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Metabolic Perturbation and Synthetic Biology Strategies for Plant Terpenoid Production—An Updated Overview

Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54874, Korea
Author to whom correspondence should be addressed.
Plants 2021, 10(10), 2179;
Submission received: 8 September 2021 / Revised: 11 October 2021 / Accepted: 12 October 2021 / Published: 14 October 2021
(This article belongs to the Special Issue Biosynthesis, Function, and Application of Plant Volatiles)


Terpenoids represent one of the high-value groups of specialized metabolites with vast structural diversity. They exhibit versatile human benefits and have been successfully exploited in several sectors of day-to-day life applications, including cosmetics, foods, and pharmaceuticals. Historically, the potential use of terpenoids is challenging, and highly hampered by their bioavailability in their natural sources. Significant progress has been made in recent years to overcome such challenges by advancing the heterologous production platforms of hosts and metabolic engineering technologies. Herein, we summarize the latest developments associated with analytical platforms, metabolic engineering, and synthetic biology, with a focus on two terpenoid classes: monoterpenoids and sesquiterpenoids. Accumulated data showed that subcellular localization of both the precursor pool and the introduced enzymes were the crucial factors for increasing the production of targeted terpenoids in plants. We believe this timely review provides a glimpse of current state-of-the-art techniques/methodologies related to terpenoid engineering that would facilitate further improvements in terpenoids research.

1. Introduction

Terpenoids are the most chemically, physically, and functionally complex family of natural chemicals found in living creatures, with over 80,000 compounds identified to date, and many more expected to exist [1]. They are the most structurally varied group of plant-derived natural compounds, and are economically significant due to their use in a variety of industrial products, including pharmaceuticals, flavoring agents, insecticides, antimicrobial agents, and perfumes [2]. They play an important role in plant–environment, plant–plant, plant–insect, and plant–animal interactions in nature [3]. Many terpenoids have a strong link to primary metabolism (e.g., phytol, the plant hormone gibberellin, and carotenoid pigments), while others are common secondary metabolites in plants [4].
Terpene biochemistry and chemistry have been studied for over a century, mainly in plants [5]. Recently, genes that encode enzymes and regulators engaged in terpene biosynthesis have been discovered, and their genomic location, mode of expression, and long-term evolution have been investigated [6]. Despite their considerable structural variations, all terpenoids are derived from the universal isoprene C5 building blocks. The terpenoid backbone is synthesized from two precursors: IPP (isopentenyl pyrophosphate), and its isomer dimethylallyl pyrophosphate (DMAPP). Terpene biosynthesis is a complex mechanism involving two independent biosynthetic pathways. The cytosolic mevalonate (MVA) pathway is found in most eukaryotes (all mammals, the cytosol and mitochondria of plants, fungi), archea, and some bacteria [7,8]. They are utilized in the production of bigger compounds, such as sesquiterpenes (C15), triterpenes (C30), sterols (C27–29), and dolichols (C40–50). The methylerythritol phosphate (MEP) pathway is found in plant chloroplasts, bacteria, algae, and cyanobacteria. This pathway predominantly produces mono-terpenes (C10), diterpenes (C20), and tetraterpenes (C40). Unlike most microbial organisms, both pathways operate in plants—the MEP pathway in the chloroplast and the MVA pathway in the cytoplasm—and the labor division between them represents a complex array of chemicals that control the development and growth of plants, and interact with plants and their surroundings to control these interactions [9].
Over recent years, efforts to generate large amounts of monoterpenes and sesquiterpenes in transgenic plants proved effective. Many plant species have been genetically modified, with the overexpression of terpene synthase under constitutive promoters being the most common. Plants with overexpressed linalool synthase genes that were produced include tomato, petunia, Arabidopsis, potato, and carnation [10,11,12,13,14]. Such plants generated and released linalool and its glycosylated or hydroxylated derivatives. Similarly, α-pinene, γ-terpinene, and limonene synthases were shown to alter the terpenoid profile of tobacco and mint plants [15]. The overexpression of gene encoding enzymes from various stages of the MEP pathway may result in even higher levels of terpenoid precursors (DXR and HDR) [16]. The genes encoding enzymes that modify the monoterpene composition have also been effectively overexpressed or knocked down in mint and tobacco. Sesquiterpene production in transgenic plants is more difficult than monoterpene production. In an effort to engineer sesquiterpenes in plants using terpene synthases, tobacco plants were transformed with Artemisia annua amorpha-4,11-diene synthase and fungal trichodiene synthase on either side, but this resulted in only a low-level yield [17,18].
Owing to their traditionally known pharmacological importance, terpenoids have at-attracted attention from plant breeders, biochemists, and pharmacologists, who have exploited them for their diverse metabolite/chemical profile. Several attempts have been made to decode their distinct metabolic profiles through high throughput metabolic fingerprinting methods including nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and capillary electrophoresis-mass spectrometry (CE-MS), and so-called metabolomics [19]. Metabolic engineering is an appropriate system to either enhance or manipulate the synthesis of terpenes in plant species that naturally produce them, or to integrate terpene biosynthetic pathways into certain plants [20]. A number of attempts have been made over the last decade to engineer the production of monoterpenoid and sesquiterpenoid compounds in various plant species and tissues. Implementing modern analytical techniques is the best way to improve the qualitative and quantitative aspects of terpenes and terpenoids in plants and other products [21].
All plants produce a variety of terpenoid compounds that serve as phytohormones and anti-oxidants, or have other functions. Hundreds of distinct terpenoids are synthesized by different plant lineages, with the overall number of such advanced plant terpenoids estimated to be in the thousands [22]. A clear understanding of a plant’s chemical composition enables a more accurate assessment of its medicinal potential. Modern chemistry can unravel the primary metabolic functions of plants, including cell division, development, respiration, storage, and reproduction. It helps improve our understanding of process components, such as glycolysis, the cancer or cycles of citric acid, and photosynthesis [23]. Small molecules, such as sugars, amino acids, proteins, nucleic acids, and polysaccharides, are examples of primary metabolites [24,25]. Secondary plant metabolites are a diverse group of chemical compounds synthesized by the plant cell through metabolic pathways derived from primary metabolic pathways [26]. By the mid-twentieth century, advancements in analytical techniques such as GC-MS, LC-MS, CE-MS, and NMR enabled the recovery of an increasing number of these molecules, providing a basis for the development of phytochemistry. Secondary plant metabolites are classified into specific categories: phenolics, alkaloids, saponins, terpenes, lipids, and carbohydrates [27]. Terpenes are the major and most varied category of secondary metabolites found in plants. Green plants, especially flowering plants, have an extraordinarily high number of terpenoids compared to other living organisms [22]. Isoprene units in the molecules, mono-terpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), and triterpenoids (C30) are considered secondary metabolites. Many terpenoids are commercially interesting because of their use as flavors and fragrances in foods and cosmetics (e.g., menthol, nootkatone, and sclareol) or because they are important for the quality of agricultural products, for example, with the flavor of fruits and the fragrance of flowers (e.g., linalool) [28]. In addition, terpenoids have medicinal properties including anti-carcinogenic (e.g., taxol and perilla alcohol), antimalarial (e.g., artemisinin), anti-ulcer, hepaticidal, antimicrobial, and diuretic (e.g., glycyrrhizin) activities [29]. Terpenoids have also been shown to be of ecological significance. Compounds, such as the bitter triterpenoid cucurbitacins and the pungent diterpenoid polygodial, have been shown to be involved in insect resistance [30]. Other terpenoid substances are active in plant interactions, plant–microorganism interactions, and plant–arthropod herbivore interactions (for example, spider mite feeding induces (E, E)-a-farnesene in cucumber) [31]. In this review, we provide an overview of the recent developments in relation to different biosynthetic and regulatory aspects of monoterpenoid and sesquiterpenoid metabolism in plants. Moreover, we report on the remarkable improvement in metabolic engineering for advanced terpenoid production in various platforms.

2. Biosynthesis and Precursors of Terpenoids

Plants possess two distinct pathways to produce terpenoids: the plastidial 2-c-methyl-d-erythritol-4-phosphate (MEP) pathway and the acetyl-CoA dependent cytosolic mevalonate (MVA) pathway (Figure 1). The C5 unit’s IPP (isopentenyl pyrophosphate) and its allylic isomer dimethylallyl pyrophosphate (DMAPP), the fundamental terpenoid biosynthesis building blocks, are produced through a metabolic assembly of multiples in both terpenoids pathways. Prenyl transferases use DMAPP and IPP in condensation reactions to generate bigger prenyl diphosphates, including the sesquiterpene precursor FPP (farnesyl pyrophosphate), monoterpene precursor GPP (geranyl pyrophosphate), and C40 carotenoid and diterpene precursor GGPP (geranylgeranyl pyrophosphate) in both compartments (Figure 1). Although there is increasing evidence that there is an exchange of intermediates between these compartments [32,33], the cytoplasmic MVA pathway is generally considered to supply the precursors for the production of sesquiterpenes. In the plastids, the MEP pathway supplies the precursors for the production of monoterpenes (Figure 1). The MVA pathway consists of seven enzymatic processes that convert the precursor acetyl-CoA to IPP and DMAPP (Figure 1). The MEP pathway requires eight enzymatic steps to convert the initial materials pyruvate and glyceraldehyde-3-phosphate to IPP and DMAPP (Figure 1). Even though IPP and DMAPP are separated spatially, they are exchanged between cytosol and plastids during the production of various terpenoids [34]. Using precursors such as GPP, FPP and GGPP are cyclized or rearranged by different terpene synthase enzymes, and are responsible for the synthesis of different classes of terpenoids; they can easily acquire new catalytic properties by minor changes in the structure [35].
Monoterpenoids are C10 compounds derived from GPP through the enzymatic activity of geranyl pyrophosphate synthase (GPS). Through the cyclization process, the range of monoterpenes is rapidly increased, and monocyclic or bicyclic compounds can be synthesized. The dephosphorylation and ionization of geranyl diphosphate to geranyl carbo-cation initiates the production of monoterpenes [36]. Linalyl pyrophosphate and neryl pyrophosphate are isomers of GPP by ionization to the allylic cation. This allows for changes in the attachment of the diphosphate group or changes in stereochemistry at the double bond. Monoterpene synthase (or monoterpene cyclase) is an enzyme that catalyzes the formation of cyclic monoterpenoids. There is an essential enzyme involved in the synthesis of each monoterpenoids, such as linalool synthase for linalool, limonene synthase for limonene, pinene synthase for pinene, and myrcene synthase for myrcene [37,38] (Figure 1).
C15 sesquiterpenoids are derived from FPP by the action of farnesyl pyrophosphate synthase (FPS), and is the common precursor of all sesquiterpenoid lactones (STLs). Sesquiterpene synthase (STPS) catalyzes the cyclisation of FPP in the first step of STL biosynthesis [39]. They are mainly located in the cytosol and are characterized by their plasticity, showing the capacity of multiple substrate utilization. Germacrene A synthase (GAS) is one of the best-characterized STPS, converting FPP into germacrene A (GA). Germacrene A oxidase (GAO), a cytochrome P450-like enzyme, converts GA into germacrene A acid (GAA). To produce costunolide, GAA is further oxidized by the costunolide synthase. Furthermore, to produce the end product of parthenolide, this costunolide catalyzes the epoxidation of the C4-C5 double bond. Figure 1 depicts the overall biosynthetic pathway and intermediate chemical structure of monoterpenoids and sesquiterpenoids [40].

