Dual factors required for cytochrome-P450-mediated hydrocarbon ring contraction in bacterial gibberellin phytohormone biosynthesis.

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction.

Origin and early evolution of the plant terpene synthase family.

As a midsized gene family conserved more by lineage than function, the typical plant terpene synthases (TPSs) could be a valuable tool to examine plant evolution. TPSs are pivotal in biosynthesis of gibberellins and related phytohormones as well as in formation of the extensive arsenal of specialized plant metabolites mediating ecological interactions whose production is often lineage specific. Yet the origin and early evolution of the TPS family is not well understood. Systematic analysis of an array of transcriptomes and sequenced genomes indicated that the TPS family originated after the divergence of land plants from charophytic algae. Phylogenetic and biochemical analyses support the hypothesis that the ancestral TPS gene encoded a bifunctional class I and II diterpene synthase producing the ent-kaurene required for phytohormone production in all extant lineages of land plants. Moreover, the ancestral TPS gene likely underwent duplication at least twice early in land plant evolution. Together these two gave rise to three TPS lineages leading to the extant TPS-c, TPS-e/f, and the remaining TPS (h/d/a/b/g) subfamilies, with the latter dedicated to secondary rather than primary metabolism while the former two contain those genes involved in ent-kaurene production. Nevertheless, parallel evolution from the ent-kaurene­producing class I and class II diterpene synthases has led to roles for TPS-e/f and -c subfamily members in secondary metabolism as well. These results clarify TPS evolutionary history and provide context for the role of these genes in producing the vast diversity of terpenoid natural products observed today in various land plant lineages.

Interdependent evolution of biosynthetic gene clusters for momilactone production in rice.

Plants can contain biosynthetic gene clusters (BGCs) that nominally resemble those found in microbes. However, while horizontal gene transmission is often observed in microbes, plants are limited to vertical gene transmission, implying that their BGCs may exhibit distinct inheritance patterns. Rice (Oryza sativa) contains two unlinked BGCs involved in diterpenoid phytoalexin metabolism, with one clearly required for momilactone biosynthesis, while the other is associated with production of phytocassanes. Here, in the process of elucidating momilactone biosynthesis, genetic evidence was found demonstrating a role for a cytochrome P450 (CYP) from the other "phytocassane" BGC. This CYP76M8 acts after the CYP99A2/3 from the "momilactone" BGC, producing a hemiacetal intermediate that is oxidized to the eponymous lactone by a short-chain alcohol dehydrogenase also from this BGC. Thus, the "momilactone" BGC is not only incomplete, but also fractured by the need for CYP76M8 to act in between steps catalyzed by enzymes from this BGC. Moreover, as supported by similar activity observed with orthologs from the momilactone-producing wild-rice species Oryza punctata, the presence of CYP76M8 in the other "phytocassane" BGC indicates interdependent evolution of these two BGCs, highlighting the distinct nature of BGC assembly in plants.

Plant (di)terpenoid evolution: from pigments to hormones and beyond.

Covering: up to 2014-2022.Diterpenoid biosynthesis in plants builds on the necessary production of (E,E,E)-geranylgeranyl diphosphate (GGPP) for photosynthetic pigment production, with diterpenoid biosynthesis arising very early in land plant evolution, enabling stockpiling of the extensive arsenal of (di)terpenoid natural products currently observed in this kingdom. This review will build upon that previously published in the Annual Review of Plant Biology, with a stronger focus on enzyme structure-function relationships, as well as additional insights into the evolution of (di)terpenoid metabolism since generated.

Diterpene synthases from Leonurus japonicus elucidate epoxy-bridge formation of spiro-labdane diterpenoids.

Spiro-9,13-epoxy-labdane diterpenoids are commonly found in Leonurus species, particularly in Leonurus japonicus Houtt., which is a medicinal herb of long-standing use in Asia and in which such spiro-heterocycles are present in at least 38 diterpenoids. Here, through generation of a transcriptome and functional characterization of six diterpene synthases (diTPSs) from L. japonicus, including three class II diTPSs (LjTPS1, LjTPS3, and LjTPS4) and three class I diTPSs (LjTPS5, LjTPS6, and LjTPS7), formation of the spiro-9,13-epoxy-labdane backbone was elucidated, along with identification of the relevant diTPSs for production of other labdane-related diterpenes. Similar to what has been found with diTPSs from other plant species, while LjTPS3 specifically produces the carbon-9 (C9) hydroxylated bicycle peregrinol diphosphate (PPP), the subsequently acting LjTPS6 yields a mixture of four products, largely labda-13(16),14-dien-9-ol, but with substantial amounts of viteagnusin D and the C13-S/R epimers of 9,13-epoxy-labda-14-ene. Notably, structure-function analysis identified a critical residue in LjTPS6 (I420) in which single site mutations enable specific production of the 13S epimer. Indeed, extensive mutagenesis demonstrated that LjTPS6:I420G reacts with PPP to both specifically and efficiently produce 9,13S-epoxy-labda-14-ene, providing a specialized synthase for further investigation of derived diterpenoid biosynthesis. The results reported here provide a strong foundation for future studies of the intriguing spiro-9,13-epoxy-labdane diterpenoid metabolism found in L. japonicus.

