Terpene Production Research Articles

Functional characterization of an Indian sandalwood (Santalum album L.) dual-localized bifunctional nerolidol/linalool synthase gene involved in stress response

Essential oils extracted from the heartwood of Indian sandalwood (Santalum album L.) contain linalool and nerolidol as minor components. However, nerolidol/linalool synthase (NES/LIS), which produce linalool and nerolidol, have yet to be characterized in sandalwood. Using a transcriptomic-based approach, a terpene synthase gene was screened from unigenes of transcriptome data derived from S. album seedlings exposed to low temperature (4 °C). The enzyme encoded by these complementary DNAs belongs to the TPS-b clade. Recombinant SaNES/LIS is a bifunctional enzyme that can catalyze the formation of (E)-nerolidol from farnesyl diphosphate and linalool from geranyl diphosphate, respectively. Whereas SaNES/LIS was primarily localized in chloroplastids, both as granular fluorescence and as diffuse fluorescence, it was also detected in the cytosol of a limited number of cells. Agrobacterium tumefaciens-mediated transient gene expression in planta produced the same terpene products as those obtained in vitro. Real-time PCR analysis showed the highest expression of SaNES/LIS in fruits, with about a three-fold higher level than in leaves, followed by flowers, heartwood and roots. SaNES/LIS transcripts were differentially activated in different tissues in response to methyl jasmonate, cold, high temperature, strong illumination, and drought stress. Our results provide novel insight into the role of sandalwood terpenoids in response to various environmental stresses.

Evolution of the Polyphenol and Terpene Content, Antioxidant Activity and Plant Morphology of Eight Different Fiber-Type Cultivars of Cannabis Sativa L. Cultivated at Three Sowing Densities.

The chemical composition of the inflorescences of eight different fibre-type Cannabis sativa L. cultivars grown in Switzerland was monitored for different sowing densities over the season 2019. HPLC-MS, GC-MS and GC-FID, as well as spectrophotometric techniques were used to measure the total phenolic content (TPC) and the antioxidative activity of the inflorescence extracts, and to characterise and quantify the flavonoids and terpenes produced by the different cultivars over different sowing densities from July to September 2019. The main finding of the present study is that the TPC, as well as the individual flavonoids and terpenes, were mainly influenced by the harvest period and the phenological stage of the plant. The content of polyphenols and flavonoids decrease during the flower development for all cultivars studied. The terpene content increased with maturation. The monoterpenes/sesquiterpenes ratio also changed between the early flowering (majority of sesquiterpenes) and the end of flowering (majority of monoterpenes). The sowing density showed an impact on plant morphology, a low density such as 30 seeds/m2 influencing the production of bigger flowers, thus increasing the yield of polyphenols and terpenes production. Therefore, hemp inflorescences can be regarded as valuable by-products of fibre production, for their valorisation in the food and beverage industry in addition to cosmetics and perfumery.

Whole-Cell Production of Patchouli Oil Sesquiterpenes in Escherichia coli: Metabolic Engineering and Fermentation Optimization in Solid-Liquid Phase Partitioning Cultivation.

Patchouli oil is a major ingredient in perfumery, granting a dark-woody scent due to its main constituent (−)-patchoulol. The growing demand for patchouli oil has raised interest in the development of a biotechnological process to assure a reliable supply. Herein, we report the production of patchouli oil sesquiterpenes by metabolically engineered Escherichia coli strains, using solid–liquid phase partitioning cultivation. The (−)-patchoulol production was possible using the endogenous methylerythritol phosphate pathway and overexpressing a (−)-patchoulol synthase isoform from Pogostemon cablin but at low titers. To improve the (−)-patchoulol production, the exogenous mevalonate pathway was overexpressed in the multi-plasmid PTS + Mev strain, which increased the (−)-patchoulol titer 5-fold. Fermentation was improved further by evaluating several defined media, and optimizing the pH and temperature of culture broth, enhancing the (−)-patchoulol titer 3-fold. To augment the (−)-patchoulol recovery from fermentation, the solid–liquid phase partitioning cultivation was analyzed by screening polymeric adsorbers, where the Diaion HP20 adsorber demonstrated the highest (−)-patchoulol recovery from all tests. Fermentation was scaled-up to fed-batch bioreactors, reaching a (−)-patchoulol titer of 40.2 mg L–1 and productivity of 20.1 mg L–1 d–1. The terpene profile and aroma produced from the PTS + Mev strain were similar to the patchouli oil, comprising (−)-patchoulol as the main product, and α-bulnesene, trans-β-caryophyllene, β-patchoulene, and guaia-5,11-diene as side products. This investigation represents the first study of (−)-patchoulol production in E. coli by solid–liquid phase partitioning cultivation, which provides new insights for the development of sustainable bioprocesses for the microbial production of fragrant terpenes.

