Sesquiterpene Synthase Research Articles

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch

AbstractSesquiterpene synthases (STPSs) catalyze carbocation‐driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation‐reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (−)‐cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (−)‐cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo‐electron microscopy‐derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure‐guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311‐S419‐C458]/[M311‐V419‐A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Whole-Transcriptome Profiling and Functional Prediction of Long Non-Coding RNAs Associated with Cold Tolerance in Japonica Rice Varieties.

Low-temperature chilling is a major abiotic stress leading to reduced rice yield and is a significant environmental threat to food security. Low-temperature chilling studies have focused on physiological changes or coding genes. However, the competitive endogenous RNA mechanism in rice at low temperatures has not been reported. Therefore, in this study, antioxidant physiological indices were combined with whole-transcriptome data through weighted correlation network analysis, which found that the gene modules had the highest correlation with the key antioxidant enzymes superoxide dismutase and peroxidase. The hub genes of the superoxide dismutase-related module included the UDP-glucosyltransferase family protein, sesquiterpene synthase and indole-3-glycerophosphatase gene. The hub genes of the peroxidase-related module included the WRKY transcription factor, abscisic acid signal transduction pathway-related gene plasma membrane hydrogen-ATPase and receptor-like kinase. Therefore, we selected the modular hub genes and significantly enriched the metabolic pathway genes to construct the key competitive endogenous RNA networks, resulting in three competitive endogenous RNA networks of seven long non-coding RNAs regulating three co-expressed messenger RNAs via four microRNAs. Finally, the negative regulatory function of the WRKY transcription factor OsWRKY61 was determined via subcellular localization and validation of the physiological indices in the mutant.

Biochemical analysis of the TPS-a subfamily in Medicago truncatula.

Terpenes are important mediators of plant chemical response to environmental cues. Here, we describe the genome-wide identification and biochemical characterization of TPS-a members in Medicago truncatula, a model legume crop. Genome mining identified thirty-nine full-length terpene synthases with a significant number predicted to produce monoterpenes and sesquiterpenes. Biochemical characterization of the TPS-a subfamily associated with sesquiterpene biosynthesis revealed such compounds, that exhibit substantial biological activity in other plants. Gene expression analysis using qPCR and the Medicago gene atlas illustrated distinct tissue and time-based variation in expression in leaves and roots. Together our work establishes the gene-to-metabolite relationships for sesquiterpene synthases in M. truncatula. Understanding the biosynthetic capacity is a foundational step to defining the ecological roles of this important family of compounds.

Terpene Synthase Gene Family in Chinese Chestnut (Castanea mollissima BL.) Harbors Two Sesquiterpene Synthase Genes Implicated in Defense against Gall Wasp Dryocosmus kuriphilus.

Chinese chestnut (Castanea mollissima BL.) is a well-known fruit tree that has been cultivated in East Asia for millennia. Leaves and buds of the plant can become seriously infested by the gall wasp Dryocosmus kuriphilus (GWDK), which results in gall formation and associated significant losses in fruit production. Herbivore-induced terpenes have been reported to play an important role in plant-herbivory interactions, and in this study, we show that upon herbivory by GWDK, four terpene-related compounds were significantly induced, while the concentrations of these four compounds in intact buds were relatively low. Among these compounds, (E)-nerolidol and (E, E)-α-farnesene have frequently been reported to be involved in plant herbivory defenses, which suggests direct and/or indirect functions in chestnut GWDK defenses. Candidate terpene synthase (TPS) genes that may account for (E)-nerolidol and (E, E)-α-farnesene terpene biosynthesis were characterized by transcriptomics and phylogenetic approaches, which revealed altered transcript levels for two TPSs: CmAFS, a TPS-g subfamily member, and CmNES/AFS, a TPS-b clade member. Both genes were dramatically upregulated in gene expression upon GWDK infestation. Furthermore, Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana showed that CmAFS catalyzed the formation of (E, E)-α-farnesene, while CmNES/AFS showed dual (E)-nerolidol and (E, E)-α-farnesene synthase activity. Biochemical assays of the recombinant CmAFS and CmNES/AFS proteins confirmed their catalytic activity in vitro, and the enzymatic products were consistent with two of the major volatile compounds released upon GWDK-infested chestnut buds. Subcellular localization demonstrated that CmAFS and CmNES/AFS were both localized in the cytoplasm, the primary compartment for sesquiterpene synthesis. In summary, we show that two novel sesquiterpene synthase genes CmAFS and CmNES/AFS are inducible by herbivory and can account for the elevated accumulation of (E, E)-α-farnesene and (E)-nerolidol upon GWDK infestation and may be implicated in chestnut defense against GWDK herbivores.

