P450s controlling metabolic bifurcations in plant terpene specialized metabolism
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.
KeywordsTerpenoid specialized metabolites Pathway bifurcation Regio-specificity Stereo-specificity Promiscuity Orthologs Biosynthetic Metabolic diversity Synthetic Biology Metabolic engineering Biotechnology
The few plant pathways to specialized metabolites found in text books appear as discrete cascades to end-point molecules of interest (e.g. morphine biosynthesis). A paradigm of linear pathways still appears applicable to the universally shared pathways providing the C5 building blocks for terpenoid metabolism across all life kingdoms. However, plant pathways of the specialized metabolism have impressively diversified throughout evolution. Growing evidence, in recent years increasingly fueled by deep sequencing technologies, indicates highly branched networks, complex anastomosing metabolic grids in rapidly evolving pathways. Because P450s typically catalyze irreversible reactions, they represent ideal points of control over metabolic bifurcations. In contrast to the view of P450s as highly specialized enzymes dedicated to specific pathways, evidence is emerging for substantial promiscuity of the enzymes. This may allow re-purposing and rapid evolution of novel pathways in plants and sets the stage for their combinatorial reconstitution in neo-natural pathways in biotechnology. Below we introduce the characteristics, applications and progress for each class of terpenoids individually, followed by a discussion of illustrative examples.
Oxidative monoterpenoid metabolism
At more than 4000 known structures with over 92% carrying two, or more oxygen atoms (Dictionary of Natural Products 23.1, Pateraki et al. 2015), plant monoterpenoids show an impressive structural diversity. The short C10 hydrocarbon backbone, typically1 derived from geranyl diphosphate (GDP) establishes their physicochemical properties and, with that, their industrial applications. Limonene, for example, a simple cyclic monoterpene alkene, is used as a green solvent with a boiling point of 176 °C. A high standard enthalpy of combustion (−6100 kJ Mol−1) means limonene is a potential high-density biofuel. Oxygenations increase the polarity and modulate the properties and applications. Carvone or perrilyl alcohol, oxidized derivatives of limonene, have a broadened spectrum of applications, such as flavor additives or emerging cancer therapeutics (Chen et al. 2015; de Carvalho and da Fonseca 2006), emphasizing the industrial relevance of the conversion.
Within the same plant system, there is a second instance of pathway control by P450s. The hepatotoxic monoterpene (R)-(+)-menthofuran, which also negatively affects commercial value of the essential oil, can accumulate to substantial levels under adverse growth conditions (Bertea et al. 2001). Specifically, a new P450 of the subfamily CYP71A was discovered from the peppermint oil glands. Functional characterization of heterologously expressed recombinant CYP71A32 indicated a role in formation of menthofuran by converting of (R)-(+)-pulegone to (R)-(+)-menthofuran via an allylic hydroxylation and spontaneous rearrangement yielding the furan ring (Bertea et al. 2001; Mizutani and Sato 2011). (R)-(+)-pulegone is also an intermediate in the route to (−)-menthol, with (+)-pulegone reductase catalyzing the conversion into the intermediate (−)-menthone (Ringer et al. 2003), effectively competing for the substrate with CYP71A32. In addition, under low-ambient light, mint plants selectively sequestered menthofuran in the oil glands at concentrations sufficient for competitive inhibition of the pulegone reductase (Mahmoud and Croteau 2001; Rios-Estepa et al. 2008). In the mint-system, improvement of the yield and purity from the natural sources was addressed by a multipronged approach, assisted by mathematical modeling: (i) overexpression of three steps of the plastidial precursor pathway provided increased isoprene C5 building blocks, (ii) a heterologous variant from a different species of the geranyl diphosphate synthase for the production of the general monoterpene precursor, geranyl diphosphate (GDP), (iii) anti-sense mediated suppression of the above menthofuran shunt-pathway, and (iv) introduction of an engineered variant of a (+)-limonene synthase. This latter feature is non-native to this system and introduces a chemical watermark permitting convenient identification of origin of the natural products of this engineered biosynthetic platform (Lange et al. 2011).
