Annabel C. Murphy

A Natural Plasmid Uniquely Encodes Two Biosynthetic Pathways Creating a Potent Anti-MRSA Antibiotic

Background Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin. Methodology/Principal Findings High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via “mutasynthesis” that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates. Conclusions/Significance Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.

Evolving Trends in the Dereplication of Natural Product Extracts. 2. The Isolation of Chrysaibol, an Antibiotic Peptaibol from a New Zealand Sample of the Mycoparasitic Fungus Sepedonium chrysospermum

By the application of an HPLC bioactivity profiling/microtiter technique in conjunction with capillary NMR instrumentation and access to the AntiMarin database the conventional evaluation/isolation dereplication/characterization procedures can be dramatically truncated. This approach is illustrated using the isolation of a new peptaibol, chrysaibol (1), from a New Zealand isolate of the mycoparasitic fungus Sepedonium chrysospermum. The unique nature of chrysaibol was recognized by bioactivity-guided fractionation using HPLC bioactivity profiling/microtiter plate analysis in conjunction with capillary NMR instrumentation and the AntiMarin database. 2D NMR techniques, in combination with MS fragmentation experiments, determined the planar structure of chrysaibol (1), while the absolute configurations of the amino acid residues were defined by Marfeys method. Chrysaibol (1) was cytotoxic against the P388 murine leukemia cell line (IC50 6.61 microM) and showed notable activity against Bacillus subtilis (IC50 1.54 microM).

Engineered Thiomarinol Antibiotics Active against MRSA Are Generated by Mutagenesis and Mutasynthesis of Pseudoalteromonas SANK73390

The obligate marine bacterium Pseudoalteromonas spp. SANK73390 produces a series of hybrid antibiotics, thiomarinols A–G (Scheme 1), in which a pyrrothine moiety is linked through an amide to close analogues of the clinically significant antibiotic mupirocin (pseudomonic acids, for example, 7–9) produced by Pseudomonas fluorescens. The pyrrothine-containing holomycin (10), N-propionylholothin (11), thiolutin (12), and aureothricin (13) are also antibiotics but the thiomarinols and mupirocin display particularly potent activity against Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) (MIC< 0.01 mgmL ). Pseudomonic acid A (7) was one of the first of an extensive family of antibiotics produced by the “transAT” class of modular polyketide synthases (PKSs). Identification of the thiomarinol (tml) biosythetic gene cluster by full genome sequencing of SANK73390 showed that it is contained on a 97 kb plasmid consisting almost entirely of the thiomarinol biosynthetic genes. These consist of trans-AT PKSs and associated tailoring genes with high homology to the mupirocin (mup) cluster, along with a nonribosomal peptide synthetase (NRPS) linked to a set of tailoring enzymes similar to that recently shown to control holomycin biosynthesis in Streptomyces clavuligerus. In contrast to thiomarinol A (1), the major mupirocin component, pseudomonic acid A (7) has the 9,10-alkene epoxidized which makes it susceptible to intramolecular rearrangements outside a narrow pH range and limits its clinical utility. Mupirocin inhibits isoleucyl-transfer RNA synthetase. The appended pyrrothine moiety in thiomarinol A improves inhibition of this target, but it is yet to be established whether it also imparts an additional mode of antibacterial action.

Biosynthesis of thiomarinol A and related metabolites of Pseudoalteromonas sp. SANK 73390

The biosynthesis of the mixed PKS-FAS-NRPS hybrid antibiotic thiomarinol A was investigated using feeding studies to both wild type and mutant strains of the marine bacterium Pseudoalteromonas. Particularly interesting features of the pathway include assembly of the 8-hydroxyoctanoic acid side-chain via chain extension of a C4-precursor (4-hydroxybutyrate), and construction of the pyrrothine unit from cysteine via (HolA-D, F-H) prior to intact incorporation into thiomarinol (catalysed by TmlU). A series of thiomarinol-related and other minor metabolites have been isolated from wild-type and mutant strains. The results of these investigations are rationalised in terms of the overall biosynthetic pathway.

Concise, Stereoselective Route to the Four Diastereoisomers of 4-Methylproline

The full stereochemical characterization of 4-methylproline, a rare amino acid found in a number of peptidic secondary metabolites, has often been hindered by long reaction sequences or low stereoselectivity in the synthesis leading to reference samples. The preparation of the four diastereoisomers of 4-methylproline by a concise and stereoselective route is presented and features a six-step route with late-stage stereodivergence, good stereoselectivity for both cis- and trans-series (75% and 88% de, respectively), and good overall yields (cumulative yields of 30-40%). Additional data on the Marfeys derivatives of the stereoisomers are also presented.

Biosynthesis of Mupirocin by Pseudomonas fluorescens NCIMB 10586 Involves Parallel Pathways

Mupirocin, a clinically important antibiotic produced via a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens, consists of a mixture of mainly pseudomonic acids A, B, and C. Detailed metabolic profiling of mutant strains produced by systematic inactivation of PKS and tailoring genes, along with re-feeding of isolated metabolites to mutant stains, has allowed the isolation of a large number of novel metabolites, identification of the 10,11-epoxidase, and full characterization of the mupirocin biosynthetic pathway, which proceeds via major (10,11-epoxide) and minor (10,11-alkene) parallel pathways.

Enantioselective syntheses of alpha-Fmoc-Pbf-[2-(13)C]-L-arginine and Fmoc-[1,3-(13)C2]-L-proline and Incorporation into the neurotensin receptor 1 ligand, NT(8-13).

Enantioselective syntheses of selectively labeled, orthogonally protected [2-(13)C]-L-arginine and [1,3-(13)C(2)]-L-proline are described from the commercially available precursors [2-(13)C]bromoacetic acid and potassium [(13)C]cyanide. Interestingly the enhanced signal assigned to C-2 in the (13)C NMR spectrum of alpha-Fmoc-Pbf-[2-(13)C]-L-arginine was very broad at room temperature. The two Fmoc-labeled amino acids were used to prepare [2-(13)C]-Arg9 and [1,3-(13)C(2)]-Pro10 labeled ligand (NT(8-13)) by manual Fmoc-SPSS.