Enhancing structural diversity of terpenoids by multisubstrate terpene synthases

• Min Li and
• Hui Tao

Beilstein J. Org. Chem. 2024, 20, 959–972, doi:10.3762/bjoc.20.86

• multisubstrate terpene synthases (MSTSs) and highlight their potential applications. Keywords: noncanonical terpene; substrate promiscuity; synthetic biology; terpene synthase; terpenoid; Introduction Terpenoids constitute the largest class of natural products with more than 80000 known structures [1] and a

• terpenes are shown. MEP: methylerythritol 4-phosphate; MVA: mevalonate; PT: prenyltransferase; TS: terpene synthase; MT: methyltransferase. Representative terpenoids produced by plant MSTSs. a) 6 and 7 are products of PamTps1 from Plectranthus amboinicus, 8–10 are products of CoTPS5 from Cananga odorata; b

Functions of enzyme domains in 2-methylisoborneol biosynthesis and enzymatic synthesis of non-natural analogs

• Binbin Gu,
• Lin-Fu Liang and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2023, 19, 1452–1459, doi:10.3762/bjoc.19.104

• Binbin Gu Lin-Fu Liang Jeroen S. Dickschat Kekulé-Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany 10.3762/bjoc.19.104 Abstract Two aspects of the biosynthesis of the non-canonical terpene synthase for 2-methylisoborneol have been

• by two sequential cyclisation reactions to A and B, and terminal quenching with water. This hypothesis was confirmed by the discovery of the biosynthetic genes coding for a GPP methyltransferase (GPPMT) and a type I terpene synthase termed 2-methylisoborneol synthase (2MIBS) [23][24]. Interestingly

Functional characterisation of twelve terpene synthases from actinobacteria

• Anuj K. Chhalodia,
• Houchao Xu,
• Georges B. Tabekoueng,
• Binbin Gu,
• Kizerbo A. Taizoumbe,
• Lukas Lauterbach and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2023, 19, 1386–1398, doi:10.3762/bjoc.19.100

• they may be bifunctional and composed of two domains. In these enzymes a prenyltransferase domain catalyses the formation of an oligoprenyl pyrophosphate precursor from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) that is subsequently cyclised by the terpene synthase domain

• Phylogenetic analysis A phylogenetic tree was constructed from 4018 bacterial terpene synthase homologs (Figure 2). In this tree all branches of homologous enzymes for which at least one representative was functionally characterised are shown in blue, whereas the uncharacterised enzymes are shown in grey

• , revealing that the functions of still many terpene synthase homologs are unknown. Some of the largest branches in this tree represent the homologs of epi-isozizaene synthase from Streptomyces coelicolor [24], caryolan-1-ol synthase from Streptomyces griseus [25], selina-4(15),7(11)-diene synthase from

Anti-inflammatory aromadendrane- and cadinane-type sesquiterpenoids from the South China Sea sponge Acanthella cavernosa

• Shou-Mao Shen,
• Qing Yang,
• Yi Zang,
• Jia Li,
• Xueting Liu and
• Yue-Wei Guo

Beilstein J. Org. Chem. 2022, 18, 916–925, doi:10.3762/bjoc.18.91

• ). Different enantiomeric ratios could explain the properties of the active sites in the corresponding terpene synthases, which remain unclear for further investigations [22]. The diversified structures of terpenes were constructed by terpene synthase [26] along with the post-modification enzymes, such as P450

Volatile emission and biosynthesis in endophytic fungi colonizing black poplar leaves

• Christin Walther,
• Pamela Baumann,
• Katrin Luck,
• Beate Rothe,
• Peter H. W. Biedermann,
• Jonathan Gershenzon,
• Tobias G. Köllner and
• Sybille B. Unsicker

Beilstein J. Org. Chem. 2021, 17, 1698–1711, doi:10.3762/bjoc.17.118

• Cladosporium sp. emitted (E)-β-caryophyllene (1) in culture (Table 2, Figure 1). As this sesquiterpene is also a characteristic VOC in the constitutive and herbivore-induced blends of black poplar [57][58][59], we wanted to identify and characterize the responsible fungal terpene synthase, as this enzyme could