Mechanistic characterisation of a sesquiterpene synthase for asterisca-1,6-diene from the liverwort Radula lindenbergiana and implications for pentalenene biosynthesis.

A sesquiterpene synthase from the liverwort Radula lindenbergiana was characterised and shown to produce the new sesquiterpene hydrocarbon (3R,9R)-asterisca-1,6-diene, besides small amounts of pentalenene. The biosynthesis of asterisca-1,6-diene was studied through isotopic labelling experiments, giving additional insights into the long discussed biosynthesis of pentalenene.

β-Terrecyclene synthase constructs the quadrane backbone in terrecyclic acid biosynthesis.

Quadrane sesquiterpenes featuring a distinctive tricyclic skeleton exhibit potent antimicrobial and anticancer activities. Although extensive studies have attempted to reveal the multistep carbocation rearrangement involved in the formation of the tricyclic quadrane scaffold, the exact biosynthetic pathway and chemical logic to generate the quadrane structure remains mysterious. Here we identified a novel sesquiterpene synthase that is capable of generating β-terrecyclene possessing the quadrane scaffold and characterized the biosynthetic pathway of a representative fungal quadrane terrecyclic acid. Further mutagenesis coupled with isotopically sensitive branching studies of this β-terrecyclene synthase provided insight into the mechanism involved in the formation of the quadrane scaffold.

Isolation and in silico analysis of a transcript variant for a putative terpene synthase (HbTPS6L-X1) from the bark tissues of rubber Hevea brasiliensis RRIV 209

Hevea brasiliensis is the only tree species grown to produce natural rubber. The method of harvesting latex by slicing a groove into the bark of the tree mimics a mechanical wounding. In addition, rubber trees could be frequently exposed to pest and pathogen attacks through the scraped bark region. Terpenoids are a group of specialised compounds that play an important role in plant-environment interactions, especially in plant response to stress factors. The diversity of terpenoids is mainly determined by enzymes of the terpene synthase (TPS) family. Screening the RNA-seq data of H. brasiliensis revealed the presence of many putative TPS genes expressed in the bark tissues of rubber trees; however, their function has not been studied. This paper presents the identification of a TPS gene transcript variant (designated as HbTPS6L-X1) from the bark tissues of H. brasiliensis RRIV 209. Through the analysis of the phylogenetic tree, the presence of conserved motifs, and the signal peptide sequence region of the protein, as well as structural homology to known sesquiterpene synthases from other plants, HbTPS6L-X1 was predicted to belong to the TPS-a subfamily, be localised in the cytoplasm and catalyse the conversion of farnesyl diphosphate (FPP) to sesquiterpenes. These in silico functional characteristics are the basis for designing experiments to confirm the in planta biological function of HbTPS6L-X1 protein.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

The Integration of the Metabolome and Transcriptome for Dendrobium nobile Lindl. in Response to Methyl Jasmonate

Dendrobium nobile Lindl., as an endangered medicinal plant within the genus Dendrobium, is widely distributed in southwestern China and has important ecological and economic value. There are a variety of metabolites with pharmacological activity in D. nobile. The alkaloids and polysaccharides contained within D. nobile are very important active components, which mainly have antiviral, anti-tumor, and immunity improvement effects. However, the changes in the compounds and functional genes of D. nobile induced by methyl jasmonate (MeJA) are not clearly understood. In this study, the metabolome and transcriptome of D. nobile were analyzed after exposure to MeJA. A total of 377 differential metabolites were obtained through data analysis, of which 15 were related to polysaccharide pathways and 35 were related to terpenoids and alkaloids pathways. Additionally, the transcriptome sequencing results identified 3256 differentially expressed genes that were discovered in 11 groups. Compared with the control group, 1346 unigenes were differentially expressed in the samples treated with MeJA for 14 days (TF14). Moreover, the expression levels of differentially expressed genes were also significant at different growth and development stages. According to GO and KEGG annotations, 189 and 99 candidate genes were identified as being involved in terpenoid biosynthesis and polysaccharide biosynthesis, respectively. In addition, the co-expression analysis indicated that 238 and 313 transcription factors (TFs) may contribute to the regulation of terpenoid and polysaccharide biosynthesis, respectively. Through a heat map analysis, fourteen terpenoid synthetase genes, twenty-three cytochrome P450 oxidase genes, eight methyltransferase genes, and six aminotransferase genes were identified that may be related to dendrobine biosynthesis. Among them, one sesquiterpene synthase gene was found to be highly expressed after the treatment with MeJA and was positively correlated with the content of dendrobine. This study provides important and valuable metabolomics and transcriptomic information for the further understanding of D. nobile at the metabolic and molecular levels and provides candidate genes and possible intermediate compounds for the dendrobine biosynthesis pathway, which lays a certain foundation for further research on and application of Dendrobium.