Connecting moss lipid droplets to patchoulol biosynthesis.

Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. Here, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Three different LD-proteins: Oleosin (PpOLE1), Lipid Droplet Associated Protein (AtLDAP1) and Seipin (PpSeipin325) were tested as anchors. Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis. The expression of PTS physically linked to Seipin increased the LD size and the retention of patchoulol in the cell. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants.

The heterologous production of terpenes by the thermophile Parageobacillus thermoglucosidasius in a consolidated bioprocess using waste bread

Parageobacillus thermoglucosidasius is a genetically tractable thermophile that grows rapidly at elevated temperatures, with a doubling time at 65 °C comparable to the shortest doubling times of Escherichia coli. It is capable of using a wide variety of substrates, including carbohydrate oligomers, and has been developed for the industrial production of ethanol. In this study, P. thermoglucosidasius NCIMB11955 has been engineered to produce the sesquiterpene τ-muurolol by introduction of a heterologous mevalonate pathway constructed using genes from several thermophilic archaea together with a recently characterised thermostable τ-muurolol synthase. P. thermoglucosidasius naturally uses the methylerythritol phosphate pathway for production of the terpene precursor, isopentenyl pyrophosphate, while archaea use a version of the mevalonate pathway. By introducing the orthogonal archaeal pathway it was possible to increase the flux through to sesquiterpene biosynthesis. Construction of such a large metabolic pathway created problems with genetic vector introduction and stability, so recombinant plasmids were introduced by conjugation, and a thermostable serine integrase system was developed for integration of large pathways onto the chromosome. Finally, by making the heterologous pathway maltose-inducible we demonstrate that the new strain is capable of using waste bread directly as an autoinduction carbon source for the production of terpenes in a consolidated bioprocess.

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks

BackgroundIn an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass.ResultsWe developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A.ConclusionOur findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

Probing of the plasticity of the active site in pinene synthase elucidates its potential evolutionary mechanism

Terpenes form a class of highly diverse natural products. The diversity of terpenes is created by terpene synthases. During the reaction, carbocation intermediates form and their rearrangement could lead to the formation of various products. Terpene synthases determine the final product profile by controlling the conformation of the intermediate or stabilizing the carbocation. Pinene synthase is a monoterpene synthase catalyzing the cyclization of geranyl pyrophosphate (GPP) to form pinene. Our study suggests that by mutating the aromatic residue F482 to Ala, Val, Ile and Thr, the enzyme can be converted to sabinene synthase, with more than 90% of its total terpene products becoming sabinene, which indicates the aromaticity of this residue is essential for stabilizing the pinyl carbocation. We also identified a mutation S491A that could cause an about 29% increase in the overall activity of the enzyme without altering its produce selectivity. Molecular dynamic simulation indicates this mutation could decrease the flexibility of the enzyme when it forms a complex with the pinyl carbocation. Our study suggests the active pocket of pinene synthase has a certain level of plasticity, making it relatively easy to change the product selectivity or overall activity. This property could have an important implication in the evolution of terpene synthases and thereby terpene diversity, as by changing a few residues an enzyme could synthesize a completely different terpene product in a significant amount, which allows selection to take place.

Lavandula dentata from Italy: Analysis of Trichomes and Volatiles.

This study presented a micromorphological and phytochemical survey on Lavandula dentata L. cultivated at the Ghirardi Botanic Garden (Toscolano Maderno, BS, Italy). The morphological investigation revealed the presence of peltate, short- and medium-stalked capitate trichomes. The histochemical survey showed terpene production by peltates and medium-stalked capitates, hydrophilic secretions by short-stalked capitates. The phytochemical survey was developed on leaf and flower volatile organic compounds (VOCs) and on the essential oil (EO) from the flowering aerial parts. The VOC profiles represented an element of novelty and were dominated by oxygenated monoterpenes, among which 1,8-cineole and β-pinene were the most abundant (77.40 %, 7.11 % leaves; 81.08 %, 10.46 % flowers). The EO of L. dentata was dominated by oxygenated monoterpenes with a high percentage of 1,8-cineole (69.08 %), followed by β-pinene, trans-pinocarveol and myrtenal. Evaluations about the ecological role, the potential biological activity and the sensory attributes were proposed, based on literature contributions.