• contribute to the overall (E)-β-caryophyllene emission from the tree. To identify terpene synthase genes potentially involved in volatile terpene formation in Cladosporium, we sequenced the transcriptome and performed a de novo assembly of the obtained reads. A TBLASTN analysis with Aspergillus terreus

• known terpene synthases from plant-associated Ascomycota exhibiting a pathogenic, endophytic or saprophytic lifestyle. One clade was indeed evident that contained only terpene synthases from endophytes. However, a close relationship between fungal lifestyle and their terpene synthase sequences is not

On the mass spectrometric fragmentations of the bacterial sesterterpenes sestermobaraenes A–C

• Anwei Hou and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2020, 16, 2807–2819, doi:10.3762/bjoc.16.231

• bacterial sesterterpenes that were recently discovered by us from the actinomycete Streptomyces mobaraensis through a genome mining approach (Figure 1) [1]. All seven compounds are produced by a canonical terpene synthase, representing the first reported sesterterpene synthase of the classical type I from

• (Scheme 6B). The inductive ring opening produces v3•+ that, upon α-cleavage with hydrogen rearrangement, leads to w3•+ (m/z = 122). The base peak ion x3•+ then results by the loss of two hydrogens. Conclusion In this work we demonstrated that 13C-labellings can efficiently be introduced by terpene

• synthase catalysed reactions into each single position of a terpene, which is useful for the deep investigations on mass spectrometric fragmentation reactions. The present study provides the first example for such investigations on sesterterpene fragmentations. The applied method, once the synthetic 13C

Understanding the role of active site residues in CotB2 catalysis using a cluster model

• Keren Raz,
• Ronja Driller,
• Thomas Brück,
• Bernhard Loll and
• Dan T. Major

Beilstein J. Org. Chem. 2020, 16, 50–59, doi:10.3762/bjoc.16.7

• ]. Theoretical quantum mechanical (QM) investigations on the chemistry of terpenes in the gas phase have provided a detailed understanding of the carbocation mechanisms underlying terpene synthase function [25][26][27]. Further, we have used multiscale modeling tools to study the effects of the enzyme

• highlight the importance of taking into account the active site residues while modeling terpene synthase mechanisms, as we have proposed previously [28][29][30][31][32][33][34][35][36][42]. We found that the energy surface in the active site model was significantly perturbed compared to the gas phase

• reaction energetics in an active site cluster model, suggesting that reaction control in terpene synthase is obtained via a combination of inherent reactivity, initial substrate folding, and enzyme environmental effects. Specifically, the results using the active site model revealed the significant effect

Emission and biosynthesis of volatile terpenoids from the plasmodial slime mold Physarum polycephalum

• Xinlu Chen,
• Tobias G. Köllner,
• Wangdan Xiong,
• Guo Wei and
• Feng Chen

Beilstein J. Org. Chem. 2019, 15, 2872–2880, doi:10.3762/bjoc.15.281

• . At the 18th day after the transfer, essentially the same profile of volatiles was detected (Figure 1A). Four terpene synthase genes were identified in P. polycephalum With the identification of terpenes from the headspace of P. polycephalum (Figure 1), the next question was how they are synthesized

• To determine whether PpolyTPS genes encode functional terpene synthases, full-length cDNAs were amplified by RT-PCR and cloned into the protein expression vector pEXP-5-CT/TOPO. Recombinant PpolyTPSs were heterologously expressed in Escherichia coli and then tested for terpene synthase activities

• to ask whether volatile terpenoids emitted from P. polycephalum are involved in chemotaxis. Conclusion In this study, we have successfully identified and characterized terpene synthase (TPS) genes that are involved in making volatile terpenoids from a plasmodial slime mold Physarum polycephalum. The

Nanangenines: drimane sesquiterpenoids as the dominant metabolite cohort of a novel Australian fungus, Aspergillus nanangensis