Cloning, Expression and Functional Characterization of a Novel α-Humulene Synthase, Responsible for the Formation of Sesquiterpene in Agarwood Originating from Aquilaria malaccensis.

This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 3' RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and β-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future.

Knockout of OsWRKY71 impairs Bph15-mediated resistance against brown planthopper in rice.

The Bph15 gene, known for its ability to confer resistance to the brown planthopper (BPH; Nilaparvata lugens Stål), has been extensively employed in rice breeding. However, the molecular mechanism by which Bph15 provides resistance against BPH in rice remains poorly understood. In this study, we reported that the transcription factor OsWRKY71 was highly responsive to BPH infestation and exhibited early-induced expression in Bph15-NIL (near-isogenic line) plants, and OsWRKY71 was localized in the nucleus of rice protoplasts. The knockout of OsWRKY71 in the Bph15-NIL background by CRISPR-Cas9 technology resulted in an impaired Bph15-mediated resistance against BPH. Transcriptome analysis revealed that the transcript profiles responsive to BPH differed between the wrky71 mutant and Bph15-NIL, and the knockout of OsWRKY71 altered the expression of defense genes. Subsequent quantitative RT-PCR analysis identified three genes, namely sesquiterpene synthase OsSTPS2, EXO70 family gene OsEXO70J1, and disease resistance gene RGA2, which might participate in BPH resistance conferred by OsWRKY71 in Bph15-NIL plants. Our investigation demonstrated the pivotal involvement of OsWRKY71 in Bph15-mediated resistance and provided new insights into the rice defense mechanisms against BPH.

Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases

Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases

Molecular and Functional Analyses of Characterized Sesquiterpene Synthases in Mushroom-Forming Fungi.

Sesquiterpenes are a type of abundant natural product with widespread applications in several industries. They are biosynthesized by sesquiterpene synthases (STSs). As valuable and abundant biological resources, mushroom-forming fungi are rich in new sesquiterpenes and STSs, which remain largely unexploited. In the present study, we collected information on 172 STSs from mushroom-forming fungi with experimentally characterized products from the literature and sorted them to develop a dataset. Furthermore, we analyzed and discussed the phylogenetic tree, catalytic products, and conserved motifs of STSs. Phylogenetic analysis revealed that the STSs were clustered into four clades. Furthermore, their cyclization reaction mechanism was divided into four corresponding categories. This database was used to predict 12 putative STS genes from the edible fungi Flammulina velutipes. Finally, three FvSTSs were selected to experimentally characterize their functions. FvSTS03 predominantly produced Δ-cadinol and FvSTS08 synthesized β-barbatene as the main product; these findings were consistent with those of the functional prediction analysis. A product titer of 78.8 mg/L β-barbatene was achieved in Saccharomyces cerevisiae via metabolic engineering. Our study findings will help screen or design STSs from fungi with specific product profiles as functional elements for applications in synthetic biology.

Purification of artemisinin precursor amorphadiene from microbial metabolites containing isomeride farnesene: Solid-Pd chemocatalysis of farnesene to squalene with high boiling point

Due to complex chemical mechanism of sesquiterpene synthases, amorpha-4, 11-diene (AD) from microbial fermentation contained isomeride β-farnesene (FA) with similar boiling point. For purifying AD, FA was converted to biofuel-valued squalene (SQ) with high boiling point using solid Pd catalysts. (NHT)OS-Pd catalysts resulted in FA conversion of >99.9% and SQ selectivity of >99.0% at 80 °C for 5 h. Moreover, this catalyst exhibited great stability and recycling performance. When (NHT)OS-Pd was applied to AD mixture, almost FA was converted to SQ and AD remained in the pre-reaction state. As SQ had a higher boiling point, AD was easily distilled out.

Functional characterisation of twelve terpene synthases from actinobacteria.

Fifteen type I terpene synthase homologs from diverse actinobacteria that were selected based on a phylogenetic analysis of more than 4000 amino acid sequences were investigated for their products. For four enzymes with functions not previously reported from bacterial terpene synthases the products were isolated and their structures were elucidated by NMR spectroscopy, resulting in the discovery of the first terpene synthases for (+)-δ-cadinol and (+)-α-cadinene, besides the first two bacterial (-)-amorpha-4,11-diene synthases. For other terpene synthases with functions reported from bacteria before the products were identified by GC-MS. The characterised enzymes include a new epi-isozizaene synthase with monoterpene synthase side activity, a 7-epi-α-eudesmol synthase that also produces hedycaryol and germacrene A, and four more sesquiterpene synthases that produce mixtures of hedycaryol and germacrene A. Three phylogenetically related enzymes were in one case not expressed and in two cases inactive, suggesting pseudogenisation in the respective branch of the phylogenetic tree. Furthermore, a diterpene synthase for allokutznerene and a sesterterpene synthase for sesterviolene were identified.