Dissecting the labdane-related diterpenoid biosynthetic gene clusters in rice reveals directional cross-cluster phytotoxicity.

Rice (Oryza sativa) is a staple food crop and serves as a model cereal plant. It contains two biosynthetic gene clusters (BGCs) for the production of labdane-related diterpenoids (LRDs), which serve important roles in combating biotic and abiotic stress. While plant BGCs have been subject to genetic analyses, these analyses have been largely confined to the investigation of single genes. CRISPR/Cas9-mediated genome editing was used to precisely remove each of these BGCs, as well as simultaneously knock out both BGCs. Deletion of the BGC from chromosome 2 (c2BGC), which is associated with phytocassane biosynthesis, but not that from chromosome 4 (c4BGC), which is associated with momilactone biosynthesis, led to a lesion mimic phenotype. This phenotype is dependent on two closely related genes encoding cytochrome P450 (CYP) mono-oxygenases, CYP76M7 and CYP76M8, from the c2BGC. However, rather than being redundant, CYP76M7 has been associated with the production of phytocassanes, whereas CYP76M8 is associated with momilactone biosynthesis. Intriguingly, the lesion mimic phenotype is not present in a line with both BGCs deleted. These results reveal directional cross-cluster phytotoxicity, presumably arising from the accumulation of LRD intermediates dependent on the c4BGC in the absence of CYP76M7 and CYP76M8, further highlighting their interdependent evolution and the selective pressures driving BGC assembly.

Rice diterpenoid phytoalexins are involved in defence against parasitic nematodes and shape rhizosphere nematode communities.

Rice diterpenoid phytoalexins (DPs) are secondary metabolites with a well known role in resistance to foliar pathogens. As DPs are also known to be produced and exuded by rice roots, we hypothesised that they might play an important role in plant-nematode interactions, and particularly in defence against phytoparasitic nematodes. We used transcriptome analysis on rice roots to analyse the effect of infection by the root-knot nematode Meloidogyne graminicola or treatment with resistance-inducing chemical stimuli on DP biosynthesis genes, and assessed the susceptibility of mutant rice lines impaired in DP biosynthesis to M. graminicola. Moreover, we grew these mutants and their wild-type in field soil and used metabarcoding to assess the effect of impairment in DP biosynthesis on rhizosphere and root nematode communities. We show that M. graminicola suppresses DP biosynthesis genes early in its invasion process and, conversely, that resistance-inducing stimuli transiently induce the biosynthesis of DPs. Moreover, we show that loss of DPs increases susceptibility to M. graminicola. Metabarcoding on wild-type and DP-deficient plants grown in field soil reveals that DPs significantly alter the composition of rhizosphere and root nematode communities. Diterpenoid phytoalexins are important players in basal and inducible defence against nematode pathogens of rice and help shape rice-associated nematode communities.

Rice contains a biosynthetic gene cluster associated with production of the casbane-type diterpenoid phytoalexin ent-10-oxodepressin.

Diterpenoids play important roles in rice microbial disease resistance as phytoalexins, as well as acting in allelopathy and abiotic stress responses. Recently, the casbane-type phytoalexin ent-10-oxodepressin was identified in rice, but its biosynthesis has not yet been elucidated. Here ent-10-oxodepressin biosynthesis was investigated via co-expression analysis and biochemical characterisation, with use of the CRISPR/Cas9 technology for genetic analysis. The results identified a biosynthetic gene cluster (BGC) on rice chromosome 7 (c7BGC), containing the relevant ent-casbene synthase (OsECBS), and four cytochrome P450 (CYP) genes from the CYP71Z subfamily. Three of these CYPs were shown to act on ent-casbene, with CYP71Z2 able to produce a keto group at carbon-5 (C5), while the closely related paralogues CYP71Z21 and CYP71Z22 both readily produce a keto group at C10. Together these C5 and C10 oxidases can elaborate ent-casbene to ent-10-oxodepressin (5,10-diketo-ent-casbene). OsECBS knockout lines no longer produce casbane-type diterpenoids and exhibit impaired resistance to the rice fungal blast pathogen Magnaporthe oryzae. Elucidation of ent-10-oxodepressin biosynthesis and the associated c7BGC provides not only a potential target for molecular breeding, but also, gives the intriguing parallels to the independently assembled BGCs for casbene-derived diterpenoids in the Euphorbiaceae, further insight into plant BGC evolution, as discussed here.

