Discovery of non-squalene triterpenes.

All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene1. This approach is fundamentally different from the biosynthesis of short-chain (C10-C25) terpenes that are formed from polyisoprenyl diphosphates2-4. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.

The Stereochemical Course of DmdC, an Enzyme Involved in the Degradation of Dimethylsulfoniopropionate.

The acyl-CoA dehydrogenase DmdC is involved in the degradation of the marine sulfur metabolite dimethylsulfonio propionate (DMSP) through the demethylation pathway. The stereochemical course of this reaction was investigated through the synthesis of four stereoselectively deuterated substrate surrogates carrying stereoselective deuterations at the α- or the ß-carbon. Analysis of the products revealed a specific abstraction of the 2-pro-R proton and of the 3-pro-S hydride, establishing an anti elimination for the DmdC reaction.

Functional and Mechanistic Characterization of the 4,5-diepi-Isoishwarane Synthase from the Liverwort Radula lindenbergiana.

The microbial type sesquiterpene synthase RlMTPSL4 from the liverwort Radula lindenbergiana was investigated for its products, showing the formation of several sesquiterpene hydrocarbons. The main product was structurally characterized as the new compound 4,5-diepi-isoishwarane, while the side products included the known hydrocarbons germacrene A, α-selinene, eremophilene and 4,5-diepi-aristolochene. The cyclization mechanism towards 4,5-diepi-isoishwarane catalyzed by RlMTPSL4 was investigated through isotopic labeling experiments, revealing the stereochemical course for the deprotonation step to the neutral intermediate germacrene A, a reprotonation for its further cyclization, and a 1,2-hydride shift along the cascade. The absolute configuration of 4,5-diepi-isoishwarane was determined using a stereoselective deuteration approach, revealing an absolute configuration typically observed for a microbial type sesquiterpene.

Enzymatic Synthesis of Diterpenoids from iso-GGPP†III: A Geranylgeranyl Diphosphate Analog with a Shifted Double Bond.

The analog of the diterpene precursor geranylgeranyl diphosphate with a double bond shifted from C14=C15 to C15=C16 (named iso-GGPP III) has been synthesized and enzymatically converted with six bacterial diterpene synthases; this allowed the isolation of nine unnatural diterpenes. For some of the enzyme-substrate combinations, the different reactivity implemented in the substrate analog iso-GGPP III opened reaction pathways that are not observed with natural GGPP, resulting in the formation of diterpenes with novel skeletons. A stereoselective deuteration strategy was used to assign the absolute configurations of the isolated diterpenes.

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

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

Enantioselective synthesis of all stereoisomers of geosmin and of biosynthetically related natural products.

Synthetic routes to geosmin and its enantiomer are well established, but the enantioselective synthesis of stereoisomers of geosmin is unknown. Here a stereoselective synthesis of all stereoisomers of geosmin is reported, yielding all compounds in high enantiomeric purity. Furthermore, the stereoselective synthesis of a geosmin derivative isolated from a mangrove associated streptomycete was performed, establishing the absolute configuration of the natural product. Finally, a new side product of the geosmin synthase from Streptomyces ambofaciens was isolated and its structure was elucidated by NMR spectroscopy. The absolute configuration of this new compound was determined through a stereoselective synthesis.

Common Biosynthesis of Non-canonical C16 Terpenes through a Fragmentation-Recombination Mechanism.

The biosynthesis of six recently reported non-canonical C16 sesquiterpenoids named after ancient Greek philosophers including archimedene, aristotelene, eratosthenene, pythagorene, a-democritene and anaximandrene was investigated through density functional theory (DFT) calculations and isotopic labeling experiments. The results revealed for all compounds except archimedene a unique fragmentation-recombination mechanism as previously demonstrated for sodorifen biosynthesis, in addition to a remarkable "dancing" mechanism for anaximandrene biosynthesis.

The Diterpenoid Substrate Analogue 19-nor-GGPP Reveals Pronounced Methyl Group Effects in Diterpene Cyclisations.

The substrate analogue 19-nor-geranylgeranyl diphosphate (19-nor-GGPP) was synthesised and incubated with 20 diterpene synthases, resulting in the formation of diterpenoids in all cases. A total of 23 different compounds were isolated from these enzyme reactions and structurally characterised, if possible including the experimental determination of absolute configurations through a stereoselective deuteration approach. In several cases the missing 19-Me group in the substrate analogue resulted in opening of completely new reaction paths towards compounds with novel skeletons. DFT calculations were applied to gain a deeper understanding of these observed methyl group effects in diterpene biosynthesis.

