Combinatorial biosynthesis and the basis for substrate promiscuity in class I diterpene synthases.

Terpene synthases are capable of mediating complex reactions, but fundamentally simply catalyze lysis of allylic diphosphate esters with subsequent deprotonation. Even with the initially generated tertiary carbocation this offers a variety of product outcomes, and deprotonation further can be preceded by the addition of water. This is particularly evident with labdane-related diterpenes (LRDs) where such lysis follows bicyclization catalyzed by class II diterpene cyclases (DTCs) that generates preceding structural variation. Previous investigation revealed that two diterpene synthases (DTSs), one bacterial and the other plant-derived, exhibit extreme substrate promiscuity, but yet still typically produce exo-ene or tertiary alcohol LRD derivatives, respectively (i.e., demonstrating high catalytic specificity), enabling rational combinatorial biosynthesis. Here two DTSs that produce either cis or trans endo-ene LRD derivatives, also plant and bacterial (respectively), were examined for their potential analogous utility. Only the bacterial trans-endo-ene forming DTS was found to exhibit significant substrate promiscuity (with moderate catalytic specificity). This further led to investigation of the basis for substrate promiscuity, which was found to be more closely correlated with phylogenetic origin than reaction complexity. Specifically, bacterial DTSs exhibited significantly more substrate promiscuity than those from plants, presumably reflecting their distinct evolutionary context. In particular, plants typically have heavily elaborated LRD metabolism, in contrast to the rarity of such natural products in bacteria, and the lack of potential substrates presumably alleviates selective pressure against such promiscuity. Regardless of such speculation, this work provides novel biosynthetic access to almost 19 LRDs, demonstrating the power of the combinatorial approach taken here.

A Pair of Residues That Interactively Affect Diterpene Synthase Product Outcome.

The labdane-related diterpenoids (LRDs) are an important superfamily of natural products whose structural diversity critically depends on the hydrocarbon skeletal structures generated, in large part, by class I diterpene synthases. In the plant kingdom, where the LRDs are predominantly found, the relevant class I diterpene synthases are clearly derived from the ent-kaurene synthase (KS) required in all land plants for phytohormone biosynthesis and, hence, are often termed KS-like (KSL). Previous work, initiated by the distinct function of two alleles of a KSL from rice, OsKSL5, identified a single residue switch with a profound effect on not only OsKSL5 product outcome but also that of land plant KSs more broadly, specifically, replacement of a key isoleucine with threonine, which interrupts formation of the tetracyclic ent-isokaurene at the tricyclic stage, leading to production of ent-pimaradiene instead. Here, further studies of these alleles led to discovery of another, nearby residue that tunes product outcome. Substitution for this newly identified residue is additionally shown to exert an epistatic effect in KSs, altering product distribution only if combined with replacement of the key isoleucine. On the other hand, this pair of residues was found to exert additive effects on the product outcome mediated by distantly related KSLs from the eudicot castor bean. Accordingly, it was possible to use a rational combination of substitutions for this pair of residues to engineer significantly increased (dominant) selectivity for novel 8α-hydroxy-ent-pimar-15-ene product outcome in the KS from the dicot Arabidopsis thaliana, demonstrating the utility of these results.