Isoprenyl Diphosphate Synthases Research Articles

Juvenile hormone III induction reveals key genes in general metabolism, pheromone biosynthesis, and detoxification in Eurasian spruce bark beetle

IntroductionIn recent years, bark beetle Ips typographus, has caused extensive damage to European Norway spruce forests through widespread outbreaks. This pest employs pheromone-assisted aggregation to overcome tree defense, resulting in mass attacks on host spruce. Many morphological and behavioral processes in I. typographus are under the regulation of juvenile hormone III (JH III), including the biosynthesis of aggregation pheromones and associated detoxification monoterpene conjugates.Objectives and MethodsIn this study, we topically applied juvenile hormone III (JH III) and performed metabolomics, transcriptomics, and proteomics in I. typographus both sexes, with focused aims; 1. Highlight the JH III-regulated metabolic processes; 2. Identify pheromone biosynthesis-linked genes; and 3. Investigate JH III’s impact on detoxification conjugates linked to pheromonal components.ResultsNumerous gene families were enriched after JH III treatment, including genes associated with catalytic and oxidoreductase activity, esterases, phosphatases, and membrane transporters. Sex-specific enrichments for reproduction-related and detoxification genes in females and metabolic regulation genes in males were observed. On the protein level were enriched metal ion binding and transferase enzymes in male beetles. After JHIII treatment, mevalonate pathway genes, including terminal isoprenyl diphosphate synthase (IPDS), were exclusively 35- folds upregulated in males, providing evidence of de novo biosynthesis of pheromone components 2-methyl-3-buten-2-ol and ipsdienol. In addition, cytochrome P450 genes likely involved in the biosynthesis of cis/trans-verbenol, detoxification, and formation of ipsdienol, were 3-fold upregulated in the male gut. The increase in gene expression correlated with the heightened production of the respective metabolites. Detoxification conjugates, verbenyl oleate in the beetle fat body and verbenyl diglycosides in the gut, were induced by JHIII application, which confirms the hormone regulation of their formation. The JH III induction also increased the gene contigs esterase and glycosyl hydrolase up to proteins from male gut tissue. The esterase was proposed to release pheromone cis-verbenol in adult males by breaking down verbenyl oleate. The correlating analyses confirmed a reduction in the abundance of verbenyl oleate in the induced male beetle.ConclusionThe data provide evidence of JH III’s regulatory role in the expression of genes and enzymes related to fundamental beetle metabolism, pheromone biosynthesis, and detoxification in Ips typographus.

Metal-dependent enzyme symmetry guides the biosynthetic flux of terpene precursors

Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential.

Terpene synthases and pathways in animals: enzymology and structural evolution in the biosynthesis of volatile infochemicals.

Covering: up to the beginning of 2023Many animals release volatile or semi-volatile terpenes as semiochemicals in intra- and inter-specific interactions. Terpenes are important constituents of pheromones and serve as chemical defenses to ward off predators. Despite the occurrence of terpene specialized metabolites from soft corals to mammals, the biosynthetic origin of these compounds has largely remained obscure. An increasing number of animal genome and transcriptome resources is facilitating the identification of enzymes and pathways that allow animals to produce terpenes independent of their food sources or microbial endosymbionts. Substantial evidence has emerged for the presence of terpene biosynthetic pathways such as in the formation of the iridoid sex pheromone nepetalactone in aphids. In addition, terpene synthase (TPS) enzymes have been discovered that are evolutionary unrelated to canonical plant and microbial TPSs and instead resemble precursor enzymes called isoprenyl diphosphate synthases (IDSs) in central terpene metabolism. Structural modifications of substrate binding motifs in canonical IDS proteins presumably facilitated the transition to TPS function at an early state in insect evolution. Other arthropods such as mites appear to have adopted their TPS genes from microbial sources via horizontal gene transfer. A similar scenario likely occurred in soft corals, where TPS families with closer resemblance to microbial TPSs have been discovered recently. Together, these findings will spur the identification of similar or still unknown enzymes in terpene biosynthesis in other lineages of animals. They will also help develop biotechnological applications for animal derived terpenes of pharmaceutical value or advance sustainable agricultural practices in pest management.

