Journal articles: 'Sesquiterpene synthases'

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Yan, Xiaoguang, Yukun Li, Weiguo Li, Dongmei Liang, Shengxin Nie, Ruiqi Chen, Jianjun Qiao, Mingzhang Wen, and Qinggele Caiyin. "Transcriptome Analysis and Identification of Sesquiterpene Synthases in Liverwort Jungermannia exsertifolia." Bioengineering 10, no. 5 (May 9, 2023): 569. http://dx.doi.org/10.3390/bioengineering10050569.

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The liverwort Jungermannia exsertifolia is one of the oldest terrestrial plants and rich in structurally specific sesquiterpenes. There are several sesquiterpene synthases (STSs) with non-classical conserved motifs that have been discovered in recent studies on liverworts; these motifs are rich in aspartate and bind with cofactors. However, more detailed sequence information is needed to clarify the biochemical diversity of these atypical STSs. This study mined J. exsertifolia sesquiterpene synthases (JeSTSs) through transcriptome analysis using BGISEQ-500 sequencing technology. A total of 257,133 unigenes was obtained, and the average length was 933 bp. Among them, a total of 36 unigenes participated in the biosynthesis of sesquiterpenes. In addition, the in vitro enzymatic characterization and heterologous expression in Saccharomyces cerevisiae showed that JeSTS1 and JeSTS2 produced nerolidol as the major product, while JeSTS4 could produce bicyclogermacrene and viridiflorol, suggesting a specificity of J. exsertifolia sesquiterpene profiles. Furthermore, the identified JeSTSs had a phylogenetic relationship with a new branch of plant terpene synthases, the microbial terpene synthase-like (MTPSL) STSs. This work contributes to the understanding of the metabolic mechanism for MTPSL-STSs in J. exsertifolia and could provide an efficient alternative to microbial synthesis of these bioactive sesquiterpenes.

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Beran, Franziska, Peter Rahfeld, Katrin Luck, Raimund Nagel, Heiko Vogel, Natalie Wielsch, Sandra Irmisch, et al. "Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle." Proceedings of the National Academy of Sciences 113, no. 11 (March 2, 2016): 2922–27. http://dx.doi.org/10.1073/pnas.1523468113.

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Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.

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Cong, Zhanren, Qiang Yin, Kunhong Tian, Njeru Joe Mukoma, Liming Ouyang, Tom Hsiang, Lixin Zhang, Lan Jiang, and Xueting Liu. "Genome Mining of Fungal Unique Trichodiene Synthase-like Sesquiterpene Synthases." Journal of Fungi 10, no. 5 (May 13, 2024): 350. http://dx.doi.org/10.3390/jof10050350.

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Sesquiterpenoids served as an important source for natural product drug discovery. Although genome mining approaches have revealed numerous novel sesquiterpenoids and biosynthetic enzymes, the comprehensive landscape of fungal sesquiterpene synthases (STSs) remains elusive. In this study, 123 previously reported fungal STSs were subjected to phylogenetic analysis, resulting in the identification of a fungi-specific STS family known as trichodiene synthase-like sesquiterpene synthases (TDTSs). Subsequently, the application of hidden Markov models allowed the discovery of 517 TDTSs from our in-house fungi genome library of over 400 sequenced genomes, and these TDTSs were defined into 79 families based on a sequence similarity network. Based on the novelty of protein sequences and the completeness of their biosynthetic gene clusters, 23 TDTS genes were selected for heterologous expression in Aspergillus oryzae. In total, 10 TDTSs were active and collectively produced 12 mono- and sesquiterpenes, resulting in the identification of the first chamipinene synthase, as well as the first fungi-derived cedrene, sabinene, and camphene synthases. Additionally, with the guidance of functionally characterized TDTSs, we found that TDTSs in Family 1 could produce bridged-cyclic sesquiterpenes, while those in Family 2 could synthesize spiro- and bridged-cyclic sesquiterpenes. Our research presents a new avenue for the genome mining of fungal sesquiterpenoids.

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Silva, Waldeyr M. C. da, Jakob L. Andersen, Maristela T. Holanda, Maria Emília M. T. Walter, Marcelo M. Brigido, Peter F. Stadler, and Christoph Flamm. "Exploring Plant Sesquiterpene Diversity by Generating Chemical Networks." Processes 7, no. 4 (April 25, 2019): 240. http://dx.doi.org/10.3390/pr7040240.

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Plants produce a diverse portfolio of sesquiterpenes that are important in their response to herbivores and the interaction with other plants. Their biosynthesis from farnesyl diphosphate depends on the sesquiterpene synthases that admit different cyclizations and rearrangements to yield a blend of sesquiterpenes. Here, we investigate to what extent sesquiterpene biosynthesis metabolic pathways can be reconstructed just from the knowledge of the final product and the reaction mechanisms catalyzed by sesquiterpene synthases. We use the software package MedØlDatschgerl (MØD) to generate chemical networks and to elucidate pathways contained in them. As examples, we successfully consider the reachability of the important plant sesquiterpenes β -caryophyllene, α -humulene, and β -farnesene. We also introduce a graph database to integrate the simulation results with experimental biological evidence for the selected predicted sesquiterpenes biosynthesis.

