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 15 mg/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.

Abietane-Type Diterpenoids Inhibit Protein Tyrosine Phosphatases by Stabilizing an Inactive Enzyme Conformation

“…Recently developed methods for using E. coli for the combinatorial synthesis of structurally varied labdane-related diterpenoids provide a starting point for this approach. 25,27,64 We note that the highly nonpolar nature of the diterpenoids examined in this study necessitated the use of high concentrations of co-solvent that reduced their apparent potencies (SI Note 7) and precluded detailed X-ray crystallo-graphic studies; future studies of the binding mode of more soluble and/or potent analogues (e.g., those discovered via synthetic biology) might enable rational, rather than screen-based, approaches to inhibitor optimization.…”

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

“…Additional findings were provided by Jia and co-workers [67], who demonstrated high substrate promiscuity of a plant and a fungal Class I diterpene synthase. This study involved general substrates of diterpene cyclases like GGPP and its cis -isomer nerylneryl diphosphate (NNPP) [68] but also new combinations with 12 known and available products of plant Class II diterpene synthases.…”

Functional Diversification of Kaurene Synthase-Like Genes in Isodon rubescens

“…It is interesting to note that the recent emergence enzymes MvELS in M. vulgare (12.6 Ma; Roy and Lindqvist, 2015) and SsScS in S. sclarea (10 Ma; Drew and Sytsma, 2012) exhibit more diversified enzymatic activity than other KSLs Schalk et al, 2012;Andersen-Ranberg et al, 2016;Jia et al, 2016). This alludes to the fast evolution of KSL enzymes in Lamiaceae.…”

“…This alludes to the fast evolution of KSL enzymes in Lamiaceae. To date, all of the identified ba bidomain KSL enzymes have dual substrate activities (Caniard et al, 2012;Brückner et al, 2014;Zerbe et al, 2014;Andersen-Ranberg et al, 2016;Jia et al, 2016), highlighting their roles in contributing to the diversity of diterpenoids in Lamiaceae. The proposed intermediate steps in KSL protein evolution studied here can be uncovered when additional genera are explored at the sequence level.…”

“…Many of these enzymes have lost the characteristic internal/g domain that is responsible for metabolite specialization, a deletion event found in KSLs in Lamiaceae (Hillwig et al, 2011;Caniard et al, 2012;Schalk et al, 2012;Brückner et al, 2014;Pateraki et al, 2014;Zerbe et al, 2014;Cui et al, 2015;Bo zi c et al, 2015;Trikka et al, 2015). A recent work has shown the plasticity of class I KSLs, such as SsScS from S. sclarea, that can react with any available CPS product (Andersen-Ranberg et al, 2016;Jia et al, 2016). These studies demonstrate the value of Lamiaceae as a useful system to study diterpenoid chemical diversity evolution.…”