Cyanobacteria produce a diverse array of natural products with significant potential in many biotechnological, clinical and commercial applications. These include pharmaceuticals, such as antitumour products, antibiotics, immunosuppressants, anticholesterolemics and anti-parasitic agents, as well as veterinary therapies and agrochemicals. These compounds are synthesised by complex secondary metabolism pathways involving polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS), both of which require an essential phosphopantetheinyl transferase (PPT) for their activity. PPTs activate the acyl, aryl and peptidyl carrier proteins within various biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. Phylogenetic analysis of the large superfamily of PPTs has revealed two separate families based on substrate specificity, which have been designated AcpS and Sfp-like. The AcpS PPT family activate acyl carrier proteins of fatty acid synthesis, while the Sfp-like PPT family, typified by the Bacillus subtilis PPT Sfp, has diverse roles in primary and secondary metabolism. The majority of cyanobacterial PPTs are of the Sfp-like PPT family and occur in genomes lacking an AcpS PPT. Phylogenetic analysis uncovered a distinct clade of cyanobacterial PPTs involved in heterocyst differentiation. Heterologous expression and functional analysis of NsPPT, the heterocyst-associated PPT in Nodularia spumigena NSOR10, represented the first characterisation of a cyanobacterial PPT. PCR-based screening was utilised to identify NsPPT and Southern hybridisation suggested this was the only PPT encoded by the N.spumigena NSOR10 genome. Enzymatic analyses demonstrated the ability of NsPPT to phosphopantetheinylate PKS and NRPS carrier proteins from a range of metabolism pathways and cyanobacterial species. Nostoc punctiforme ATCC 29133 encodes three PPTs. One of these PPTs, NgcS, is also a heterocyst-associated PPT and is homologous to NsPPT of N.spumigena NSOR10. Expression and enzymatic analysis of NgcS from N. punctiforme ATCC 29133, revealed contrasting phosphopantetheinylation activity to that seen for NsPPT, and indicated that NgcS may have evolved to have a strict specificity for the glycolipid biosynthesis pathway. Although the Sfp-like family of PPTs are normally associated with secondary metabolite biosynthesis, Synechocystis sp. PCC 6803 harbours a unique Sfp-like PPT (Sppt) but does not produce NRPS or PKS compounds. Genetic disruption of Sppt was attempted and expression of Sppt allowed the characterisation of its enzyme kinetics. Sppt displayed the ability to activate non-cognate cyanobacterial carrier proteins from NRPS and PKS biosynthetic pathways, although only at a low level of activity. This suggested that wild-type Synechocystis sp. PCC6803 would not be suitable for heterologous expression of cyanobacterial secondary metabolites. These results have important implications regarding the expression and manipulation of cyanobacterial bioactive compounds in heterologous hosts. Applications of this research may provide a biotechnological platform for the sustainable production of cyanobacterial natural products.
| Identifer | oai:union.ndltd.org:ADTP/187657 |
| Date | January 2005 |
| Creators | Copp, Janine Naomi, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Janine Naomi Copp, http://unsworks.unsw.edu.au/copyright |
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