Ancient origin and conserved gene function in terpene pheromone and defense evolution of stink bugs and hemipteran insects

Insects use diverse arrays of small molecules such as metabolites of the large class of terpenes for intra- and inter-specific communication and defense. These molecules are synthesized by specialized metabolic pathways; however, the origin of enzymes involved in terpene biosynthesis and their evolution in insect genomes is still poorly understood. We addressed this question by investigating the evolution of isoprenyl diphosphate synthase (IDS)-like genes with terpene synthase (TPS) function in the family of stink bugs (Pentatomidae) within the large order of piercing–sucking Hemipteran insects. Stink bugs include species of global pest status, many of which emit structurally related 15-carbon sesquiterpenes as sex or aggregation pheromones. We provide evidence for the emergence of IDS-type TPS enzymes at the onset of pentatomid evolution over 100 million years ago, coinciding with the evolution of flowering plants. Stink bugs of different geographical origin maintain small IDS-type families with genes of conserved TPS function, which stands in contrast to the diversification of TPS genes in plants. Expanded gene mining and phylogenetic analysis in other hemipteran insects further provides evidence for an ancient emergence of IDS-like genes under presumed selection for terpene-mediated chemical interactions, and this process occurred independently from a similar evolution of IDS-type TPS genes in beetles. Our findings further suggest differences in TPS diversification in insects and plants in conjunction with different modes of gene functionalization in chemical interactions.

Structural insights to a bi-functional isoprenyl diphosphate synthase that can catalyze head-to-tail and head-to-middle condensation

Isoprenoids represent the largest group of natural products, whose basal skeletons are synthesized by various isoprenyl diphosphate synthases (IDSs). As majority of IDSs catalyze head-to-tail reaction to produce linear form isoprenoids, some catalyze head-to-middle reaction to produce branched form products. In a previous study, an IDS termed MA1831 from Methanosarcina acetivorans was found to be capable of catalyzing both types of reaction. In addition to the canonical linear product of C35 in length, MA1831 also catalyzes head-to-middle condensation of farnesyl diphosphate (FPP) and dimethylallyl diphosphate (DMAPP) to produce geranyllavandulyl diphosphate. In order to investigate the mechanism of action of MA1831, we determined its crystal structures in apo-form and in complex with substrates and analogues. The complex structures that contain isopentenyl S-thiolodiphosphate and DMAPP as homoallylic substrates were also reported, which should represent the reaction modes of MA1831-mediated head-to-tail and head-to-middle reaction, respectively. Based on the structural information, the mechanism of MA1831 catalyze head-to-tail and head-to-middle condensation reaction was proposed.

Bark Beetle Attack History Does Not Influence the Induction of Terpene and Phenolic Defenses in Mature Norway Spruce (Picea abies) Trees by the Bark Beetle-Associated Fungus Endoconidiophora polonica.

Terpenes and phenolics are important constitutive and inducible conifer defenses against bark beetles and their associated fungi. In this study, the inducible defenses of mature Norway spruce (Picea abies) trees with different histories of attack by the spruce bark beetle, Ips typographus were tested by inoculation with the I. typographus-associated fungus Endoconidiophora polonica. We compared trees that had been under previous attack with those under current attack and those that had no record of attack. After fungal inoculation, the concentrations of mono-, sesqui-, and diterpenes in bark increased 3- to 9-fold. For the phenolics, the flavan-3-ols, catechin, and gallocatechin, increased significantly by 2- and 5-fold, respectively, while other flavonoids and stilbenes did not. The magnitudes of these inductions were not influenced by prior bark beetle attack history for all the major compounds and compound classes measured. Before fungal inoculation, the total amounts of monoterpenes, diterpenes, and phenolics (constitutive defenses) were greater in trees that had been previously attacked compared to those under current attack, possibly a result of previous induction. The transcript levels of many genes involved in terpene formation (isoprenyl diphosphate synthases and terpene synthases) and phenolic formation (chalcone synthases) were significantly enhanced by fungal inoculation suggesting de novo biosynthesis. Similar inductions were found for the enzymatic activity of isoprenyl diphosphate synthases and the concentration of their prenyl diphosphate products after fungal inoculation. Quantification of defense hormones revealed a significant induction of the jasmonate pathway, but not the salicylic acid pathway after fungal inoculation. Our data highlight the coordinated induction of terpenes and phenolics in spruce upon infection by E. polonica, a fungal associate of the bark beetle I. typographus, but provide no evidence for the priming of these defense responses by prior beetle attack.

Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity.

We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non‐head‐to‐tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild‐type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis‐orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans‐isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non‐head‐to‐tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.

Diversity and Biosynthesis of Volatile Terpenoid Secondary Metabolites in the Chrysanthemum Genus

Chrysanthemum is a significant genus belonging to one of the largest plant families, the Asteraceae. Among the over 40 recognized species, C. morifolium is best-known for its long history of cultivation and countless varieties. Like some other genera of this family, many Chrysanthemum species are producers of high levels of secondary metabolites. This review focuses on the largest class of plant secondary metabolites, namely terpenoids produced by Chrysanthemum species. An extensive literature search revealed a total of 390 terpenoids from these species, which include 183 monoterpenoids and 207 sesquiterpenoids. While some terpenes are produced by almost all of the investigated varieties/species of Chrysanthemum, many terpenoids show specificity to certain varieties or species, indicating species-specific functions. The enzymes and genes participating in the pathways for terpene biosynthesis are also described. In addition to a general overview, in this review we focus on isoprenyl diphosphate synthases and terpene synthases, two enzyme classes pivotal for generating the chemical diversity of terpenes, as established by our analysis of two sequenced genomes of Chrysanthemum. While our knowledge about the biological functions of terpenoids in Chrysanthemum is limited, we discuss their functions in chemical defenses against insects and microbial pathogens. Finally, we present the prospects of understanding the molecular basis of terpene biosynthesis in Chrysanthemum and elucidating its biological significance, as well as the prospects of translational research on terpene-based applications.

Non-canonical substrates for terpene synthases in bacteria are synthesized by a new family of methyltransferases.

The 'biogenetic isoprene rule', formulated in the mid 20th century, predicted that terpenoids are biosynthesized via polymerization of C5 isoprene units. The polymerizing enzymes have been identified to be isoprenyl diphosphate synthases, products of which are catalyzed by terpene synthases (TPSs) to achieve vast structural diversity of terpene skeletons. Irregular terpenes (e.g, C11, C12, C16 and C17) are also frequently observed, and they have presumed to be synthesized by the modification of terpene skeletons. This review highlights the exciting discovery of an additional route to the biosynthesis of irregular terpenes which involves the action of a newly discovered enzyme family of isoprenyl diphosphate methyltransferases (IDMTs). These enzymes methylate, and sometimes cyclize, the classical isoprenyl diphosphate substrates to produce modified, non-canonical substrates for specifically evolved TPSs. So far, this new pathway has been found only in bacteria. Structure and sequence comparisons of the IDMTs strongly indicate a conservation of their active pockets and overall topologies. Some bacterial IDMTs and TPSs appear in small gene clusters, which may facilitate future mining of bacterial genomes for identification of irregular terpene-producing enzymes. The IDMT-TPS route for terpenoid biosynthesis presents another example of nature's ingenuity in creating chemical diversity, particularly terpenoids, for organismal fitness.

Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases

Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20), and geranylfarnesyl diphosphate (GFPP; C25) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C5), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed using radiolabelled substrates, and the products formed are identified by employing expensive instruments such as phosphor imager, radio-GC, or radio-HPLC. Though a non-radioactive assay for measuring IDS activity in crude plant extract has been reported, it requires a complex methodology utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS). Here, we describe a non-radioactive and simple inexpensive assay for determining the IDS assay products using non-radiolabeled IPP and its co-allylic substrates DMAPP, GPP, and FPP. The detection of prenyl diphosphate products generated in the assay was highly efficient and spots corresponding to prenyl alcohols were visible at >40 µM concentrations of IPP and DMAPP/GPP/FPP substrates. The protocol described here is sensitive, reliable, and technically simple, which could be used for functional characterization of IDS candidates.

Versatile cis-isoprenyl Diphosphate Synthase Superfamily Members in Catalyzing Carbon–Carbon Bond Formation

Isoprenyl diphosphate synthases (IDSs) catalyze condensation reactions of isoprene units to produce isoprenoids or terpenoids, the largest class of natural products on earth. IDSs are divided into ...

Short-chain isoprenyl diphosphate synthases of lavender (Lavandula).

We reported the functional characterization of cDNAs encoding short-chain isoprenyl diphosphate synthases that control the partitioning of precursors for lavender terpenoids. Lavender essential oil is composed of regular and irregular monoterpenes, which are derived from linear precursors geranyl diphosphate (GPP) and lavandulyl diphosphate (LPP), respectively. Although this plant strongly expresses genes responsible for the biosynthesis of both monoterpene classes, it is unclear why regular monoterpenes dominate the oil. Here, we cloned and characterized Lavandula x intermedia cDNAs encoding geranyl diphosphate synthase (LiGPPS), geranylgeranyl diphosphate synthase (LiGGPPS) and farnesyl diphosphate synthase (LiFPPS). LiGPPS was heteromeric protein, consisting of a large subunit (LiGPPS.LSU) and a small subunit for which two different cDNAs (LiGPPS.SSU1 and LiGPPS.SSU2) were detected. Neither recombinant LiGPPS subunits was active by itself. However, when co-expressed in E. coli LiGPPS.LSU and LiGPPS.SSU1 formed an active heteromeric GPPS, while LiGPPS.LSU and LiGPPS.SSU2 did not form an active protein. Recombinant LiGGPPS, LiFPPS and LPP synthase (LPPS) proteins were active individually. Further, LiGPPS.SSU1 modified the activity of LiGGPPS (to produce GPP) in bacterial cells co-expressing both proteins. Given this, and previous evidence indicating that GPPS.SSU can modify the activity of GGPPS to GPPS in vitro and in plants, we hypothesized that LiGPPS.SSU1 modifies the activity of L. x intermedia LPP synthase (LiLPPS), thus accounting for the relatively low abundance of LPP-derived irregular monoterpenes in this plant. However, LiGPPS.SSU1 did not affect the activity of LiLPPS. These results, coupled to the observation that LiLPPS transcripts are more abundant than those of GPPS subunits in L. x intermedia flowers, suggest that regulatory mechanisms other than transcriptional control of LPPS regulate precursor partitioning in lavender flowers.

A Comprehensive Survey on the Terpene Synthase Gene Family Provides New Insight into Its Evolutionary Patterns.

Terpenes are organic compounds and play important roles in plant growth and development as well as in mediating interactions of plants with the environment. Terpene synthases (TPSs) are the key enzymes responsible for the biosynthesis of terpenes. Although some species were employed for the genome-wide identification and characterization of the TPS family, limited information is available regarding the evolution, expansion, and retention mechanisms occurring in this gene family. We performed a genome-wide identification of the TPS family members in 50 sequenced genomes. Additionally, we also characterized the TPS family from aromatic spearmint and basil plants using RNA-Seq data. No TPSs were identified in algae genomes but the remaining plant species encoded various numbers of the family members ranging from 2 to 79 full-length TPSs. Some species showed lineage-specific expansion of certain subfamilies, which might have contributed toward species or ecotype divergence or environmental adaptation. A large-scale family expansion was observed mainly in dicot and monocot plants, which was accompanied by frequent domain loss. Both tandem and segmental duplication significantly contributed toward family expansion and expression divergence and played important roles in the survival of these expanded genes. Our data provide new insight into the TPS family expansion and evolution and suggest that TPSs might have originated from isoprenyl diphosphate synthase genes.