Oxidized derivatives of linalool
Diversity in sesquiterpenoid metabolism
The regio- and stereospecificity of various P450s involved in sesquiterpene biosynthesis leads to broad diversification of oxygenated sesquiterpenes. Here, our current knowledge indicates that the sesquiterpene synthases, rather than the P450s, contribute more significantly to pathway bifurcations, leading from the most common precursor of farnesyl diphosphate (FDP) to distinct families of products. However, the substrate specificity and/or promiscuity of the relevant P450s typically drive chemical diversification of the different families of sesquiterpenoids. Illustrative examples of biotechnologically motivated discovery of P450s yielding the industrially relevant sesquiterpene feedstock nootkatone and the P450 driven diversification of the structurally intriguing sesquiterpene lactones, including the flagship molecule artemisinin, have been recently reviewed (e.g. Hamberger and Bak 2013; Pateraki et al. 2015). Here we discuss selected examples of sesquiterpenes metabolite pathways, where the activity of P450s plays a key role, often at the end of the pathway.
Capsidol is a bicyclic, dihydroxylated sesquiterpene produced by many solanaceous species in response to various environmental cues including pathogen attack, elicitor challenge or exposure to UV light (Ralston et al. 2001; Takahashi et al. 2005). Biosynthesis of capsidol involves the 5-epi-aristolochene synthase, catalyzing formation of the bicyclic sesquiterpene olefin intermediate, 5-epi-aristolochene (5-EA), followed by P450-mediated dihydroxylation of 5-EA to capsidol (Facchini and Chappell 1992; Takahashi et al. 2005). The corresponding enzyme CYP71D20 from Nicotiana tabacum was found to catalyze the unique stereo- and regio-selective sequential dihydroxylation of 5-EA at C-1 and C-3 to afford capsidol (Greenhagen et al. 2003; Ralston et al. 2001; Takahashi et al. 2005). Investigation of the kinetic behavior of CYP71D20 established the putative sequence of oxidation of 5-EA at the C-1 position followed by the C-3 position, generating stereoselectively 1β-hydroxylated EA followed by 1β,3α-capsidol (Takahashi et al. 2005). CYP71D20 has also been found to catalyze the conversion of premnaspirodiene to solavetivone, albeit at very low rates (Greenhagen et al. 2003). Although the pathway bifurcation for the sesquiterpene hydrocarbon intermediates 5-EA and premnaspirodiene depends on the corresponding sesquiterpene synthase, the stereo-and regio-selective catalysis by CYP71D20 of the individual sesquiterpene hydrocarbon intermediate plays an important role in creating the diversity of the oxygenated sesquiterpenes (Fig. 5).
Solavetivone, a potent antifungal phytoalexin, plays a role as defense molecule in the solanaceous plants henbane (Hyoscyamus muticus) and potato. It is biochemically synthesized from the vetispirane-type sesquiterpene based scaffold premnaspirodiene, which is formed by the H. muticus premnaspirodiene synthase. Another member of the CYP71 subfamily, CYP71D55, was found to catalyze the successive regio-selective oxidation at the carbon atom C-2 position of premnaspirodiene to yield solavetivone (Fig. 5). In vitro assays demonstrated that CYP71D55 also converted valencene and 5-EA with an eremophilane based sesquiterpene scaffold, but only to their corresponding mono-oxygenated product (Takahashi et al. 2007), unlike the related P450s catalyzing successive oxidations such as CYP71D20 and CYP71AV8 (Cankar et al. 2011; Ralston et al. 2001). As with previous examples, CYP71D55 is involved in increasing the biodiversity of the oxygenated sesquiterpenes.