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Ker, De-Sheng, Sze Lei Pang, Noor Farhan Othman, Sekar Kumaran, Ee Fun Tan, Thiba Krishnan, Kok Gan Chan, Roohaida Othman, Maizom Hassan, and Chyan Leong Ng. "Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase." PeerJ 5 (February 28, 2017): e2961. http://dx.doi.org/10.7717/peerj.2961.

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Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, addition of 15% (v/v) glycerol to the protein purification buffer and removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

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Ee, Su-Fang, Zeti-Azura Mohamed-Hussein, Roohaida Othman, Noor Azmi Shaharuddin, Ismanizan Ismail, and Zamri Zainal. "Functional Characterization of Sesquiterpene Synthase fromPolygonum minus." Scientific World Journal 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/840592.

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Polygonum minusis an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene fromP. minus.P. minussesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function ofPmSTS, we expressed this gene inArabidopsis thaliana. Two transgenic lines, designated asOE3andOE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production ofβ-sesquiphellandrene.

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Yamada, Yuuki, Tomohisa Kuzuyama, Mamoru Komatsu, Kazuo Shin-ya, Satoshi Omura, David E. Cane, and Haruo Ikeda. "Terpene synthases are widely distributed in bacteria." Proceedings of the National Academy of Sciences 112, no. 3 (December 22, 2014): 857–62. http://dx.doi.org/10.1073/pnas.1422108112.

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Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequencedStreptomycetaceaemicroorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologousStreptomyceshost and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.

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Sun, Xiang, You-Sheng Cai, Yujie Yuan, Guangkai Bian, Ziling Ye, Zixin Deng, and Tiangang Liu. "Genome mining in Trichoderma viride J1-030: discovery and identification of novel sesquiterpene synthase and its products." Beilstein Journal of Organic Chemistry 15 (August 28, 2019): 2052–58. http://dx.doi.org/10.3762/bjoc.15.202.

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Sesquiterpene synthases in Trichoderma viride have been seldom studied, despite the efficiency of filamentous fungi for terpenoid production. Using the farnesyl diphosphate-overexpressing Saccharomyces cerevisiae platform to produce diverse terpenoids, we herein identified an unknown sesquiterpene synthase from T. viride by genome mining and determined the structure of its corresponding products. One new 5/6 bicyclic sesquiterpene and its esterified derivative were characterised by GC–MS and 1D and 2D NMR spectroscopy. To the best of our knowledge, this is the first well-identified sesquiterpene synthase from T. viride to date.

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Liang, Dongmei, Weiguo Li, Xiaoguang Yan, Qinggele Caiyin, Guangrong Zhao, and Jianjun Qiao. "Molecular and Functional Evolution of the Spermatophyte Sesquiterpene Synthases." International Journal of Molecular Sciences 22, no. 12 (June 14, 2021): 6348. http://dx.doi.org/10.3390/ijms22126348.

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Sesquiterpenes are important defense and signal molecules for plants to adapt to the environment, cope with stress, and communicate with the outside world, and their evolutionary history is closely related to physiological functions. In this study, the information of plant sesquiterpene synthases (STSs) with identified functions were collected and sorted to form a dataset containing about 500 members. The phylogeny of spermatophyte functional STSs was constructed based on the structural comparative analysis to reveal the sequence–structure–function relationships. We propose the evolutionary history of plant sesquiterpene skeletons, from chain structure to small rings, followed by large rings for the first time and put forward a more detailed function-driven hypothesis. Then, the evolutionary origins and history of spermatophyte STSs are also discussed. In addition, three newly identified STSs CaSTS2, CaSTS3, and CaSTS4 were analyzed in this functional evolutionary system, and their germacrene D products were consistent with the functional prediction. This demonstrates an application of the structure-based phylogeny in predicting STS function. This work will help us to understand evolutionary patterns and dynamics of plant sesquiterpenes and STSs and screen or design STSs with specific product profiles as functional elements for synthetic biology application.

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Manczak, Tom, and Henrik Toft Simonsen. "Insight into Biochemical Characterization of Plant Sesquiterpene Synthases." Analytical Chemistry Insights 11s1 (January 2016): ACI.S40292. http://dx.doi.org/10.4137/aci.s40292.

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A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/ KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data.

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Göpfert, Jens, Anna-Katharina Bülow, and Otmar Spring. "Identification and Functional Characterization of a new Sunflower Germacrene A Synthase (HaGAS3)." Natural Product Communications 5, no. 5 (May 2010): 1934578X1000500. http://dx.doi.org/10.1177/1934578x1000500507.

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Sesquiterpenes and sesquiterpene lactones are major natural compounds found in linear and capitate glandular trichomes of sunflower, Helianthus annuus L. In addition to two recently identified germacrene A synthases HaGAS1 and HaGAS2, found in capitate trichome gland cells, reverse transcription-PCR experiments have now allowed identification of a third enzyme of this type, HaGAS3. Its cDNA sequence was established and its functional characterization as a germacrene A synthase was achieved through in vitro expression in engineered yeast, and by GC-MS experiments. PCR and RT-PCR experiments with cDNA from different plant organs revealed that the new enzyme is expressed independently from the other two. While these latter two were expressed in plant organs bearing capitate glandular trichomes and in roots, the new enzyme occurred in plant tissues not linked to the presence of specific trichomes (for example, cotyledons), and was absent in roots. The experiments show that independently regulated pathways for the first cyclic sesquiterpene, germacrene A, are present in sunflower.