• Heather J. Lacey,
• Cameron L. M. Gilchrist,
• Andrew Crombie,
• John A. Kalaitzis,
• Daniel Vuong,
• Peter J. Rutledge,
• Peter Turner,
• John I. Pitt,
• Ernest Lacey,
• Yit-Heng Chooi and
• Andrew M. Piggott

Beilstein J. Org. Chem. 2019, 15, 2631–2643, doi:10.3762/bjoc.15.256

• terpene synthase family, showing similarity to haloacid dehalogenase (HAD)-like hydrolases [21]. Thus, we suspected that a related enzyme may be involved in the biosynthesis of the nanangenines, and used the amino acid sequence of AstC to probe the A. nanangensis genome. We also hypothesised that the acyl

• . insuetus CBS 107.25 (Figure 2). A homologous cluster was also found in A. pseudodeflectus CBS 756.74, though it is located on a truncated assembly scaffold. In total, six genes are conserved across these species: the HR-PKS (FE257_006541), HAD-like terpene synthase (FE257_006542) and FAD-binding

• analyses above, a biosynthetic pathway to the nanangenines was proposed (Figure 3). Unlike the ast cluster, where there are multiple HAD-like enzymes encoded (one terpene synthase and two phosphatases), the putative nanangenine cluster only encodes one such enzyme, FE257_006542. However, given that

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

• Ronja Driller,
• Daniel Garbe,
• Norbert Mehlmer,
• Monika Fuchs,
• Keren Raz,
• Dan Thomas Major,
• Thomas Brück and
• Bernhard Loll

Beilstein J. Org. Chem. 2019, 15, 2355–2368, doi:10.3762/bjoc.15.228

• mutagenesis have exciting applications for the sustainable production of high value bioactive substances. Keywords: biotechnology; CotB2; crystal structure; cyclooctatin; diterpene; reaction mechanism; terpene synthase; Introduction Terpenes represent one of the most diverse groups of natural biomolecules

• , antibiotic, neuroprotective and even insecticidal activities, which makes these compounds high-value commercial targets for the chemical and pharmaceutical industry [9][10]. Structural diversity of diterpenes is created by the terpene synthase (TPS) enzyme family, which use acyclic isoprenoid precursors to

• ) [12] to the acyclic terpene synthase substrate geranylgeranyl diphosphate 3 (GGDP) [1][13][14][15][16]. Following initial substrate binding and folding in a product-like conformation, the cyclization reaction can be subdivided into three steps: (1) generation of a reactive allyl carbocation as a

Genome mining in Trichoderma viride J1-030: discovery and identification of novel sesquiterpene synthase and its products

• Xiang Sun,
• You-Sheng Cai,
• Yujie Yuan,
• Guangkai Bian,
• Ziling Ye,
• Zixin Deng and
• Tiangang Liu

Beilstein J. Org. Chem. 2019, 15, 2052–2058, doi:10.3762/bjoc.15.202

• the combination of genome mining and heterologous expression of predicted terpene synthases for detecting unknown terpenoids from rarely studied fungi. Results and Discussion Prediction and analysis of terpene synthase genes in T. viride J1-030 Through genome sequencing of T. viride J1-030 and

• prediction of the potential terpene synthases in J1-030 genome, gene Tvi09626 was selected and the following bioinformatics analysis of the function of this unidentified terpene synthase was performed. A protein blast search against the NCBI database was performed with Tvi09626, revealing sequence identities

• Trichoderma, such as trichodiene synthase homologous gene isolation and characterisation in T. harzianum [32] and functional identification of terpene synthase vir4 in T. virens [33]. However, owing to the general lack of previous studies of terpene synthases in T. viride, Tvi09626 is the first terpene

Bipolenins K–N: New sesquiterpenoids from the fungal plant pathogen Bipolaris sorokiniana

• Chin-Soon Phan,
• Hang Li,
• Simon Kessler,
• Peter S. Solomon,
• Andrew M. Piggott and
• Yit-Heng Chooi