2.1. Monoterpenoid Chemical Compounds

Monoterpenoids are substances that can be found in essential oils derived from different plants, including vegetables, fruits, herbs, and spices [41,42,43]. Monoterpenes are C10 molecules that can be acyclic, monocyclic, or bicyclic. Monoterpene synthase uses GPP (geranyl pyrophosphate) as a substrate to produce them. Additionally, GPP is also a substrate for the production of GGPP (geranyl-geranyl pyrophosphate) and FPP (farnesyl pyrophosphate), two important substances in animal, plant, and yeast cell metabolism [22]. Monoterpenoid compounds are classified as acyclic (e.g., linalool, geraniol, β-myrcene, (+)-citronellol, nerol), monocyclic (thymol, (−)-menthol, limonene, eugenol, γ-terpinene, terpinolene, and piperitone), bicyclic (α-pinene, (−)-β-pinene, camphene, sabinene, myrtenol, (+)-camphor, (−)-borneol, (+)-cis-verbenol, ∆3-terpinene, eucalyptol, sabinene hydrate, and fenchone), and others (α-phellandrene, ρ-cymene, ocimene, fenchol, (−)-isopulegol terpinen-4-ol, α-terpineol, (+)-dihydrocarvone, pulegone, carvone, geranyl acetate, methol-isomer, and safranal) [44]. Monoterpenoids are found mostly in the taxonomic groups Asteraceae, Apiaceae, Verbenaceae, Poaceae, Myrtaceae, Lamiaceae, Pinaceae, Rutaceae, Lauraceae, and Cannabaceae. The following monoterpenoid compounds are significant in plant species: α- and β- pinene (Pinus palustris), δ-3-carene, α-phellandrene, and myrcene (Lippia citriodora) are found as complex mixtures in most essential oils, particularly in those extracted from plant leaves, while seed and flower oils contain more specialized monoterpenes and present fruity or flowery odors. Linalool has two stereoisomers are present: (R)-(−)-linalool and (S)-(+)-linalool. S-linalool is found in major constituents of the essential oils of coriander (Coriandrum sativum L.) seed, palmarosa (Cymbopogon martinii var martinii (Roxb.) Wats), and sweet orange (Citrus sinensis Osbeck) flowers. Meanwhile, (R)-Linalool is present in Ho oils from Cinnamomun camphora, rosewood oil, lavender (Lavandula officinalis Chaix), laurel (Laurus nobilis), and sweet basil (Ocimum basilicum) [2]. D-carvone from caraway (Carum carvi), with its spicy and bread-like fragrance; menthol is derived from wild mint (Mentha arvensis) and has a strong minty aroma; D-limonene from citrus species with a fresh orange peel odor; citral from lemongrass (Cymbopogon citratus) having a fresh lemon peel odor; Eucalyptol, also known as 1,8-cineole from eucalyptus (Eucalyptus globulus) having a camphoraceous cool odor. Menthone and isomenthone are components of essential oils such as pennyroyal, peppermint, Pelargonium geraniums, and others. Thymol (Thymus vulgaris); camphor derived from camphor tee (Cinnamomun camphora) [45].

2.2. Sesquiterpenoid Chemical Compounds

Sesquiterpenoids are C15 compounds with three isoprene (C5H8) units that are primarily present in fresh raw plant materials. They form the most diverse terpenoids group, and the biosynthesis of sesquiterpenoids is synthesized using mevalonic acid [46,47,48]. There are about 150 known sesquiterpenes compounds including artemisinin, (−)-β-elemene, β-caryophyllene, aromaadendrene, trans-β-farnesene, α-humulene, valencene, ledene, trans-nerollidol, caryophyllene oxide, globulol, viridiflorol, (−)-guaiol, (+)-cedrol, β-eudesmol, α-bisabolol, cis-muurola-4(15), 5-diene, germacrene D, costunolide, parthenolide, guatterin A, dihydromadolin, madolin-K, madolin-W, malayscaphiol, sarcanolides A and B, perovskanol, eudesmane-type I and II, chrysanolide A, isocyperotun-done, and 1,4-epoxy-4-hydroxy-4-5-seco-guain-11-en-5-1 [49]. Sesquiterpenoids are predominantly found in the taxonomic groups Poaceae, Solanaceae, Araceae, Rutaceae, Zingiberaceae, Cannabaceae, Myrtaceae, and Cupressaceae. However, they are most common in the Asteraceae family, where they are almost ubiquitous. Sesquiterpenoid compounds are associated with the following plant species: Farnesol (Cymbopogon species), β-nerolidol (Citrus aurantium), α-humlene (Humulus lupulus), Zingiberene (Zingiber officinale), β-Santol (Santalum album), artemisinin (Artemisia annua), nootkaton (Citrus paradisi), costunolide (Saussurea costus), and parthenolide (Rosmarinus officinalis) [50].

3. Analytical Platforms for Terpenoids

3.1. Chromatographic Techniques

A number of methods used for analyzing terpenoids have already been developed; the most common approach is chromatographic analysis, specifically GC-MS or LC-MS. Compounds of monoterpenoids and sesquiterpenoids were determined by various methods such as GC-MS, GC-MS/MS, GC-FID (GC-Flame Ionization Detection), and GC-GC [51]. Each method has its own advantages and disadvantages. The main advantage of using GC-MS is that it is sensitive and robust, and also capable of routinely and reproducibly measuring hundreds of analytes across thousands of samples. Among the different methods, the best one for analyzing terpenes is solvent extraction followed by GC-FID analysis. The FID is a powerful instrument for quality control because of its low cost, accuracy, and simple interface. But the main disadvantage is that it provides little information other than the retention time. For better identification and characterization, various types of columns, dimensions, and oven programs were used (Table 1) [52,53,54,55,56,57,58,59]. Despite this, the optimum GC detector for terpene analysis is still unknown. The column ZB-5 PLUSTM is used selectivity for high temperature limitations, allowing for a high resolution of essential terpenoids. Phenomenex TM groups that used this column in GC-FID identified 33 cannabis-derived primary and secondary terpenes [56]. Another reliable method for determining terpenoids is HS-GC-FID. Headspace sampling is a technique that involves heating a solid or liquid sample inside a sealed vial (which converts the volatile substance to the gas phase). This approach increases column lifetime and minimizes inlet maintenance by preventing nonvolatile material from entering the GC system. Apart from the aforementioned approaches, the use of GC-MS, another frequently used technique, has the additional advantage of spectral peak identification to ensure that selection is accurate. However, it may not be the optimal detector for terpene analysis due to the structural and functional similarity of terpene class molecules. Moreover, GC-MS provides a different level of sensitivity. GC-MS with high-temperature headspace sampling was used to quantify selected terpenoids using a TG-624 SilMS column [53]. The separated constituents were tentatively identified by comparing their mass spectra with those in the available MS library such as: Wiley, Flavors and Fragrances of Natural and Synthetic Compounds (FFNSC) and NIST08 (National Institute of Standards and Technology, Gaithersburg, MD, USA) and by comparing their retention indices (RIs) with literature values. Each constituent was quantified based on the comparison of its peak area with that of the internal standard, and the contents are expressed as ng/g FW. Despite the advantages of various GC methods, the main disadvantage is that degraded chemicals cannot be quantified effectively. In this case, researchers prefer LC-MS/MS for the characterization of degraded chemicals such as GPP and FPP. It is challenging to isolate these compounds using HPLC due to the ionic nature of the phosphate groups. MS, on the other hand, has an adequate sensitivity and specificity. These metabolites have been determined using HPLC-tandem MS (MS/MS) and ultra HPLC-MS/MS [60,61]. Furthermore, obtaining the proper analytical result of terpene analysis is a difficult task that necessitates the evaluation of a number of critical factors, such as equipment selection, instrument parameters, and the optimization of extraction methods.
The general process of GC-MS analysis includes the injections of extracted compounds directly into the GC, and the separation of different components using capillary columns or similar columns of 30m in length or greater. Helium (99.99%), the carrier gas, will continuously pass through at a flow rate of 1 mL/min. Throughout all GC strokes, the oven temperature is typically set to begin at 60 °C and then ramped up to 160 °C (at an increasing rate of 5−10 °C/min). Following this initial stage, the temperature is often raised to 360 °C in order to elute the chemicals. Electron ionization (EI) sources were used in the GC-MS setup, with collision energies ranging from 15 to 70 eV depending on the target molecules. Total GC analysis time ranged from 2 to 50 min. Despite the similarity of several terpenes MS spectra, further identification was performed using the retention index. The retention indices for the chemicals detected in each sample were determined using an n-alkane standard analyzed on the same GC–MS instrument under identical GC conditions [52,53,54,55,56,57,58,59]. Here, metabolic profiles of monoterpenoid (linalool, limonene, and alpha-pinene) and sesquiterpenoid (costunolide, parthenolide, and trans-caryophyllene) groups, injected individually at a concentration of 100 µg/mL in GC-Q-Orbitrap-MS, are shown in Figure 2.

3.2. Metabolic Profiling of Plant Terpenoids

In classic plant biotechnology, the metabolomic approach is generally used for metabolic profiling of a target set of metabolites, such as fatty acids, alkaloids, terpenes, phenolics, etc., in plant physiology and genetic research, along with other tools including genomics, transcriptomics, and proteomics [62]. Different analytical approaches were employed to determine the metabolomic compounds, and were utilized to estimate targeted metabolites such as NMR, GC-MS, LC-MS, and CE-MS. In plant extracts, there are no specific chemical technologies for the identification of monoterpenoids and sesquiterpenoids, unlike for other secondary metabolites such as triterpenes, carotenoids, phytosterols, flavonoids, and other primary metabolites [63]. Volatile compounds can be classified as endogenous or emitted. Solvents such as hexane, pentane, diethyl ether, dichloromethane, chloroform, ethyl acetate, and solvent mixtures can be used to extract endogenous volatiles. The use of an organic solvent has the advantage of reducing the activation of enzymes, which may alter the original composition of volatile compounds. A solid-phase extraction (SPE) column has been used in some cases to remove nonvolatile compounds from the organic solvent extracts. On the other hand, static headspace sampling techniques such as solid phase microextraction, monotrap, and a dynamic headspace sampling system have been used for emitted volatiles [64]. Earlier studies of terpenoid metabolite profiling from different plant species are as follows: twenty-three terpenes were recently analyzed using different accelerated solvent extraction (ASE) methods [65]. In a comprehensive analysis of terpenes and terpenoids in medicinal cannabis biomass, a total of 49 distinct individual compounds were detected [52]. Using 12 cannabis samples, 30 terpene compounds were detected in another study [66]. Nguyen et al. used a robust testing method to quantify 30 selected terpenoids in dry plant materials, with 30 monoterpenoids and sesquiterpenoids quantified [53]. Using a solid-phase microextraction method, 28 terpenes were identified and quantified in Muscat grape cultivars [67]. In another study of Citrus medica (finger citron), a total of 62 volatiles were detected, among which monoterpenoid limonene and γ-terpinene were most abundant [68]. The different developmental stages of eight Artemisia plants were analyzed by GC-MS profiles, which consisted of 40–90% of monoterpene and sesquiterpene derivatives [54]. Another study comparing the terpene profiles of four important fruits (gooseberry, crab apple, cherry silver berry, and scarlet haw-thorn) identified 79 terpenoid compounds [69]. Three different extraction methods were used, and a total of 81 volatile compounds were identified in Exocarpium citri Grandis, around 58% of which were terpenes [70]. Similarly, different methods were tested on Ocimum basilicum leaves and 18 terpenoid compounds were identified [71]. Ma et al. performed metabolite profiling on three different ginger (Zingiber officinale) lines containing 102 compounds, among which 29 monoterpenoids and 47 sesquiterpenoids were identified [72]. Table 2 shows the metabolite profiling and identified terpenoid compounds in the different plant species.

