Identification of volatiles from six marine Celeribacter strains.

The volatiles emitted from six marine Rhodobacteraceae species of the genus Celeribacter were investigated by GC-MS. Besides several known compounds including dimethyl trisulfide and S-methyl methanethiosulfonate, the sulfur-containing compounds ethyl (E)-3-(methylsulfanyl)acrylate and 2-(methyldisulfanyl)benzothiazole were identified and their structures were verified by synthesis. Feeding experiments with [methyl-2H3]methionine, [methyl-13C]methionine and [34S]-3-(dimethylsulfonio)propanoate (DMSP) resulted in the high incorporation into dimethyl trisulfide and S-methyl methanethiosulfonate, and revealed the origin of the methylsulfanyl group of 2-(methyldisulfanyl)benzothiazole from methionine or DMSP, while the biosynthetic origin of the benzothiazol-2-ylsulfanyl portion could not be traced. The heterocyclic moiety of this compound is likely of anthropogenic origin, because 2-mercaptobenzothiazole is used in the sulfur vulcanization of rubber. Also in none of the feeding experiments incorporation into ethyl (E)-3-(methylsulfanyl)acrylate could be observed, questioning its bacterial origin. Our results demonstrate that the Celeribacter strains are capable of methionine and DMSP degradation to widespread sulfur volatiles, but the analysis of trace compounds in natural samples must be taken with care.

Mechanistic divergence between (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol synthases from Dictyostelium purpureum and Streptomyces coelicolor.

The main product of DpTPS9 from the social amoeba Dictyostelium purpureum was identified as (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol that is also known as an intermediate of bacterial geosmin synthase, but the experimentally verified cyclisation mechanisms differ. Together with the low sequence identity this points to convergent evolution. The functionality of selected residues in DpTPS9 was investigated via site-directed mutagenesis experiments.

Mechanistic Studies on Trichoacorenol Synthase from Amycolatopsis benzoatilytica.

Isotopic labeling experiments performed with a newly identified bacterial trichoacorenol synthase established a 1,5-hydride shift occurring in the cyclization mechanism. During EI-MS analysis, major fragments of the sesquiterpenoid were shown to arise via cryptic hydrogen movements. Therefore, the interpretation of earlier results regarding the cyclization mechanism obtained by feeding experiments in Trichoderma is revised.

A Family of Related Fungal and Bacterial Di- and Sesterterpenes: Studies on Fusaterpenol and Variediene.

The absolute configuration of fusaterpenol (GJ1012E) has been revised by an enantioselective deuteration strategy. A bifunctional enzyme with a terpene synthase and a prenyltransferase domain from Aspergillus brasiliensis was characterised as variediene synthase, and the absolute configuration of its product was elucidated. The uniform absolute configurations of these and structurally related di- and sesterterpenes together with a common stereochemical course for the geminal methyl groups of GGPP unravel a similar conformational fold of the substrate in the active sites of the terpene synthases. For variediene, a thermal reaction observed during GC/MS analysis was studied in detail for which a surprising mechanism was uncovered.

Characterization of Micromonocyclol Synthase from the Marine Actinomycete Micromonospora marina.

As a member of a large phylogenetic clade of enzymes in Micromonospora, a terpene synthase from M. marina is functionally characterized to produce micromonocyclol. This diterpene alcohol features a rare 15-membered ring, which prevented elucidation of the only stereocenter by labeling experiments. This problem was addressed by chemical transformation into bicyclic brominated derivatives, whose rigidified skeletons allowed for a stereochemical assignment. Using this strategy, a complete stereochemical model of the cyclization mechanism was also elaborated.

Characterisation of three terpene synthases for ß-barbatene, ß-araneosene and nephthenol from social amoebae.

The products of three terpene synthases from two social amoebae, Dictyostelium discoideum and Dictyostelium purpureum, were identified, showing sesquiterpene synthase activity for one and diterpene synthase activity for the other two enzymes. Site-directed mutagenesis experiments revealed the importance of a newly identfied highly conserved residue for catalytic activity. For one of the enzyme products, ß-araneosene, a bromonium ion induced transannular cyclisation was investigated, yielding the first brominated derivatives of this diterpene.

Mechanistic investigations on multiproduct ß-himachalene synthase from Cryptosporangium arvum.

A bacterial terpene synthase from Cryptosporangium arvum was characterised as a multiproduct ß-himachalene synthase. In vitro studies showed not only a high promiscuity with respect to its numerous sesquiterpene products, including the structurally demanding terpenes longicyclene, longifolene and α-longipinene, but also to its substrates, as additional activity was observed with geranyl- and geranylgeranyl diphosphate. In-depth mechanistic investigations using isotopically labelled precursors regarding the stereochemical course of both 1,11-cyclisation and 1,3-hydride shift furnished a detailed catalytic model suggesting the molecular basis of the observed low product selectivity. The enzyme's synthetic potential was also exploited in the preparation of sesquiterpene isotopomers, which provided insights into their EIMS fragmentation mechanisms.

