Terpene Production Research Articles

Intraspecific correlations between growth and defence vary with resource availability and differ within and among populations

Abstract A paradigm in the plant defence literature is that defending against herbivores comes at a cost to growth, resulting in a growth–defence trade‐off. However, while there is strong evidence for growth–defence trade‐offs across species, evidence is mixed within species. Several mechanisms can account for this equivocal support within species, but teasing them apart requires examining growth–defence relationships both within and among populations, an approach seldom employed. We examined correlations between plant biomass (growth) and terpene production (defence) within and among populations of Monarda fistulosa, a perennial herb. We sampled populations from Montana and Wisconsin, regions that differ in resource availability characterized by different summer precipitation and associated abiotic conditions that influence plant productivity. We found negative, neutral and positive growth–defence correlations, depending on the scale examined. Negative correlations occurred across populations originating from divergent regions, positive correlations occurred across populations originating from within the high‐resource region and neutral correlations were found within single populations. Collectively, these results challenge the general expectation of ubiquitous trade‐offs and support emerging views that resource availability (as it affects productivity) shapes the evolution of defence at different scales. A free Plain Language Summary can be found within the Supporting Information of this article.

Storage-related changes of terpene constituents in caraway (Carum carvi L.) under real-time storage conditions

Caraway is a widespread agricultural crop that has been utilized by humans since ancient times. It is known as a kitchen spice, medicinal plant in phytotherapy and, due to its high essential oil content, as a fragrant plant and raw material for terpene production. Limonene and carvone, which in turn are used as precursors for the synthesis of further flavorings and fine chemicals, account for more than 90 % of the essential oil. However, the quality of essential oils can quickly deteriorate due to inadequate storage and by external impacts such as heat, oxygen, and light. In the present study, caraway seeds, caraway essential oil as well as pure standards of limonene and carvone were stored in parallel under real-time conditions at 25 °C and 40 °C to assess the effect of the plant matrix and the natural blend of compounds on essential oil stability. GC and HPLC analyses were used to show that the volatile compounds were well protected in the caraway seeds as no significant changes were determined. In comparison, a remarkable loss of limonene (22.4 %) due to oxidation-induced alterations was observed for the pure substance, while carvone contents remained almost constant over the entire storage period. The decline of limonene (9.2 %) as part of the essential oil was much lower while carvone again remained unchanged. The formation of novel minor compounds such as limonene-1,2-epoxide, carveol, and limonene-1,2-diol was mainly due to limonene downstream oxidation and increased with elevated storage temperatures. Volatile compounds were best protected in the intact plant seeds, and storage of the entire essential oil is preferable compared with isolated terpenes due to higher stability as representatively demonstrated for the major compound limonene.

Production of C20, C30 and C40 terpenes in the engineered phototrophic bacterium Rhodobacter capsulatus

Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as food-additives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic α-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production. Recently, we have developed modular tools allowing to engineer the biosynthesis of terpene precursors. These tools were now applied to boost the biosynthesis of the diterpene casbene, the triterpene squalene and the tetraterpene β-carotene in R. capsulatus SB1003. Selected enzymes of the intrinsic isoprenoid pathway and the heterologous mevalonate (MVA) pathway were co-expressed together with the respective terpene synthases in various combinations. Remarkably, co-expression of genes ispA, idi and dxs enhanced the synthesis of casbene and β-carotene. In contrast, co-expression of precursor biosynthetic genes with the squalene synthase from Arabidopsis thaliana reduced squalene titers. Therefore, we further employed four alternative pro- and eukaryotic squalene synthases. Here, the synthase from Methylococcus capsulatus enabled highest product levels of 90 mg/L squalene upon co-expression with ispA. In summary, we demonstrate the applicability of R. capsulatus for the heterologous production of diverse terpene classes and provide relevant insights for further development of such platforms.