A (conditional) role for labdane-related diterpenoid natural products in rice stomatal closure.

Rice (Oryza sativa) is the staple food for over half the world's population. Drought stress imposes major constraints on rice yields. Intriguingly, labdane-related diterpenoid (LRD) phytoalexins in maize (Zea mays) affect drought tolerance, as indicated by the increased susceptibility of an insertion mutant of the class II diterpene cyclase ZmCPS2/An2 that initiates such biosynthesis. Rice also produces LRD phytoalexins, utilizing OsCPS2 and OsCPS4 to initiate a complex metabolic network. For genetic studies of rice LRD biosynthesis the fast-growing Kitaake cultivar was selected for targeted mutagenesis via CRISPR/Cas9, with an initial focus on OsCPS2 and OsCPS4. The resulting cps2 and cps4 knockout lines were further crossed to create a cps2x4 double mutant. Both CPSs also were overexpressed. Strikingly, all of the cv Kitaake cps mutants exhibit significantly increased susceptibility to drought, which was associated with reduced stomatal closure that was evident even under well-watered conditions. However, CPS overexpression did not increase drought resistance, and cps mutants in other cultivars did not alter susceptibility to drought, although these also exhibited lesser effects on LRD production. The results suggest that LRDs may act as a regulatory switch that triggers stomatal closure in rice, which might reflect the role of these openings in microbial entry.

Inferring Roles in Defense from Metabolic Allocation of Rice Diterpenoids.

Among their responses to microbial infection, plants deploy an arsenal of natural antibiotic products. Historically these have been identified on the basis of their antibiotic activity in vitro, which leaves open the question of their relevance to defense in planta. The vast majority of such natural products from the important crop plant rice (Oryza sativa) are diterpenoids whose biosynthesis proceeds via either ent- or syn-copalyl diphosphate (CPP) intermediates, which were isolated on the basis of their antibiotic activity against the fungal blast pathogen Magnaporthe oryzae However, rice plants in which the gene for the syn-CPP synthase Os-CPS4 is knocked out do not exhibit increased susceptibility to M. oryzae Here, we show that knocking out or knocking down Os-CPS4 actually decreases susceptibility to the bacterial leaf blight pathogen Xanthomonas oryzae By contrast, genetic manipulation of the gene for the ent-CPP synthase Os-CPS2 alters susceptibility to both M. oryzae and X. oryzae Despite the secretion of diterpenoids dependent on Os-CPS2 or Os-CPS4 from roots, neither knockout exhibited significant changes in the composition of their rhizosphere bacterial communities. Nevertheless, rice plants allocate substantial metabolic resources toward syn- as well as ent-CPP derived diterpenoids upon infection/induction. Further investigation revealed that Os-CPS4 plays a role in fungal non-host disease resistance. Thus, examination of metabolic allocation provides important clues into physiological function.

Mining of the Catharanthus roseus Genome Leads to Identification of a Biosynthetic Gene Cluster for Fungicidal Sesquiterpenes.

Characterization of cryptic biosynthetic gene clusters (BGCs) from microbial genomes has been proven to be a powerful approach to the discovery of new natural products. However, such a genome mining approach to the discovery of bioactive plant metabolites has been muted. The plant BGCs characterized to date encode pathways for antibiotics important in plant defense against microbial pathogens, providing a means to discover such phytoalexins by mining plant genomes. Here is reported the discovery and characterization of a minimal BGC from the medicinal plant Catharanthus roseus, consisting of an adjacent pair of genes encoding a terpene synthase (CrTPS18) and cytochrome P450 (CYP71D349). These two enzymes act sequentially, with CrTPS18 acting as a sesquiterpene synthase, producing 5-epi-jinkoheremol (1), which CYP71D349 further hydroxylates to debneyol (2). Infection studies with maize revealed that 1 and 2 exhibit more potent fungicidal activity than validamycin. Accordingly, this study demonstrates that characterization of such cryptic plant BGCs is a promising strategy for the discovery of potential agrochemical leads. Moreover, despite the observed absence of 1 and 2 in C. roseus, the observed transcriptional regulation is consistent with their differential fungicidal activity, suggesting that such conditional coexpression may be sufficient to drive BGC assembly in plants.