Terpene Synthase Genes Originated from Bacteria through Horizontal Gene Transfer Contribute to Terpenoid Diversity in Fungi.

Fungi are successful eukaryotes of wide distribution. They are known as rich producers of secondary metabolites, especially terpenoids, which are important for fungi-environment interactions. Horizontal gene transfer (HGT) is an important mechanism contributing to genetic innovation of fungi. However, it remains unclear whether HGT has played a role in creating the enormous chemical diversity of fungal terpenoids. Here we report that fungi have acquired terpene synthase genes (TPSs), which encode pivotal enzymes for terpenoid biosynthesis, from bacteria through HGT. Phylogenetic analysis placed the majority of fungal and bacterial TPS genes from diverse taxa into two clades, indicating ancient divergence. Nested in the bacterial TPS clade is a number of fungal TPS genes that are inferred as the outcome of HGT. These include a monophyletic clade of nine fungal TPS genes, designated as BTPSL for bacterial TPS-like genes, from eight species of related entomopathogenic fungi, including seven TPSs from six species in the genus Metarhizium. In vitro enzyme assays demonstrate that all seven BTPSL genes from the genus Metarhizium encode active enzymes with sesquiterpene synthase activities of two general product profiles. By analyzing the catalytic activity of two resurrected ancestral BTPSLs and one closely related bacterial TPS, the trajectory of functional evolution of BTPSLs after HGT from bacteria to fungi and functional divergence within Metarhizium could be traced. Using M. brunneum as a model species, both BTPSLs and typical fungal TPSs were demonstrated to be involved in the in vivo production of terpenoids, illustrating the general importance of HGT of TPS genes from bacteria as a mechanism contributing to terpenoid diversity in fungi.

Stereochemical investigations on the biosynthesis of achiral (Z)-γ-bisabolene in Cryptosporangium arvum.

A newly identified bacterial (Z)-γ-bisabolene synthase was used for investigating the cyclisation mechanism of the sesquiterpene. Since the stereoinformation of both chiral putative intermediates, nerolidyl diphosphate (NPP) and the bisabolyl cation, is lost during formation of the achiral product, the intriguing question of their absolute configurations was addressed by incubating both enantiomers of NPP with the recombinant enzyme, which resolved in an exclusive cyclisation of (R)-NPP, while (S)-NPP that is non-natural to the (Z)-γ-bisabolene synthase was specifically converted into (E)-ß-farnesene. A hypothetical enzyme mechanistic model that explains these observations is presented.

Diterpene Biosynthesis in Actinomycetes: Studies on Cattleyene Synthase and Phomopsene Synthase.

Three diterpene synthases from actinomycetes have been studied. The first enzyme from Streptomyces cattleya produced the novel compound cattleyene. The other two enzymes from Nocardia testacea and Nocardia rhamnosiphila were identified as phomopsene synthases. The cyclisation mechanism of cattleyene synthase and the EIMS fragmentation mechanism of its product were extensively studied by incubation experiments with isotopically labelled precursors. Oxidative transformations expanded the chemical space of these unique diterpenes.

Addressing the Chemistry of Germacrene A by Isotope Labeling Experiments.

Despite the central role of germacrene A in sesquiterpene biosynthesis and its widespread occurrence in nature, its complete NMR characterization is still pending. This problem was solved through enzymatic preparation of germacrene A isotopomers that allowed for a full signal assignment to all three conformers. The obtained materials gave insights into the stereochemical course of the Cope rearrangement to ß-elemene and uncovered the Cope rearrangement as a new EI-MS fragmentation reaction.

A Branched Diterpene Cascade: The Mechanism of Spinodiene Synthase from Saccharopolyspora spinosa.

A diterpene synthase from Saccharopolyspora spinosa was found to convert geranylgeranyl diphosphate into the new natural products spinodiene A and B, accompanied by 2,7,18-dolabellatriene. The structures and the formation mechanism of the enzyme products were investigated by extensive isotopic labelling experiments, which revealed an unusual branched isomerisation mechanism towards the neutral intermediate 2,7,18-dolabellatriene. A Diels-Alder reaction was used to convert the main diterpene product with its rare conjugated diene moiety into formal sesterterpene alcohols.

Volatiles from the hypoxylaceous fungi Hypoxylon griseobrunneum and Hypoxylon macrocarpum.