Eine modulare In‐vitro‐Plattform für die Produktion von Terpenen und Polyketiden aus CO2

AbstractEines der Ziele für die Realisierung nachhaltiger chemischer Syntheseverfahren ist die direkte Nutzung des Treibhausgases CO2 als Rohstoff für die Produktion von Grund‐ und Feinchemikalien, wie z. B. Pharmazeutika, Duftstoffe und Lebensmittelzusatzstoffe. In dieser Studie präsentieren wir eine modulare In‐vitro‐Plattform für die kontinuierliche Umwandlung von CO2 in komplexe Kohlenstoff‐Verbindungen, wie Monoterpene (C10), Sesquiterpene (C15) und Polyketide. Durch Kombination von natürlichen und synthetischen Stoffwechselwegen etablierten wir eine neue Route von CO2 zu den Schlüsselintermediaten Acetyl‐ und Malonyl‐CoA, welche anschließend durch verschiedene Terpen‐ und Polyketidsynthasen diversifiziert werden können. In unserer Machbarkeitsstudie demonstrieren wir die Kombination verschiedener Stoffwechselmodule mit bis zu 29 Enzymen in einem Ansatz. Dies ebnet den Weg für die weitere Entwicklung und Optimierung von Syntheserouten für die Erzeugung komplexer CO2‐basierter Chemikalien.

A Modular In Vitro Platform for the Production of Terpenes and Polyketides from CO2.

A long‐term goal in realizing a sustainable biocatalysis and organic synthesis is the direct use of the greenhouse gas CO2 as feedstock for the production of bulk and fine chemicals, such as pharmaceuticals, fragrances and food additives. Here we developed a modular in vitro platform for the continuous conversion of CO2 into complex multi‐carbon compounds, such as monoterpenes (C10), sesquiterpenes (C15) and polyketides. Combining natural and synthetic metabolic pathway modules, we established a route from CO2 into the key intermediates acetyl‐ and malonyl‐CoA, which can be subsequently diversified through the action of different terpene and polyketide synthases. Our proof‐of‐principle study demonstrates the simultaneous operation of different metabolic modules comprising of up to 29 enzymes in one pot, which paves the way for developing and optimizing synthesis routes for the generation of complex CO2‐based chemicals in the future.

Inside Cover: A Modular In Vitro Platform for the Production of Terpenes and Polyketides from CO2 (Angew. Chem. Int. Ed. 30/2021)

Direct conversion of the greenhouse gas CO2 into value-added compounds remains a major challenge. Simultaneous one-pot coupling of a synthetic CO2-fixing cycle with a natural glyoxylate assimilation module and different natural product biosynthetic pathways enables the production of valuable terpenes and polyketides directly from CO2. Demonstration of this modular in vitro platform sets the stage for harnessing the greenhouse gas CO2 as source for the sustainable synthesis of multi-carbon compounds, as reported by Tobias J. Erb et al. in their Communication on page 16420.

Innentitelbild: Eine modulare In‐vitro‐Plattform für die Produktion von Terpenen und Polyketiden aus CO2 (Angew. Chem. 30/2021)

Die direkte Umwandlung des Treibhausgases CO2 in wertschöpfende Verbindungen bleibt eine große Herausforderung. Die simultane Kopplung eines synthetischen CO2-Fixierungszyklus mit einem natürlichen Glyoxylat-Assimilationsmodul und verschiedenen Naturstoff-Biosynthesewegen ermöglicht die Produktion von wertvollen Terpenen und Polyketiden direkt aus CO2. Die Demonstration dieser modularen In-vitro-Plattform schafft die Voraussetzungen für die Nutzung des Treibhausgases CO2 als Quelle für die nachhaltige Synthese von Multi-Kohlenstoff-Verbindungen, wie Tobias J. Erb et al. in ihrer Zuschrift auf S. 16556 schildern.

Sub-Tissue Localization of Phytochemicals in Cinnamomum camphora (L.) J. Presl. Growing in Northern Italy.

