among these, the diterpene alcohol sclareol is the major industrial precursor obtained from cultivated clary sage (Salvia sclarea). In plants, a large family of diterpene synthase (diTPS) enzymes controls key reactions in diterpenoid biosynthesis. Advanced metabolite profiling and high-throughput sequencing of fragrant and medicinal plants have accelerated discovery of novel diTPS functions, providing a resource for combinatorial synthetic biology and metabolic engineering approaches. This chapter highlights recent progress on the discovery, characterization, and engineering of plant diTPSs with potential uses in ambroxide production. It features biosynthesis of sclareol, cis-abienol, and diterpene resin acids, as sources of genes and enzymes for diterpenoid bioproducts.

Exploring diterpene metabolism in non‐model species: transcriptome‐enabled discovery and functional characterization of labda‐7,13E‐dienyl diphosphate synthase from Grindelia robusta

“…Previously characterized plant class II diTPSs include enzymes for the formation of ent ‐CPP, syn ‐CPP, normal CPP [i.e. (+)‐CPP], LPP and peregrinol diphosphate (for review see Zerbe and Bohlmann, ).…”

Whole-cell (+)-ambrein production in the yeast Pichia pastoris

“…Beyond application in the perfume industry, animal studies have also demonstrated the potential anti-nociceptive ( Taha, 1992 ) and aphrodisiac ( Taha et al, 1995 ) properties of (+)-ambrein as well as possible effects on the cardiovascular system ( Raza et al, 1999 ) and on smooth muscle response ( Taha et al, 1998 ). As the natural ambergris supply is highly limited, total or partial syntheses (reviewed by Zerbe and Bohlmann, 2015 ) have been developed for production of (+)-ambrein, (-)-ambrox and related compounds. A recent study by Ueda et al (2013) described the possibility to produce (+)-ambrein from squalene applying only two enzymes ( Fig.…”

Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products

“…For example, DRAs were used in large quantities in the traditional naval stores industry to protect wooden ships and boats against fouling (Langenheim, 2003). In modern industrial applications, DRAs are converted into an array of biochemicals and bioproducts such as coatings, polymers, inks, flavors, and fragrances Hillwig et al, 2011;Zerbe and Bohlmann, 2015a). Conifer DRAs are of considerable structural diversity, which results from the combined activities of multienzyme families of diterpene synthases (diTPSs) and cytochrome P450 monooxygenases (P450s) in a modular biosynthetic system (Hamberger et al, 2011;Bohlmann, 2015a, 2015b).…”

“…S1) to produce various bicyclic or, more commonly, tricyclic diterpene structures (Peters, 2010;Zerbe and Bohlmann, 2015a). Families of diTPSs, including the isopimaradiene synthases (ISO) and levopimaradiene/abietadiene synthases (LAS), have been characterized in several spruce and pine species (Martin et al, 2004;Ro and Bohlmann, 2006;Keeling et al, 2011;Zerbe et al, 2012;Hall et al, 2013b).…”