The founding member of the subfamily, CYP71AV1, from Artemisia annua catalyzes a three-step oxidation of amorphadiene to artemisinic acid, off-product of the pathway to the anti-malaria pharmaceutical artemisinin (Ro et al. 2006). The homology-based identification of orthologous sequences in closely related Asteraceae led to the discovery of enzymes with lowered regioselectivity (i.e. formation of a distinct amorphadienol isomer), in addition to a lack of activity for the formation of artemisinic acid (Komori et al. 2013). The initial number of different amino acids over the entire sequence was narrowed down to a section carrying nine residues through functional testing of chimeric variants with swapped domains. Using structural modeling, four amino acids emerged, putatively residing in the catalytic site. Following site directed mutagenesis and functional testing, conversion of a serine to phenylalanine in position 479 reduced the conversion of the alcohol to the aldehyde nearly 20-fold and limited the activity of the P450 to a single step (Komori et al. 2013). While not of immediate biotechnological relevance, as the pathway to Artemisinin proceeds via the aldehyde, this approach highlights the rational engineering of the activity of a P450 with an important role at this metabolic junction, guided by natural variation and structural modeling.
Oxidation of labdane-type and macrocyclic diterpenes
With backbones consisting of 20 carbon atoms, the theoretically possible structural complexity of diterpenes far exceeds that of the classes using two or three C5 building blocks. Yet, among documented plant terpenoids, the number of diterpenoids is in the same range of sesquiterpenoids, with substantially fewer monoterpenoids known (diterpenoids, 12,505 vs. sesquiterpenoids 13,981 vs. monoterpenoids, 4129; Dictionary of Natural Products 23.1; Pateraki et al. 2015). On the other hand, over 95% of known diterpenoid structures are carrying two or more oxygen atoms (sesquiterpenoids, 90%; monoterpenoids, 92%), indicating increased relevance of P450s in generation of structural complexity and chirality in the respective biosynthetic routes.
Casbene based diterpenes
Carnosic acid, tanshinones, steviol and forskolin
Independent, complementary research found that CYP76AH24, CYP76AK6 from S.- pomifera and CYP76AH4, CYP76AK8 from R. officinalis account for the set of oxidations in the pathway from dehydroabietadiene to carnosic acid (Ignea et al. 2016). Functional analysis of the P450s in engineered yeast showed in addition that yet another member of a related subfamily, CYP71BE52, can oxidize ferruginol in the C-2 position to salviol. Hence, CYP76AH22/CYP76AH24/CYP76AH4, CYP76AK6/CYP76AK7/CYP76AK8, and CYP71BE52 control multiple pathway bifurcations leading to chemical diversification in the biosynthesis of dehydroabietadiene based diterpenoid metabolism.
Salvia miltiorrhiza, another member of the family of Lamiaceae, is well known for accumulating tanshinones, which have expansive uses in traditional Chinese medicine, but having also attracted interest due to anti-bacterial and a range of therapeutic activities. Tanshinones, like carnosic acid, are abietane-type diterpenoids, derived from ferruginol and through a biosynthetic route involving members of subfamilies CYP76AH and CYP76AK: founding members CYP76AH1 and CYP76AK1 of both subfamilies were discovered in S. miltiorrhiza. CYP76AH1 was established as ferruginol synthase, however, a rather obscure mechanism was proposed to explain the apparent conversion of the diterpene olefin miltiradiene to ferruginol (Guo et al. 2013). This misperception was later corrected when the orthologous CYP76AH4 from R. officinalis was functionally characterized demonstrating that ferruginol is produced by CYP76AH4 mediated oxidation of dehydroabietadiene (abietatriene), which is a spontaneous oxidation product from miltiradiene (Zi and Peters 2013). Subsequent identification and characterization of CYP76AH3 and CYP76AK1 by expression in yeast led to demonstration of a pathway with at least one intersection and bifurcation controlled by these P450s. CYP76AH3 catalyzed the oxidation to yield a C-11 hydroxyl function, as well as formation of 7-keto ferruginol (sugiol) and 7-keto-11-hydroxyferruginol (11-hydroxy sugiol). In contrast, CYP76AK1 showed regio-selectivity for oxidation at carbon atom C-20 of both 11-hydroxy ferruginol and 11-hydroxy sugiol (Guo et al. 2016).
Another group of labdane-type diterpenoids with commercial relevance are steviol-glucoside sweeteners accumulating in the leaves of the Asteraceae Stevia rebaudiana. These are based on a bifurcation, leading from the GA12 intermediate ent-kaurenoic acid through a single hydroxylation at carbon atom C-13 to steviol (Fig. 6). Recombinant Arabidopsis CYP714A2, with a role in GA metabolism, was shown to also yield steviol, when incubated with ent-kaurenoic acid (Nomura et al. 2013). Inspired by this finding, an active ent-kaurenoic acid hydroxylase was identified in S. rebaudiana. An engineered variant of CYP714A2 finally yielded over 15 mg L−1 of steviol, when expressed in a strain of E. coli dedicated for production of ent-kaurenoic acid (Fig. 6; Wang et al. 2016).
In the Lamiaceae Coleus forskohlii, diterpenes carrying both abietane and epoxy-labdane (13R-manoyl oxide) scaffolds are prevalent, and their biosynthetic origins have been elucidated (Pateraki et al. 2014). Of interest in this plant species is the root-specific accumulation of the 13R-manoyl oxide-derived structurally complex diterpene forskolin, which consists of an oxygen heteroatom-containing labdane scaffold, with five functionalized positions. Identification of a substantial bloom of subfamily CYP76AH yielded an intriguing number of enzymes with, in part multifunctional activity towards 13R-manoyl oxide and, concomitant, extensive chemical diversification of the product palette. Ultimately, combinatorial testing led to a minimal set sufficient to catalyze regio- and stereo-specific formation of deacetyl-forskolin. Isolation of a regio-selective acetyl-transferase completed a biosynthetic route to forskolin, which was stably integrated in an engineered yeast strain optimized for production of 13R-manoyl oxide (Pateraki et al. 2017; patents Andersen-Ranberg and Pateraki 2016; Hamberger et al. 2015, 2016).
Diterpene Resin Acids
Oxidative pathways of triterpene specialized metabolism
Recruitment of highly diverse P450s for triterpenoid oxidation
Studies involving selection of candidate genes by co-expression analysis in Medicago truncatula followed by their in vivo and in vitro functional characterization demonstrated that CYP716A12 acts as β-amyrin 28-oxidase (Carelli et al. 2011; Fukushima et al. 2011; Naoumkina et al. 2010). CYP716A12 catalyzes three sequential oxidation steps at C-28 position of β-amyrin to produce oleanolic acid, which gets further decorated by other P450s to produce hemolytic sapogenins. In transgenic yeast, CYP716A12 is also shown to oxidize α-amyrin and lupeol to ursolic acid and betulinic acid, respectively (CYP716A175 and CYP716A179 from apple and licorice, respectively, have identical activity, Fig. 11). On the contrary, CYP93E2 from M. truncatula has been found to oxidize β-amyrin at C-24 forming 24-hydroxy β-amyrin (and also probably β-amyrin-24-oic acid) which further leads to the biosynthesis of non-hemolytic sapogenins (soyasapogenols) (Fukushima et al. 2011). This highlights the recruitment of highly divergent P450s from different clans for controlling the metabolic junction in these routes and has implications for pathway discovery driven by identification of recent expansions of gene families. Specifically, CYP93E2 is a member of the notoriously enriched CYP71 clan, where numerous functions in terpenoid specialized metabolism have spawned. In contrast, CYP716A12 resides in the CYP85 clan, with broader involvement in terpenoid general metabolism, but which also carries evolutionarily old examples of specialized metabolism such as in the conifer lineage (Kaspera and Croteau 2006; Ro et al. 2005). In recent advances, M. truncatula CYP72A67 was identified through TILLING in a mutagenized