The Application of Synthetic Biology to Elucidation of Plant Mono-, Sesqui-, and Diterpenoid Metabolism

Abstract

Plants synthesize a huge variety of terpenoid natural products, including photosynthetic pigments, signaling molecules and defensive substances. These are often produced as complex mixtures, presumably shaped by selective pressure over evolutionary timescales, some of which have been found to have pharmaceutical and other industrial uses. Elucidation of the relevant biosynthetic pathways can provide increased access (e.g., via molecular breeding or metabolic engineering), and enable reverse genetic approaches towards understanding the physiological role of these natural products in plants as well. While such information can be obtained via a variety of approaches, this review describes the emerging use of synthetic biology to recombinantly reconstitute plant terpenoid biosynthetic pathways in heterologous host organisms as a functional discovery tool, with a particular focus on incorporation of the historically problematic cytochrome P450 mono-oxygenases. Also falling under the synthetic biology rubric and discussed here is the nascent application of genome-editing tools to probe physiological function.

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…To test for the activity of ZmKSL4 in specialized diterpenoid metabolism, we utilized the combinatorial functionality of class II and class I diTPSs of different plant species, which can be exploited to probe diTPS functions (Brückner and Tissier 2013;Zerbe et al, 2013;Kitaoka et al, 2015). An established Escherichia coli coexpression system (Morrone et al, 2010) was used to analyze the coupled activities of ZmKSL4 with (1) the ent-CPP synthase ZmAN2, (2) the rice syn-CPP synthase (OsCPS4; Xu et al, 2004), and (3) the grand fir (Abies grandis) abietadiene synthase variant D621A that produces CPP of normal [i.e.…”

“…The functional coexpression of enzymes was carried out using a previously described E. coli system engineered for enhanced diterpenoid production (Morrone et al, 2010;Kitaoka et al, 2015). For biochemical characterization of ZmKSL4, the N-terminally truncated gene in the expression vector pET28b(+) or pCOLA-DUET was cotransformed in E. coli BL21DE3-C41 cells (Lucigen) with a plasmid carrying a geranylgeranyl diphosphate synthase and constructs of class II diTPSs with different products: ZmAN2 forming ent-CPP (pGGeC), rice (Oryza sativa) CPS4 producing syn-CPP (pGGsC), or a variant of grand fir (Abies grandis) abietadiene synthase forming (+)-CPP (pGGnC; Morrone et al, 2010).…”

“…Modular enzyme networks largely determine the variations of diterpene profiles in closely and distantly related plant species (Peters, 2010;Hamberger and Bak, 2013;Zerbe and Bohlmann, 2015). Naturally occurring modularity in diterpenoid biosynthesis further enables the engineering of combinatorial diTPS and P450 expression systems in microbial and plant host systems to accelerate the discovery of terpenoid pathways (Brückner and Tissier, 2013;Lange and Ahkami, 2013;Kitaoka et al, 2015;Zerbe and Bohlmann, 2015).…”

Probing the promiscuity of ent -kaurene oxidases via combinatorial biosynthesis

“…We have developed a modular metabolic engineering system that enables facile transformation of Escherichia coli to engineer the production of any terpene for which the corresponding synthase or synthases are known (14). This has been further coupled to strategies for functional expression of plant CYPs in E. coli, to (re)create terpenoid biosynthetic pathways via a synthetic biology approach (15). Given the difficulty of obtaining an extended range of relevant substrates for in vitro reactions, for example, by (retro)synthesis, here we have adopted this approach to screen AtKO and OsKO2 activity against a range of LRD olefins in E. coli.…”

“…The relevant combinations of diterpene synthases are described in the SI Appendix (SI Appendix, Table S1). This system is further amendable to incorporation of CYPs, in particular, using synthetic genes, codon-optimized for expression in E. coli along with replacement of the N-terminal transmembrane helix sequence with a leader peptide (15), as has been previously reported for AtKO and OsKO2 (9, 13). Table S14).…”