In the present paper, we focused our attention on Cinnamomum camphora (L.) J. Presl. (Lauraceae), studied at three levels: (i) micromorphological, with the analysis of the secretory structures and a novel in-depth histochemical characterization of the secreted compounds; (ii) phytochemical, with the characterization of the essential oils from young stems, fruits, and leaves, subjected to different conservation procedures (fresh, dried, stored at −20 °C, stored at −80 °C) and collected in two different years; (iii) bioactive, consisting of a study of the potential antibacterial activity of the essential oils. The micromorphological investigation proved the presence of secretory cells characterized by a multi-layered wall in the young stems and leaves. They resulted in two different types: mucilage cells producing muco-polysaccharides and oil cells with an exclusive terpene production. The phytochemical investigations showed a predominance of monoterpenes over sesquiterpene derivatives; among them, the main components retrieved in all samples were 1,8-cineole followed by α-terpineol and sabinene. Conservation procedures seem to only influence the amounts of specific components, i.e., 1,8-cineole and α-terpineol, while analyses on each plant part revealed the presence of some peculiar secondary constituents for each of them. Finally, the evaluation of the antibacterial activity of the essential oil showed a promising activity against various microorganisms, as Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. In conclusion, we combined a micromorphological and phytochemical approach of the study on different plant parts of C. camphora, linking the occurrence of secretory cells to the production of essential oils. We compared, for the first time, the composition of essential oils derived from different plant matrices conserved with different procedures, allowing us to highlight a relation between the conservation technique and the main components of the profiles. Moreover, the preliminary antibacterial studies evidenced the potential activity of the essential oils against various microorganisms potentially dangerous for plants and humans.

Micromorphological and phytochemical survey of Ballota acetabulosa (L.) Benth.

Within the Open Science project entitled ‘Botanic Garden, factory of molecules’, a multidisciplinary study approach was applied to Ballota acetabulosa (L.) Benth., at the Ghirardi Botanic Garden (Toscolano Maderno, BS, Italy).Micromorphological and histochemical investigations were performed on the secreting structures of the vegetative and reproductive organs under light, fuorescence and electronic microscopy. Concurrently the characterization of the volatiles spontaneously emitted from leaves and flowers were examined.Four trichome morphotypes were identified: peltate and short‐stalked, medium‐stalked and long‐stalked capitate trichomes, each with a specific distribution pattern. The histochemical analysis was confirmed using ultrastructural observations, with the peltates and long‐stalked capitates as the main sites responsible for terpene production. The head‐space characterization revealed that sesquiterpene hydrocarbons dominated both in leaves and flowers, with γ‐muurolene, β‐caryophyllene and (E)‐nerolidol as the most abundant compounds. Moreover, a comparison with literature data concerning the ecological roles of the main compounds suggested their dominant roles in defence, both at the leaf and flower level.Hence, we correlated the trichome morphotypes with the production of secondary metabolites in an attempt to link these data to their potential ecological roles. Finally, we made the obtained scientific knowledge available to visitors of the Botanic Garden through the realization of new labelling dedicated to B. acetabulosa that highlights the ‘invisible’, microscopic features of the plant.

First time \u03b2-farnesene production by the versatile bacterium Cupriavidus necator

BackgroundTerpenes are remarkably diverse natural structures, which can be formed via two different pathways leading to two common intermediates. Among those, sesquiterpenes represent a variety of industrially relevant products. One important industrially produced product is β-farnesene as a precursor for a jet fuel additive. So far, microbial terpene production has been mostly limited to known production hosts, which are only able to grow on heterotrophic substrates.ResultsIn this paper, we for the first time describe β-farnesene production by the versatile bacterial host Cupriavidus necator on fructose, which is known to grow hetero- and autotrophically and even in bioelectrochemical systems. We were able to show a growth-dependent production of β-farnesene by expressing the β-farnesene synthase from Artemisia annua in C. necator H16 PHB-4. Additionally, we performed a scale-up in a parallel reactor system with production titers of 26.3 ± 1.3 µM β-farnesene with a fed-batch process.ConclusionsThe β-farnesene production titers reported in this paper are not in the same range as titers published with known heterotrophic producers E. coli or S. cerevisiae. However, this proof-of-principle study with C. necator as production host opens new synthesis routes toward a sustainable economy and leaves room for further optimizations, which have been already performed with the known production strains.

