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Covering: 2015 to 2022 In 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. [ABSTRACT FROM AUTHOR]

The multiproduct chimeric sesterterpene synthase AcAS from Aspergillus calidoustus yielded spirocyclic calidoustene, which exhibits a novel skeleton, besides five known sesterterpenes. The complex cyclisation mechanism to all six compounds was investigated by isotopic labelling experiments in combination with DFT calculations. Chemically synthesised 8‐hydroxyfarnesyl diphosphate was converted with isopentenyl diphosphate and AcAS into four oxygenated sesterterpenoids that structurally resemble cytochrome P450 oxidation products of the sesterterpene hydrocarbons. Protein engineering of AcAS broadened the substrate scope and gave significantly improved enzyme yields. [ABSTRACT FROM AUTHOR]

Die chimäre Multiprodukt‐Sesterterpensynthase AcAS aus Aspergillus calidoustus lieferte neben fünf bekannten Sesterterpenen das spirocyclische Calidousten, welches ein neues Gerüst aufweist. Der komplexe Cyclisierungsmechanismus zu allen sechs Verbindungen wurde durch Isotopenmarkierungsexperimente in Kombination mit DFT‐Rechnungen untersucht. Chemisch synthetisiertes 8‐Hydroxyfarnesyldiphosphat wurde mit Isopentenyldiphosphat und AcAS in vier oxygenierte Sesterterpenoide umgewandelt, die strukturell den Cytochrom‐P450‐Oxidationsprodukten der Sesterterpen‐Kohlenwasserstoffe ähneln. Das Proteinengineering von AcAS erweiterte dessen Substratspektrum und führte zu signifikant verbesserten Enzymausbeuten. [ABSTRACT FROM AUTHOR]

The fragmentation mechanisms leading to the formation of several pronounced fragment ions formed during EI mass spectrometry of the diterpene hydrocarbons polytrichastrene A and wanju‐2,5‐diene were investigated by isotopic labelling experiments. All twenty singly 13C‐labelled isotopomers of these diterpenes were enzymatically synthesised from the twenty isotopomers of (13C)geranylgeranyl diphosphate using the polytrichastrene synthase from Chryseobacterium polytrichastri. Their mass spectrometric investigation allowed for conclusions on the fragmentation mechanisms leading to the observed fragment ions. [ABSTRACT FROM AUTHOR]

Four synthetic farnesyl diphosphate analogues were enzymatically converted with three bacterial sesquiterpene synthases, including β‐himachalene synthase (HcS) and (Z)‐γ‐bisabolene synthase (BbS) from Cryptosporangium arvum, and germacrene A synthase (SmTS6) from Streptomyces mobaraensis. These enzyme reactions not only yielded several previously unknown compounds, showing that this approach opened the door to a new chemical space, but substrates with blocked or altered reactivities also gave interesting insights into the cyclisation mechanisms and the potential to catalyse reactions with different initial cyclisation modes. [ABSTRACT FROM AUTHOR]

A reinvestigation of the linalool synthase from Chryseobacterium polytrichastri uncovered its diterpene synthase activity, yielding polytrichastrene A and polytrichastrol A with new skeletons, besides known wanju‐2,5‐diene and thunbergol. The enzyme mechanism was investigated by isotopic labeling experiments and DFT calculations to explain an unusual ethyl group formation. Rationally designed exchanges of active site residues showed major functional switches, resulting for I66F in the production of five more new compounds, including polytrichastrene B and polytrichastrol B, while A87T, A192V and the double exchange A87T, A192V gave a product shift towards wanju‐2,5‐diene. [ABSTRACT FROM AUTHOR]

Eine erneute Untersuchung der Linaloolsynthase aus Chryseobacterium polytrichastri zeigte dessen Aktivität als Diterpensynthase, wodurch neben Wanju‐2,5‐dien und Thunbergol Polytrichastren A und Polytrichastrol A mit neuen Skeletten erhalten wurden. Der Enzymmechanismus wurde durch Isotopenmarkierungsexperimente und DFT‐Rechnungen untersucht, um eine ungewöhnliche Ethylgruppenbildung zu studieren. Rational ausgewählte Austausche von Residuen des aktiven Zentrums führten zu bedeutenden Funktionsänderungen und resultierten für I66F zur Produktion fünf weiterer neuer Verbindungen, einschließlich Polytrichastren B und Polytrichastrol B, während A87T, A192V und der doppelte Austausch A87T,A192V eine Produktverschiebung zu Wanju‐2,5‐dien ergaben. [ABSTRACT FROM AUTHOR]

The dauc‐8‐en‐11‐ol synthase from Streptomyces venezuelae was investigated for its catalytic activity towards alternative terpene precursors, specifically designed to enable new cyclisation pathways. Exchange of aromatic amino acid residues at the enzyme surface by site‐directed mutagenesis led to a 4‐fold increase of the yield in preparative scale incubations, which likely results from an increased enzyme stability instead of improved enzyme kinetics. [ABSTRACT FROM AUTHOR]