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Agger, Sean A., Fernando Lopez-Gallego, Thomas R. Hoye, and Claudia Schmidt-Dannert. "Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120." Journal of Bacteriology 190, no. 18 (July 25, 2008): 6084–96. http://dx.doi.org/10.1128/jb.00759-08.

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ABSTRACT Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.

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Chhalodia, Anuj K., Houchao Xu, Georges B. Tabekoueng, Binbin Gu, Kizerbo A. Taizoumbe, Lukas Lauterbach, and Jeroen S. Dickschat. "Functional characterisation of twelve terpene synthases from actinobacteria." Beilstein Journal of Organic Chemistry 19 (September 15, 2023): 1386–98. http://dx.doi.org/10.3762/bjoc.19.100.

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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.

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Fang, Xin, Jian-Xu Li, Jin-Quan Huang, You-Li Xiao, Peng Zhang, and Xiao-Ya Chen. "Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase." Biochemical Journal 474, no. 13 (June 16, 2017): 2191–202. http://dx.doi.org/10.1042/bcj20170060.

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Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7 and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399, and Thr447, which play a major role in directing catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of the substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we substantially increased amorpha-4,11-diene production in a near-additive manner by engineering Thr399 and Thr447 for product release. Our results provide new insight for the rational design of enzyme activity using synthetic biology.

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Püth, Nils, Franziska Ersoy, Ulrich Krings, and Ralf G. Berger. "Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor." Catalysts 11, no. 11 (November 12, 2021): 1361. http://dx.doi.org/10.3390/catal11111361.

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Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity.

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Huang, Jin-Quan, Dong-Mei Li, Jian-Xu Li, Jia-Ling Lin, Xiu Tian, Ling-Jian Wang, Xiao-Ya Chen, and Xin Fang. "1,10/1,11-Cyclization catalyzed by diverged plant sesquiterpene synthases is dependent on a single residue." Organic & Biomolecular Chemistry 19, no. 30 (2021): 6650–56. http://dx.doi.org/10.1039/d1ob00827g.

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A conserved amino acid residue responsible for the 1,10- or 1,11-cyclization of FDP was identified in sesquiterpene synthases of different sources and functions, shedding new light on the mechanisms of sesquiterpene cyclization.

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Yu, Cuicui, Shixi Gao, Mei Rong, Mengjun Xiao, Yanhong Xu, and Jianhe Wei. "Identification and characterization of novel sesquiterpene synthases TPS9 and TPS12 from Aquilaria sinensis." PeerJ 11 (August 30, 2023): e15818. http://dx.doi.org/10.7717/peerj.15818.

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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.

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Wang, Shengli, Ruiqi Chen, Lin Yuan, Chenyang Zhang, Dongmei Liang, and Jianjun Qiao. "Molecular and Functional Analyses of Characterized Sesquiterpene Synthases in Mushroom-Forming Fungi." Journal of Fungi 9, no. 10 (October 14, 2023): 1017. http://dx.doi.org/10.3390/jof9101017.

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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.

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Pu, Qingyu, Jin Liang, Qinqin Shen, Jingye Fu, Zhien Pu, Jiang Liu, Xuegui Wang, and Qiang Wang. "A Wheat β-Patchoulene Synthase Confers Resistance against Herbivory in Transgenic Arabidopsis." Genes 10, no. 6 (June 10, 2019): 441. http://dx.doi.org/10.3390/genes10060441.

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Terpenoids play important roles in plant defense. Although some terpene synthases have been characterized, terpenoids and their biosynthesis in wheat (Triticum aestivum L.) still remain largely unknown. Here, we describe the identification of a terpene synthase gene in wheat. It encodes a sesquiterpene synthase that catalyzes β-patchoulene formation with E,E-farnesyl diphosphate (FPP) as the substrate, thus named as TaPS. TaPS exhibits inducible expression in wheat in response to various elicitations. Particularly, alamethicin treatment strongly induces TaPS gene expression and β-patchoulene accumulation in wheat. Overexpression of TaPS in Arabidopsis successfully produces β-patchoulene, verifying the biochemical function of TaPS in planta. Furthermore, these transgenic Arabidopsis plants exhibit resistance against herbivory by repelling beet armyworm larvae feeding, thereby indicating anti-herbivory activity of β-patchoulene. The catalytic mechanism of TaPS is also explored by homology modeling and site-directed mutagenesis. Two key amino acids are identified to act in protonation and stability of intermediates and product formation. Taken together, one wheat sesquiterpene synthase is identified as β-patchoulene synthase. TaPS exhibits inducible gene expression and the sesquiterpene β-patchoulene is involved in repelling insect infestation.

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Barreto, Ighor C., Anderson S. de Almeida, and José G. Sena Filho. "Taxonomic Insights and Its Type Cyclization Correlation of Volatile Sesquiterpenes in Vitex Species and Potential Source Insecticidal Compounds: A Review." Molecules 26, no. 21 (October 23, 2021): 6405. http://dx.doi.org/10.3390/molecules26216405.