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

BackgroundMitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates.ResultsThis study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement.ConclusionsThis work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

In Silico Mining of Terpenes from Red-Sea Invertebrates for SARS-CoV-2 Main Protease (Mpro) Inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic, which generated more than 1.82 million deaths in 2020 alone, in addition to 83.8 million infections. Currently, there is no antiviral medication to treat COVID-19. In the search for drug leads, marine-derived metabolites are reported here as prospective SARS-CoV-2 inhibitors. Two hundred and twenty-seven terpene natural products isolated from the biodiverse Red-Sea ecosystem were screened for inhibitor activity against the SARS-CoV-2 main protease (Mpro) using molecular docking and molecular dynamics (MD) simulations combined with molecular mechanics/generalized Born surface area binding energy calculations. On the basis of in silico analyses, six terpenes demonstrated high potency as Mpro inhibitors with ΔGbinding ≤ −40.0 kcal/mol. The stability and binding affinity of the most potent metabolite, erylosides B, were compared to the human immunodeficiency virus protease inhibitor, lopinavir. Erylosides B showed greater binding affinity towards SARS-CoV-2 Mpro than lopinavir over 100 ns with ΔGbinding values of −51.9 vs. −33.6 kcal/mol, respectively. Protein–protein interactions indicate that erylosides B biochemical signaling shares gene components that mediate severe acute respiratory syndrome diseases, including the cytokine- and immune-signaling components BCL2L1, IL2, and PRKC. Pathway enrichment analysis and Boolean network modeling were performed towards a deep dissection and mining of the erylosides B target–function interactions. The current study identifies erylosides B as a promising anti-COVID-19 drug lead that warrants further in vitro and in vivo testing.

Carbon sources to enhance the biosynthesis of useful secondary metabolites in Fusarium verticillioides submerged cultures

Fusarium verticillioides is a prolific producer of useful secondary metabolites such as naphthoquinone pigments, monoterpenes, and sesquiterpenes, as well as the harmful mycotoxins fumonisins. A strategy to increase their production includes creating a proper nutritional environment that enables the fungus to produce the compounds of interest. The aim of the present work was to study the effect of different carbon sources (glucose, fructose, xylose, sucrose, and lactose) on secondary metabolites biosynthesis in F. verticillioides submerged cultures. The production of volatile terpenes was evaluated through gas chromatography coupled to mass spectrometry. The quantification and identification of pigments was conducted using a UV/VIS spectrophotometer and NMR spectrometer, respectively. The quantification of fumonisin B1 and fumonisin B2 was performed by high-performance liquid chromatography. Our results showed that the biosynthesis of naphthoquinone pigments, monoterpenes, and sesquiterpenes was highest in cultures with fructose (13.00 ± 0.71mmol/g), lactose [564.52 × 10-11 ± 11.50 × 10-11μg/g dry weight (DW)], and xylose (54.41 × 10-11 ± 1.55 × 10-11μg/g DW), respectively, with fumonisin being absent or present in trace amounts in the presence of these carbon sources. The highest biosynthesis of fumonisins occurred in sucrose-containing medium (fumonisin B1: 7.85 × 103 ± 0.25 × 103μg/g DW and fumonisin B2: 0.38 × 103 ± 0.03 × 103μg/g DW). These results are encouraging since we were able to enhance the production of useful fungal metabolites without co-production with harmful mycotoxins by controlling the carbon source provided in the culture medium.

