P450s controlling metabolic bifurcations in plant terpene specialized metabolism

Abstract

Abstract: Catalyzing stereo- and regio-specific oxidation of inert hydrocarbon backbones, and a range of more exotic reactions inherently difficult in formal chemical synthesis, cytochromes P450 (P450s) offer outstanding potential for biotechnological engineering. Plants and their dazzling diversity of specialized metabolites have emerged as rich repository for functional P450s with the advances of deep transcriptomics and genome wide discovery. P450s are of outstanding interest for understanding chemical diversification throughout evolution, for gaining mechanistic insights through the study of their structure-function relationship, and for exploitation in Synthetic Biology. In this review, we highlight recent developments and examples in the discovery of plant P450s involved in the biosynthesis of industrially relevant monoterpenoids, sesquiterpenoids, diterpenoids and triterpenoids, throughout 2016 and early 2017. Examples were selected to illustrate the spectrum of value from commodity chemicals, flavor and fragrance compounds to pharmacologically active terpenoids. We focus on a recently emerging theme, where P450s control metabolic bifurcations and chemical diversity of the final product profile, either within a pathway, or through neo-functionalization in related species. The implications may inform approaches for rational assembly of recombinant pathways, biotechnological production of high value terpenoids and generation of novel chemical entities.

Keywords: Biosynthetic; Biotechnology; Metabolic diversity; Metabolic engineering; Orthologs; Pathway bifurcation; Promiscuity; Regio-specificity; Stereo-specificity; Synthetic Biology; Terpenoid specialized metabolites.

Fig. 1

Regio-specific hydroxylation of (−)-4S-limonene by cytochromes P450. *Recombinant chimeric variant of CYP71D174 yields (−)-perillyl alcohol along with (−)-trans-isopiperitenol and (−)-trans-carveol in an in vitro assay

Fig. 2

Cytochrome P450 mediated linalool metabolism in Arabidopsis. With exception of CYP76C4, the P450s are expressed in flowers. CYP76C4 has very low expression in roots. TPS: terpene synthase

Fig. 3

Cytochrome P450 mediated regio-specific oxidation of geraniol takes place at the marked C-8 and C-9 positions

Fig. 4

Cytochrome P450 mediated santalol metabolism in S. album. Stereo-specificity of heartwood specific CYP736A167 channels the olefin precursor into the (Z)-isomers of the constituent santalols of the sandalwood oil. SaSSY: S. album santalene/bergamotene synthase

Fig. 5

Cytochrome P450 mediated biodiversity of oxygenated sesquiterpenes in various plants. CYP71BE5 has been found to be active in Syrah grape exocarp, CYP71D20 in tobacco, CYP71D55 in henbane. Vv: Vitis vinifera, TPS: terpene synthase, EAS: 5-epi-aristolochene synthase, HPS: Hyoscyamus muticus premnaspirodiene synthase

Fig. 6

Cytochrome P450 mediated regio-specific oxidation of ent-kaurene in GA₁₂ biosynthesis. Regio-specificity of P450s (CYP714A2 and CYP88) leads to pathway bifurcation between general and engineered specialized metabolism

Fig. 7

Regio-specific oxidation of casbene by different orthologous members of cytochromes P450. ADH alcohol dehydrogenase

Fig. 8

Regio-specific oxidation of miltiradiene by different orthologous members of cytochromes P450. Regio-specificity of P450s leads to pathway bifurcation and metabolic diversity. CPS copalyl diphosphate synthase, MiS miltiradiene synthase

Fig. 9

Cytochrome P450 mediated regio-specific oxidation of various diterpene scaffolds leads to chemical diversity of diterpene resin acids in conifers. Example of P450s from different clades in subfamily CYP720B which yield the same products through distinct routes. *The pathway forming dehydroabietadiene from GGDP is not known in conifers. LAS levopimaradiene/abietadiene synthase, ISO isopimaradiene synthase, PIM pimaradiene synthase, diTPS diterpene synthase

Fig. 10

CYP725A4 mediated regio-specific oxidation of taxadiene takes place at the marked C-5 position

Fig. 11

Regio-specific oxidation of β-amyrin by different orthologous members of cytochromes P450. *Key cytochrome P450s controlling the regio-specific C-2 oxidation of various intermediates involved in sapogenin biosynthesis

Fig. 12

Regio-specific oxidation of α-amyrin, δ-amyrin, germanicol, and lupeol by various orthologous members of cytochromes P450

Fig. 13

Regio-specificity of orthologous members of cytochromes P450 in the metabolism of cucurbitadienol. SgCbQ S. grosvenorii cucurbitadienol synthase