The volatiles emitted by the ascomycetes Hypoxylon griseobrunneum and Hypoxylon macrocarpum (Hypoxylaceae, Xylariales) were collected by use of a closed-loop stripping apparatus (CLSA) and analysed by GC-MS. The main compound class of both species were polysubstituted benzene derivatives. Their structures could only be unambiguously determined by comparison to all isomers with different substitution patterns. The substitution pattern of the main compound from H. griseobrunneum, the new natural product 2,4,5-trimethylanisole, was explainable by a polyketide biosynthesis mechanism that was supported by a feeding experiment with (methyl-2H3)methionine.

A Clade II-D Fungal Chimeric Diterpene Synthase from Colletotrichum gloeosporioides Produces Dolasta-1(15),8-diene.

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.

Sesquiterpene Cyclisations Catalysed inside the Resorcinarene Capsule and Application in the Short Synthesis of Isolongifolene and Isolongifolenone.

Terpenes constitute the largest class of natural products and serve as an important source for medicinal treatments. Despite constant progress in chemical synthesis, the construction of complex polycyclic sesqui- and diterpene scaffolds remains challenging. Natural cyclase enzymes, however, are able to form the whole variety of terpene structures from just a handful of linear precursors. Man-made catalysts able to mimic such natural enzymes are lacking. Here, we describe the examples of sesquiterpene cyclisations inside an enzyme-mimicking supramolecular catalyst. This strategy allowed the formation of the tricyclic sesquiterpene isolongifolene in only four steps. The mechanism of the catalysed cyclisation reaction was elucidated using 13C-labelling studies and DFT calculations.

Acyl-group specificity of AHL synthases involved in quorum-sensing in Roseobacter group bacteria.

N-Acylhomoserine lactones (AHLs) are important bacterial messengers, mediating different bacterial traits by quorum sensing in a cell-density dependent manner. AHLs are also produced by many bacteria of the marine Roseobacter group, which constitutes a large group within the marine microbiome. Often, specific mixtures of AHLs differing in chain length and oxidation status are produced by bacteria, but how the biosynthetic enzymes, LuxI homologs, are selecting the correct acyl precursors is largely unknown. We have analyzed the AHL production in Dinoroseobacter shibae and three Phaeobacter inhibens strains, revealing strain-specific mixtures. Although large differences were present between the species, the fatty acid profiles, the pool for the acyl precursors for AHL biosynthesis, were very similar. To test the acyl-chain selectivity, the three enzymes LuxI1 and LuxI2 from D. shibae DFL-12 as well as PgaI2 from P. inhibens DSM 17395 were heterologously expressed in E. coli and the enzymes isolated for in vitro incubation experiments. The enzymes readily accepted shortened acyl coenzyme A analogs, N-pantothenoylcysteamine thioesters of fatty acids (PCEs). Fifteen PCEs were synthesized, varying in chain length from C4 to C20, the degree of unsaturation and also including unusual acid esters, e.g., 2E,11Z-C18:2-PCE. The latter served as a precursor of the major AHL of D. shibae DFL-12 LuxI1, 2E,11Z-C18:2-homoserine lactone (HSL). Incubation experiments revealed that PgaI2 accepts all substrates except C4 and C20-PCE. Competition experiments demonstrated a preference of this enzyme for C10 and C12 PCEs. In contrast, the LuxI enzymes of D. shibae are more selective. While 2E,11Z-C18:2-PCE is preferentially accepted by LuxI1, all other PCEs were not, except for the shorter, saturated C10-C14-PCEs. The AHL synthase LuxI2 accepted only C14 PCE and 3-hydroxydecanoyl-PCE. In summary, chain-length selectivity in AHLs can vary between different AHL enzymes. Both, a broad substrate acceptance and tuned specificity occur in the investigated enzymes.

Two Bacterial Diterpene Synthases from Allokutzneria albata Produce Bonnadiene, Phomopsene, and Allokutznerene.

Two diterpene synthases from Allokutzneria albata were studied for their products, resulting in the identification of the new compound bonnadiene from the first enzyme. Although phylogenetically unrelated to fungal phomopsene synthase, the second enzyme produced a mixture of phomopsene and a biosynthetically linked new compound, allokutznerene, as well as spiroviolene. Both enzymes were subjected to in-depth mechanistic studies involving isotopic labelling experiments, metal-cofactor variation, and site-directed mutagenesis. Oxidation products of phomopsene and allokutznerene are also discussed.

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.

From lignin to nylon: Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida.

Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L-1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L-1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.

Two Diterpene Synthases for Spiroalbatene and Cembrene†A from Allokutzneria albata.

Two bacterial diterpene synthases from the actinomycete Allokutzneria albata were investigated, resulting in the identification of the structurally unprecedented compound spiroalbatene from the first and cembrene A from the second enzyme. Both enzymes were thoroughly investigated in terms of their mechanisms by isotope labeling experiments, site-directed mutagenesis, and variation of the metal cofactors and pH value. For spiroalbatene synthase, the pH- and Mn2+ -dependent formation of the side product thunbergol was observed, which is biosynthetically linked to spiroalbatene.