Characterization of the Mupirocin Biosynthesis Gene Cluster from Pseudomonas fluorescens NCIMB 10586

Abstract

The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.

Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl- S -carrier proteins in the pksX pathway of Bacillus subtilis

“…Consistent with this proposed series of events, when we treated [2-14 C]Mal-S-AcpK and Acac-S-PksL-T2 with PksF, PksG, PksH, and PksI, we observed stable radiolabeling of PksL-T2. In contrast, when the same experiment was performed with [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-SAcpK, we observed significant loss of radiolabel from PksL-T2 when PksH and PksI were added ( Fig. 5 and Fig.…”

“…No decarboxylation was detected when Mal-S-PksL-T2 was incubated with PksF, demonstrating that it is selective for Mal-S-AcpK (data not shown). Treatment of [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-S-AcpK with PksF gave similar results, with rapid PksF-dependent loss of radiolabel observed, to form [1-14 C]Ac-S-AcpK at a rate of 5.5 M⅐min Ϫ1 (Figs. 8 and 9, which are published as supporting information on the PNAS web site).…”

“…We also identify roles for the freestanding carrier protein AcpK and the tandem T-T didomain in PksL. Collections of proteins similar to AcpK, PksFGHI, and tandem carrier domains such as those found in PksL have also been found in the curacin, jamaicamide, mupriocin, antibiotic TA, and pederin biosynthetic clusters (10)(11)(12)(13)(14)(15)(16)(17)(18)(19), and they appear to encode the biosyntheses of natural products that universally contain a single-carbon ␤-branch (18).…”

“…Other biosynthetic gene clusters, including those responsible for the biosyntheses of leinamycin (16) and onnamide (17), contain subsets of these features. By comparison with the known product structures of homologous gene clusters, it has been suggested that this series of domains is responsible for the incorporation of a ␤-carbon (12,13,(17)(18)(19), which may subsequently be elaborated to diverse structures including vinylic chloride or cyclopropyl branches. We used a combination of biochemical and high-resolution mass spectrometric techniques to test this hypothesis, and we conclude that these proteins from the pksX cluster act to form a ⌬ 2 -isoprenyl unit atypically tethered to a PKS carrier protein domain.…”

The Biosynthesis of Polyketide Metabolites by Dinoflagellates

“…In the biosynthesis of curacin A (Chang et al, 2004) and jamaicamide (Edwards et al, 2004), the incorporation of C2 of acetate is performed by an HMG-CoA synthetase-like enzyme via an aldol condensation followed by a decarboxylation. The pathways for the antibiotic TA (Paitan et al, 1999), mupirocin (El-Sayed et al, 2003), leinamycin (Cheng et al, 2003), and difficidin believed to be made by PksX (Albertini et al, 1995) also contain HMG-CoA synthase-like genes. These genes show high homology to each other and lower homology to HMG-CoA synthase genes associated with terpene biosynthesis.…”