4. Metabolic Engineering of Terpenoids in Plants

Changing the availability of precursors is one method used to control terpenoid levels in plants. It is possible that altering the terpenoid precursor pool alone is not enough to elevate levels of target terpenoids, and that simultaneously engineering the downstream genes would also be needed. In metabolic engineering, the availability of precursors is a key concern. The level of an isoprenoid precursor that is limiting for the synthesis of terpenoids is likely to vary depending on the plant tissue, species, and physiological condition [16]. A previous study of the metabolic engineering of monoterpenoids focused on producing heterologous monoterpenes in spearmint, and the knocking down of limonene synthase resulted in a huge reduction of limonene and carvone synthesis, while RNAi plants showed an increased sesquiterpenoid level [73]. The overexpression of geranyl diphosphate synthase small subunit 1(GPS SSU1) in Listea cubeba (Lour) plants showed a significant increase in monoterpene levels [74]. Two potential genes in the MEP pathway, namely, DXS and DXR, were cloned and developed in transgenic tobacco plants, which resulted in an increased content of monoterpenoid and sesquiterpenoid linalool and caryophyllene, respectively [75]. The co-expression of Solanum lycoperscum DXP, Arabidopsis thaliana GPS1, and Mentha X piperita GPS SSU through transient expression in Nicotiana benthamiana plants enhanced the production of monoterpenes such as limonene, linalool, alpha/beta-pinene, and myrcene [76]. The monoterpene key enzyme terpene synthase (TPS) subfamily was expressed in Osmanthus fragrans and the transient expression of leaves exhibited a high level of linalool and trans-β-ocimene, hence TPS played an important role in monoterpene production [77]. The development of transgenic Mentha spicata by Agrobacterium tumefaciens mediated transformation with isopentyldiphospahte (IPP) isomerase, and limonene synthase gene resulted in high levels of terpenoid production [78]. The overexpression of HMG CoA reductase in Lavandula angustifolia resulted in high levels of linalool production [79]. In another study, snapdragon (Antirrhinum majus) GPPS-SSU was overexpressed in tomato fruits, resulting in the production of monoterpenes including geraniol, geranial, neral, citronella, and citronellal [80]. Lucker et al. found that the production level corresponding to three different monoterpene synthases was high in transgenic tobacco plant flowers exhibiting three separate monoterpene synthases, but that the expression of endogenous linalool production was not affected [81]. In Arabidopsis, to produce monoterpenes, the strawberry gene nerolidol synthase 1 was used, and this resulted in the formation of linalool; this gene also encodes the dual function for the production of monoterpenes and sesquiterpenoids [14]. In another study, Perilla frutescens limonene synthase was introduced to tobacco plants, and limonene formation was detected in the plastids and cytosol of transgenic plants [82]. In petunia, the overexpression of S-linalool synthase resulted in the monoterpene production of newly formed linalool [10]. The overexpression of the S-linalool synthase gene in tomato (Lycopersicon esculentum) resulted in high levels of monoterpenes [11]. The results of many of the studies reported to date suggest that, in general, the direct precursor for monoterpene biosynthesis (i.e., GPP) is largely available to introduced monoterpene synthases.
The metabolic engineering and overexpression of sesquiterpenoids is limited to certain sources; β-caryophyllene synthase from Artemisia annua was introduced to a viral vector and transferred into Agrobacterium, which was then agroinfiltrated into N. benthamiana leaves and produced 26.5 mg of β-caryophyllene [83]. In an in vitro regeneration study, sesquiterpene cyclase was transformed in the medicinal plant A. annua, and resulted in a 30% atremisinin content [84]. The co-expression of the parthenolide pathway candidate genes was reconstituted by transient co-expression in N. benthamiana, and up to 1.4 µg of the final product was produced [85]. The key enzyme amorpha-4,11-diene synthase was transformed into Agrobacterium and agroinfiltrated into N. benthamiana, which resulted in various sesquiterpene products [86]. Co-expression with different plant-specific genes, such as feverfew germacrene A synthase (GAS), chicory germacrene A oxidase (GAO), and chicory costunolide synthase (COS), was reconstituted and agroinfiltrated into N. benthamiana, and results showed 60 ng g−1 of costunolide production [87]. Furthermore, different plant species have been used successfully to produce valuable sesquiterpenoid compounds using plant suspension cell culture technology [88,89]. Hitherto, five mevalonate and artemisinin pathway genes were expressed in tobacco plant cell cultures using a single vector, yielding 0.48–6.8 μg/g of artemisinin [90]. Similarly, constructing four genes (amorpha-4,11-diene synthase, amorphadiene monooxygenase, aldehyde ∆ (13) reductase, and aldehyde dehydrogenase) in N. tabacum leaf cells resulted in 0.01 mg/g of artemisinic alcohol [91]. Likewise, five artemisinin biosynthesis genes were transferred into Physcomitrella patens, which produced 0.21 mg/g of artemisinin after three days of cultures [92]. Table 3 summarizes the overall documented list of metabolic engineering of mono-terpenoids and sesquiterpenoids targeting genes and upregulated metabolites.

5. Synthetic Biology of Terpenoids

Synthetic biology is defined as the ‘design and building of novel biological components such as enzymes, genetic circuits, or restructuring of existing biological system’. Various monoterpenoids and sesquiterpenoids were synthesized over the last decade using engineered bacteria and yeast [93,94]. The most well-known example of synthetic biology-based high value chemical production is artemisinic acid, the precursor of the antimalarial drug arteminisin, which was synthesized in engineered Escherichia coli and baker’s yeast, Saccharomyces cerevisiae, following ten years of optimization [95,96]. Furthermore, a specialized limonene and perillyl alcohol manufacturing system was established in E. coli by co-expression of heterologous, codon-optimized, Staphylococus aureus, and S. cerevisiae MVA pathway genes into E. coli with Abies grandis GPP synthase and Mentha spicata limonene synthase genes. Optimum gene regulation and growing circumstance resulted in a 400 mg/L limonene titre [97]. However, alternative strategies in E. coli focused on the MEP pathway genes DXS, and because IDI was overexpressed, the resulting strains provided a poor titre of 35.8 mg/L limonene [98]. Similarly, using Yarrowia lipolytica yeast, sesquiterpenes (+)-nootkatone was synthesized by heterologous co-expressing genes such as valencene synthase, nootkatone synthase, and NADPH-cytochrome P450 reductase, resulting in 978.2 μg/L (+)-nootkatone [99]. As an application, most synthetic biology research on monoterpenoids and sesquiterpenes has focused on the high level of production in few compounds. However, a number of other monoterpenoids and sesquiterpenes have been produced in E. coli or yeast, and other microbial systems by assembling and optimizing biosynthetic pathways that contain a heterologous MVA or MEP pathway, as well as GPP and FPP.
Moreover, the field of synthetic biology in plants is still in its infancy. Synthetic biology research on plants needs reliable and effective techniques for compiling and transforming multi-component DNA constructions, such as a promoter and terminator. Reporter gene fusion is also a regular task in this field. Hence, optimizing this procedure is likely to result in significant productivity improvements. Traditionally, the required DNA construct has been designed using the restriction of endonuclease-mediated cleavage together with T4 DNA ligase-mediated joining. However, this approach takes a long time, and reliability along the sequence is not good for a large number of structures in a very efficient assembly [100,101,102]. Synthetic biology, which was inspired by engineering and mechanical assembly lines, uses standardized components to construct genetic creations. By using a Type IIS cloning system such as Golden Gate, MoClo, GoldenBraid, Loop Assembly, Fragment exchange, and others strategies for assembling synthetic constructs, high-throughput combinatorial libraries of synthetic constructs were easily constructed [103,104]. Internal occurrences of the recognition sequence are a constraint of Golden Gate Cloning. Type IIS restricted enzymes are employed in Golden Gate Cloning. Such enzymes only cut at a single site beyond their recognition-binding site sequence. BsaI detects the sequence 5′-GGTCTC-3′. Each fragment that will be integrated into the target vector is bounded by BsaI sites in the usual cloning process. [105]. Many of the reports included in the parts kit are also applicable to non-plant species. Such parts apply directly to multiple systems. Combinatorial pathway reconstruction includes all of the vector backbones and sequences needed for domesticating additional sequences and assembling them into single and multigene binary constructs [106]. These types of constructs are widely utilized in synthetic biology, and are a vital part of combinatorial bioengineering in plants. Additionally, with the emergence of CRISPR-Cas9 technology, large scale genome editing has become more feasible and affordable. All of these technological developments have largely eliminated the bottleneck associated with multi-gene transfer in plants. The development of new transformation technologies to generate stable transformed vector and cell lines with multiple constructs should be a high priority for commercial success in sustainable plant terpenoid metabolite production using synthetic biology approaches. Together, these tools will improve the bio-industrial production of monoterpenoids and sesquiterpenoids.

6. Terpenoids Pharmacological Activity

Terpenoids have been extensively utilized as raw materials in medicines because they have anti-inflammatory, antitumor, antiviral, antibacterial, and antimalarial properties, and they have the ability to increase transdermal absorption, prevent and cure cardiovascular disease, and exhibit hypoglycemic properties.

6.1. Monoterpenoids

Monoterpenes are C10 terpenoids produced from plastids that have high volatility. As a result, they are often found in plant essential oils. Below, we describe the three major classes of monoterpenoids (linalool, limonene, and alpha-pinene) with well-documented pharmacological properties.

6.1.1. Linalool

Linalool (C10H18O), also known as 3,7-dimethyl-1,6-octadien-3-ol, is an acyclic monoterpene tertiary alcohol found in the essential oils of various plant species [107]. Linalool is the primary element of various essential oils, which have been shown to have a range of biological activities, including antibacterial as well as anti-plasmodial properties [108]. Linalool has been shown to have anti-hyperalgesia, anti-inflammatory, and anti-inociceptive properties in a variety of animal models [109]. The anti-oxidant properties of Cinnamomum osmophloeum (Lauraceae) oil scavenged the DPPH radical (IC50 value: 29.7 g/mL), and this action was linked to the main component linalool, which made up 73% of the whole [110]. In the traditional medicinal system, different species of linalool and linalyl acetate are utilized to alleviate symptoms and treat many chronic and acute ailments [111]. Linalool-producing species have been shown to have anti-inflammatory properties and a peripheral analgesic impact [112,113].

6.1.2. Limonene

Limonene (C10H16)—(R)-4-Isopropenyl-1-methylethenyl-cyclohexene is a monocyclic monoterpene found in a variety of plants and a common essential oil component of aromatic plants [114]. Limonene has significant uses in cosmetics, soft drinks, and a variety of flavoring products. It has received increased interest due to its anticancer, antimicrobial, toxicity, and antiparasitic activities, among others [114]. Dabbah et al. reported the antimicrobial activity of pure limonene and the oil to be extremely effective when comparing the inhibitory impact of the essential oils from the fruits of lemon, orange, mandarin, grapefruit, and d-limonene [115]. According to Keinan et al., the anti-oxidant activity of limonene may readily saturate the pulmonary membrane, and therefore protect lung cells against both endogenous and exogenous ozone [116].