Probing Enzymatic Structure and Function in the Dihydroxylating Sesquiterpene Synthase ZmEDS.

Terpene synthases (TPSs) play a vital role in forming the complex hydrocarbon backbones that underlie terpenoid diversity. Notably, some TPSs can add water prior to terminating the catalyzed reaction, leading to hydroxyl groups, which are critical for biological activity. A particularly intriguing example of this is the maize (Zea mays) sesquiterpene TPS whose major product is eudesmanediol, ZmEDS. This production of dual hydroxyl groups is presumably enabled by protonation of the singly hydroxylated transient stable intermediate hedycaryol. To probe the enzymatic structure-function relationships underlying this unusual reaction, protein modeling and docking were used to direct mutagenesis of ZmEDS. Previously, an F303A mutant was shown to produce only hedycaryol, suggesting a role in protonation. Here this is shown to be dependent on the steric bulk positioning of hedycaryol, including a supporting role played by the nearby F299, rather than π-cation interaction. Among the additional residues investigated here, G411 at the conserved kink in helix G is of particular interest, as substitution of this leads to predominant production of the distinct (-)-valerianol, while substitution for the aliphatic I279 and V306 can lead to significant production of the alternative eudesmane-type diols 2,3-epi-cryptomeridiol and 3-epi-cryptomeridol, respectively. Altogether, nine residues that are important for this unusual reaction were investigated here, with the results not only emphasizing the importance of reactant positioning suggested by the stereospecificity observed among the various product types but also highlighting the potential role of the Mg2+-diphosphate complex as the general acid for the protonation-initiated (bi)cyclization of hedycaryol.

The genome of the medicinal plant Andrographis paniculata provides insight into the biosynthesis of the bioactive diterpenoid neoandrographolide.

Andrographis paniculata is a herbaceous dicot plant widely used for its anti-inflammatory and anti-viral properties across its distribution in China, India and other Southeast Asian countries. A. paniculata was used as a crucial therapeutic treatment during the influenza epidemic of 1919 in India, and is still used for the treatment of infectious disease in China. A. paniculata produces large quantities of the anti-inflammatory diterpenoid lactones andrographolide and neoandrographolide, and their analogs, which are touted to be the next generation of natural anti-inflammatory medicines for lung diseases, hepatitis, neurodegenerative disorders, autoimmune disorders and inflammatory skin diseases. Here, we report a chromosome-scale A. paniculata genome sequence of 269 Mb that was assembled by Illumina short reads, PacBio long reads and high-confidence (Hi-C) data. Gene annotation predicted 25 428 protein-coding genes. In order to decipher the genetic underpinning of diterpenoid biosynthesis, transcriptome data from seedlings elicited with methyl jasmonate were also obtained, which enabled the identification of genes encoding diterpenoid synthases, cytochrome P450 monooxygenases, 2-oxoglutarate-dependent dioxygenases and UDP-dependent glycosyltransferases potentially involved in diterpenoid lactone biosynthesis. We further carried out functional characterization of pairs of class-I and -II diterpene synthases, revealing the ability to produce diversified labdane-related diterpene scaffolds. In addition, a glycosyltransferase able to catalyze O-linked glucosylation of andrograpanin, yielding the major active product neoandrographolide, was also identified. Thus, our results demonstrate the utility of the combined genomic and transcriptomic data set generated here for the investigation of the production of the bioactive diterpenoid lactone constituents of the important medicinal herb A. paniculata.

The honeysuckle genome provides insight into the molecular mechanism of carotenoid metabolism underlying dynamic flower coloration.

