In vivo Mutational Analysis of the Mupirocin Gene Cluster Reveals Labile Points in the Biosynthetic Pathway: the “Leaky Hosepipe” Mechanism

Abstract

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.

Biosynthesis of polyketides by trans-AT polyketide synthases

“…17). 150,151 Similar phenotypes were also observed for mutants in which mupS, mupQ and macpD were deleted, supporting the hypothesis that they might be involved in biosynthesis of 3-hydroxypropionate, the suspected starter unit of 9-hydroxynonanoic acid (see above). A 'leaky hosepipe' mechanism was proposed, according to which biosynthetic bottlenecks in the deletion strains lead to accumulation of intermediates on the PKS.…”

Biosynthesis of Mupirocin by Pseudomonas fluorescens NCIMB 10586 Involves Parallel Pathways

“…The ∆mupC/∆mupF double mutant is identical to the sin--gle mutant ∆mupC confirming that MupC acts before MupF. Interestingly, re--examination of the strain in which the KR domain (KR6) responsible for initial reduction at C7 during PKS assembly has been deleted, led to isolation of the previously reported 19 truncated product mupiric acid 13, along with the 7--keto analogues 21 and 22 of mupirocin W4 and W5. Thus a 7--hydroxyl group is clearly required for action of MmpEOR and MupW/T.…”

Polyketide Proofreading by an Acyltransferase-like Enzyme

“…This phenomenon—which has, with one exception (Yu et al, 1999), not been reported for textbook ( cis -AT) PKSs—suggests the existence of an as-yet uncharacterized proofreading mechanism that releases stalled intermediates from the enzyme. Such truncated products were also observed for the rhizoxin ( 4 ) (Kusebauch et al, 2009) and, for some elongation stages, the mupirocin (Wu et al, 2008) pathways, indicating that intermediate hydrolysis is a more general phenomenon for trans -AT PKSs. Here, we report genetic, biochemical, and chemical studies of a member of the AT2 group, identifying it as a thioester hydrolase for structurally diverse N -acetylcysteamine- or ACP-bound acyl units.…”