Beilstein J. Org. Chem. 2019, 15, 2020–2028, doi:10.3762/bjoc.15.198

• Cochliobolus sp.) [1][26], were reported in B. sorokiniana for the first time, while, known metabolites 6 and 7 [20], 9 [34], 11 [2], and 12 [16] were previously reported from B. sorokiniana (syn. C. sativus and H. sativum). The terpene synthase responsible for the biosynthesis of the sativene/longifolene

Phylogenomic analyses and distribution of terpene synthases among Streptomyces

• Lara Martín-Sánchez,
• Kumar Saurabh Singh,
• Mariana Avalos,
• Gilles P. van Wezel,
• Jeroen S. Dickschat and
• Paolina Garbeva

Beilstein J. Org. Chem. 2019, 15, 1181–1193, doi:10.3762/bjoc.15.115

• analysis of Streptomyces species, and compared the distribution of terpene synthase genes among them. Overall, our study revealed that ten major types of terpene synthases are present within the genus Streptomyces, namely those for geosmin, 2-methylisoborneol, epi-isozizaene, 7-epi-α-eudesmol, epi-cubenol

• revealed that terpenoids can be produced by all kingdoms of life including bacteria, fungi and protists [6][7][8][9][10]. The ability of an organism to produce terpenoids relies on whether the organism contains terpene synthase genes. Biosynthetically, the production of the different types of terpenes

• , little is known about the distribution of terpene synthase encoding genes among Streptomyces. Are terpene synthase genes specific for certain species or randomly distributed among Streptomyces? To address this question, phylogenomic analyses of Streptomyces species were performed, using complete genomes

Mechanistic investigations on multiproduct β-himachalene synthase from Cryptosporangium arvum

• Jan Rinkel and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2019, 15, 1008–1019, doi:10.3762/bjoc.15.99

• Jan Rinkel Jeroen S. Dickschat Kekulé-Institute for Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany 10.3762/bjoc.15.99 Abstract A bacterial terpene synthase from Cryptosporangium arvum was characterised as a multiproduct β-himachalene synthase

• ). Possible EI-fragmentation mechanisms connected to them are discussed in Schemes S1–S3 (Supporting Information File 1). Conclusion In summary, a new terpene synthase from C. arvum was characterised as a multiproduct (+)-β-himachalene synthase. Accepting GPP, FPP and GGPP, HcS is a promiscuous enzyme, whose

Volatiles from three genome sequenced fungi from the genus Aspergillus

• Jeroen S. Dickschat,
• Ersin Celik and
• Nelson L. Brock

Beilstein J. Org. Chem. 2018, 14, 900–910, doi:10.3762/bjoc.14.77

• ][30], but geosmin could not be observed as a volatile of A. fischeri. The bacterial geosmin synthase is a class I terpene synthase (TS) with two domains [31] that occurs in many actinomycetes, cyanobacteria and myxobacteria, but fungal geosmin biosynthesis must require a different enzyme, because no

• ). A phylogenetic analysis of 878 fungal terpene synthase homologs (Figure S1 in Supporting Information File 1) demonstrates that this enzyme is closely related to the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase from Fusarium fujikuroi [33]. The N-terminal domain shows the DXDD

18-Hydroxydolabella-3,7-diene synthase – a diterpene synthase from Chitinophaga pinensis

• Jeroen S. Dickschat,
• Jan Rinkel,
• Patrick Rabe,
• Arman Beyraghdar Kashkooli and
• Harro J. Bouwmeester

Beilstein J. Org. Chem. 2017, 13, 1770–1780, doi:10.3762/bjoc.13.171

• previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase

• , revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products. Keywords: biosynthesis; Chitinophaga pinensis; Nicotiana benthamiana; structure elucidation; terpenes; Introduction Terpene synthases convert a handful of simple linear

• reports: [7][8][9][10][11][12][13][14]). One possible method to investigate the products of terpene synthases is the expression of terpene synthase genes in a heterologous host, as was recently performed for a large number of bacterial enzymes in an engineered Streptomyces avermitilis strain from which