Lonicera japonica is a widespread member of the Caprifoliaceae (honeysuckle) family utilized in traditional medical practices. This twining vine honeysuckle also is a much-sought ornamental, in part due to its dynamic flower coloration, which changes from white to gold during development. The molecular mechanism underlying dynamic flower coloration in L. japonica was elucidated by integrating whole genome sequencing, transcriptomic analysis and biochemical assays. Here, we report a chromosome-level genome assembly of L. japonica, comprising nine pseudochromosomes with a total size of 843.2 Mb. We also provide evidence for a whole-genome duplication event in the lineage leading to L. japonica, which occurred after its divergence from Dipsacales and Asterales. Moreover, gene expression analysis not only revealed correlated expression of the relevant biosynthetic genes with carotenoid accumulation, but also suggested a role for carotenoid degradation in L. japonica's dynamic flower coloration. The variation of flower color is consistent with not only the observed carotenoid accumulation pattern, but also with the release of volatile apocarotenoids that presumably serve as pollinator attractants. Beyond novel insights into the evolution and dynamics of flower coloration, the high-quality L. japonica genome sequence also provides a foundation for molecular breeding to improve desired characteristics.

Magnesium-specific ring expansion/contraction catalysed by the class II diterpene cyclase from pleuromutilin biosynthesis.

The class II diterpene cyclase (DTC) from pleuromutilin biosynthesis uniquely mediates 'A' ring contraction of the initially formed decalin bicycle, yielding mutildienyl diphosphate (MPP). Catalysis requires a divalent metal cation co-factor. Intriguingly, selectively with magnesium, this DTC catalyzes ring expansion/contraction between MPP and halimadienyl diphosphate, providing some catalytic insight.

Conserved bases for the initial cyclase in gibberellin biosynthesis: from bacteria to plants.

All land plants contain at least one class II diterpene cyclase (DTC), which utilize an acid-base catalytic mechanism, for the requisite production of ent-copalyl diphosphate (ent-CPP) in gibberellin A (GA) phytohormone biosynthesis. These ent-CPP synthases (CPSs) are hypothesized to be derived from ancient bacterial origins and, in turn, to have given rise to the frequently observed additional DTCs utilized in more specialized plant metabolism. However, such gene duplication and neo-functionalization has occurred repeatedly, reducing the utility of phylogenetic analyses. Support for evolutionary scenarios can be found in more specific conservation of key enzymatic features. While DTCs generally utilize a DxDD motif as the catalytic acid, the identity of the catalytic base seems to vary depending, at least in part, on product outcome. The CPS from Arabidopsis thaliana has been found to utilize a histidine-asparagine dyad to ligate a water molecule that serves as the catalytic base, with alanine substitution leading to the production of 8ß-hydroxy-ent-CPP. Here this dyad and effect of Ala substitution is shown to be specifically conserved in plant CPSs involved in GA biosynthesis, providing insight into plant DTC evolution and assisting functional assignment. Even more strikingly, while GA biosynthesis arose independently in plant-associated bacteria and fungi, the catalytic base dyad also is specifically found in the relevant bacterial, but not fungal, CPSs. This suggests functional conservation of CPSs from bacteria to plants, presumably reflecting an early role for derived diterpenoids in both plant development and plant-microbe interactions, eventually leading to GA, and a speculative evolutionary scenario is presented.

Direct production of dihydroxylated sesquiterpenoids by a maize terpene synthase.

The astounding structural and biological diversities of the large class of terpenoid natural products are imparted by both their complex hydrocarbon backbones and further elaboration by the addition of multiple hydroxyl groups, which provide both solubility and specific binding properties. While the role of terpene synthases (TPSs) in generating hydrocarbons with complex backbones is well known, these also are known to generate (singly) hydroxylated products by the addition of water prior to terminating deprotonation. Here a maize sesquiterpene synthase was unexpectedly found to generate dually hydroxylated products directly from (E,E)-farnesyl diphosphate, primarily eudesmane-2,11-diol, along with two closely related structural isomers. The unprecedented formation of these diols was proposed to proceed via initial addition of water to a germacradienyl+ intermediate, followed by protonation of the internal carbon-6,7-double-bond in the resulting hedycarol, with subsequent cyclization and further addition of water to an eudesmolyl+ intermediate. Evidence for the proposed mechanism was provided by labeling studies, as well as site-directed mutagenesis, based on structural modeling, which identified an active site phenylalanine required for the protonation and further elaboration of hedycaryol. This dihydroxylated sesquiterpenoid synthase was specifically expressed in maize roots and induced by pathogen infection, with its major enzymatic product only detected in root exudates or infected roots, suggesting a role in defense. Regardless of the ultimate metabolic fate or physiological role of these diols, this report not only reveals an unanticipated extension of the catalytic prowess of TPSs, but also provides insight into the underlying enzymatic mechanism.

Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii.