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Sesquiterpenes (SS) are secondary metabolites formed by the bonding of 3 isoprene (C5) units. They play an important role in the defense and signaling of plants to adapt to the environment, face stress, and communicate with the outside world, and their evolutionary history is closely related to their physiological functions. This review considers their presence and extensively summarizes the 156 sesquiterpenes identified in Vitextaxa, emphasizing those with higher concentrations and frequency among species and correlating with the insecticidal activities and defensive responses reported in the literature. In addition, we classify the SS based on their chemical structures and addresses cyclization in biosynthetic origin. Most relevant sesquiterpenes of the Vitex genus are derived from the germacredienyl cation mainly via bicyclogermacrene and germacrene C, giving rise to aromadrendanes, a skeleton with the highest number of representative compounds in this genus, and 6,9-guaiadiene, respectively, indicating the production of 1.10-cyclizing sesquiterpene synthases. These enzymes can play an important role in the chemosystematics of the genus from their corresponding routes and cyclizations, constituting a new approach to chemotaxonomy. In conclusion, this review is a compilation of detailed information on the profile of sesquiterpene in the Vitex genus and, thus, points to new unexplored horizons for future research.

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Miller, David J., and Rudolf K. Allemann. "Sesquiterpene synthases: Passive catalysts or active players?" Nat. Prod. Rep. 29, no. 1 (2012): 60–71. http://dx.doi.org/10.1039/c1np00060h.

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Steele, Christopher L., John Crock, Jörg Bohlmann, and Rodney Croteau. "Sesquiterpene Synthases from Grand Fir (Abies grandis)." Journal of Biological Chemistry 273, no. 4 (January 23, 1998): 2078–89. http://dx.doi.org/10.1074/jbc.273.4.2078.

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Durairaj, Janani, Alice Di Girolamo, Harro J. Bouwmeester, Dick de Ridder, Jules Beekwilder, and Aalt DJ van Dijk. "An analysis of characterized plant sesquiterpene synthases." Phytochemistry 158 (February 2019): 157–65. http://dx.doi.org/10.1016/j.phytochem.2018.10.020.

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Chow, Jeng-Yeong, Bo-Xue Tian, Gurusankar Ramamoorthy, Brandan S. Hillerich, Ronald D. Seidel, Steven C. Almo, Matthew P. Jacobson, and C. Dale Poulter. "Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus." Proceedings of the National Academy of Sciences 112, no. 18 (April 21, 2015): 5661–66. http://dx.doi.org/10.1073/pnas.1505127112.

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Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.

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Trapp, Susan C., and Rodney B. Croteau. "Genomic Organization of Plant Terpene Synthases and Molecular Evolutionary Implications." Genetics 158, no. 2 (June 1, 2001): 811–32. http://dx.doi.org/10.1093/genetics/158.2.811.

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Abstract Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C10), sesquiterpene (C15), and diterpene synthases (C20). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C10), (-)-limonene (C10), (E)-α-bisabolene (C15), δ-selinene (C15), and abietadiene synthase (C20) from Abies grandis and taxadiene synthase (C20) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.

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Jia, Ying, Dianren Xia, and E. S. Louzada. "Molecular Cloning and Expression Analysis of a Putative Terpene Synthase Gene from Citrus." Journal of the American Society for Horticultural Science 130, no. 3 (May 2005): 454–58. http://dx.doi.org/10.21273/jashs.130.3.454.

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A cDNA coding for a putative terpene synthase (Grtps) was isolated from `Rio Red' grapefruit (Citrus paradisi Macf.) mature fruit by differential display RT-PCR and the corresponding full-length cDNA and genomic clone were subsequently obtained. The isolated cDNA clone was 1644 bp in length encoding a protein of 548 amino acids with a predicted molecular mass of 64 kDa and of pI 5.38. The genomic clone was 3203 bp in length with 6 introns and 7 exons. This Grtps appears to be a sesquiterpene synthase based on molecular weight, genomic organization, and similarity with the other terpene synthases. Both RT-PCR and Northern blot expression analysis indicated that Grtps is not expressed in immature fruits, roots, or leaves, but only in mature fruits. Southern blot analysis of genomic DNA demonstrated that Grtps is one of the members in the family of terpene synthases.

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Walther, Christin, Pamela Baumann, Katrin Luck, Beate Rothe, Peter H. W. Biedermann, Jonathan Gershenzon, Tobias G. Köllner, and Sybille B. Unsicker. "Volatile emission and biosynthesis in endophytic fungi colonizing black poplar leaves." Beilstein Journal of Organic Chemistry 17 (July 22, 2021): 1698–711. http://dx.doi.org/10.3762/bjoc.17.118.

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Plant volatiles play a major role in plant–insect interactions as defense compounds or attractants for insect herbivores. Recent studies have shown that endophytic fungi are also able to produce volatiles and this raises the question of whether these fungal volatiles influence plant–insect interactions. Here, we qualitatively investigated the volatiles released from 13 endophytic fungal species isolated from leaves of mature black poplar (Populus nigra) trees. The volatile blends of these endophytes grown on agar medium consist of typical fungal compounds, including aliphatic alcohols, ketones and esters, the aromatic alcohol 2-phenylethanol and various sesquiterpenes. Some of the compounds were previously reported as constituents of the poplar volatile blend. For one endophyte, a species of Cladosporium, we isolated and characterized two sesquiterpene synthases that can produce a number of mono- and sesquiterpenes like (E)-β-ocimene and (E)-β-caryophyllene, compounds that are dominant components of the herbivore-induced volatile bouquet of black poplar trees. As several of the fungus-derived volatiles like 2-phenylethanol, 3-methyl-1-butanol and the sesquiterpene (E)-β-caryophyllene, are known to play a role in direct and indirect plant defense, the emission of volatiles from endophytic microbial species should be considered in future studies investigating tree-insect interactions.