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, β-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

Chemical defence responses of Norway spruce to two fungal pathogens

AbstractConstitutive and inducible terpene production is involved in conifer resistance against insects and fungal infestations. To gain knowledge about local defence responses of Norway spruce bark against pathogens and to find potential chemical markers for resistance breeding, we inoculated the stem of 8‐year‐old Norway spruce (Picea abies) clonal trees with both Endoconidiophora polonica (Ep, a common fungal pathogen associated with the spruce bark beetle Ips typographus) and Heterobasidion parviporum (Hp, a severe pathogen causing root and stem rot disease). Three weeks after inoculation, the fungal‐inoculated and intact bark from each tree was sampled. The terpenes in tree bark were extracted with hexane and characterized by gas chromatography–mass spectrometry (GC‐MS). The two fungi induced varied terpene responses in the four spruce clones used. Three of the clones showed a 2.3‐fold to 5.7‐fold stronger terpene response to Hp relative to Ep inoculation, while one clone responded similarly to inoculation with the two fungal pathogens. The amount of the diterpenes thunbergol and geranyllinalool varied between the clones. The level of thunbergol was higher in both intact and fungal‐inoculated bark from the less susceptible clones compared with the more susceptible clones. Geranyllinalool was present in higher amounts in the susceptible clones and is thus a possible marker for susceptibility. Our observations show that Norway spruce employs a similar chemical mechanism against the two fungal pathogens. Based on the present and earlier published data, we suggest that certain Norway spruce genotypes have a strong defence reaction against these two pathogens. The diterpenes thunbergol and geranyllinalool might be useful markers of susceptibility in tree‐breeding programmes and should be the focus of further detailed investigations.

Chelativorans xinjiangense sp. nov., a novel bacterial species isolated from soil in Xinjiang, China

A novel Gram-strain-negative, beige-pigmented, aerobic, rod-shaped, non-flagellated and non-gliding bacterium, designated strain lm93T, was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alar city, located in Xinjiang province, China. Growth optimally occurred at 30°C, pH 6.5-7.5, and 0-2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain lm93T belonged to the genus Chelativorans, with highest sequence similarity to Chelativorans multitrophicus DSM 9103T (96.9%). Genome sequencing revealed a genome size of 5 689 708bp and a G + C content of 64.3mol%. The ANI, POCP and the dDDH between strain lm93T and C. multitrophicus DSM 9103T were 76.4%, 54.8% and 0.8%, respectively. The prediction result of secondary metabolites based on genome showed that the strain lm93T contained one cluster of bacteriocin, one cluster of terpene production, two clusters of ectoine production, one cluster of non-ribosomal peptide synthetase, one cluster of type I polyketide synthases, three clusters of homoserine lactone production, one cluster of N-acetylglutaminylglutamine amide production and one cluster of phosphonate production. The major respiratory quinone was Q-10. The major fatty acids were C19:0 cyclo ω8c, iso-C17:0 and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, aminoglycolipid, three unknown lipids and diphosphatidylglycerol. On the basis of these data, strain lm93T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans xinjiangense sp. nov. is proposed. The type strain is lm93T (= KCTC 72857T = CCTCC AB2019376T).

Synthetic biology, systems biology, and metabolic engineering of Yarrowia lipolytica toward a sustainable biorefinery platform.

Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemicals and drop-in transportation fuels. The unique acetyl-CoA/malonyl-CoA supply mode along with the versatile carbon-utilization pathways makes this yeast a superior host to upgrade low-value carbons into high-value secondary metabolites and fatty acid-based chemicals. The expanded synthetic biology toolkits enabled us to explore a large portfolio of specialized metabolism beyond fatty acids and lipid-based chemicals. In this review, we will summarize the recent advances in genetic, omics, and computational tool development that enables us to streamline the genetic or genomic modification for Y. lipolytica. We will also summarize various metabolic engineering strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA pathway for production of complex oleochemicals, polyols, terpenes, polyketides, and commodity chemicals. We envision that Y. lipolytica will be an excellent microbial chassis to expand nature's biosynthetic capacity to produce plant secondary metabolites, industrially relevant oleochemicals, agrochemicals, commodity, and specialty chemicals and empower us to build a sustainable biorefinery platform that contributes to the prosperity of a bio-based economy in the future.