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

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.

Arginine in the FARM and SARM: A Role in Chain-Length Determination for Arginine in the Aspartate-Rich Motifs of Isoprenyl Diphosphate Synthases from Mycobacterium tuberculosis.

Isoprenyl chains are found in many important metabolites. These are derived from precursors of the appropriate length produced by isoprenyl diphosphate synthases (IDSs). The human pathogen Mycobacterium tuberculosis makes various isoprenoids/terpenoids, with important roles in their biosynthesis played by two closely related IDSs, encoded by grcC1 (Rv0562) and grcC2 (Rv0989c), with Rv0989c generating the 10-carbon precursor (E)-geranyl diphosphate (GPP), and Rv0562 the 20-carbon precursor (E,E,E)-geranylgeranyl diphosphate (GGPP). Intriguingly, while Rv0562 contains the prototypical trans-IDS first and second aspartate-rich (DDxxD) motifs (FARM and SARM, respectively), Rv0989c uniquely contains arginine in place of the second Asp in the FARM and first Asp in the SARM. Here site-directed mutagenesis of the corresponding residues in both Rv0562 and Rv0989c reveals that these play a role in determination of product chain length. Specifically, substitution of Asp for the Arg in the FARM and SARM of Rv0989c leads to increased production of the longer 15-carbon farnesyl diphosphate (FPP), while substitution of Arg for the corresponding Asp in Rv0562 leads to increased release of shorter products, both FPP and GPP. Accordingly, while the primary role of the FARM and SARM is known to be chelation of the divalent magnesium ion co-factors that assist substrate binding and catalysis, the Arg substitutions found in Rv0989c seem to provide a novel means by which product chain length is moderated, at least in these M. tuberculosis IDSs.

An IDS-Type Sesquiterpene Synthase Produces the Pheromone Precursor (Z)-α-Bisabolene in Nezara viridula.

Insects use a wide range of structurally diverse pheromones for intra-specific communication. Compounds in the class of terpenes are emitted as sex, aggregation, alarm, or trail pheromones. Despite the common occurrence of terpene pheromones in different insect lineages, their origin from dietary host plant precursors or de novo biosynthetic pathways often remains unknown. Several stink bugs (Hemiptera: Pentatomidae) release bisabolene-type sesquiterpenes for aggregation and mating. Here we provide evidence for de novo biosynthesis of the sex pheromone trans-/cis-(Z)-α-bisabolene epoxide of the Southern green stink bug, Nezara viridula. We show that an enzyme (NvTPS) related to isoprenyl diphosphate synthases (IDSs) of the core terpene metabolic pathway functions as a terpene synthase (TPS), which converts the general intermediate (E,E)-farnesyl diphosphate (FPP) to the putative pheromone precursor (+)-(S,Z)-α-bisabolene in vitro and in protein lysates. A second identified IDS-type protein (NvFPPS) makes the TPS substrate (E,E)-FPP and functions as a bona fide FPP synthase. NvTPS is highly expressed in male epidermal tissue associated with the cuticle of ventral sternites, which is in agreement with the male specific release of the pheromone from glandular cells in this tissue. Our study supports findings of the function of similar TPS enzymes in the biosynthesis of aggregation pheromones from the pine engraver beetle Ips pini, the striped flea beetle Phyllotreta striolata, and the harlequin bug Murgantia histrionica, and hence provides growing evidence for the evolution of terpene de novo biosynthesis by IDS-type TPS families in insects.

Structural characterization of geranylgeranyl pyrophosphate synthase GACE1337 from the hyperthermophilic archaeon Geoglobus acetivorans.