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Agger, Sean, Fernando Lopez-Gallego, and Claudia Schmidt-Dannert. "Diversity of sesquiterpene synthases in the basidiomyceteCoprinus cinereus." Molecular Microbiology 72, no. 5 (June 2009): 1181–95. http://dx.doi.org/10.1111/j.1365-2958.2009.06717.x.

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Agger, Sean, Fernando Lopez-Gallego, and Claudia Schmidt-Dannert. "Diversity of sesquiterpene synthases in the basidiomyceteCoprinus cinereus." Molecular Microbiology 72, no. 5 (June 2009): 1307–8. http://dx.doi.org/10.1111/j.1365-2958.2009.06743.x.

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SHEN, H. "Advances in Sesquiterpene Synthases (Cyclases) of Artemisia annua." Chinese Journal of Biotechnology 23, no. 6 (November 2007): 976–81. http://dx.doi.org/10.1016/s1872-2075(07)60059-4.

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Lauterbach, Lukas, and Jeroen S. Dickschat. "Sesquiterpene synthases for bungoene, pentalenene and epi-isozizaene from Streptomyces bungoensis." Organic & Biomolecular Chemistry 18, no. 24 (2020): 4547–50. http://dx.doi.org/10.1039/d0ob00606h.

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Smit, Samuel J., Melané A. Vivier, and Philip R. Young. "Seeing the Forest through the (Phylogenetic) Trees: Functional Characterisation of Grapevine Terpene Synthase (VviTPS) Paralogues and Orthologues." Plants 10, no. 8 (July 26, 2021): 1520. http://dx.doi.org/10.3390/plants10081520.

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Gene families involved in specialised metabolism play a key role in a myriad of ecophysiological and biochemical functions. The Vitis vinifera sesquiterpene synthases represent the largest subfamily of grapevine terpene synthase (VviTPS) genes and are important volatile metabolites for wine flavour and aroma, as well as ecophysiological interactions. The functional characterisation of VviTPS genes is complicated by a reliance on a single reference genome that greatly underrepresents this large gene family, exacerbated by extensive duplications and paralogy. The recent release of multiple phased diploid grapevine genomes, as well as extensive whole-genome resequencing efforts, provide a wealth of new sequence information that can be utilised to overcome the limitations of the reference genome. A large cluster of sesquiterpene synthases, localised to chromosome 18, was explored by means of comparative sequence analyses using the publicly available grapevine reference genome, three PacBio phased diploid genomes and whole-genome resequencing data from multiple genotypes. Two genes, VviTPS04 and -10, were identified as putative paralogues and/or allelic variants. Subsequent gene isolation from multiple grapevine genotypes and characterisation by means of a heterologous in planta expression and volatile analysis resulted in the identification of genotype-specific structural variations and polymorphisms that impact the gene function. These results present novel insight into how grapevine domestication likely shaped the VviTPS landscape to result in genotype-specific functions.

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O’Maille, Paul E., Joe Chappell, and Joseph P. Noel. "Biosynthetic potential of sesquiterpene synthases: Alternative products of tobacco 5-epi-aristolochene synthase." Archives of Biochemistry and Biophysics 448, no. 1-2 (April 2006): 73–82. http://dx.doi.org/10.1016/j.abb.2005.10.028.

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Li, Chunhong, Sreelatha Sarangapani, Qian Wang, Kumar Nadimuthu, and Rajani Sarojam. "Metabolic Engineering of the Native Monoterpene Pathway in Spearmint for Production of Heterologous Monoterpenes Reveals Complex Metabolism and Pathway Interactions." International Journal of Molecular Sciences 21, no. 17 (August 26, 2020): 6164. http://dx.doi.org/10.3390/ijms21176164.

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Spearmint produces and stores large amounts of monoterpenes, mainly limonene and carvone, in glandular trichomes and is the major natural source of these compounds. Towards producing heterologous monoterpenes in spearmint, we first reduced the flux into the native limonene pathway by knocking down the expression of limonene synthase (MsLS) by RNAi method. The MsLS RNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed that MsLS RNAi plants also showed an increase in sesquiterpene, phytosterols, fatty acids, flavonoids, and phenolic metabolites, suggesting an interaction between the MEP, MVA shikimate and fatty acid pathways in spearmint. Three different heterologous monoterpene synthases namely, linalool synthase and myrcene synthase from Picea abies and geraniol synthase from Cananga odorata were cloned and introduced independently into the MsLS RNAi mutant background. The expression of these heterologous terpene synthases resulted mainly in production of monoterpene derivatives. Of all the introduced monoterpenes geraniol showed the maximum number of derivatives. Our results provide new insights into MEP pathway interactions and regulation and reveals the existence of mechanisms for complex metabolism of monoterpenes in spearmint.