Overexpression of the Key Enzymes in the Methylerythritol 4-phosphate Pathway in Corynebacterium glutamicum for Improving Farnesyl Diphosphate-Derived Terpene Production.

A systematic and combinatorial optimization has been employed to metabolically engineer microbes for identifying key gene targets for overexpression to increase the intermediate pools for terpenoid production. Herein, the methylerythritol 4-phosphate (MEP) pathway in Corynebacterium glutamicum, an industrial host, was investigated to identify the key genes whose overexpression would improve the production of farnesyl diphosphate (FPP)-derived terpenoids (squalene and α-farnesene). Using a combinatorial approach with the single, double, and triple expression of genes in the MEP pathway in a high-throughput fermentation, overexpression of the ispDF genes, along with the known dxs and idi genes, was most effective at increasing the squalene contents, i.e., by 14-fold. The dxr gene was identified as the key target enzyme for α-farnesene production. This result could provide fundamental information for improving the metabolic engineering of C. glutamicum for terpene production via an optimized MEP pathway.

Contiguous Quaternary Carbons: A Selection of Total Syntheses.

Contiguous quaternary carbons in terpene natural products remain a major challenge in total synthesis. Synthetic strategies to overcome this challenge will be a pivotal prerequisite to the medicinal application of natural products and their analogs or derivatives. In this review, we cover syntheses of natural products that exhibit a dense assembly of quaternary carbons and whose syntheses were uncompleted until recently. While discussing their syntheses, we not only cover the most recent total syntheses but also provide an update on the status quo of modern syntheses of complex natural products. Herein, we review (±)-canataxpropellane, (+)-waihoensene, (–)-illisimonin A and (±)-11-O-debenzoyltashironin as prominent examples of natural products bearing contiguous quaternary carbons.

Isolation and functional characterization of four microbial type terpene synthases from ferns

Typical plant terpene synthases (TPSs) are responsible for the production of terpenes, a major class of plant secondary metabolites. However, various nonseed plants also harbor genes encoding microbial terpene synthase-like (MTPSL) enzymes. Here, a scan of 31 ferns transcriptomes revealed 40 sequences putatively encoding MTPSLs. Two groups of sequences were recognized based on the key conserved motifs. Four representative genes were isolated from each of the four species Adiantum capillus-veneris, Cyclosorus parasiticus, Drynaria bonii and Microlepia platyphylla. Following their heterologous expression in E. coli, the recombinant proteins were tested for monoterpene synthase and sesquiterpene synthase activity. These enzymatic products were typical monoterpenes and sesquiterpenes that have been previous shown to be generated by classical plant TPSs when provided with GPP and FPP as substrates. Subcellular localization experiments in the leaf epidermis of Nicotiana benthamiana and onion (Allium cepa) inner epidermal cells indicated that AcMTPSL1 and DbMTPSL were deposited in both the cytoplasm and nucleus, whereas CpMTPSL1 and MpMTPSL were localized in the cytoplasm, chloroplasts and nucleus. AcMTPSL1 was up-regulated in plants exposed to methyl jasmonate treatment, suggesting a role for this gene in host defense. This study provides more information about the catalytic function of MTPSLs in nonseed plants and for the first time, the subcellular localization of MTPSLs was experimentally characterized.

Production of multiple terpenes of different chain lengths by subcellular targeting of multi-substrate terpene synthase in plants