6.1.3. Alpha-Pinene

Pinene (C10H16)—(1S,5S)-2,6,6-Trimethylbicyclo (3,1,1) hept-2-ene—is a bicyclic monoterpene found in essential oils of pine (coniferous trees) [117]. Pinene has a wide range of biological activities, which means it has a wide range of applications, including in fungicidal agents, flavorings, perfumes, and antiviral agents [118]. Due to its toxic effects on membranes, pinene is also employed as an antibacterial agent [119]. Furthermore, pinene has been shown to suppress breast cancer and leukemia [120]. Moreover, pinene has potential as a natural medicine; for example, it is particularly flexible in the synthesis of polymers [121].

6.1.4. Others

Several p-menthane monoterpenoids of pharmacological relevance are found in the genus Mentha (Lamiaceae). (−)-Menthol, a key component of the essential oil of peppermint (Mentha × piperita) since the 1950s, has been recognized to serve as a full agonist of the CMR1 (Cold and Menthol Receptor 1) [122]. Cannabinoids were first used to describe a group of prenylated phenolic substances found in Cannabis spp. (Cannabaceae) but now comprise any ligand capable of interacting with human cannabinoid receptors; specifically, this includes endogenously-generated cannabinoids with no molecular resemblance to their plant-derived, terpene phenolic counterparts [123]. Cannabinoids, like p-menthane monoterpenoids in the Lamiaceae, accumulate in glandular trichomes, while cannabinoid-rich trichomes in the genus Cannabis are mainly found in the calyces and bracts of female flowers [124].

6.2. Sesquiterpenoids

Sesquiterpenoids have been reported to exhibit many pharmacological effects, including anti-inflammatory, antiviral, antibacterial, antifungal, antifeedant, antinociceptive, antileshmanial, and cytotoxic effects; they also exhibit lymphocyte proliferation and hydroxy radical scavenging. Below, we present three therapeutically important plant sesquiterpenoids.

6.2.1. Costunolide

Costunolide (CT) is a well-known sesquiterpene lactone of the germacranolides class. It is a white crystalline powder with the chemical formula C15H20O2. This chemical was first isolated from the root of costus (Saussurea lappa Clarke), and later from lettuce (Aucklandia lappa) and many other plant species [125]. Several studies have shown that CT has effects on anitbladder cancer [126], ovarian cancer [127], leukemia [128], and prostate cancer [129]. It was also reported that costunolide inhibits angiogenic responses by blocking the angiogenic factor signaling pathway and microtuble-interacting activity of costunolide [130,131].

6.2.2. Parthenolide

Parthenolide (C15H20O3) is considered to be one of the main active components in feverfew, accounting for many of the plant’s biological characteristics [132]. Recently, feverfew has been widely used for the prophylaxis of migraine headaches, relief of the pain and inflammation associated with arthritis, and the treatment of psoriasis [133]. As well as occurring in feverfew, parthenolide is the major anti-inflammatory component of tansy (Tanacetum vulgare). In animal studies, parthenolide significantly alleviated carrageenan-induced paw oedema when administered orally, with a stronger effect following intraperitoneal administration [134].

6.2.3. Trans-Caryophyllene

Trans-caryophyllene has been reported to possess many pharmacological effects. For example, it displays antimicrobial [135] and analgesic activity [136], and has a well-documented anti-inflammatory activity [137,138]. Additionally, trans-caryophyllene is an effective treatment for intestinal smooth muscle, blocking the electromechanical and ablepharmacochemical excitation–contraction coupling [139]. Those activities mean it is considered as a potential anti-spasmodic agent in tracheal smooth muscle.

6.2.4. Others

On the basis of dozens of carbon skeletons, sesquiterpene synthases act on FDP to produce hundreds of sesquiterpene hydrocarbons and alcohols. Clove, ginger (gingerol), rosemary (-caryophyllene), cannabis, sandalwood (-santalene), rain (geosmin, a bacterial sesquiterpene), and sandalwood (-santalene) are just a few examples that are responsible for flavors and fragrances. Under normal conditions, they are the heaviest of the volatile terpenes (heat is usually required to generate gases from diterpene hydrocarbons). Geraniaceae, Lamiaceae, Myrtaceae, Rutaceae, Gingeraceae, and Cannabaceae are among the plant families that generate sesquiterpene volatiles. It is widely acknowledged that such essential oils are used in traditional herbal treatments, including Ayurvedic and aromatherapy medicine [41]. Wormwood (Artemisia annua L., also known as qinghaosu, in family Asteraceae) is a Chinese plant that produces the sesquiterpene endoperoxide artemisinin, which has been proven to be efficient in the treatment of malaria [140]. It is more effective than conventional antimalarials, such as quinine, against a broader range of apicomplexan parasite life cycle stages [141].

7. Conclusions and Future Perspectives

This review mainly focused on the metabolic engineering and synthetic biology caused by the overexpression of terpenoid compounds in plants due to their diverse and biologically significant uses for humans. Over the last few years, research has taken advantage of advances in genomics, transcriptomics, and metabolomics, which has resulted in a greater understanding of the pathways and regulatory mechanisms involved in the biosynthesis of specialized terpenoids. Furthermore, the identification of regulatory factors and gene clusters involved in the biosynthesis of terpenoids in various plant species has provided a means to improve the biosynthesis of specialized terpenoids. Despite their several chemical constituents, monoterpenoids and sesquiterpenoids were investigated in detail. Particular compounds were overexpressed using single-construct vectors. Future studies should focus on the combination of multiple biosynthesis pathway genes constructed in single cloning vectors and agro-infiltrated in models, as well as original plants which are essential for the mass production of terpenoids in plants over a short period of time.

Author Contributions

V.M. and K.L. conceived and designed the conceptualization; V.M. and S.P. performed literature searching; J.A.K. and S.I.L. provided feedback and helped to improve final manuscript; V.M. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.


