Lyngbya-KENYA · Cyp-450 biosynthesis of Lyngbya majuscula natural products
FP7 — People (Marie Curie Actions)
- Duration
- 2012-04-26 → 2014-04-25
- EU contribution
- €209,033
- Participants
- 1
- Scheme
- MC-IIF
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Results in brief
Cyp-450 biosynthesis of Lyngbya majuscula natural products
EXECUTIVE SUMMARY A sustainable supply of marine natural products for potential therapeutics is one of the greatest challenges facing drug discovery efforts today, especially during clinical trials. Nearly 300 compounds with therapeutic potential have been isolated from the tropical marine cyanobacterium Lyngbya majuscula. However there are considerable concerns regarding the real source of this large number of natural products attributed to L. majuscula. This project focused on the cytochrome p450 biosynthesis of L. majuscula natural products namely the modular cyclodepsipeptides homodolastatin 16 (HMDS 16) and antanapeptin A (ANTAP A). L. majuscula was collected from Shimoni, Kenya in April 2012. The aim was to identify if these compounds originate from the cyanobacteria or bacteria cohabiting with it or both. Bacteria representing γ-proteobacteria, Firmicutes, and α–proteobacteria were isolated from the cyanobacteria. Non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) screens of bacteria with known complete genome sequences using bioinformatics tools showed in this study that the modular assembly lines for these bacteria are inconsistent with those of HMDS 16 and ANTAP A. However, Bacillus licheniformis and Marinobacterium stanieri synthesise the β amino acid dolamethleuline and Klebsiella oxytoca is involved with the biosynthesis of the unusual dolaphenvaline amino acid in HMDS 16 putatively. Profiling for the cyclodepsipeptides in bacteria supernatants using liquid chromatograph mass spectrometry (LCMS) confirmed the presence of HMDS 16, its analogue dolastatin 16, and ANTAP A in L. majuscula but not in the bacteria. Subsequently such leads aided the prospects for obtaining the complete genome sequence of L. majuscula from its metagenome and in identifying gene clusters encoding for HMDS 16 and ANTAP A. The cloning and heterologous expression of the gene clusters for these especially important anticancer agents is the goal that we aimed to attain by the end of the program.
Data: CORDIS, © European Union
Project objective
The filamentous marine cyanobacterium, Lyngbya majuscula (order Oscillatoriales) is a prolific source of modular natural products, some of which have shown promise for the treatment of cancer, diabetes, HIV and Alzheimers disease. However, the failure to grow its axenic cultures (because of the lack of the nifH gene) suggests that obtaining the natural products from the cyanobacterium in sustainable yields is enigmatic. My investigations into the Kenyan L. majuscula molecular diversity established it to be encapsulated by a sheath of brightly coloured epibiotic bacteria (EB) species, representative of a group of phyla that are an especially important source of novel natural products in drug discovery. Consistent with the paucity of natural product genes (a mere 3%) observed in the L. majuscula 3L draft genome sequence, there are serious concerns on whether the nearly 300 compounds isolated pan-tropically from L. majuscula originate from the cyanobacterium or EB cohabiting with it or both. This study therefore aims to investigate the heterologous cytochrome P450 (cyp450) mediated biosynthesis of L. majuscula natural products from EB cohabiting with it. The Kenyan L. majuscula is the source of the modular homodolastatin 16 (HMDS 16) and antanapeptin A (ANTAP A). Specifically, EB will be screened for polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) megasynthase gene clusters targeting HMDS 16 and ANTAP A. Gene clusters encoding for HMDS 16, ANTAP A and cyp450 flanking the clusters will be amplified from cDNA using specific primers for the gene models. PCR amplification products will be cloned and resulting plasmids transformed into a Saccharomyces cerevisiae shuttle vector for co-expression with cyp450. Methanolic extracts of the recombinant cultures will be analysed for modular natural products and the results compared with those of EB in direct culture and of L. majuscula. Structural elucidation of natural products will utilise LC/MS/NMR spectrometry.
Original text from CORDIS.
Participants
- UNIVERSITY OF NEWCASTLE UPON TYNE · Newcastle Upon TyneCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union