A novel type 1 geranylgeranyl pyrophosphate synthase GACE1337 has been identified within the genome of a newly identified hyperthermophilic archaeon Geoglobus acetivorans. The enzyme has been cloned and over-expressed in Escherichia coli. The recombinant enzyme has been biochemically and structurally characterized. It is able to catalyze the synthesis of geranylgeranyl pyrophosphate as a major product and of farnesyl pyrophosphate in smaller amounts, as measured by gas chromatography-mass spectrometry at an elevated temperature of 60°C. Its ability to produce two products is consistent with the fact that GACE1337 is the only short-chain isoprenyl diphosphate synthase in G.acetivorans. Attempts to crystallize the enzyme were successful only at 37°C. The three-dimensional structure of GACE1337 was determined by X-ray diffraction to 2.5Å resolution. A comparison of its structure with those of related enzymes revealed that the Geoglobus enzyme has the features of both type I and type III geranylgeranyl pyrophosphate synthases, which allow it to regulate the product length. The active enzyme is a dimer and has three aromatic amino acids, two Phe, and a Tyr, located in the hydrophobic cleft between the two subunits. It is proposed that these bulky residues play a major role in the synthetic reaction by controlling the product elongation.

Cloning and functional identification of farnesyl diphosphate synthase from Pinus massoniana Lamb

Farnesyl diphosphate synthase (FPPS) is a key isoprenyl diphosphate synthase (IDS), which provides synthetic precursors to the terpenoid metabolic pathway. We isolated and characterized a Pinus massoniana FPPS (PmFPPS) gene which encodes a putative farnesyl diphosphate synthase from P. massoniana Lamb. In silico domain analysis revealed that PmFPPS contained all five conserved IDS domains and was homologous to FPPSs from other plant species. An in vitro enzymatic activity assay resulted in an optimum pH, temperature, and Mg2+ concentration of 7.0–7.5, 25 °C, and 1.2 mM, respectively. To identify the function of PmFPPS in vivo, sense and antisense expression vectors were constructed and transformed into tobacco using a constitutive cauliflower mosaic virus-35S promoter. The overexpression of PmFPPS in transgenic plants had higher squalene contents than the control, and the downregulated transgenic plants had lower squalene contents than the control. These results indicate that PmFPPS performs a regulatory role in triterpene biosynthesis.

Optimization of thermophilic trans‐isoprenyl diphosphate synthase expression in Escherichia coli by response surface methodology

We optimized the heterologous expression of trans-isoprenyl diphosphate synthase (IDS), the key enzyme involved in the biosynthesis of trans-polyisoprene. trans-Polyisoprene is a particularly valuable compound due to its superior stiffness, excellent insulation, and low thermal expansion coefficient. Currently, trans-polyisoprene is mainly produced through chemical synthesis and no biotechnological processes have been established so far for its large-scale production. In this work, we employed D-optimal design and response surface methodology to optimize the expression of thermophilic enzymes IDS from Thermococcus kodakaraensis. The design of experiment took into account of six factors (preinduction cell density, inducer concentration, postinduction temperature, salt concentration, alternative carbon source, and protein inhibitor) and seven culture media (LB, NZCYM, TB, M9, Ec, Ac, and EDAVIS) at five different pH points. By screening only 109 experimental points, we were able to improve IDS production by 48% in close-batch fermentation.

Catalytic Functions of the Isoprenyl Diphosphate Synthase Superfamily in Plants: A Growing Repertoire

The discovery of artemisinin as an antimalarial agent by Professor Youyou Tu of China was awarded the Noble prize in Physiology and Medicine in 2015. Notably, artemisinin belongs to the terpenoids; more than 55 000 of them have been isolated from nature. Important for both cellular and ecological functions (Gershenzon and Dudareva, 2007), terpenoids are particularly rich in plants. It has been a long-standing question to chemists and biologists alike: how are enormously diverse terpenoids made by plants? One current understanding is that terpene synthases, which convert isoprenyl diphosphates to terpene skeletons, play a central role in generating structural diversity of terpenoids (Chen et al., 2011).