A biosynthetic gene cluster from Streptomyces mobaraensis encoding the first cases of a bacterial geranylfarnesyl diphosphate synthase and a type I sesterterpene synthase was identified. The structures of seven sesterterpenes produced by these enzymes were elucidated, including their absolute configurations. The enzyme mechanism of the sesterterpene synthase was investigated by extensive isotope labeling experiments. [ABSTRACT FROM AUTHOR]

Ein Biosynthesegencluster aus Streptomyces mobaraensis wurde identifiziert, das die ersten Beispiele einer bakteriellen Geranylfarnesyldiphosphatsynthase und einer Typ I Sesterterpensynthase kodiert. Die Strukturen von sieben durch diese Enzyme produzierten Sesterterpene einschließlich ihrer absoluten Konfigurationen wurden aufgeklärt. Der Enzymmechanismus der Sesterterpensynthase wurde durch extensive Isotopenmarkierungsexperimente untersucht. [ABSTRACT FROM AUTHOR]

Methylated analogues of isopentenyl diphosphate were synthesised and enzymatically incorporated into methylated terpenes. A detailed stereochemical analysis of the obtained products is presented. The methylated terpene precursors were also used in conjunction with various isotopic labellings to gain insights into the mechanisms of their enzymatic formation. [ABSTRACT FROM AUTHOR]

Based on a terpenoid overproduction platform in yeast for genome mining, a chimeric diterpene synthase from the endophytic fungus Colletotrichum gloeosporioides ES026 was characterized as the (5R,12R,14S)‐dolasta‐1(15),8‐diene synthase. The absolute configuration was independently verified through the use of enantioselectively deuterated terpene precursors, which unequivocally established the predicted C1‐III‐IV cyclization mode for this first characterized clade II‐D enzyme. Extensive isotopic labeling experiments and isolation of the intermediate (1R)‐δ‐araneosene supported the proposed cyclization mechanism. The missing link: Fungal chimeric diterpene synthases with prenyltransferase and terpene synthase domains fall into two clades (I+II) with six subclades (A–F). The present study reports the characterization of the first clade II‐D enzyme, the structures of its products, and the cyclization mechanism. The observed 12R configuration reveals a uniform stereochemistry at this carbon for all clade II enzyme products. [ABSTRACT FROM AUTHOR]

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Both enantiomers of 2-methyllinalyl diphosphate (2-Me-LPP) were synthesized enantioselectively using Sharpless epoxidation as a key step and purification of enantiomerically enriched intermediates through HPLC separation on a chiral stationary phase. Their enzymatic conversion with 2-methylisoborneol synthase (2MIBS) demonstrates that ( R )-2-Me-LPP is the on-pathway intermediate, while a minor formation of 2-methylisoborneol from ( S )-2-Me-LPP may be explained by isomerization to 2-Me-GPP and then to ( R )-2-Me-LPP., (Copyright © 2022, Gu et al.)

A barley diterpene synthase (HvKSL4) was found to produce (14 S )-cleistantha-8,12-diene ( 1 ). Formation of the nearly planar cyclohexa-1,4-diene configuration leaves the ring poised for aromatization, but necessitates a deceptively complicated series of rearrangements steered through a complex energetic landscape, as elucidated here through quantum chemical calculations and labeling studies.

The sesterterpene synthase SmTS1 from Streptomyces mobaraensis contains several unusual residues in positions that are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed, turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions., (Copyright © 2021, Hou and Dickschat.)

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O 2 into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphic Burkholderia plantarii . Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens ).

A 13 C-labelling was introduced into each individual carbon of the recently discovered sestermobaraenes by the enzymatic conversion of the correspondingly 13 C-labelled isoprenyl diphosphate precursors with the sestermobaraene synthase from Streptomyces mobaraensis . The main compounds sestermobaraenes A, B, and C were analysed by gas chromatography-mass spectrometry (GC-MS), allowing for a deep mechanistic investigation of the electron impact mass spectrometry (EIMS) fragmentation reactions of these sesterterpene hydrocarbons., (Copyright © 2020, Hou and Dickschat; licensee Beilstein-Institut.)

Based on a terpenoid overproduction platform in yeast for genome mining, a chimeric diterpene synthase from the endophytic fungus Colletotrichum gloeosporioides ES026 was characterized as the (5R,12R,14S)-dolasta-1(15),8-diene synthase. The absolute configuration was independently verified through the use of enantioselectively deuterated terpene precursors, which unequivocally established the predicted C1-III-IV cyclization mode for this first characterized clade II-D enzyme. Extensive isotopic labeling experiments and isolation of the intermediate (1R)-δ-araneosene supported the proposed cyclization mechanism., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The potential power of sesquiterpene synthase FgJ03939 from Fusarium graminearum was fully exploited in a farnesyl diphosphate-overexpressing Saccharomyces cerevisiae chassis to produce the novel sesquiterpenes fusariumdiene (1), epi-fusagramineol (2), and fusagramineol (3) with 5/7 bicyclic and 5/6/3 tricyclic ring systems, respectively, as well as five known sesquiterpenes (4-8). The structure of the unusual skeletons was characterized, and an absolute configuration was proposed. A mechanism for the biosynthesis of 1-8 was also proposed.