The authors would like to acknowledge funding through grants allocated to K.L. from the National Institute of Agricultural Sciences (Project No. PJ01495703), Rural Development Administration, Republic of Korea. This study was supported by the 2021 Postdoctoral Fellowship Program (V.M.) of the National Institute of Agricultural Sciences, RDA, Republic of Korea.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Christianson, D.W. Structural and Chemical Biology of Terpenoid Cyclases. Chem. Rev. 2017, 117, 11570–11648. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Caputi, L.; Aprea, E. Use of terpenoids as natural flavouring compounds in food industry. Recent Patents Food. Nutr. Agric. 2012, 3, 9–16. [Google Scholar] [CrossRef]
  3. Nagegowda, D.A. Plant volatile terpenoid metabolism: Biosynthetic genes, transcriptional regulation and subcellular compartmentation. FEBS Lett. 2010, 584, 2965–2973. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Gershenzon, J.; Croteau, R.B. Terpenoid biosynthesis: The basic pathway and formation of monoterpenes, sesquiterpenes and diterpenes. In Lipid Metabolism in Plants; Moore, T.S., Ed.; CRC Press: Boca Raton, FL, USA, 1993; pp. 340–388. [Google Scholar]
  5. Noriega, P. Terpenes in Essential Oils: Bioactivity and Applications; IntechOpen: Rijeka, Croatia, 2020. [Google Scholar] [CrossRef]
  6. Zhou, F.; Pichersky, E. More is better: The diversity of terpene metabolism in plants. Curr. Opin. Plant Biol. 2020, 55, 1–10. [Google Scholar] [CrossRef]
  7. Mahmoud, S.S.; Croteau, R.B. Strategies for transgenic manipulation of monoterpene biosynthesis in plants. Trends Plant Sci. 2002, 7, 366–373. [Google Scholar] [CrossRef]
  8. Rodríguez-Concepción, M.; Boronat, A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol. 2002, 130, 1079–1089. [Google Scholar] [CrossRef] [Green Version]
  9. Bick, J.A.; Lange, B.M. Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: Unidirectional transport of intermediates across the chloroplast envelope membrane. Arch. Biochem. Biophys. 2003, 415, 146–154. [Google Scholar] [CrossRef]
  10. Lücker, J.; Bouwmeester, H.J.; Schwab, W.; Blaas, J.; Van Der Plas, L.H.W.; Verhoeven, H.A. Expression of clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-β-D-glucopyranoside. Plant J. 2001, 27, 315–324. [Google Scholar] [CrossRef]
  11. Lewinsohn, E.; Schalechet, F.; Wilkinson, J.; Matsui, K.; Tadmor, Y.; Nam, K.; Amar, O.; Lastochkin, E.; Larkov, O.; Hiatt, W.; et al. Enhanced levels of the aroma and flavor compound S-Linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physol. 2001, 3, 1256–1265. [Google Scholar] [CrossRef]
  12. Lavy, M.; Zuker, A.; Lewinsohn, E.; Larkov, O.; Ravid, U.; Vainstein, A.; Weiss, D. Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene. Mol. Breed. 2002, 9, 103–111. [Google Scholar] [CrossRef]
  13. Aharoni, A.; Jongsma, M.A.; Kim, T.Y.; Ri, M.B.; Giri, A.P.; Verstappen, F.W.A.; Schwab, W.; Bouwmeester, H.J. Metabolic engineering of terpenoid biosynthesis in plants. Phytochem. Rev. 2006, 5, 49–58. [Google Scholar] [CrossRef] [Green Version]
  14. Aharoni, A.; Giri, A.P.; Deuerlein, S.; Griepink, F.; De Kogel, W.J.; Verstappen, F.W.A.; Verhoeven, H.A.; Jongsma, M.A.; Schwab, W.; Bouwmeester, H.J. Terpenoid metabolism in wild-type and transgenic Arabidopsis plants. Plant Cell 2003, 15, 2866–2884. [Google Scholar] [CrossRef] [Green Version]
  15. Krasnyanski, S.; May, R.A.; Loskutov, A.; Ball, T.M.; Sink, K.C. Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants. Theor. Appl. Genet. 1999, 99, 676–682. [Google Scholar] [CrossRef]
  16. Aharoni, A.; Jongsma, M.A.; Bouwmeester, H.J. Volatile science? Metabolic engineering of terpenoids in plants. Trends Plant Sci. 2005, 10, 594–602. [Google Scholar] [CrossRef]
  17. Wallaart, T.E.; Bouwmeester, H.J.; Hille, J.; Poppinga, L.; Maijers, N.C.A. Amorpha-4,11-diene synthase: Cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta 2001, 212, 460–465. [Google Scholar] [CrossRef] [Green Version]
  18. Hohn, T.M.; Ohlrogge, J.B. Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco. Plant Physiol. 1991, 97, 460–462. [Google Scholar] [CrossRef] [Green Version]
  19. Petzold, C.J.; Chan, L.J.G.; Nhan, M.; Adams, P.D. Analytics for metabolic engineering. Front. Bioeng. Biotechnol. 2015, 3, 1–11. [Google Scholar] [CrossRef] [Green Version]
  20. Courdavault, V.; O’Connor, S.E.; Jensen, M.K.; Papon, N. Metabolic engineering for plant natural products biosynthesis: New procedures, concrete achievements and remaining limits. Nat. Prod. Rep. 2021. [Google Scholar] [CrossRef]
  21. Gutensohn, M.; Henry, L.K.; Gentry, S.A.; Lynch, J.H.; Nguyen, T.T.H.; Pichersky, E.; Dudareva, N. Overcoming bottlenecks for metabolic engineering of sesquiterpene production in tomato fruits. Front. Plant Sci. 2021, 12, 691754. [Google Scholar] [CrossRef]
  22. Pichersky, E.; Raguso, R.A. Why do plants produce so many terpenoid compounds? New Phytol. 2018, 220, 692–702. [Google Scholar] [CrossRef]
  23. Seigler, D.S. Plant Secondary Metabolism; Springer Science Business Media: New York, NY, USA, 1995; ISBN 978-1-4613-7228-8. [Google Scholar] [CrossRef]
  24. Bohlmann, J.; Keeling, C.I. Terpenoid biomaterials. Plant J. 2008, 54, 656–669. [Google Scholar] [CrossRef]
  25. Vranová, E.; Coman, D.; Gruissem, W. Structure and dynamics of the isoprenoid pathway network. Mol. Plant 2012, 5, 318–333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Bourgaud, F.; Gravot, A.; Milesi, S.; Gontier, E. Production of plant secondary metabolites: A historical perspective. Plant Sci. 2001, 161, 839–851. [Google Scholar] [CrossRef]
  27. Ramawat, K.G.; Mérillon, J.M. Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes; Springer: Berlin, Germany, 2013; pp. 1–4242. [Google Scholar] [CrossRef]
  28. Hussein, R.A.; El-Anssary, A.A. Plants Secondary Metabolites: The Key Drivers of the Pharmacological Actions of Medicinal Plants; IntechOpen: Rijeka, Croatia, 2019. [Google Scholar] [CrossRef] [Green Version]
  29. Cox-Georgian, D.; Ramadoss, N.; Dona, C.; Basu, C. Therapeutic and medicinal uses of terpenes. In Medicinal Plants: From Farm to Pharmacy; Springer Nature: Cham, Switzerland, 2019; pp. 333–359. [Google Scholar] [CrossRef]
  30. Boncan, D.A.T.; Tsang, S.S.K.; Li, C.; Lee, I.H.T.; Lam, H.M.; Chan, T.F.; Hui, J.H.L. Terpenes and terpenoids in plants: Interactions with environment and insects. Int. J. Mol. Sci. 2020, 21, 7382. [Google Scholar] [CrossRef]
  31. Mumm, R.; Posthumus, M.A.; Dicke, M. Significance of terpenoids in induced indirect plant defence against herbivorous arthropods. Plant Cell Environ. 2008, 31, 575–585. [Google Scholar] [CrossRef]
  32. McCaskill, D.; Croteau, R. Some caveats for bioengineering terpenoid metabolism in plants. Trends Biotechnol. 1998, 16, 349–355. [Google Scholar] [CrossRef]
  33. Dudareva, N.; Andersson, S.; Orlova, I.; Gatto, N.; Reichelt, M.; Rhodes, D.; Boland, W.; Gershenzon, J. The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers. Proc. Natl. Acad. Sci. USA 2005, 102, 933–938. [Google Scholar] [CrossRef] [Green Version]
  34. Hemmerlin, A.; Harwood, J.L.; Bach, T.J. A raison d’être for two distinct pathways in the early steps of plant isoprenoid biosynthesis? Prog. Lipid Res. 2012, 51, 95–148. [Google Scholar] [CrossRef]
  35. Chen, F.; Tholl, D.; Bohlmann, J.; Pichersky, E. The family of terpene synthases in plants: A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011, 66, 212–229. [Google Scholar] [CrossRef]
  36. Huang, M.; Abel, C.; Sohrabi, R.; Petri, J.; Haupt, I.; Cosimano, J.; Gershenzon, J.; Tholl, D. Variation of herbivore-induced volatile terpenes among arabidopsis ecotypes depends on allelic differences and subcellular targeting of two terpene synthases, TPS02 and TPS03. Plant Physiol. 2010, 153, 1293–1310. [Google Scholar] [CrossRef] [Green Version]
  37. Lei, D.; Qiu, Z.; Qiao, J.; Zhao, G.R. Plasticity engineering of plant monoterpene synthases and application for microbial production of monoterpenoids. Biotechnol. Biofuels 2021, 14, 147. [Google Scholar] [CrossRef]
  38. Dewick, P.M. The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids; John Wiley & Sons, Ltd.: Chichester, UK, 2009; ISBN 9780470741689. [Google Scholar]
  39. Nagegowda, D.A.; Gupta, P. Advances in biosynthesis, regulation, and metabolic engineering of plant specialized terpenoids. Plant Sci. 2020, 294, 110457. [Google Scholar] [CrossRef] [PubMed]
  40. Sülsen, V.P.; Martino, V.S. Sesquiterpene Lactones: Advances in Their Chemistry and Biological Aspects; Springer International Publishing: Cham, Switzerland, 2018; ISBN 9783319782744. [Google Scholar]
  41. Kozioł, A.; Stryjewska, A.; Librowski, T.; Sałat, K.; Gaweł, M.; Moniczewski, A.; Lochy´nski, S. An overview of the pharmacological properties and potential applications of natural monoterpenes. Med. Chem. 2014, 14, 1156–1168. [Google Scholar] [CrossRef] [PubMed]
  42. Wojtunik-Kulesza, K.A.; Kasprzak, K.; Oniszczuk, T.; Oniszczuk, A. Natural monoterpenes: Much more than only a scent. Chem. Biodivers. 2019, 16, e1900434. [Google Scholar] [CrossRef]
  43. Barreto, R.S.S.; Albuquerque-Júnior, R.L.C.; Araújo, A.A.S.; Almeida, J.R.G.S.; Santos, M.R.V.; Barreto, A.S.; DeSantana, J.M.; Siqueira-Lima, P.S.; Quintans, J.S.S.; Quintans-Júnior, L.J. A systematic review of the wound-healing effects of monoterpenes and iridoid derivatives. Molecules 2014, 19, 846–862. [Google Scholar] [CrossRef]
  44. Zielińska-Błajet, M.; Feder-Kubis, J. Monoterpenes and their derivatives—Recent development in biological and medical applications. Int. J. Mol. Sci. 2020, 21, 7078. [Google Scholar] [CrossRef]
  45. Singh, G. Chemistry of Terpenoids and Carotenoids; Discovery Publisher Pvt. Ltd.: New Delhi, India, 2007; pp. 1–286. [Google Scholar]
  46. Hohmann, M.S.N.; Longhi-Balbinot, D.T.; Guazelli, C.F.S.; Navarro, S.A.; Zarpelon, A.C.; Casagrande, R.; Arakawa, N.S.; Verri, W.A. Sesquiterpene Lactones: Structural diversity and perspectives as anti-inflammatory molecules. Stud. Nat. Prod. Chem. 2016, 49, 243–264. [Google Scholar] [CrossRef]