Cyclodextrins: a tool in plant cell and organ culture bioprocesses for the production of secondary metabolites

Plant cell culture bioprocesses have become an alternative source for bioactive compound production to the traditional extraction technology from plants grown in nature or through agricultural techniques. Thus, environmental conditions could make the supply of these secondary metabolites intermittent and heterogeneous. Therefore, plant cell cultures assure a continuous product supply, homogeneous production, fulfilling GMP requirements. Elicitation has been an extensively used strategy to boost secondary metabolite production in plant cell cultures. Cyclodextrins, the naturally occurring cyclic oligosaccharides of glucose residues, has emerged as an elicitor that can trigger plant cell defense responses and secondary metabolite accumulation. Moreover, CDs can form complex with most of the plant secondary compounds, thus contribute to product removal, which results in the elimination of feedback inhibition of product biosynthesis and also prevents product degradation and toxicity against the plant cells. In the present manuscript, we collect and compare the effects of cyclodextrins on different secondary metabolite pathways in plant cell culture processes. We review different case studies, which includes the production of phenylpropanoids, terpenes, alkaloids, naphthoquinones and anthraquinones derivatives. In the present manuscript, we collect and compare the effects of cyclodextrins on different secondary metabolite pathways in plant cell culture, which include the production of phenylpropanoids, terpenes, alkaloids, naphthoquinones and anthraquinones derivatives. Cyclodextrins trigger secondary metabolite production in plant cell cultures and product release. Moreover, they form complexes with these bioactive compounds, reduce feedback inhibition biosynthesis and their degradation.

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

BackgroundLinalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes.ResultsHere, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic β-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in “dual-phase” fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes.ConclusionsThis study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

Chelativorans alearense sp. nov., A Novel Bacterial Species Isolated From Soil in Alear, China.

A novel Gram-strain-negative, rod-shaped, non-flagellated, non-gliding, beige-pigmented and aerobic bacterium, designated strain UJN715T, was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alear city, located in Xinjiang province, PR China. Growth optimally occurred at 37°C, pH 6.5-7.5, and 0-3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain UJN715T belonged to the genus Chelativorans, with the highest sequence similarity to Chelativorans multitrophicus DSM 9103T (97.7%). Genome sequencing revealed a genome size of 5 702 301bp and a G + C content of 64.1mol%. The ANI, POCP and the dDDH between strain UJN715T and C. multitrophicus DSM 9103T were 76.2%, 49.3%, and 20.5%, respectively. The prediction result of secondary metabolites based on genome showed that the strain UJN715T contained one cluster of ectoine production, one cluster of non-ribosomal peptide synthetase (NRPS), one cluster of type I polyketide synthases (TIPKS), one cluster of bacteriocin, one cluster of TfuA-related, one cluster of N-acetylglutaminylglutamine amide (NAGGN) production, one cluster of terpene production, two clusters of homoserine lactone (Hserlactone) production. The major respiratory quinone was Q-10. The major fatty acids were iso-C17:0, C18:0 and C19:0 cyclo ω8c and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phospholipids, unknown lipids, diphosphatidylglycerol, aminoglycolipid, unidentified aminophospholipids. On the basis of these data, strain UJN715T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans alearense sp. nov. is proposed. The type strain is UJN715T (= KCTC 72856T = CCTCC AB2019378T).

Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function

The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation-a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.

Enhanced limonene production in a fast-growing cyanobacterium through combinatorial metabolic engineering

Terpenoids are a large and diverse group of natural products with commercial applications. Microbial production of terpenes is considered as a feasible approach for the stable supply of these complex hydrocarbons. Cyanobacteria, photosynthetic prokaryotes, are attractive hosts for sustainable bioproduction, because these autotrophs require only light and CO2 for growth. Despite cyanobacteria having been engineered to produce a variety of compounds, their productivities of terpenes are generally low. Further research is needed to determine the bottleneck reactions for enhancing terpene production in cyanobacteria. In this study, we engineered the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce a commercially-used terpenoid, limonene. We identified a beneficial mutation in the gene encoding geranylgeranyl pyrophosphate synthase crtE, leading to a 2.5-fold increase in limonene production. The engineered strain produced 16.4 ​mg ​L−1 of limonene at a rate of 8.2 ​mg ​L−1 day−1, which is 8-fold higher than limonene productivities previously reported in other cyanobacterial species. Furthermore, we employed a combinatorial metabolic engineering approach to optimize genes involved in the upstream pathway of limonene biosynthesis. By modulating the expression of genes encoding the enzymes in the MEP pathway and the geranyl pyrophosphate synthase, we showed that optimization of the expression level is critical to enhance limonene production in cyanobacteria.