population. Through functional characterization by genetic approaches and heterologous expression, CYP72A67 was established in the context of other related P450s as the key enzyme controlling oxidation at C-2 of several intermediates in the hemolytic sapogenin pathway to zhantic acid (Biazzi et al. 2015). This body of work, together with numerous studies in other plant systems, solidly established the P450 families CYP93, CYP716 and CYP72 as rich repositories for candidates involved in tripterpenoid oxidation. However, isolated examples have also occurred in other families. This has inspired recent studies, where since 2015 members of CYP716 were implicated as key enzymes in C-28 oxidation of α-amyrin, β-amyrin, and lupeol leading to the corresponding acids in apple (see overview provided in Fig. 12, CYP716A175; Andre et al. 2016), and analogously in licorice, producing ursolic acid, oleanolic acid, and betulinic acid (CYP716A179, Tamura et al. 2017). Similar oxidation is also observed for germanicol to morolic acid by CYP716A175. In Artemisia annua, best known for the elucidated biosynthesis of the sesquiterpenoid artemisinin, CYP716A14v2 was shown to catalyze C-3 oxidation of α-amyrin, β-amyrin, and δ-amyrin to yield the 3-keto triterpenes (Moses et al. 2015). Founding members of the subfamily, Arabidopsis CYP716A1 and CYP716A2, were reported in a genomic cluster co-localized on chromosome 5 with a triterpene synthase. When co-expressed with the triterpene synthase, CYP716A1 displayed activity and afforded an oxidized tirucalla-7,24-dien-3β-ol (Boutanaev et al. 2015). Comprehensive testing of both P450s in yeast, engineered for production of α-amyrin, β-amyrin, and lupeol established them as multifunctional enzymes, with partially overlapping functions. CYP716A1 catalyzed multiple oxidations at specific positions of the tripterpene scaffolds toward ursolic acid and oleanolic acid but not betulinic acid. In contrast, CYP716A2 was limited to a mono-oxidation, yielding the alcohol intermediates of the triterpenes, 22-α-hydroxy α-amyrin and traces of β-amyrin oxidized at C-28 and C-16. With that, CYP716A2 contributes triterpene oxidation of carbon C-22 to the existing toolbox enabling combinatorial biosynthesis (Yasumoto et al. 2016). Formation of additional, yet unidentified oxidized triterpenes by other relatives in subfamily CYP716A (Khakimov et al. 2015) and broad phylogenetic distribution of P450s with demonstrated activity in triterpenoid oxidation over at least eight subfamilies in four clans (Yasumoto et al. 2016) highlights a promising biosynthetic potential for biotechnological production of high-value triterpenoids.
Reconstruction of a complete mogroside recombinant pathway, including optimization of production and several steps catalyzed by UDP-dependent glycosyl transferases, has been reported (Liu et al. 2014). However, the origin of the vicinal C-24 and C-25 hydroxyl groups in the mogroside aglycon remained unclear with alternatives possible (Itkin et al. 2016). C-24 and C-25 hydroxyl groups are rare among cucurbitane triterpenoids. Zhang and co-workers proposed an origin of the intermediate epoxide through activity of CYP87D18 (Zhang et al. 2016). In contrast, based on observations in yeast and transient expression in tobacco, where endogenously formed 2,3;22,23-diepoxy squalene is offered as substrate for the cucurbitadienol synthase, Itkin and co-workers suggest an initial di-epoxydation of squalene, and cyclization to 24,25-epoxy cucurbitadienol as plausible intermediate step. Hydrolytic ring opening to yield the vicinal 24,25-diol was suggested to be catalyzed by epoxide hydrolases and not to require activity of P450s (for an overview see Fig. 13; Itkin et al. 2016).