  47. Zwenger, S.; Basu, C. Plant terpenoids. Biotechnol. Mol. Biol. Rev. 2008, 3, 1–7. [Google Scholar]
  48. Tholl, D. Biosynthesis and biological functions of terpenoids in plants. Adv. Biochem. Eng. Biotechnol. 2015, 148, 63–106. [Google Scholar] [CrossRef]
  49. Ludwiczuk, A.; Skalicka-Woźniak, K.; Georgiev, M.I. Terpenoids; Academic Press: Boston, MA, USA, 2017; ISBN 9780128020999. [Google Scholar]
  50. Chadwick, M.; Trewin, H.; Gawthrop, F.; Wagstaff, C. Sesquiterpenoids lactones: Benefits to plants and people. Int. J. Mol. Sci. 2013, 14, 12780–12805. [Google Scholar] [CrossRef] [Green Version]
  51. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing: Carol Stream, IL, USA, 2007. [Google Scholar]
  52. Krill, C.; Rochfort, S.; Spangenberg, G. A high-throughput method for the comprehensive analysis of terpenes and terpenoids in medicinal Cannabis biomass. Metabolites 2020, 10, 276. [Google Scholar] [CrossRef]
  53. Nguyen, T.D.; Riordan-Short, S.; Dang, T.T.T.; O’Brien, R.; Noestheden, M. Quantitation of select terpenes/terpenoids and nicotine using gas chromatography-mass spectrometry with high-temperature headspace sampling. ACS Omega 2020, 5, 5565–5573. [Google Scholar] [CrossRef]
  54. Wang, Y.; Li, X.; Jiang, Q.; Sun, H.; Jiang, J.; Chen, S.; Guan, Z.; Fang, W.; Chen, F. GC-MS analysis of the volatile constituents in the leaves of 14 compositae plants. Molecules 2018, 23, 166. [Google Scholar] [CrossRef] [Green Version]
  55. Johnson, V.; Frost, J.M.; Clifford, B. Analysis of 21 Terpenes in 3 Cannabis Cultivars by HS-GCMS; Shimadzu Scientific Instruments: Columbia, MD, USA.
  56. Trass, M.; Anderson, T.; Krepich, S.; Ave, M. Analysis of 33 Primary and Secondary Terpenes Found in Cannabis by GC-FID; Phenomenex: Torrance, CA, USA, 2017. [Google Scholar]
  57. Fausett, A. Analysis of Terpene and Terpenoid Content in Cannabis Sativa Using Headspace with GC/MSD; Agilent Technologies: Santa Clara, CA, USA, 2020; pp. 1–7. [Google Scholar]
  58. Abualhasan, M.N.; Zaid, A.N.; Jaradat, N.; Mousa, A. GC Method validation for the analysis of menthol in suppository pharmaceutical dosage form. Int. J. Anal. Chem. 2017, 2017, 1728414. [Google Scholar] [CrossRef] [Green Version]
  59. James, A. Using Gas Chromatography for accurate terpene analysis in Cannabis. Cannabis Sci. Technol. 2019, 2. [Google Scholar]
  60. Henneman, L.; van Cruchten, A.G.; Denis, S.W.; Amolins, M.W.; Placzek, A.T.; Gibbs, R.A.; Kulik, W.; Waterham, H.R. Detection of nonsterol isoprenoids by HPLC-MS/MS. Anal. Biochem. 2008, 383, 18–24. [Google Scholar] [CrossRef] [Green Version]
  61. Henneman, L.; Van Cruchten, A.G.; Kulik, W.; Waterham, H.R. Inhibition of the isoprenoid biosynthesis pathway; Detection of intermediates by UPLC-MS/MS. Biochim. Biophys. Acta-Mol. Cell Biol. Lipids 2011, 1811, 227–233. [Google Scholar] [CrossRef]
  62. Kumar, R.; Bohra, A.; Pandey, A.K.; Pandey, M.K.; Kumar, A. Metabolomics for plant improvement: Status and prospects. Front. Plant Sci. 2017, 8, 1–27. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Jiang, Z.; Kempinski, C.; Chappell, J. Extraction and analysis of Terpenes/Terpenoids. Curr. Protoc. Plant Biol. 2016, 1, 345–358. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Fu, X.; Zhou, Y.; Zeng, L.; Dong, F.; Mei, X.; Liao, Y.; Watanabe, N.; Yang, Z. Analytical method for metabolites involved in biosynthesis of plant volatile compounds. RSC Adv. 2017, 7, 19363–19372. [Google Scholar] [CrossRef] [Green Version]
  65. Myers, C.; Herrington, J.S.; Hamrah, P.; Anderson, K. Accelerated solvent extraction of terpenes in Cannabis coupled with various injection techniques for GC-MS analysis. Front. Chem. 2021, 9, 1–13. [Google Scholar] [CrossRef]
  66. Rocha, E.D.; Silva, V.E.A.; Pereira, F.C.S.; Jean, V.M.; Costa Souza, F.L.; Baratto, L.C.; Vieira, A.C.M.; Carvalho, V.M. Qualitative terpene profiling of Cannabis varieties cultivated for medical purposes. Rodriguesia 2020, 7, 48–51. [Google Scholar] [CrossRef]
  67. Sun, L.; Zhu, B.; Zhang, X.; Wang, H.; Yan, A.; Zhang, G.; Wang, X.; Xu, H. The accumulation profiles of terpene metabolites in three Muscat table grape cultivars through HS-SPME-GCMS. Sci. Data 2020, 7, 1–6. [Google Scholar] [CrossRef]
  68. Xu, Y.; Zhu, C.; Xu, C.; Sun, J.; Grierson, D.; Zhang, B.; Chen, K. Integration of metabolite profiling and transcriptome analysis reveals genes related to volatile terpenoid metabolism in finger citron (C. Medica var. sarcodactylis). Molecules 2019, 24, 2564. [Google Scholar] [CrossRef] [Green Version]
  69. Kupska, M.; Wasilewski, T.; Jędrkiewicz, R.; Gromadzka, J.; Namieśnik, J. Determination of terpene profiles in potential superfruits. Int. J. Food Prop. 2016, 19, 2726–2738. [Google Scholar] [CrossRef]
  70. Xie, Z.; Liu, Q.; Liang, Z.; Zhao, M.; Yu, X.; Yang, D.; Xu, X. The GC/MS analysis of volatile components extracted by different methods from Exocarpium citri grandis. J. Anal. Methods Chem. 2013, 2013, 918406. [Google Scholar] [CrossRef] [Green Version]
  71. Di Carro, M.; Ianni, C.; Magi, E. Determination of terpenoids in plant leaves by GC-MS: Development of the method and application to Ocimum basilicum and Nicotiana langsdorffii. Anal. Lett. 2013, 46, 630–639. [Google Scholar] [CrossRef]
  72. Ma, X.; Gang, D.R. Metabolic profiling of in vitro micropropagated and conventionally greenhouse grown ginger (Zingiber officinale). Phytochemistry 2006, 67, 2239–2255. [Google Scholar] [CrossRef]
  73. Li, C.; Sarangapani, S.; Wang, Q.; Nadimuthu, K.; Sarojam, R. Metabolic engineering of the native monoterpene pathway in spearmint for production of heterologous monoterpenes reveals complex metabolism and pathway interactions. Int. J. Mol. Sci. 2020, 21, 6164. [Google Scholar] [CrossRef]
  74. Zhao, Y.; Chen, Y.; Gao, M.; Yin, H.; Wu, L.; Wang, Y. Overexpression of geranyl diphosphate synthase small subunit 1 (LcGPPS.SSU1) enhances the monoterpene content and biomass. Ind. Crops Prod. 2020, 143, 111926. [Google Scholar] [CrossRef]
  75. Zhang, T.; Sun, M.; Guo, Y.; Shi, X.; Yang, Y.; Chen, J.; Zheng, T.; Han, Y.; Bao, F.; Ahmad, S. Overexpression of LiDXS and LiDXR from lily (Lilium ‘siberia’) enhances the terpenoid content in tobacco flowers. Front. Plant Sci. 2018, 9, 1–12. [Google Scholar] [CrossRef]
  76. Yin, J.L.; Wong, W.S.; Jang, I.C.; Chua, N.H. Co-expression of peppermint geranyl diphosphate synthase small subunit enhances monoterpene production in transgenic tobacco plants. New Phytol. 2017, 213, 1133–1144. [Google Scholar] [CrossRef] [Green Version]
  77. Zeng, X.; Liu, C.; Zheng, R.; Cai, X.; Luo, J.; Zou, J.; Wang, C. Emission and accumulation of monoterpene and the key terpene synthase(TPS)associated with monoterpene biosynthesis in Osmanthus fragrans lour. Front. Plant Sci. 2016, 6, 1–16. [Google Scholar] [CrossRef] [Green Version]
  78. Kang, Y.M.; Park, D.J.; Lee, D.G.; Song, H.J.; Kang, S.M.; Min, J.Y.; Moon, B.C.; Lee, C.K.; Jeon, K.S.; Shivappakarigar, C.; et al. Over expression of IPP isomerase and limonene synthase enzymes in Mentha spicata and their influence on the terpenoid metabolism. Rom. Biotechnol. Lett. 2015, 20, 10358–10368. [Google Scholar]
  79. Amiri, P.; Shahpiri, A.; Asadollahi, M.A.; Momenbeik, F.; Partow, S. Metabolic engineering of Saccharomyces cerevisiae for linalool production. Biotechnol. Lett. 2016, 38, 503–508. [Google Scholar] [CrossRef]
  80. Gutensohn, M.; Orlova, I.; Nguyen, T.T.H.; Davidovich-Rikanati, R.; Ferruzzi, M.G.; Sitrit, Y.; Lewinsohn, E.; Pichersky, E.; Dudareva, N. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits. Plant J. 2013, 75, 351–363. [Google Scholar] [CrossRef]
  81. Lücker, J.; Schwab, W.; Van Hautum, B.; Blaas, J.; Van Der Plas, L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A. Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from Lemon. Plant Physiol. 2004, 134, 510–519. [Google Scholar] [CrossRef] [Green Version]
  82. Ohara, K.; Ujihara, T.; Endo, T.; Sato, F.; Yazaki, K. Limonene production in tobacco with Perilla limonene synthase cDNA. J. Exp. Bot. 2003, 54, 2635–2642. [Google Scholar] [CrossRef]
  83. Muthusamy, S.; Vetukuri, R.R.; Lundgren, A.; Ganji, S.; Zhu, L.H.; Brodelius, P.E.; Kanagarajan, S. Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro. PeerJ 2020, 2020, 1–21. [Google Scholar] [CrossRef]
  84. Mirzaee, H.; Sharafi, A.; Hashemi Sohi, H. In vitro regeneration and transient expression of recombinant sesquiterpene cyclase (SQC) in Artemisia annua L. South African J. Bot. 2016, 104, 225–231. [Google Scholar] [CrossRef]
  85. Liu, Q.; Manzano, D.; Tanić, N.; Pesic, M.; Bankovic, J.; Pateraki, I.; Ricard, L.; Ferrer, A.; de Vos, R.; van de Krol, S.; et al. Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway. Metab. Eng. 2014, 23, 145–153. [Google Scholar] [CrossRef] [PubMed]
  86. Kanagarajan, S.; Muthusamy, S.; Gliszczyńska, A.; Lundgren, A.; Brodelius, P.E. Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana. Plant Cell Rep. 2012, 31, 1309–1319. [Google Scholar] [CrossRef] [PubMed]
  87. Liu, Q.; Majdi, M.; Cankar, K.; Goedbloed, M.; Charnikhova, T.; Verstappen, F.W.A.; de Vos, R.C.H.; Beekwilder, J.; van der Krol, S.; Bouwmeester, H.J. Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana. PLoS ONE 2011, 6, e23255. [Google Scholar] [CrossRef] [PubMed]
  88. Wu, T.; Kerbler, S.M.; Fernie, A.R.; Zhang, Y. Plant cell cultures as heterologous bio-factories for secondary metabolite production. Plant Commun. 2021, 2, 100235. [Google Scholar] [CrossRef]
  89. Zhu, X.; Liu, X.; Liu, T.; Wang, Y.; Ahmed, N.; Li, Z.; Jiang, H. Synthetic biology of plant natural products: From pathway elucidation to engineered biosynthesis in plant cells. Plant Commun. 2021, 2, 100229. [Google Scholar] [CrossRef]
  90. Farhi, M.; Marhevka, E.; Ben-Ari, J.; Algamas-Dimantov, A.; Liang, Z.; Zeevi, V.; Edelbaum, O.; Spitzer-Rimon, B.; Abeliovich, H.; Schwartz, B.; et al. Generation of the potent anti-malarial drug artemisinin in tobacco. Nat. Biotechnol. 2011, 29, 1072–1074. [Google Scholar] [CrossRef]
  91. Zhang, Y.; Nowak, G.; Reed, D.W.; Covello, P.S. The production of artemisinin precursors in tobacco. Plant Biotechnol. J. 2011, 9, 445–454. [Google Scholar] [CrossRef] [Green Version]