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Li, Jian-Xu, Xin Fang, Qin Zhao, Ju-Xin Ruan, Chang-Qing Yang, Ling-Jian Wang, David J. Miller, et al. "Rational engineering of plasticity residues of sesquiterpene synthases from Artemisia annua: product specificity and catalytic efficiency." Biochemical Journal 451, no. 3 (April 12, 2013): 417–26. http://dx.doi.org/10.1042/bj20130041.

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Most TPSs (terpene synthases) contain plasticity residues that are responsible for diversified terpene products and functional evolution, which provide a potential for improving catalytic efficiency. Artemisinin, a sesquiterpene lactone from Artemisia annua L., is widely used for malaria treatment and progress has been made in engineering the production of artemisinin or its precursors. In the present paper, we report a new sesquiterpene synthase from A. annua, AaBOS (A. annua α-bisabolol synthase), which has high sequence identity with AaADS (A. annua amorpha-4,11-diene synthase), a key enzyme in artemisinin biosynthesis. Comparative analysis of the two enzymes by domain-swapping and structure-based mutagenesis led to the identification of several plasticity residues, whose alteration changed the product profile of AaBOS to include γ-humulene as the major product. To elucidate the underlying mechanisms, we solved the crystal structures of AaBOS and a γ-humulene-producing AaBOS mutant (termed AaBOS-M2). Among the plasticity residues, position 399, located in the substrate-binding pocket, is crucial for both enzymes. In AaBOS, substitution of threonine for leucine (AaBOSL339T) is required for γ-humulene production; whereas in AaADS, replacing the threonine residue with serine (AaADST399S) resulted in a substantial increase in the activity of amorpha-4,11-diene production, probably as a result of accelerated product release. The present study demonstrates that substitution of plasticity residues has potential for improving catalytic efficiency of the enzyme.

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Ali, Mohammed, Elsayed Nishawy, Walaa A. Ramadan, Mohamed Ewas, Mokhtar Said Rizk, Ahmed G. M. Sief-Eldein, Mohamed Abd S. El-Zayat, et al. "Molecular characterization of a Novel NAD+-dependent farnesol dehydrogenase SoFLDH gene involved in sesquiterpenoid synthases from Salvia officinalis." PLOS ONE 17, no. 6 (June 3, 2022): e0269045. http://dx.doi.org/10.1371/journal.pone.0269045.

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Salvia officinalis is one of the most important medicinal and aromatic plants in terms of nutritional and medicinal value because it contains a variety of vital active ingredients. Terpenoid compounds, particularly monoterpenes (C10) and sesquiterpenes, are the most important and abundant among these active substances (C15). Terpenes play a variety of roles and have beneficial biological properties in plants. With these considerations, the current study sought to clone theNAD+-dependent farnesol dehydrogenase (SoFLDH, EC: 1.1.1.354) gene from S. officinalis. Functional analysis revealed that, SoFLDH has an open reading frame of 2,580 base pairs that encodes 860 amino acids.SoFLDH has two conserved domains and four types of highly conserved motifs: YxxxK, RXR, RR (X8) W, TGxxGhaG. However, SoFLDH was cloned from Salvia officinalis leaves and functionally overexpressed in Arabidopsis thaliana to investigate its role in sesquiterpenoid synthases. In comparison to the transgenic plants, the wild-type plants showed a slight delay in growth and flowering formation. To this end, a gas chromatography-mass spectrometry analysis revealed that SoFLDH transgenic plants were responsible for numerous forms of terpene synthesis, particularly sesquiterpene. These results provide a base for further investigation on SoFLDH gene role and elucidating the regulatory mechanisms for sesquiterpene synthesis in S. offcinalis. And our study paves the way for the future metabolic engineering of the biosynthesis of useful terpene compounds in S. offcinalis.

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Tholl, Dorothea, Feng Chen, Jana Petri, Jonathan Gershenzon, and Eran Pichersky. "Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers." Plant Journal 42, no. 5 (April 26, 2005): 757–71. http://dx.doi.org/10.1111/j.1365-313x.2005.02417.x.

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Chen, Xinlu, Tobias G. Köllner, Wangdan Xiong, Guo Wei, and Feng Chen. "Emission and biosynthesis of volatile terpenoids from the plasmodial slime mold Physarum polycephalum." Beilstein Journal of Organic Chemistry 15 (November 28, 2019): 2872–80. http://dx.doi.org/10.3762/bjoc.15.281.

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Terpene synthases (TPSs) are pivotal enzymes for the production of diverse terpenes, including monoterpenes, sesquiterpenes, and diterpenes. In our recent studies, dictyostelid social amoebae, also known as cellular slime molds, were found to contain TPS genes for making volatile terpenes. For comparison, here we investigated Physarum polycephalum, a plasmodial slime mold also known as acellular amoeba. Plasmodia of P. polycephalum grown on agar plates were found to release a mixture of volatile terpenoids consisting of four major sesquiterpenes (α-muurolene, (E)-β-caryophyllene, two unidentified sesquiterpenoids) and the monoterpene linalool. There were no qualitative differences in terpenoid composition at two stages of young plasmodia. To understand terpene biosynthesis, we analyzed the transcriptome and genome sequences of P. polycephalum and identified four TPS genes designated PpolyTPS1–PpolyTPS4. They share 28–73% of sequence identities. Full-length cDNAs for the four TPS genes were cloned and expressed in Escherichia coli to produce recombinant proteins, which were tested for sesquiterpene synthase and monoterpene synthase activities. While neither PpolyTPS2 nor PpolyTPS3 was active, PpolyTPS1 and PpolyTPS4 were able to produce sesquiterpenes and monoterpenes from the respective substrates farnesyl diphosphate and geranyl diphosphate. By comparing the volatile profile of P. polycephalum plasmodia and the in vitro products of PpolyTPS1 and PpolyTPS4, it was concluded that most sesquiterpenoids emitted from P. polycephalum were attributed to PpolyTPS4. Phylogenetic analysis placed the four PpolyTPSs genes into two groups: PpolyTPS1 and PpolyTPS4 being one group that was clustered with the TPSs from the dictyostelid social amoeba and PpolyTPS2 and PpolyTPS3 being the other group that showed closer relatedness to bacterial TPSs. The biological role of the volatile terpenoids produced by the plasmodia of P. polycephalum is discussed.