Regulation and organization
Two recent studies shed light on a previously unrecognized level of metabolic regulation and organization. Parage and co-workers suggest that control of the monoterpene indole alkaloid pathway in C. roseus and activity of the large group of P450s involved, including CYP76B6, is administrated through the enzyme providing the reduction equivalents, the NADPH-dependent cytochrome P450 oxidoreductase (POR, synonym CPR) (Parage et al. 2016). With exceptions (Apiaceae, Andersen et al. 2016), PORs are typically represented by two distinct classes. Specifically, in planta, but not in reconstituted or in vitro systems, a member of the C. roseus class II POR was shown to be essential for the specialized metabolism responding to external stimuli. In contrast, the class I POR was found to be associated with the general metabolism, with no measurable contribution in the metabolism of monoterpene indole alkaloids, as shown by deep co-expression and silencing studies (Parage et al. 2016). Further highlighting a critical mechanistic importance of the POR in specialized metabolism, a recent study of the protein–protein interaction in context of the local lipid environment demonstrated the existence of a dynamically assembled and disassembled metabolon (Laursen et al. 2016). The team led by Jean-Étienne Bassard proposed an operative stabilization of the metabolon when all enzymes are co-expressed and interact. The work establishes a higher complexity order, including homo- and heterodimers of the POR, a soluble glycosyl transferase, and the two involved P450s, resulting in a highly efficient biosynthetic pathway. Despite using an experimental model outside of terpenoid metabolism, it is suggested that the principle of organization in dynamic metabolons may apply more broadly to biosynthetic pathways involved in specialized metabolism (Laursen et al. 2016). This principle may also have implications for the rational engineering of orchestrated pathways or scaffolded complexes in Synthetic Biology, where a critical goal is to enable effective channeling of intermediates while avoiding disadvantageous metabolic shunt pathways, leakage of labile intermediates or unspecific endogenous activities in the chassis organism.
The insights discussed in this review highlight only a few of the complexities encountered during the engineering of biotechnological production platforms, and when depending on heterologous enzymes which plausibly evolved to drive chemical diversification in their plant source species. On the other hand, the biotechnological host species, or chassis, presents conceptual challenges, which need to be overcome (reviewed in Renault et al. 2014). The use of heterotrophic microbes has been comprehensively reviewed (e.g. Li and Pfeifer 2014) and efficient gene-stacking of P450s remains a limitation. A recent study elegantly demonstrated the production of di-oxygenated and acetylated taxadiene through a synthetic consortium of both E. coli and S. cerevisiae. The novel approach successfully engineered interdependency between the species and split the pathway into two segments, each expressed in one of the microbes (Zhou et al. 2015). Photosynthetic hosts offer several potential advantages, including presence of reducing equivalents (electrons) derived from photosynthesis and a source of carbon. Even though there is currently no photosynthetic platform with reported scaled and stable production of multiple oxidized terpenoids at industrially relevant level, recent proof-of-concept studies in cyanobacteria, algae, the lower land plant Physcomitrella patens and chloroplast engineering (reviewed in Nielsen et al. 2016) highlight the potential for these platforms. Finally, vascular land plants have evolved highly specialized anatomical structures dedicated for terpenoid storage, such as glandular trichomes, laticifers and resin ducts. This structural repertoire was recently suggested to include lipid droplets for storage of both simple and highly functionalized terpenoids (Pateraki et al. 2014). As terpenes were shown to potentially cause critical perturbations on the thermotropic and structural properties of lipid bilayers (Jagalski et al. 2016), the coordinated engineering of both terpenoid biosynthetic pathways and the formation of intracellular storage organelles may relieve this potential bottleneck.
For monoterpenes, GDP (the trans-isomer/E-isomer) is the most common precursor, although the cis-/E-isomer, neryl diphosphate was also reported in Solanaceae, but with terpene synthases accepting the substrate also outside the family. Similarly, the prenyl diphosphate in trans-configuration E,E-farnesyl diphosphate (FDP) and E,E,E-geranylgeranyl diphosphate (GGDP) are the most common precursors for sesqui- and diterpenes, respectively. The corresponding cis-isomers, i.e. all-cis Z,Z-farnesyl diphosphate and nerylneryl diphosphate (Solanaceae), can also serve as precursors for some sesqui- and diterpene synthases (Jones et al. 2011; Matsuba et al. 2013; Schilmiller et al. 2009).
We thank Dr. Sean Johnson (Michigan State University, East Lansing, MI) for critical reading of the manuscript. Bj.H. and A.B. are supported by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) and Bj.H. gratefully acknowledges the Strategic Partnership Grant (15-SPG-Full-3101), MSU Foundation, startup funding from the Department of Molecular Biology and Biochemistry, Michigan State University and support from Michigan State University AgBioResearch (MICL02454).
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