  92. Malhotra, K.; Subramaniyan, M.; Rawat, K.; Kalamuddin, M.; Qureshi, M.I.; Malhotra, P.; Mohmmed, A.; Cornish, K.; Daniell, H.; Kumar, S. Compartmentalized metabolic engineering for Artemisinin biosynthesis and effective malaria treatment by oral delivery of plant cells. Mol. Plant. 2016, 9, 1464–1477. [Google Scholar] [CrossRef] [Green Version]
  93. Zebec, Z.; Wilkes, J.; Jervis, A.J.; Scrutton, N.S.; Takano, E.; Breitling, R. Towards synthesis of monoterpenes and derivatives using synthetic biology. Curr. Opin. Chem. Biol. 2016, 34, 37–43. [Google Scholar] [CrossRef]
  94. Mai, J.; Li, W.; Ledesma-Amaro, R.; Ji, X.J. Engineering Plant Sesquiterpene Synthesis into Yeasts: A Review. J. Agric. Food Chem. 2021, 69, 9498–9510. [Google Scholar] [CrossRef]
  95. Paddon, C.J.; Keasling, J.D. Semi-synthetic artemisinin: A model for the use of synthetic biology in pharmaceutical development. Nat. Rev. Microbiol. 2014, 12, 355–367. [Google Scholar] [CrossRef]
  96. Paddon, C.J.; Westfall, P.J.; Pitera, D.J.; Benjamin, K.; Fisher, K.; McPhee, D.; Leavell, M.D.; Tai, A.; Main, A.; Eng, D.; et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 2013, 496, 528–532. [Google Scholar] [CrossRef] [Green Version]
  97. Alonso-Gutierrez, J.; Chan, R.; Batth, T.S.; Adams, P.D.; Keasling, J.D.; Petzold, C.J.; Lee, T.S. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab. Eng. 2013, 19, 33–41. [Google Scholar] [CrossRef]
  98. Du, F.L.; Yu, H.L.; Xu, J.H.; Li, C.X. Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. Coli. Bioresour. Bioprocess. 2014, 1, 1–10. [Google Scholar] [CrossRef] [Green Version]
  99. Guo, X.; Sun, J.; Li, D.; Lu, W. Heterologous biosynthesis of (+)-nootkatone in unconventional yeast Yarrowia lipolytica. Biochem. Eng. J. 2018, 137, 125–131. [Google Scholar] [CrossRef]
  100. Hartley, J.L.; Temple, G.F.; Brasch, M.A. DNA cloning using in vitro site-specific recombination. Genome Res. 2000, 10, 1788–1795. [Google Scholar] [CrossRef] [Green Version]
  101. Gibson, D.G. Enzymatic assembly of overlapping DNA fragments. Methods Enzymol. 2011, 498, 349–361. [Google Scholar] [CrossRef]
  102. Quan, J.; Tian, J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat. Protoc. 2011, 6, 242–251. [Google Scholar] [CrossRef]
  103. Irfan, M.; Chavez, B.; Rizzo, P.; D’Auria, J.C.; Moghe, G.D. Evolution-aided engineering of plant specialized metabolism. aBIOTECH 2021, 2, 240–263. [Google Scholar] [CrossRef]
  104. Engler, C.; Marillonnet, S. Generation of families of construct variants using golden gate shuffling. Methods Mol. Biol. 2011, 729, 167–181. [Google Scholar] [CrossRef]
  105. Emami, S.; Yee, M.C.; Dinneny, J.R. A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology. Front. Plant Sci. 2013, 4, 1–6. [Google Scholar] [CrossRef] [Green Version]
  106. Engler, C.; Youles, M.; Gruetzner, R.; Ehnert, T.M.; Werner, S.; Jones, J.D.G.; Patron, N.J.; Marillonnet, S. A Golden Gate modular cloning toolbox for plants. ACS Synth. Biol. 2014, 3, 839–843. [Google Scholar] [CrossRef]
  107. Ilc, T.; Parage, C.; Boachon, B.; Navrot, N.; Werck-Reichhart, D. Monoterpenol oxidative metabolism: Role in plant adaptation and potential applications. Front. Plant Sci. 2016, 7, 1–16. [Google Scholar] [CrossRef] [Green Version]
  108. Van Zyl, R.L.; Seatlholo, S.T.; Van Vuuren, S.F.; Viljoen, A.M. The biological activities of 20 nature identical essential oil constituents. J. Essent. Oil Res. 2006, 18, 129–133. [Google Scholar] [CrossRef]
  109. Kamatou, G.P.P.; Viljoen, A.M. Linalool—A Review of a biologically active compound of commercial importance. Nat. Prod. Commun. 2008, 3, 1934578X0800300727. [Google Scholar] [CrossRef] [Green Version]
  110. Lin, K.H.; Yeh, S.Y.; Lin, M.Y.; Shih, M.C.; Yang, K.T.; Hwang, S.Y. Major chemotypes and antioxidative activity of the leaf essential oils of Cinnamomum osmophloeum Kaneh. from a clonal orchard. Food Chem. 2007, 105, 133–139. [Google Scholar] [CrossRef]
  111. Peana, A.T.; D’Aquila, P.S.; Panin, F.; Serra, G.; Pippia, P.; Moretti, M.D.L. Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils. Phytomedicine 2002, 9, 721–726. [Google Scholar] [CrossRef]
  112. Moretti, M.D.L.; Peana, A.T.; Satta, M. A study on anti-inflammatory and peripheral analgesic action of salvia sclareaoil and its main components. J. Essent. Oil Res. 1997, 9, 199–204. [Google Scholar] [CrossRef]
  113. Peana, A.T.; Moretti, M.D.L. Pharmacological activities and applications of Salvia sclarea and Salvia desoleana essential oils. Stud. Nat. Prod. Chem. 2002, 26, 391–423. [Google Scholar] [CrossRef]
  114. Erasto, P.; Viljoen, A.M. Limonene—A review: Biosynthetic, ecological and pharmacological relevance. Nat. Prod. Commun. 2008, 3, 1934578X0800300728. [Google Scholar] [CrossRef] [Green Version]
  115. Dabbah, R.; Edwards, V.M.; Moats, W.A. Antimicrobial action of some citrus fruit oils on selected food-borne bacteria. Appl. Microbiol. 1970, 19, 27–31. [Google Scholar] [CrossRef] [PubMed]
  116. Keinan, E.; Alt, A.; Amir, G.; Bentur, L.; Bibi, H.; Shoseyov, D. Natural ozone scavenger prevents asthma in sensitized rats. Bioorganic Med. Chem. 2005, 13, 557–562. [Google Scholar] [CrossRef] [PubMed]
  117. Winnacker, M. Pinenes: Abundant and renewable building blocks for a variety of sustainable polymers. Angew. Chemie-Int. Ed. 2018, 57, 14362–14371. [Google Scholar] [CrossRef] [PubMed]
  118. da Silva, A.C.R.; Lopes, P.M.; De Azevedo, M.M.B.; Costa, D.C.; Alviano, C.S.; Alviano, D.S. Biological activities of α-pinene and β-pinene enantiomers. Molecules 2012, 17, 6305–6316. [Google Scholar] [CrossRef] [Green Version]
  119. Sybilska, D.; Kowalczyk, J.; Asztemborska, M.; Ochocka, R.J.; Lamparczyk, H. Chromatographic studies of the enantiomeric composition of some therapeutic compositions applied in the treatment of liver and kidney diseases. J. Chromatogr. A 1994, 665, 67–73. [Google Scholar] [CrossRef]
  120. Zhou, J.Y.; Di Tang, F.; Mao, G.G.; Bian, R.L. Effect of α-pinene on nuclear translocation of NF-κB in THP-1 cells. Acta Pharmacol. Sin. 2004, 25, 480–484. [Google Scholar]
  121. Thomsett, M.R.; Moore, J.C.; Buchard, A.; Stockman, R.A.; Howdle, S.M. New renewably-sourced polyesters from limonene-derived monomers. Green Chem. 2019, 21, 149–156. [Google Scholar] [CrossRef]
  122. Farco, J.A.; Grundmann, O. Menthol—Pharmacology of an important naturally medicinal “Cool”. Mini Rev. Med. Chem. 2012, 13, 124–131. [Google Scholar] [CrossRef]
  123. Zou, S.; Kumar, U. Cannabinoid receptors and the endocannabinoid system: Signaling and function in the central nervous system. Int. J. Mol. Sci. 2018, 19, 833. [Google Scholar] [CrossRef] [Green Version]
  124. Hanuš, L.O.; Hod, Y. Terpenes/Terpenoids in Cannabis: Are They Important? Med. Cannabis Cannabinoids 2020, 3, 25–60. [Google Scholar] [CrossRef]
  125. Kim, D.Y.; Choi, B.Y. Costunolide—A bioactive Sesquiterpene Lactone with Diverse Therapeutic Potential. Int. J. Mol. Sci. 2019, 20, 2926. [Google Scholar] [CrossRef] [Green Version]
  126. Rasul, A.; Parveen, S.; Ma, T. Costunolide: A novel anti-cancer sesquiterpene lactone. Bangladesh J. Pharmacol. 2012, 7, 6–13. [Google Scholar] [CrossRef] [Green Version]
  127. Yang, Y.I.; Kim, J.H.; Lee, K.T.; Choi, J.H. Costunolide induces apoptosis in platinum-resistant human ovarian cancer cells by generating reactive oxygen species. Gynecol. Oncol. 2011, 123, 588–596. [Google Scholar] [CrossRef]
  128. Choi, J.H.; Lee, K.T. Costunolide-induced apoptosis in human leukemia cells: Involvement of c-Jun N-terminal kinase activation. Biol. Pharm. Bull. 2009, 32, 1803–1808. [Google Scholar] [CrossRef] [Green Version]
  129. Hsu, J.L.; Pan, S.L.; Ho, Y.F.; Hwang, T.L.; Kung, F.L.; Guh, J.H. Costunolide induces apoptosis through nuclear calcium2+ overload and DNA damage response in human prostate cancer. J. Urol. 2011, 185, 1967–1974. [Google Scholar] [CrossRef]
  130. Jeong, S.J.; Itokawa, T.; Shibuya, M.; Kuwano, M.; Ono, M.; Higuchi, R.; Miyamoto, T. Costunolide, a sesquiterpene lactone from Saussurea lappa, inhibits the VEGFR KDR/Flk-1 signaling pathway. Cancer Lett. 2002, 187, 129–133. [Google Scholar] [CrossRef]
  131. Bocca, C.; Gabriel, L.; Bozzo, F.; Miglietta, A. A sesquiterpene lactone, costunolide, interacts with microtubule protein and inhibits the growth of MCF-7 cells. Chem. Biol. Interact. 2004, 147, 79–86. [Google Scholar] [CrossRef]
  132. Agatonovic-Kustrin, S.; Morton, D.W. The Current and Potential Therapeutic Uses of Parthenolide, 1st ed.; Elsevier B.V.: Amsterdam, The Netherlands, 2018; Volume 58, ISBN 9780444640567. [Google Scholar]
  133. Brown, A.M.G.; Lowe, K.C.; Davey, M.R.; Brian Power, J.; Knight, D.W.; Heptinstall, S. Comparison of extraction procedures for parthenolide in Tanacetum parthenium. Phytochem. Anal. 1996, 7, 86–91. [Google Scholar] [CrossRef]
  134. Schinella, G.R.; Giner, R.M.; Del Carmen Recio, M.; De Buschiazzo, P.M.; Ríos, J.L.; Máñez, S. Anti-inflammatory effects of South American Tanacetum vulgare. J. Pharm. Pharmacol. 1998, 50, 1069–1074. [Google Scholar] [CrossRef]
  135. Schnitzler, P.; Astani, A.; Reichling, J. Screening for antiviral activities of isolated compounds from essential oils. Evid.based Complement. Altern. Med. 2011, 2011, 253643. [Google Scholar] [CrossRef] [Green Version]
  136. Chavan, M.J.; Wakte, P.S.; Shinde, D.B. Analgesic and anti-inflammatory activity of Caryophyllene oxide from Annona squamosa L. bark. Phytomedicine 2010, 17, 149–151. [Google Scholar] [CrossRef]
  137. Fernandes, E.S.; Passos, G.F.; Medeiros, R.; da Cunha, F.M.; Ferreira, J.; Campos, M.M.; Pianowski, L.F.; Calixto, J.B. Anti-inflammatory effects of compounds alpha-humulene and (−)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea. Eur. J. Pharmacol. 2007, 569, 228–236. [Google Scholar] [CrossRef]
  138. Medeiros, R.; Passos, G.F.; Vitor, C.E.; Koepp, J.; Mazzuco, T.L.; Pianowski, L.F.; Campos, M.M.; Calixto, J.B. Effect of two active compounds obtained from the essential oil of Cordia verbenacea on the acute inflammatory responses elicited by LPS in the rat paw. Br. J. Pharmacol. 2007, 151, 618–627. [Google Scholar] [CrossRef] [Green Version]
