Extreme promiscuity of a bacterial and a plant diterpene synthase enables combinatorial biosynthesis

Abstract

Diterpenes are widely distributed across many biological kingdoms, where they serve a diverse range of physiological functions, and some have significant industrial utility. Their biosynthesis involves class I diterpene synthases (DTSs), whose activity can be preceded by that of class II diterpene cyclases (DTCs). Here, a modular metabolic engineering system was used to examine the promiscuity of DTSs. Strikingly, both a bacterial and plant DTS were found to exhibit extreme promiscuity, reacting with all available precursors with orthogonal activity, producing an olefin or hydroxyl group, respectively. Such DTS promiscuity enables combinatorial biosynthesis, with remarkably high yields for these unoptimized non-native enzymatic combinations (up to 15mg/L). Indeed, it was possible to readily characterize the 13 unknown products. Notably, 16 of the observed diterpenes were previously inaccessible, and these results provide biosynthetic routes that are further expected to enable assembly of more extended pathways to produce additionally elaborated 'non-natural' diterpenoids.

Keywords: Diterpene synthases; Diterpenes; Metabolic engineering; Sclareol; Substrate specificity; ent-kolavelool; ent-manool; ent-manoyl oxide; ent-sclareol; iso-abienol; kolavelool; nosyberkol; viteagnusin D; vitexifolin A.

Fig. 1

Basic DTC products from bicyclization and subsequent rearrangement (PP = diphosphate). Also shown are known stereoisomers for the initial bicycle, and derived products for which DTCs are known are shown by superscript (ⁿnormal, ^eent, ^ssyn).

Fig. 2

SsSS and KgTS readily react with 1. (A) GC-MS chromatograms of extracts from E. coli engineered for production of GGPP (1) and expressing either KgTS or SsSS (numbers correspond to chemical structures defined in text, with prime’ notation used to indicate the dephosphorylated derivative, where relevant). (B) Reactions catalyzed by KgTS and SsSS with GGPP (1). The KgTS reaction arrow and products are shown in gray and boxed, with product ratio shown in parentheses, while the SsSS reaction arrow and product are shown in black.

Fig. 3

GC-MS chromatograms of extracts from E. coli engineered for production of one of the 12 distinct DTC products that are currently accessible (3 – 14) by introducing a relevant DTC (Table 1) into E. coli engineered to produce 1. The ability of KgTS and SsSS to react with these is demonstrated by their additional co-expression, as indicated in the corresponding chromatograms (numbers correspond to chemical structures defined in text, with prime’ notation used to indicate the dephosphorylated derivative, where relevant).

Fig. 4

Scheme depicting cyclization of 1 to peregrinol diphosphate (11). With elucidation of the C8α-methyl conformation reported here, it can be appreciated that this derives from a pro-chair-boat conformation of 1 that leads to an initial syn-labda-13E-en-8-yl⁺ intermediate, which undergoes a C9 → C8 hydride transfer and addition of water to the resulting 9-yl carbocation before terminating deprotonation.

Fig. 5

Prototypical reactions catalyzed by KgTS (gray arrow and product) or SsSS (black arrow and product) with various diterpene precursors, either 1 or DTC products, via lysis/ionization of the trans allylic diphosphate ester to a common tertiary carbocation intermediate.

Fig. 6

Summary of the combinatorial biosynthesis enabled by the extreme promiscuity of KgTS and SsSS. Show are the 12 bicyclic DTC products (3–14), derived from the general diterpene precursor GGPP (1), and the subsequent reactions catalyzed by KgTS (grey arrows and products) and SsSS (black arrows and products), with product ratio shown in the parenthesis when multiple products were observed (numbering as in the text, with the 12 unknown products boxed and the 5 for which only inefficient biosynthetic access was previously available circled).