Two Sesterterpene Synthases from Lentzea atacamensis Demonstrate the Role of Conformational Variability in Terpene Biosynthesis.

Mining of two multiproduct sesterterpene synthases from Lentzea atacamensis resulted in the identification of the synthases for lentzeadiene (LaLDS) and atacamatriene (LaATS). The main product of LaLDS (lentzeadiene) is a new compound, while one of the side products (lentzeatetraene) is the enantiomer of brassitetraene B and the other side product (sestermobaraene F) is known from a surprisingly distantly related sesterterpene synthase. LaATS produces six new compounds, one of which is the enantiomer of the known sesterterpene Bm1. Notably, for both enzymes the products cannot all be explained from one and the same starting conformation of geranylfarnesyl diphosphate, demonstrating the requirement of conformational flexibility of the substrate in the enzymes' active sites. For lentzeadiene an intriguing thermal [1,5]-sigmatropic rearrangement was discovered, reminiscent of the biosynthesis of vitamin D3. All enzyme reactions and the [1,5]-sigmatropic rearrangement were investigated through isotopic labeling experiments and DFT calculations. The results also emphasize the importance of conformational changes during terpene cyclizations.

Biosynthesis of the Non-canonical C17 Sesquiterpenoids Chlororaphen A and B from Pseudomonas chlororaphis.

Chlororaphens A and B are structurally unique non-canonical C17 sesquiterpenoids from Pseudomonas chlororaphis that are made by two SAM-dependent methyltransferases and a type I terpene synthase. This study addresses the mechanism of their formation in isotopic labelling experiments and DFT calculations. The results demonstrate an astonishing complexity with distribution of labellings within a cyclopentane core that is reversely connected to two acyclic fragments in chlororaphen A and B. In addition, the uptake of up to 14 deuterium atoms from D2O was observed. These findings are explainable by a repeated late stage multistep rearrangement sequence. The absolute configurations of the chlororaphens and their biosynthetic intermediates were elucidated in stereoselective labelling experiments.

Structural Insights Into the Terpene Cyclization Domains of Two Fungal Sesterterpene Synthases and Enzymatic Engineering for Sesterterpene Diversification.

Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg2+), clearly defining the catalytic active sites. A combination of theory and experiments including carbocationic intermediates modeling, site-directed mutagenesis, and isotope labeling provided detailed insights into the structural basis for their catalytic mechanisms. Structure-based engineering of NfSS and PbSS resulted in the formation of 20 sesterterpenes including 13 new compounds and four pairs of epimers with different configurations at C18. These results expand the structural diversity of sesterterpenes and provide important insights for future synthetic biology research.

A Functional Switch Between Asperfumene and Fusicoccadiene Synthase and Entrance to Asperfumene Biosynthesis through a Vicinal Deprotonation-Reprotonation Process.

The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and - according to a structural model - active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.

Substrate specificity of a ketosynthase domain involved in bacillaene biosynthesis.

An isotopic labelling method was developed to investigate substrate binding by ketosynthases, exemplified by the second ketosynthase of the polyketide synthase BaeJ involved in bacillaene biosynthesis (BaeJ-KS2). For this purpose, both enantiomers of a 13C-labelled N-acetylcysteamine thioester (SNAC ester) surrogate of the proposed natural intermediate of BaeJ-KS2 were synthesised, including an enzymatic step with glutamate decarboxylase, and incubated with BaeJ-KS2. Substrate binding was demonstrated through 13C NMR analysis of the products against the background of various control experiments.

Structural Insights into Three Sesquiterpene Synthases for the Biosynthesis of Tricyclic Sesquiterpenes and Chemical Space Expansion by Structure-Based Mutagenesis.

The cyclization of farnesyl diphosphate (FPP) into highly strained polycyclic sesquiterpenes is challenging. We here determined the crystal structures of three sesquiterpene synthases (STSs, namely, BcBOT2, DbPROS, and CLM1) catalyzing the biosynthesis of the tricyclic sesquiterpenes presilphiperfolan-8ß-ol (1), Δ6-protoilludene (2), and longiborneol (3). All three STS structures contain a substrate mimic, the benzyltriethylammonium cation (BTAC), in their active sites, providing ideal templates for quantum mechanics/molecular mechanics (QM/MM) analyses toward their catalytic mechanisms. The QM/MM-based molecular dynamics (MD) simulations revealed the cascade reactions toward the enzyme products, and different key active site residues that play important roles in stabilizing reactive carbocation intermediates along the three pathways. Site-directed mutagenesis experiments confirmed the roles of these key residues and concomitantly resulted in 17 shunt products (4-20). Isotopic labeling experiments addressed the key hydride and methyl migrations toward the main and several shunt products. These combined methods provided deep insights into the catalytic mechanisms of the three STSs and demonstrated how the chemical space of STSs can rationally be expanded, which may facilitate applications in synthetic biology approaches toward pharmaceutical and perfumery agents.