  139. Pinho-Da-Silva, L.; Mendes-Maia, P.V.; Do Nascimento Garcia Teófilo, T.M.; Barbosa, R.; Ceccatto, V.M.; Coelho-De-Souza, A.N.; Cruz, J.S.; Leal-Cardoso, J.H. Trans-caryophyllene, a natural sesquiterpene, causes tracheal smooth muscle relaxation through blockade of voltage-dependent Ca2+ channels. Molecules 2012, 17, 11965–11977. [Google Scholar] [CrossRef]
  140. Kayani, W.K.; Kiani, B.H.; Dilshad, E.; Mirza, B. Biotechnological approaches for artemisinin production in Artemisia. World J. Microbiol. Biotechnol. 2018, 34, 54. [Google Scholar] [CrossRef] [Green Version]
  141. Bryant, L.; Flatley, B.; Patole, C.; Brown, G.D.; Cramer, R. Proteomic analysis of Artemisia annua—Towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin. BMC Plant Biol. 2015, 15, 175. [Google Scholar] [CrossRef] [Green Version]
Figure 1. Terpenoid biosynthesis in plants. There are two distinct pathways in plants for the synthesis of the universal precursors isopentenyl pyrophosphate (IPP) and dimethyl-allyl pyrophosphate (DMAPP): the cytoplasm-localized mevalonate (MVA) pathway and the plastid-localized methyl erythritol phosphate (MEP) pathway. The brown color indicates intermediate precursors of plastids (geranyl pyrophosphate) and cytosol (farnesyl pyrophosphate). AACT acetoacetyl-CoA thiolase; HMGCAS 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR 3-hydroxy-3-methylglutaryl-CoA reductase; MK mevalonate-5-kinase; PMK phosphomevalonate kinase; MPD mevalonate-5-phpshate decarboxylase; MPPD mevalonate pyrophosphate decarboxylase; IPK isopentyl pyrophosphate kinase; FPS farnesyl pyrophosphate synthase. DXS 1-deoxy-d-xylulose-5-phosphate synthase; DXR 1-deoxy-d-xylulose 5-phosphate reductoisomerase; CMS 2-c-methyl-d-erythritol 4-phosphate cytidylyltransferase; CMK 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase; MCS 2-c-methyl-d-erythritol 2, 4-cyclodiphosphate synthase; HDS 4-hydroxy-3-methyl-but-2-enyldiphosphate synthase; HDR 4-hydroxy-3-methyl-but-2-enyldiphosphate reductase; GPPS geranyl pyrophosphate synthase; GGPPS geranylgeranyl pyrophosphate synthase; STPS sesquiterpene synthase; LPP linalyl pyrophosphate; NPP neryl pyrophosphate; GAO germacrene A oxidase; GAS germacrene A synthase; CTS costunolide synthase; PTS parthenolide synthase; LNS linalool synthase; LMS limonene synthase; PES pinene synthase; MCS myrcene synthase. As representative examples of terpenoids: linalool, limonene, α-pinene, β-myrcene, germacrene A, germacrene A acid, costunolide and parthenolide are illustrated in chemical structure.
Figure 1. Terpenoid biosynthesis in plants. There are two distinct pathways in plants for the synthesis of the universal precursors isopentenyl pyrophosphate (IPP) and dimethyl-allyl pyrophosphate (DMAPP): the cytoplasm-localized mevalonate (MVA) pathway and the plastid-localized methyl erythritol phosphate (MEP) pathway. The brown color indicates intermediate precursors of plastids (geranyl pyrophosphate) and cytosol (farnesyl pyrophosphate). AACT acetoacetyl-CoA thiolase; HMGCAS 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR 3-hydroxy-3-methylglutaryl-CoA reductase; MK mevalonate-5-kinase; PMK phosphomevalonate kinase; MPD mevalonate-5-phpshate decarboxylase; MPPD mevalonate pyrophosphate decarboxylase; IPK isopentyl pyrophosphate kinase; FPS farnesyl pyrophosphate synthase. DXS 1-deoxy-d-xylulose-5-phosphate synthase; DXR 1-deoxy-d-xylulose 5-phosphate reductoisomerase; CMS 2-c-methyl-d-erythritol 4-phosphate cytidylyltransferase; CMK 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase; MCS 2-c-methyl-d-erythritol 2, 4-cyclodiphosphate synthase; HDS 4-hydroxy-3-methyl-but-2-enyldiphosphate synthase; HDR 4-hydroxy-3-methyl-but-2-enyldiphosphate reductase; GPPS geranyl pyrophosphate synthase; GGPPS geranylgeranyl pyrophosphate synthase; STPS sesquiterpene synthase; LPP linalyl pyrophosphate; NPP neryl pyrophosphate; GAO germacrene A oxidase; GAS germacrene A synthase; CTS costunolide synthase; PTS parthenolide synthase; LNS linalool synthase; LMS limonene synthase; PES pinene synthase; MCS myrcene synthase. As representative examples of terpenoids: linalool, limonene, α-pinene, β-myrcene, germacrene A, germacrene A acid, costunolide and parthenolide are illustrated in chemical structure.
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Figure 2. GC-Q-Orbitrap-MS profiling of three major standard compounds from monoterpenoids (alpha-pinene, linalool and limonene) and sesquiterpenoids (trans-caryophyllene, costunolide and parthenolide).
Figure 2. GC-Q-Orbitrap-MS profiling of three major standard compounds from monoterpenoids (alpha-pinene, linalool and limonene) and sesquiterpenoids (trans-caryophyllene, costunolide and parthenolide).
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Table 1. List of GC-MS column and oven program for terpenoid analysis.
Table 1. List of GC-MS column and oven program for terpenoid analysis.
InstrumentColumn NameDimension (Length, Inner Diameter & Thickness)Oven ProgramReferences
GC-MSDB-5MS-DG, DB-17, VF-3530 m × 0.25 µm ID × 0.25 µm, 30 m × 0.25 µm ID × 1.0 µm60 °C (2 min), Ramp: 5 °C/min to 200 °C[52]
GC-MSTG-624 SilMS30 m × 0.25 mm ID × 1.4 µm60 °C (30 s), Ramp 1: 15 °C/min to 130 (3 min); Ramp 2 5 °C/min to 140 °C (1 min); Ramp 3: 22° C/min 280 °C (3 min)[53]
GC-MSHP-530 m × 0.25 µm ID × 0.25 µm50 °C (2 min), Ramp 1: 5 °C min to 180 °C, Ramp 2: 20 °C/min to 270 °C[54]
GC-MSRxi-624 Sil MS30 m × 0.25 mm ID × 1.4 µm80 °C (1 min), Ramp 1: 12 °C/min to 150 (1 min); Ramp 2 9 °C/min to 250 (1 min)[55]
GC-FIDZB-5 PLUSTM20 m × 0.18 mm ID × 0.36 µmRamp 1: 35 °C to 105 °C 10 °C/min to 205 °C Ramp 2: 15 °C/min to 360 °C Ramp 3: 35 °C/min for 1.9 min[56]
GC-MSDDB-HeavyWax30 m × 250 µm ID × 1.4 µm50 °C (0.75 min), Ramp 1: 80 °C (0 min); Ramp 2: 240 °C (5 min)[57]
GC-FIDVF-624 ms60 m × 0.32 mm ID × 1.8 µm90° C (1 min), Ramp 1: 15 °C min to 181° C (3 min)[58]
GC-MSElite-530 m × 0.25 µm ID × 0.25 µm100 °C (5 min), Ramp 1: 20 C/min (200° C), Ramp 2: 10 °C/min (270 °C)[59]
Table 2. Identification of terpenoid metabolite compounds and analytical methods used.
Table 2. Identification of terpenoid metabolite compounds and analytical methods used.
SourceInstrument *MethodIdentified TerpenesReferences
CannabisGC-MSHeadspace, SPME and Liquid injection49[52]
Cannabis varietiesGC-MSHS-SPME30[66]
Muscat grapeGC-MSHS-SPME28[67]
Finger CirtonGC-MSSPME62[68]
Fourteen Compositae plantsGC-MSn-hexane213[54]
Goose berry, crabapple, cherry silver berry, scarlet hawthornqGC × GC-TOF-MSSPME79[69]
Exocarpium citri GrandisGC-MSSP, HS-SPME & solvent extraction81[70]
Basil & TobaccoGC-MSSP, Ultrasound-Assisted18[71]
* GC-MS—Gas chromatography-mass spectrometry; GC-TOF-MS—Gas chromatography time of flight-mass spectrometry; LC-ESI-MS—Liquid chromatography electrospray ionization mass spectrometry; ASE—Accelerated solvent extraction; HS-SPME—Headspace solid-phase microextraction; SPME—Solid-phase microextraction; SP—Solid phase.
Table 3. Reports on the metabolic engineering of monoterpenoids, sesquiterpenoid targeted genes, their derivatives, as well as their precursors and upregulated metabolites are listed.
Table 3. Reports on the metabolic engineering of monoterpenoids, sesquiterpenoid targeted genes, their derivatives, as well as their precursors and upregulated metabolites are listed.
SourceSpeciesTargeted GenesUp/DownregulatedReferences
Monoterpenoids (C15)
MenthaMentha spicataLimonene synthaseIncresead in sesquiterpenoid[73]
LourLitsea cubebaGeranyl diphosphate synthase small subunit 1Increase in monoterpene content[74]
LiliumLilium “Siberia”1-deoxy-d-xylulose-5-phosphate synthase, 1-deoxy-d-xylulose-5-phosphate reductoisomeraseLinalool (mono), Caryophyllene (sesqui)[75]
Mentha X piperitaNicotiania benthamiana & Nicotiania tabacumGeranyl diphosphate synthase small subunit(−) Limonene, (−)-Linalool, (−)-β-pinene, (−)-α-pinene, Myrcene[76]
Sweet osmanthusOsmanthus fragransTerpene synthaseβ-linalool, trans-β-ocimene, α-farnesene[77]
MenthaMentha spicataIPP isomerase & limonen synthase1,8-cineole, linalool, camphor, terpinene, lomonene,
borneol, safranal, geraniol, thymol, 1-α-terpineol, methyl eugenol, menthone, menthol-isomer, thymol, piperitone
English lavenderLavandula angustifoliaLinalool synthase, HMG-CoA reductaseLinalool[79]
SnapdragonAntirrhinum majusGeranyl diphosphate synthase small sub unitIncrease in monoterpene and sesquiterpene content[80]
TobaccoNicotiania tabacumϒ-Terpinene synthaseϒ-Terpinene, limonene, β-pinene and side products[81]
ArabidopsisArabidopsis thalinaLinalool/nerolidol synthaseLinalool, hydroxylated and glycosylated linalool[14]
TobaccoNicotiania tabacumLimonene synthaseLimonene[82]
PetuniaPetunia hybridaLinalool synthaseLinalool glycoside[10]
TomatoLycopersicon esculentumLinalool synthaseFruit-Linalool & hydroxylated linalool[11]
Sesquiterpenoids (C15)
Sweet wormwoodNicotiania benthamianaβ-caryophyllene synthaseβ-caryophyllene[83]
Sweet wormwoodArtemisia annuaSesquiterpene cyclaseAtremisinin[84]
FeverfewTanacetum partheniumParthenolide synthaseParthenolide[85]
Sweet wormwoodNicotiania benthamianaSesquiterpene synthaseAmorpha-4,11-diene & epi-cedrol[86]
LettuceLactuca sativaCostunolide synthaseCostunolide[87]
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Mani, V.; Park, S.; Kim, J.A.; Lee, S.I.; Lee, K. Metabolic Perturbation and Synthetic Biology Strategies for Plant Terpenoid Production—An Updated Overview. Plants 2021, 10, 2179.

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Mani V, Park S, Kim JA, Lee SI, Lee K. Metabolic Perturbation and Synthetic Biology Strategies for Plant Terpenoid Production—An Updated Overview. Plants. 2021; 10(10):2179.

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Mani, Vimalraj, Soyoung Park, Jin A Kim, Soo In Lee, and Kijong Lee. 2021. "Metabolic Perturbation and Synthetic Biology Strategies for Plant Terpenoid Production—An Updated Overview" Plants 10, no. 10: 2179.

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