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

• Katarina Kemper,
• Max Hirte,
• Markus Reinbold,
• Monika Fuchs and
• Thomas Brück

Beilstein J. Org. Chem. 2017, 13, 845–854, doi:10.3762/bjoc.13.85

• synthases do not comprise combinations of Class I/Class II domains but contain both a prenyltransferase domain and a terpene synthase moiety. This combination of catalytic modules allows the direct formation of the isoprenyl diphosphate substrate for the terpene synthase in a single biocatalyst. An unusual

• example of these bifunctional enzymes was published by Chen and coworkers [60], who managed to crystalize catalytic domains of PaFS, a diterpene synthase from Phomopsis amygdali. The formation of GGPP is located in a C-terminal α-domain with very low sequence identity to the N-terminal Class I terpene

• synthase domain indicating different catalytical properties. The natural product of PaFS is fusicocca-2,10(14)-diene, an intermediate in the biosynthesis of the phytotoxin fusicoccin A by P. amygdali. Interestingly, a recent work by Qin and co-workers [71] even revealed the conversion of a fungal diterpene

Mechanistic investigations on six bacterial terpene cyclases

• Patrick Rabe,
• Thomas Schmitz and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2016, 12, 1839–1850, doi:10.3762/bjoc.12.173

• relative of the enzyme from S. viridochromogenes is found in Streptomyces sp. NRRL S-481 (86% identity). Incubation of FPP with a recombinant terpene synthase from Roseiflexus castenholzii DSM 13941 (accession number WP_012119179) resulted in the formation of the sesquiterpene alcohol 2, previously

• . Heterologous expression of a third terpene synthase from Streptosporangium roseum DSM 43021 (accession number WP_043653400) and its incubation with FPP yielded the sesquiterpene alcohol 3, identified as 4-epi-cubebol by GC–MS, and minor amounts of cubebol, germacrene D-4-ol and δ-cadinene (Figure S1

• relative (94% identical sites). Another terpene synthase from Chitinophaga pinensis DSM 2588 (accession number WP_012792334) converted FPP into a terpene hydrocarbon 5, identified as γ-cadinene by GC–MS [32], besides traces of α- and δ-cadinene (Figure S1, Supporting Information File 1), while no reaction

Dynamic behavior of rearranging carbocations – implications for terpene biosynthesis

• Stephanie R. Hare and
• Dean J. Tantillo

Beilstein J. Org. Chem. 2016, 12, 377–390, doi:10.3762/bjoc.12.41

• tendencies, i.e., the dynamical behavior of carbocations in the absence of an enzyme, tend to be reflected in product distributions for enzyme-promoted reactions. Consequently, the problem of elucidating the role of terpene synthase enzymes in terpene formation has been redefined. In addition, these studies

• the absence of specifically oriented noncovalent interactions with groups in terpene synthase active sites. Molecular dynamics calculations using the full bornyl diphosphate synthase enzyme were also carried out (here using a combination of DFT and molecular mechanics) [21][22]. These simulations

Recent highlights in biosynthesis research using stable isotopes

• Jan Rinkel and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2015, 11, 2493–2508, doi:10.3762/bjoc.11.271

• interesting insights into terpene synthase catalyzed cyclizations. Labeled oligoprenyl diphosphates, the substrates for these enzymes, can be made available by synthesis and provide an excellent tool for such investigations, as recently demonstrated for sesquiterpenes by the synthesis of all 15 singly 13C

• rearrangement in a concerted process leads to 42 and 43. The protonation of C-5 was shown by using (2-13C)FPP as a substrate for an in vitro incubation of the terpene synthase in D2O leading to characteristic strongly enhanced triplets for the labeled carbons of 42 and 43 in the 13C NMR spectrum. As an

• signals mentioned above. To investigate the 1,5-hydride shift, (8,8-2H2)GGPP and IPP were used for an in vitro reaction with the recombinant terpene synthase, utilizing the bifunctional character of the enzyme to form (12,12-2H2)GFPP and its subsequent cyclization to (2H2)-59. NMR data of the obtained