Engineering fungal terpene biosynthesis.

Covering: 2015 to 2022Fungal terpenoids are of large structural diversity and often exhibit interesting biological activities. Recent work has focused on two main aspects: (1) the discovery and understanding of unknown biosynthetic genes and pathways, and (2) the usage of already known biosynthetic genes in the construction of high yielding production strains. Both aspects will be covered in this review article that aims to summarise the most important work of the past few years.

Labelling studies in the biosynthesis of polyketides and non-ribosomal peptides.

Covering: 2015 to 2022In this review, we discuss the recent advances in the use of isotopically labelled compounds to investigate the biosynthesis of polyketides, non-ribosomally synthesised peptides, and their hybrids. Also, we highlight the use of isotopes in the elucidation of their structures and investigation of enzyme mechanisms. The biosynthetic pathways of selected examples are presented in detail to reveal the principles of the discussed labelling experiments. The presented examples demonstrate that the application of isotopically labelled compounds is still the state of the art and can provide valuable information for the biosynthesis of natural products.

Volatile sensation: The chemical ecology of the earthy odorant geosmin.

Geosmin may be the most familiar volatile compound, as it lends the earthy smell to soil. The compound is a member of the largest family of natural products, the terpenoids. The broad distribution of geosmin among bacteria in both terrestrial and aquatic environments suggests that this compound has an important ecological function, for example, as a signal (attractant or repellent) or as a protective specialized metabolite against biotic and abiotic stresses. While geosmin is part of our everyday life, scientists still do not understand the exact biological function of this omnipresent natural product. This minireview summarizes the current general observations regarding geosmin in prokaryotes and introduces new insights into its biosynthesis and regulation, as well as its biological roles in terrestrial and aquatic environments.

A Detailed View on Geosmin Biosynthesis.

The bacterial geosmin synthase is a fascinating bifunctional enzyme that has been discovered almost two decades ago. Several aspects of the cyclisation mechanism from FPP to geosmin are known, but a detailed picture of the stereochemical course of this reaction is unknown. This article reports on a deep investigation of the mechanism of geosmin synthase through isotopic labelling experiments. Furthermore, the effects of divalent cations on geosmin synthase catalysis were investigated. The addition of cyclodextrin to enzymatic reactions, a molecule that can capture terpenes, suggests that the biosynthetic intermediate (1(10)E,5E)-germacradien-11-ol produced by the N-terminal domain is passed to the C-terminal domain not through a tunnel, but rather through release into the medium and uptake by the C-terminal domain.

A Case of Convergent Evolution: The Bacterial Sesquiterpene Synthase for 1-epi-Cubenol from Nonomuraea coxensis.

A terpene synthase from Nonomuraea coxensis was identified as (+)-1-epi-cubenol synthase. The enzyme is phylogenetically unrelated to the known enzyme of the same function that is widespread in streptomycetes. Isotopic labelling experiments were performed to unambiguously assign the NMR data and to investigate hydrogen migrations during terpene cyclisations. Epoxidations of (+)-1-epi-cubenol and of the plant derived compounds (-)-cubenol and (-)-1-epi-cubenol confirmed the structure of a natural product isolated from the brown alga Dictyopteris divaricata and allowed to conclude on its absolute configuration. The crystal structures of the epoxides from (+)- and (-)-1-epi-cubenol and the acid catalysed conversion into an isomeric ketone are reported.

Cladosporin, A Highly Potent Antimalaria Drug?

Cladosporin, a unique natural product from the fungus Cladosporium cladosporioides, exhibits nanomolar inhibitory activity against Plasmodium falciparum by targeting its cytosolic lysyl-tRNA synthetase (PfKRS) to inhibit protein biosynthesis. Due to its exquisite selectivity towards pathogenic parasites, cladosporin has become a very promising lead compound for developing antiparasitic drugs to treat drug-resistant malaria and cryptosporidiosis infections. Here we review the recent research progress of cladosporin covering aspects of the chemical synthesis, biosynthesis, bioactivity, cellular target and structure-activity relationship.