Revision of the Cyclisation Mechanism for the Diterpene Spiroviolene and Investigations of Its Mass Spectrometric Fragmentation

The diterpene spiroviolene, its diterpene synthase from Streptomyces violens and the experimentally determined terpene cyclisation mechanism were reported in 2017. Recently, the structure of spiroviolene was revised based on a total synthesis, with consequences for the cyclisation mechanism. Herein, a reinvestigation of the terpene cyclisation to spiroviolene and the mass spectrometric fragmentation mechanism investigated by ¹³C-labelling experiments are presented.     

Geographic Variation and Tissue Distribution of Endogenous Terpenoids in the Northeastern Pacific Ocean Dorid Nudibranch Cadlina luteomarginata: Implications for the Regulation of De Novo Biosynthesis

Gas chromatography (GC) analyses of whole animal skin extracts and individual tissue extracts obtained from specimens of Cadlina luteomarginata collected in British Columbia and southern California were used to determine if concentrations of the nudibranch's biosynthetic products—albicanyl acetate (1), cadlinaldehyde (2), and luteone (3)—vary significantly between two populations, among individuals of a population, and among body tissues of individual specimens. The major biosynthetic product, albicanyl acetate (1), has the same concentration in both British Columbia and California populations, while the British Columbia population contains greater total amounts of 2 and 3 than the California population. Within individuals from one population, the largest proportion of endogenous metabolites is in the dorsum, specifically in the mantle dermal formations and margins. The GC analyses show that across geographically separated populations and within geographically localized populations the concentration of endogenous metabolites is inversely correlated with availability of structurally similar compounds from dietary sources. This suggests that the de novo biosynthesis of defensive compounds might be regulated according to need.     

Polymerization of β-pinene with ethylaluminum dichloride (C2H5AlCl2)

The main goal of this work is to study the cationic polymerization of terpenes, particularly of β-pinene, with alkylaluminum catalysts. Some experiments at different polymerization temperatures (10, 20, and 50°C) were carried out with ethylaluminum dichloride catalyst, C₂H₅AlCl₂. From the results, it is possible to draw some general conclusions about the evolution of chain growth during polymerization. This work also includes the influence of reaction temperature on some properties of the final resin, particularly the molecular weight distribution, the softening point, and the Gardner color index. The role of reducing agents, such as iodine, during the distillation of the final resin is also evaluated with respect to color and softening point. As a result of this investigation, the Mark–Howink constants for terpene resins in toluene and dicloromethane as solvents have also been experimentally determined, thus allowing a more precise use of size exclusion chromatography in the characterization of such natural based products. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 2558–2565, 2001     

Chimeric Terpene Synthases Possessing both Terpene Cyclization and Prenyltransfer Activities

Prenyltransferase (PT) and terpene synthase (TPS) are key enzymes in the formation of the basic carbon skeletons of terpenoids. The PTs determine the prenyl carbon chain length, whereas TPSs generate the structural complexity of the molecular scaffolds, forming various ring structures. Normally, PTs and TPSs are separate, independent enzymes. However, in 2007, a chimeric enzyme, in which the PT was fused with the TPS, was found in a fungus. Recent studies have revealed that such chimeric TPSs are widely distributed in fungi and function in the biosyntheses of various terpene natural products, including sesterterpenes, which are a relatively rare group of terpenoids. This review summarizes the accumulated knowledge of these recently discovered, unique, chimeric TPSs.     

Biosynthesis of Sesterterpenes,Head‐to‐Tail Triterpenes,and Sesquarterpenes in Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isoprenoid Metabolites

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head‐to‐tail triterpenes, and sesquarterpenes. We have identified an (all‐E)‐isoprenyl diphosphate synthase (E‐IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl‐β‐curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E‐IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone‐7, respectively.     

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1–geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X‐ray diffraction were carried out by employing an Escherichia coli whole‐cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)‐eremophilene synthase. The very tight binding of (+)‐eremophilene (～0.40 μM ), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1–geoA gene cluster is required for the biosynthesis of (+)‐eremophilene derivatives.     

New Sesquiterpene Oxidations with CYP260A1 and CYP264B1 from Sorangium cellulosum So ce56

Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non‐activated C?H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum So ce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)‐nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes.     

Terpenoids are widespread in actinomycetes: a correlation of secondary metabolism and genome data

The genomes of all bacteria with publicly available sequenced genomes have been screened for the presence of sesquiterpene cyclase homologues, resulting in the identification of 55 putative geosmin synthases, 23 homologues of 2-methylisoborneol synthases, and 98 other sesquiterpene cyclase homologues. Most of these enzymes by far were found in actinomycetes. The terpenoid volatiles from 35 strains, including 31 actinomycetes and four strains from other taxa, were collected by using a closed-loop stripping apparatus and identified by GC-MS. All of these bacteria apart from one strain encode sesquiterpene cyclase homologues in their genomes. The identified volatile terpenoids were grouped according to structural similarities and their biosynthetic relationship, and the results of these analyses were correlated to the available genome information, resulting in valuable new insights into bacterial terpene biosynthesis.     

An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes

The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for the enzymatic oxidation of the sesquiterpene alcohols T‐muurolol and isodauc‐8‐en‐11‐ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro‐scale reactions, for the determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450‐catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes.