Search

Nudibranchia, marine soft-bodied organisms, developed, due to the absence of a protective shell, different strategies to protect themselves against putative predators and fouling organisms. One strategy is to use chemical weapons to distract predators, as well as pathogenic microorganisms. Hence, these gastropods take advantage of the incorporation of chemical molecules. Thereby the original source of these natural products varies; it might be the food source, de novo synthesis from the sea slug, or biosynthesis by associated bacteria. These bioactive molecules applied by the slugs can become important drug leads for future medicinal drugs. To test the potential of the associated bacteria, the latter were isolated from their hosts, brought into culture and extracts were prepared and tested for antimicrobial activities. From 49 isolated bacterial strains 35 showed antibiotic activity. The most promising extracts were chosen for further testing against relevant pathogens. In that way three strains showing activity against methicillin resistant Staphylococcus aureus (MRSA) and one strain with activity against enterohemorrhagic Escherichia coli (EHEC), respectively, were identified. The obtained results indicate that the sea slug associated microbiome is a promising source for bacterial strains, which hold the potential for the biotechnological production of antibiotics.

An FAD-dependent monooxygenase encoding gene (SorbC) was cloned from Penicillium chrysogenum E01-10/3 and expressed as a soluble protein in Escherichia coli. The enzyme efficiently performed the oxidative dearomatisation of sorbicillin and dihydrosorbicillin to give sorbicillinol and dihydrosorbicillinol respectively. Bioinformatic examination of the gene cluster surrounding SorbC indicated the presence of two polyketide synthase (PKS) encoding genes designated sorbA and sorbB. The gene sorbA-encodes a highly reducing iterative PKS while SorbB encodes a non-reducing iterative PKS which features a reductive release domain usually involved in the production of polyketide aldehydes. Using these observations and previously reported results from isotopic feeding experiments a new and simpler biosynthetic route to the sorbicillin class of secondary metabolites is proposed which is consistent with all reported experimental results.

Piecing it together: Aspergillus niger produces kotanin by dimerization of the monomeric, polyketide-synthase-derived (PKS) 7-demethylsiderin. A combined approach, comprising bioinformatics and gene-deletion experiments, identified the biosynthetic cluster responsible for kotanin production. Homology modeling and substrate docking provide a rationale for the regio- and stereoselective phenol coupling reaction.

The mechanism of programming of iterative highly reducing polyketide synthases remains one of the key unsolved problems of secondary metabolism. We conducted rational domain swaps between the polyketide synthases encoding the biosynthesis of the closely related compounds tenellin and desmethylbassianin. Expression of the hybrid synthetases in Aspergillus oryzae led to the production of reprogrammed compounds in which the changes to the methylation pattern and chain length could be mapped to the domain swaps. These experiments reveal for the first time the origin of programming in these systems. Domain swaps combined with coexpression of two cytochrome P450 encoding genes from the tenellin biosynthetic gene cluster led to the resurrection of the extinct metabolite bassianin.

The fungus Trichoderma viride was isolated from the Caribbean sponge Agelas dispar, which was collected from waters around the island of Dominica. Its EtOAc extract, exhibiting mild radical scavenging properties, was mass cultivated and found to produce a new pyranone derivative, trichopyrone (1), and ten compounds, namely four sorbicillinoid polyketide derivatives, trichodermanone A-D (2 - 5), two hexaketide derivatives, rezishanone (6) and vertinolide (7), three known dodecaketides, trichodimerol (8), bislongiquinolide (trichotetronine, 9), and bisvertinol (10), as well as 2-furancarboxylic acid (11). The structures of all compounds were determined by interpretation of their spectroscopic data (1D and 2D NMR, MS, UV and IR). The biological activities of all isolates were evaluated in a series of bioassays (radical scavenging, antioxidant, antimicrobial, inhibition of HIV-1 RT). The majority had very weak or no effects in the applied test systems

A marine fungal isolate, identified as Acremonium sp., was mass cultivated and found to produce two novel hydroquinone derivatives, 7-isopropenylbicyclo[4.2.0]octa-1,3,5-triene-2,5-diol (1) and 7-isopropenylbicyclo[4.2.0]octa-1,3,5-triene-2,5-diol-5-beta-D-glucopyranoside (2). Compound 1 and its glucoside 2 possess a most unusual ring system. The new natural products (3R,4S)-3,4-dihydroxy-7-methyl-3,4-dihydro-1(2H)naphthalenone (3) and (3S,4S)-3,4-dihydroxy-7-methyl-3,4-dihydro-1(2H)-naphthalenone (4) were obtained as a 1:0.8 mixture. 2-(1-Methylethylidene)pentanedioic acid (5) was isolated for the first time as a natural product and its structure proven by X-ray analysis. In addition to these compounds an inseparable mixture of three new isomeric compounds, pentanedioic acid 2-(1-methylethylidene)-5-methyl ester (6), pentanedioic acid 2-(1-methylethylidene)-1-methyl ester (7), and pentanedioic acid 2-(1-methylethenyl)-5-methyl ester (8), was also obtained. Isolated together with the new compounds were three known hydroquinone derivatives, 9, 10, and 11. The structures of all compounds were determined by interpretation of their spectroscopic data (1D and 2D NMR, MS, UV, and IR). Each isolate was tested for its antioxidant properties, and compounds 1 and 9-11 were found to have significant activity.

A new biosynthetic pathway to the sorbicillinoid natural products is proposed based on the observation of oxidative dearomatisation of dihydrosorbicillin 10b., An FAD-dependent monooxygenase encoding gene (SorbC) was cloned from Penicillium chrysogenum E01-10/3 and expressed as a soluble protein in Escherichia coli. The enzyme efficiently performed the oxidative dearomatisation of sorbicillin and dihydrosorbicillin to give sorbicillinol and dihydrosorbicillinol respectively. Bioinformatic examination of the gene cluster surrounding SorbC indicated the presence of two polyketide synthase (PKS) encoding genes designated sorbA and sorbB. The gene sorbA-encodes a highly reducing iterative PKS while SorbB encodes a non-reducing iterative PKS which features a reductive release domain usually involved in the production of polyketide aldehydes. Using these observations and previously reported results from isotopic feeding experiments a new and simpler biosynthetic route to the sorbicillin class of secondary metabolites is proposed which is consistent with all reported experimental results.

The in vivo activity of truncated forms of methylorcinaldehyde synthase shows that the synthase retains a hydrolytic release activity in the absence of reductive chain release and that chain-length is not controlled by the reductive release domain; experiments using a methyltransferase inhibitor suggest that methylation occurs prior to aromatisation.

Formation of tumor cell adhesion and following extravasation are limiting steps in organ specific metastasis. Focal Adhesion Kinase (FAK) plays a pivotal role in the intracellular signaling during cell adhesion and migration, which can be modulated by shear forces acting on circulating tumor cells. The intrinsic FAK inhibitor FRNK (FAK-related-non-kinase) was overexpressed in HEP G2 and HT-29 cells to investigate the role of FAK during early steps of metastasis. Static adhesion was not influenced by FRNK expression, whereas dynamic adhesion under flow conditions was dramatically reduced, apparent by significant reduced dynamic adhesion rates DAR (FRNK+: 12 ± 9 cells vs. FRNK-: 32 ± 10 cells; p 80%, p < 0.001) and complete inhibition of migration of the cells to the parenchyma. Our results indicate that FAK is involved in stabilization of cell adhesion on extracellular matrix in vitro and in vivo, which allows circulating tumor cells to resist the shear forces. This kinase is therefore involved in the regulation of rate limiting steps in distant metastasis formation.

The fungus Epicoccum sp., was isolated from the marine brown alga Fucus vesiculosus. After cultivation the fungus was investigated for its secondary metabolite content, and found to contain the new natural product 4,5,6-trihydroxy-7-methylphthalide (1, epicoccone), together with 5-(acetoxymethyl)-furan-2-carboxylic acid (2), furan-2-carboxylic acid (3), 5-(hydroxymethyl)-furan-2-carboxylic acid (4), (-)-(3 R,4 S)-4-hydroxymellein (5), and (-)-(3 R)-5-hydroxymellein (6). The structures of all compounds were determined by interpretation of their spectroscopic data (1D and 2D NMR, MS, UV, optical rotation and IR). Each isolate was tested for its antioxidative properties. Compound 1 was found to be potently active, showing 95 % DPPH radical scavenging effects at 25 microg/mL. Compound 1 also inhibited the peroxidation of linolenic acid in the TBARS assay (62 % inhibition at 37 microg/mL).

Six new tetraprenyltoluquinol derivatives (1-6), two new triprenyltoluquinol derivatives (7 and 8), and two new tetraprenyltoluquinone derivatives (9 and 10) were isolated from the brown alga Cystoseira crinita Duby together with four known tetraprenyltoluquinol derivatives (11-14). All structures were elucidated by employing spectroscopic techniques (NMR, MS, UV, and IR). Each compound was evaluated for its antioxidative properties in the TBARS and DPPH assay, and compounds 1, 2, 6, and 10-14 were additionally assessed in the TEAC and PCL assay. Hydroquinones were found to have powerful antioxidant activity.

This review discusses the biosynthesis of natural products generated by iterative hybrid polyketide synthases–non ribosomal peptide synthetases (PKS–NRPS) from fungi and bacteria, the programming of the enzymes and bioengineering approaches.

A novel polyketide synthase nonribosomal peptide synthetase (PKS-NRPS) genecluster was isolated from Beauveria bassiania 992.05. The cluster encodes the enzymes responsible for the biosynthesis of the new 2-pyridone desmethylbassianin (DMB). DMB is structurally related to tenellin from B. bassiana 110.25 but it differs in chain length and degree of methylation. Despite these programming differences the 20 kb DMB biosynthetic genecluster has 90% sequence identity to the tenellingenecluster. Silencing of the PKS-NRPS gene, dmbS, resulted in total loss of DMB production. Co-expression of dmbS in Aspergillus oryzae with its cognate trans-actingenoyl reductase gene, dmbC, produced predesmethylbassianin A, the first isolable precursor in the biosynthetic pathway. Expression of dmbS with the tenellintrans-actingenoyl reductase gene, tenC, also resulted in the production of predesmethylbassianin A. Co-expression of tenS, the tenellin PKS-NRPS, with dmbC produced pretenellin A. These results show that the tenS and dmbS encoded PKS-NRPS contains the programme for polyketide biosynthesis, while the trans-actingERs appear to control the fidelity of the programme. Expression of a hybrid synthetase in which the PKS of the tenellin synthetase was fused to the NRPS from DMBS produced prototenellins A to C, indicating that the NRPS does not act as a selecting gatekeeper to affect the PKS programme.