Transcriptomics Integrated with Free and Bound Terpenoid Aroma Profiling during "Shine Muscat" (Vitis labrusca × V. vinifera) Grape Berry Development Reveals Coordinate Regulation of MEP Pathway and Terpene Synthase Gene Expression.

Terpenes and their derivatives are important biomarkers of grape quality as they contribute to the flavor and aroma of grapes. However, the molecular basis of terpene biosynthesis throughout the grapevine phenological developmental cycle remains elusive. Our current study investigates the free and bound terpene biosynthesis of berries at different phenological stages from preveraison to harvest. Detailed gene expression (transcriptomics) analysis, terpenoid volatile production by gas chromatography-mass spectrometry (GC-MS), and in planta transient expression were employed. Our results show that concentrations of most individual terpenes at different stages are distinctive and increase from preveraison to the veraison stage followed by a decrease from veraison to maturity. The combined transcriptomic analysis and terpene profiling revealed that 22 genes belonging to the MEP pathway and multiple classes of transcription factor family members including bHLH and several hormone biosynthesis- or signaling-related genes likely participate in the regulation of terpenoid biosynthesis according to their specific expression patterns in berries. Quantitative real-time polymerase chain expression analysis of 8 key differentially expressed genes in MEP pathways and further 12 randomly selected genes was performed during 8 sampling stages and validated the RNA-seq-derived expression profiles. To further confirm the function of a subset of the differentially expressed genes, we investigated the effects of combined overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (VvDXS1-LOC100249323), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (VvDXR-LOC100248516), and terpene synthase (VvTPS56-LOC100266449) on the production of terpenes by transient overexpression in Nicotiana benthamiana leaves. The overall developmental patterns of total terpenes and gene expression profiles will help guide the functional analyses of further candidate genes important for terpene biosynthesis of grape as well as identifying the master transcriptional and hormonal regulators of this pathway in the future.

Glandular Trichome-Derived Mono- and Sesquiterpenes of Tomato Have Contrasting Roles in the Interaction with the Potato Aphid Macrosiphum euphorbiae.

Secondary metabolites produced in glandular trichomes of tomato are involved in interactions with herbivores. In cultivated tomato (Solanum lycopersicum) glandular trichomes accumulate a blend of abundant monoterpenes and smaller amounts of a few sesquiterpenes. These mono- and sesquiterpenes are synthesized by three terpene synthases, TPS20 as well as TPS9 and TPS12, respectively. To study effects of these terpenes on performance and choice behavior of potato aphid (Macrosiphum euphorbiae), we utilized two tomato trichome mutants, hairless and odorless-2, that are differently affected in mono- and sesquiterpene production. Non-choice assays demonstrated that longevity and fecundity of M. euphorbiae were increased when kept on the trichome mutants. A principal component analysis of these aphid performance parameters and terpene production in the trichome mutants indicated that longevity and fecundity of M. euphorbiae were negatively correlated with production of the TPS12-derived sesquiterpenes β-caryophyllene and α-humulene. While we had previously shown that addition of pure β-caryophyllene/α-humulene to an artificial feeding diet affected M. euphorbiae apterae survivorship and feeding behavior, no such effects were observed here upon addition of a mixture of pure TPS20-derived monoterpenes. In olfactometer assays M. euphorbiae alates displayed differential choice behaviors towards the hairless and odorless-2 mutants suggesting a role of TPS20-derived monoterpenes in aphid attraction, which was further confirmed using a mixture of pure monoterpenes. Our analyses revealed contrasting roles of glandular trichome-derived terpenes in S. lycopersicum. While TPS12-derived sesquiterpenes contribute to host plant resistance against M. euphorbiae, TPS20-derived monoterpenes appear to be exploited as cue for host plant orientation by aphids.