Turning a Sesquiterpene Synthase into a Di- and Sesterterpene Synthase

Turning a Sesquiterpene Synthase into a Di- and Sesterterpene Synthase

A "Magic Mushroom" Multi-Product Sesquiterpene Synthase.

Psilocybe "magic mushrooms" are chemically well understood for their psychotropic tryptamines. However, the diversity of their other specialized metabolites, in particular terpenoids, has largely remained an open question. Yet, knowledge on the natural product background is critical to understand if other compounds modulate the psychotropic pharmacological effects. CubA, the single clade II sesquiterpene synthase of P. cubensis, was heterologously produced in Escherichia coli and characterized in vitro, complemented by in vivo product formation assays in Aspergillus niger as a heterologous host. Extensive GC-MS analyses proved a function as multi-product synthase and, depending on the reaction conditions, cubebol, β-copaene, δ-cadinene, and germacrene D were detected as the major products of CubA. In addition, mature P. cubensis carpophores were analysed chromatographically which led to the detection of β-copaene and δ-cadinene. Enzymes closely related to CubA are encoded in the genomes of various Psilocybe species. Therefore, our results provide insight into the metabolic capacity of the entire genus.

Identification and characterization of novel sesquiterpene synthases TPS9 and TPS12 from Aquilaria sinensis.

Sesquiterpenes are characteristic components and important quality criterions for agarwood. Although sesquiterpenes are well-known to be biosynthesized by sesquiterpene synthases (TPSs), to date, only a few TPS genes involved in agarwood formation have been reported. Here, two new TPS genes, namely, TPS9 and TPS12, were isolated from Aquilaria sinensis (Lour.) Gilg, and their functions were examined in Escherichia coli BL21(DE3), with farnesyl pyrophosphate (FPP) and geranyl pyrophosphate (GPP) as the substrate of the corresponding enzyme activities. They were both identified as a multiproduct enzymes. After incubation with FPP, TPS9 liberated β-farnesene and cis-sesquisabinene hydrate as main products, with cedrol and another unidentified sesquiterpene as minor products. TPS12 catalyzes the formation of β-farnesene, nerolidol, γ-eudesmol, and hinesol. After incubation with GPP, TPS9 generated citronellol and geraniol as main products, with seven minor products. TPS12 converted GPP into four monoterpenes, with citral as the main product, and three minor products. Both TPS9 and TPS12 showed much higher expression in the two major tissues emitting floral volatiles: flowers and agarwood. Further, RT-PCR analysis showed TPS9 and TPS12 are typical genes mainly expressed during later stages of stress response, which is better known than that of chromone derivatives. This study will advance our understanding of agarwood formation and provide a solid theoretical foundation for clarifying its mechanism in A. sinensis.

Identification and characterization of a novel sativene synthase from Fischerella thermalis

Sesquiterpene synthases (TPS) determine the structural diversity of terpenoids, which are species specific. In this study, we report a TPS from Fischerella thermalis (named as FtTPS), recombinantly expressed as a soluble protein in Escherichia coli BL21(DE3) strain. The FtTPS protein could catalyze the conversion of farnesyl pyrophosphate (FPP) to sativene, a kind of tricyclic sesquiterpene. The optimal pH and temperature of FtTPS were 7.5 and 30 °C, respectively. The KM and Vmax values of FtTPS for FPP were 1.846 μM and 0.372 μM/min, respectively. By constructing an engineered E. coli strain carrying the FtTPS and the heterologous mevalonate (MVA) pathway genes, sativene could be detected and its yield reached 24 mg/L after 96 h cultivation. The highest yield of sativene was obtained when E.coli BL21 Star was used as the host with SBMSN medium. These results exhibited the biosynthesis of sativene for the first time.

Highly Efficient Biosynthesis of γ-Bisabolene with a New Sesquiterpene Synthase AcTPS5 by Dual Cytoplasmic-Peroxisomal Engineering in Saccharomyces cerevisiae

γ-bisabolene is a monocyclic sesquiterpene with various biological activities; it has also been approved as a food additive. Additionally, the hydrogenated form of bisabolene is considered as a potential alternative to D2 diesel. Saccharomyces cerevisiae has the ability to produce a large amount of acetyl-CoA in both cytosol and peroxisomes, which serves as a precursor in terpene biosynthesis. In this study, AcTPS5 was identified as a new γ-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, γ-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene atg36 further improved γ-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted γ-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of ERG9, γ-bisabolene production was achieved at 584.14 mg/L in shake-flask fermentation and 2.69 g/L in fed-batch fermentation, which is the highest reported production of γ-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in S. cerevisiae.