Multi-substrate terpene synthases (TPSs) are distinct from typical TPSs that react with a single substrate. Although in vitro activity of few multi-substrate TPSs have been reported, in vivo characterization has not been well investigated for most of them. Here, a new TPS from Cananga odorata, CoTPS5, belonging to TPS-f subfamily was functionally characterized in vitro as well as in vivo. CoTPS5 reacted with multiple prenyl-pyrophosphate substrates of various chain lengths as a multi-substrate TPS. It catalyzed the formation of (E)-β-ocimene, (E,E)-α-farnesene and α-springene from geranyl pyrophosphate, (E,E)-farnesyl pyrophosphate and geranylgeranyl pyrophosphate, respectively. Upon transient expression in Nicotiana benthamiana, CoTPS5 localized to cytosol and produced only (E,E)-α-farnesene. However, expression of plastid-targeted CoTPS5 in N. benthamiana resulted in biosynthesis of all three compounds, (E)-β-ocimene, (E,E)-α-farnesene and α-springene. Similarly, transgenic Arabidopsis plants overexpressing plastid-targeted CoTPS5 showed stable and sustainable production of (E)-β-ocimene, (E,E)-α-farnesene and α-springene. Moreover, their production did not affect the growth and development of transgenic Arabidopsis plants. Our results demonstrate that redirecting multi-substrate TPS to a different intracellular compartment could be an effective way to prove in vivo activity of multi-substrate TPSs and thereby allowing for the production of multiple terpenoids simultaneously in plants.

Natural product-inspired aryl isonitriles as a new class of antimalarial compounds against drug-resistant parasites

Malaria is a prevalent and deadly disease. The fast emergence of drug-resistant malaria parasites makes the situation even worse. Thus, developing new chemical entities, preferably with novel mechanisms of action, is urgent and important. Inspired by the complex and scarce isonitrile-containing terpene natural products, we evaluated a collection of easily prepared synthetic mono- and bis-isonitrile compounds, most of which feature a simple, but rigid stilbene backbone. From this collection, potent antimalarial lead compounds with EC50 value ranging from 27 to 88 nM against the Dd2 strain using a blood stage proliferation assay were identified. Preliminary SAR information showed that the isonitrile group is essential for the observed activity against the Dd2 strain and the bis-isonitrile compounds in general perform better than the corresponding mono-isonitrile compounds.

Photoreactivity of Oil and Alcoholic Diterpenic Varnishes: Key Role of the Polymerization Process

AbstractThe conservation of artworks is a key issue in cultural heritage. The alterations of the varnish could be linked mainly to the photopolymerization of the natural resin. The photodegradation of sandarac and rosin under simulated solar light was studied using a multianalytical approach using a second derivative FT‐IR spectroscopy and gas chromatography coupled to mass spectroscopy. In all resins, main terpene molecules underwent esterification and perestererifications via the formation of hydroperoxides preferentially formed on tertiary carbons. This polymerization could be associated with the advice of important art manuals and treatises recommending exposing varnishes to the sunlight while drying. In a second step, these esters and perester bonds underwent photoscissions by Norrish I and II reactions, leading to the production of free terpenes in the varnishes. In sandarac varnishes, the vinyl bonds showed a strong reactivity associated with the formation of trans and cis secondary alkenes. These photochemical reactions could explain an important part of the problem of conservation of the varnish layers experienced by museums all over the world.

Increased glycosidase activities improved the production of wine varietal odorants in mixed fermentation of P. fermentans and high antagonistic S. cerevisiae.

A selected Pichia fermentans strain was simultaneously and sequentially inoculated in synthetic and real juice with S. cerevisiae strains of different antagonistic activities in a ratio 1:1 to observe the correlation between varietal odorants and glycosidase activities. Fermentations using pure S. cerevisiae strains were used for comparison. Yeast biomass and glycosidase activities were monitored, varietal odorants were detected using HS-SPME-GC/MS during fermentation. The final wine aroma attributes were analyzed by trained panelists. Results showed that co-inoculation with high antagonistic S. cerevisiae resulted in higher glycosidase activities than others. Pearson correlation analysis indicated that yeast biomass was positively related to glycosidase activities during fermentation. The increase in glycosidase activities was the main reason for the higher production of terpenes and C13-norisoprenoids, and for the lower C6 compound content, which lead to superior fruity and floral aromas in the final wine samples of the high antagonistic S. cerevisiae group.

Production of the Inaccessible Sesquiterpene (-)-5-Epieremophilene by Metabolically Engineered Escherichia coli.

(-)-5-Epieremophilene, an epimer of the versatile sesquiterpene (+)-valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1-3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (-)-5-epieremophilene production with yields of 42.4-76.0 mg/L in shake-flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1-FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (-)-5-epieremophilene were achieved. Biological assay indicated that (-)-5-epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC50 =1.25 μg/cm2 ), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (-)-5-epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology.