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.

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 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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

Characterisation of the l-Cystine ß-Lyase PatB from Phaeobacter inhibens: An Enzyme Involved in the Biosynthesis of the Marine Antibiotic Tropodithietic Acid.

The l-cystine ß-lyase from Phaeobacter inhibens is involved in the biosynthesis of the sulfur-containing antibiotic tropodithietic acid. The recombinant enzyme was obtained by heterologous expression in Escherichia coli and biochemically characterised by unambiguous chemical identification of the products formed from the substrate l-cystine, investigation of the substrate spectrum, determination of the enzyme kinetics, sequence alignment with closely related homologues and site-directed mutagenesis to identify a highly conserved lysine residue that is critical for functionality. PatB from P. inhibens is a new member of the small group of characterised l-cystine ß-lyases and the first example of an enzyme with such an activity that is required for the biosynthesis of an antibiotic. A comparison of PatB to previously reported enzymes with l-cystine ß-lyase activity from bacteria and plants is given.

Mechanistic Characterization of Two Chimeric Sesterterpene Synthases from Penicillium.

The products of two bifunctional fungal sesterterpene synthases (StTPS), with prenyl transferase (PT) and terpene synthase (TPS) domains from Penicillium, were structurally characterized and their mechanisms studied in detail by labeling experiments. A phylogenetic analysis of the TPS domains of the new and previously characterized enzymes revealed six distinct clades. Enzymes from the same clade catalyze a common initial cyclization step, which suggests the potential for structural predictions from amino acid sequences.

Mechanisms of the Diterpene Cyclases ß-Pinacene Synthase from Dictyostelium discoideum and Hydropyrene Synthase from Streptomyces clavuligerus.

Two diterpene cyclases, one from the social amoeba Dictyostelium discoideum and the other from the bacterium Streptomyces clavuligerus, with products containing a Z-configured double bond between the original C2 and C3 of geranylgeranyl diphosphate, were extensively investigated for their mechanisms through isotopic labelling experiments. The participation of geranyllinalyl diphosphate, in analogy to the role of linalyl and nerolidyl diphosphate for mono- and sesquiterpene biosynthesis, as an intermediate towards diterpenes with a Z-configured C2=C3 double bond is discussed.

Spata-13,17-diene Synthase-An Enzyme with Sesqui-, Di-, and Sesterterpene Synthase Activity from Streptomyces xinghaiensis.

A terpene synthase from the marine bacterium Streptomyces xinghaiensis has been characterised, including a full structure elucidation of its products from various substrates and an in-depth investigation of the enzyme mechanism by isotope labelling experiments, metal cofactor variations, and mutation experiments. The results revealed an interesting dependency of Mn2+ catalysis on the presence of Asp-217, a residue that is occupied by a highly conserved Glu in most other bacterial terpene synthases.

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase.

The mechanisms of two diterpene cyclases from streptomycetes-one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product tsukubadiene-were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.