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Li, Yuanjun, Fangfang Chen, Zhenqiu Li, Changfu Li, and Yansheng Zhang. "Identification and Functional Characterization of Sesquiterpene Synthases fromXanthium strumarium." Plant and Cell Physiology 57, no. 3 (February 8, 2016): 630–41. http://dx.doi.org/10.1093/pcp/pcw019.

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Durairaj, Janani, Elena Melillo, Harro J. Bouwmeester, Jules Beekwilder, Dick de Ridder, and Aalt D. J. van Dijk. "Integrating structure-based machine learning and co-evolution to investigate specificity in plant sesquiterpene synthases." PLOS Computational Biology 17, no. 3 (March 22, 2021): e1008197. http://dx.doi.org/10.1371/journal.pcbi.1008197.

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Sesquiterpene synthases (STSs) catalyze the formation of a large class of plant volatiles called sesquiterpenes. While thousands of putative STS sequences from diverse plant species are available, only a small number of them have been functionally characterized. Sequence identity-based screening for desired enzymes, often used in biotechnological applications, is difficult to apply here as STS sequence similarity is strongly affected by species. This calls for more sophisticated computational methods for functionality prediction. We investigate the specificity of precursor cation formation in these elusive enzymes. By inspecting multi-product STSs, we demonstrate that STSs have a strong selectivity towards one precursor cation. We use a machine learning approach combining sequence and structure information to accurately predict precursor cation specificity for STSs across all plant species. We combine this with a co-evolutionary analysis on the wealth of uncharacterized putative STS sequences, to pinpoint residues and distant functional contacts influencing cation formation and reaction pathway selection. These structural factors can be used to predict and engineer enzymes with specific functions, as we demonstrate by predicting and characterizing two novel STSs from Citrus bergamia.

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Zhang, Tao, Jianjv Feng, Wenni He, Xiaoting Rong, Hui Lv, Jun Li, Xinxin Li, et al. "Genomic and Transcriptomic Approaches Provide a Predictive Framework for Sesquiterpenes Biosynthesis in Desarmillaria tabescens CPCC 401429." Journal of Fungi 9, no. 4 (April 17, 2023): 481. http://dx.doi.org/10.3390/jof9040481.

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Terpenoids constitute a structurally diverse class of secondary metabolites with wide applications in the pharmaceutical, fragrance and flavor industries. Desarmillaria tabescens CPCC 401429 is a basidiomycetous mushroom that could produce anti-tumor melleolides. To date, no studies have been conducted to thoroughly investigate the sesquiterpenes biosynthetic potential in Desarmillaria or related genus. This study aims to unravel the phylogeny, terpenome, and functional characterization of unique sesquiterpene biosynthetic genes of the strain CPCC 401429. Herein, we report the genome of the fungus containing 15,145 protein-encoding genes. MLST-based phylogeny and comparative genomic analyses shed light on the precise reclassification of D. tabescens suggesting that it belongs to the genus Desarmillaria. Gene ontology enrichment and pathway analyses uncover the hidden capacity for producing polyketides and terpenoids. Genome mining directed predictive framework reveals a diverse network of sesquiterpene synthases (STSs). Among twelve putative STSs encoded in the genome, six ones are belonging to the novel minor group: diverse Clade IV. In addition, RNA-sequencing based transcriptomic profiling revealed differentially expressed genes (DEGs) of the fungus CPCC 401429 in three different fermentation conditions, that of which enable us to identify noteworthy genes exemplified as STSs coding genes. Among the ten sesquiterpene biosynthetic DEGs, two genes including DtSTS9 and DtSTS10 were selected for functional characterization. Yeast cells expressing DtSTS9 and DtSTS10 could produce diverse sesquiterpene compounds, reinforced that STSs in the group Clade IV might be highly promiscuous producers. This highlights the potential of Desarmillaria in generating novel terpenoids. To summarize, our analyses will facilitate our understanding of phylogeny, STSs diversity and functional significance of Desarmillaria species. These results will encourage the scientific community for further research on uncharacterized STSs of Basidiomycota phylum, biological functions, and potential application of this vast source of secondary metabolites.

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Jones, Christopher G., Jessie Moniodis, Katherine G. Zulak, Adrian Scaffidi, Julie A. Plummer, Emilio L. Ghisalberti, Elizabeth L. Barbour, and Jörg Bohlmann. "Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases." Journal of Biological Chemistry 287, no. 45 (November 2, 2012): 37713–14. http://dx.doi.org/10.1074/jbc.a111.231787.