Tripterygium wilfordii, which has long been used as a medicinal plant, exhibits impressive and effective anti-inflammatory, immunosuppressive and anti-tumor activities. The main active ingredients are diterpenoids and triterpenoids, such as triptolide and celastrol, respectively. A major challenge to harnessing these natural products is that they are found in very low amounts in planta. Access has been further limited by the lack of knowledge regarding their underlying biosynthetic pathways, particularly for the abeo-abietane tri-epoxide lactone triptolide. Here suspension cell cultures of T. wilfordii were found to produce triptolide in an inducible fashion, with feeding studies indicating that miltiradiene is the relevant abietane olefin precursor. Subsequently, transcriptome data were used to identify eight putative (di)terpene synthases that were then characterized for their potential involvement in triptolide biosynthesis. This included not only biochemical studies which revealed the expected presence of class II diterpene cyclases that produce the intermediate copalyl diphosphate (CPP), along with the more surprising finding of an atypical class I (di)terpene synthase that acts on CPP to produce the abietane olefin miltiradiene, but also their subcellular localization and, critically, genetic analysis. In particular, RNA interference targeting either both of the CPP synthases, TwTPS7v2 and TwTPS9v2, or the subsequently acting miltiradiene synthase, TwTPS27v2, led to decreased production of triptolide. Importantly, these results then both confirm that miltiradiene is the relevant precursor and the relevance of the identified diterpene synthases, enabling future studies of the biosynthesis of this important bioactive natural product.

Isoprenyl diphosphate synthases: the chain length determining step in terpene biosynthesis.

MAIN CONCLUSION: This review summarizes the recent developments in the study of isoprenyl diphosphate synthases with an emphasis on analytical techniques, product length determination, and the physiological consequences of manipulating expression in planta. The highly diverse structures of all terpenes are synthesized from the five carbon precursors dimethylallyl diphosphate and a varying number of isopentenyl diphosphate units through 1'-4 alkylation reactions. These elongation reactions are catalyzed by isoprenyl diphosphate synthases (IDS). IDS are classified depending on the configuration of the ensuing double bond as trans- and cis-IDS. In addition, IDS are further stratified by the length of their prenyl diphosphate product. This review discusses analytical techniques for the determination of product length and the factors that control product length, with an emphasis on alternative mechanisms. With recent advances in analytics, multiple IDS of Arabidopsis thaliana have been recently reinvestigated and demonstrated to yield products of different lengths than originally reported, which is summarized here. As IDS dictate prenyl diphosphate length and thereby which class of terpenes is ultimately produced, another focus of this review is the impact that altering IDS expression has on terpenoid natural product accumulation. Finally, recent findings regarding the ability of a few IDS to not catalyze 1'-4 alkylation reactions, but instead produce irregular products, with unusual connectivity, or act as terpene synthases, are also discussed.

Combinatorial biosynthesis and the basis for substrate promiscuity in class I diterpene synthases.

Terpene synthases are capable of mediating complex reactions, but fundamentally simply catalyze lysis of allylic diphosphate esters with subsequent deprotonation. Even with the initially generated tertiary carbocation this offers a variety of product outcomes, and deprotonation further can be preceded by the addition of water. This is particularly evident with labdane-related diterpenes (LRDs) where such lysis follows bicyclization catalyzed by class II diterpene cyclases (DTCs) that generates preceding structural variation. Previous investigation revealed that two diterpene synthases (DTSs), one bacterial and the other plant-derived, exhibit extreme substrate promiscuity, but yet still typically produce exo-ene or tertiary alcohol LRD derivatives, respectively (i.e., demonstrating high catalytic specificity), enabling rational combinatorial biosynthesis. Here two DTSs that produce either cis or trans endo-ene LRD derivatives, also plant and bacterial (respectively), were examined for their potential analogous utility. Only the bacterial trans-endo-ene forming DTS was found to exhibit significant substrate promiscuity (with moderate catalytic specificity). This further led to investigation of the basis for substrate promiscuity, which was found to be more closely correlated with phylogenetic origin than reaction complexity. Specifically, bacterial DTSs exhibited significantly more substrate promiscuity than those from plants, presumably reflecting their distinct evolutionary context. In particular, plants typically have heavily elaborated LRD metabolism, in contrast to the rarity of such natural products in bacteria, and the lack of potential substrates presumably alleviates selective pressure against such promiscuity. Regardless of such speculation, this work provides novel biosynthetic access to almost 19 LRDs, demonstrating the power of the combinatorial approach taken here.