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Jones, Christopher G., Jessie Moniodis, Katherine G. Zulak, Adrian Scaffidi, Julie A. Plummer, Emilio L. Ghisalberti, Elizabeth L. Barbour, and Jörg Bohlmann. "Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases." Journal of Biological Chemistry 286, no. 20 (March 24, 2011): 17445–54. http://dx.doi.org/10.1074/jbc.m111.231787.

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Chen, Xinlu, Marcin Nowicki, Phillip A. Wadl, Chi Zhang, Tobias G. Köllner, Miriam Payá‐Milans, Matthew L. Huff, Margaret E. Staton, Feng Chen, and Robert N. Trigiano. "Chemical profile and analysis of biosynthetic pathways and genes of volatile terpenes in Pityopsis ruthii, a rare and endangered flowering plant." PLOS ONE 18, no. 6 (June 23, 2023): e0287524. http://dx.doi.org/10.1371/journal.pone.0287524.

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It is critical to gather biological information about rare and endangered plants to incorporate into conservation efforts. The secondary metabolism of Pityopsis ruthii, an endangered flowering plant that only occurs along limited sections of two rivers (Ocoee and Hiwassee) in Tennessee, USA was studied. Our long-term goal is to understand the mechanisms behind P. ruthii’s adaptation to restricted areas in Tennessee. Here, we profiled the secondary metabolites, specifically in flowers, with a focus on terpenes, aiming to uncover the genomic and molecular basis of terpene biosynthesis in P. ruthii flowers using transcriptomic and biochemical approaches. By comparative profiling of the nonpolar portion of metabolites from various tissues, P. ruthii flowers were rich in terpenes, which included 4 monoterpenes and 10 sesquiterpenes. These terpenes were emitted from flowers as volatiles with monoterpenes and sesquiterpenes accounting for almost 68% and 32% of total emission of terpenes, respectively. These findings suggested that floral terpenes play important roles for the biology and adaptation of P. ruthii to its limited range. To investigate the biosynthesis of floral terpenes, transcriptome data for flowers were produced and analyzed. Genes involved in the terpene biosynthetic pathway were identified and their relative expressions determined. Using this approach, 67 putative terpene synthase (TPS) contigs were detected. TPSs in general are critical for terpene biosynthesis. Seven full-length TPS genes encoding putative monoterpene and sesquiterpene synthases were cloned and functionally characterized. Three catalyzed the biosynthesis of sesquiterpenes and four catalyzed the biosynthesis of monoterpenes. In conclusion, P. ruthii plants employ multiple TPS genes for the biosynthesis of a mixture of floral monoterpenes and sesquiterpenes, which probably play roles in chemical defense and attracting insect pollinators alike.

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LÃ¯Â¿Â½pez-Gallego, Fernando, GraysonT Wawrzyn, and Claudia Schmidt-Dannert. "Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis." Applied and Environmental Microbiology 76, no. 23 (October 1, 2010): 7723–33. http://dx.doi.org/10.1128/aem.01811-10.

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ABSTRACT Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.

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Burkhardt, Immo, Nina B. Kreuzenbeck, Christine Beemelmanns, and Jeroen S. Dickschat. "Mechanistic characterization of three sesquiterpene synthases from the termite-associated fungus Termitomyces." Organic & Biomolecular Chemistry 17, no. 13 (2019): 3348–55. http://dx.doi.org/10.1039/c8ob02744g.

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Sundaraj, Yasotha, Hasdianty Abdullah, Nima Ghahremani Nezhad, Kenneth Francis Rodrigues, Suriana Sabri, and Syarul Nataqain Baharum. "Cloning, Expression and Functional Characterization of a Novel α-Humulene Synthase, Responsible for the Formation of Sesquiterpene in Agarwood Originating from Aquilaria malaccensis." Current Issues in Molecular Biology 45, no. 11 (November 10, 2023): 8989–9002. http://dx.doi.org/10.3390/cimb45110564.

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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.

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Lancaster, Jason, Ashot Khrimian, Sharon Young, Bryan Lehner, Katrin Luck, Anna Wallingford, Saikat Kumar B. Ghosh, et al. "De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug." Proceedings of the National Academy of Sciences 115, no. 37 (August 23, 2018): E8634—E8641. http://dx.doi.org/10.1073/pnas.1800008115.

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Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.

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Köpke, Diana, Ivo Beyaert, Jonathan Gershenzon, Monika Hilker, and Axel Schmidt. "Species-specific responses of pine sesquiterpene synthases to sawfly oviposition." Phytochemistry 71, no. 8-9 (June 2010): 909–17. http://dx.doi.org/10.1016/j.phytochem.2010.03.017.

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Koo, Hyun Jo, Christopher R. Vickery, Yi Xu, Gordon V. Louie, Paul E. O'Maille, Marianne Bowman, Charisse M. Nartey, Michael D. Burkart, and Joseph P. Noel. "Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants." Journal of Antibiotics 69, no. 7 (June 22, 2016): 524–33. http://dx.doi.org/10.1038/ja.2016.68.

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Journal articles on the topic 'Sesquiterpene synthases'

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