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J. Biol. Chem., Vol. 282, Issue 21, 15451-15461, May 25, 2007

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Mutational Analysis Reveals That All Tailoring Region Genes Are Required for Production of Polyketide Antibiotic Mupirocin by Pseudomonas fluorescens

PSEUDOMONIC ACID B BIOSYNTHESIS PRECEDES PSEUDOMONIC ACID A^*

Joanne Hothersall, Ji'en Wu, Ayesha S. Rahman¹, Jennifer A. Shields², James Haddock, Nicola Johnson, Sian M. Cooper³, Elton R. Stephens, Russell J. Cox, John Crosby, Christine L. Willis, Thomas J. Simpson, and Christopher M. Thomas⁴

From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom and the School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom

The Pseudomonas fluorescens mupirocin biosynthetic cluster encodes six proteins involved in polyketide biosynthesis and 26 single polypeptides proposed to perform largely tailoring functions. In-frame deletions in the tailoring open reading frames demonstrated that all are required for mupirocin production. A bidirectional promoter region was identified between mupF, which runs counter to other open reading frames and its immediate neighbor macpC, implying the 74-kb cluster consists of two transcriptional units. mupD/E and mupJ/K must be cotranscribed as pairs for normal function implying co-assembly during translation. MupJ and K belong to a widely distributed enzyme pair implicated, with MupH, in methyl addition. Deletion of mupF, a putative ketoreductase, produced a mupirocin analogue with a C-7 ketone. Deletion of mupC, a putative dienoyl CoA reductase, generated an analogue whose structure indicated that MupC is also implicated in control of the oxidation state around the tetrahydropyran ring of monic acid. Double mutants with mupC and mupO, mupU, mupV, or macpE produced pseudomonic acid B but not pseudomonic acid A, as do the mupO, U, V, and macpE mutants, indicating that MupC must work after MupO, U, and V.

Received for publication, February 20, 2007 , and in revised form, March 21, 2007.

^* This work was supported by BBSRC Grants P15257 [GenBank] and P07071 [GenBank] (to J. H. and E. S.) as well as EPSRC Grant S78124 (to J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Tables S1 and S2.

¹ Supported by a Scholarship from the Darwin Trust of Edinburgh.

² Supported by a BBSRC DTA studentship.

³ Supported by a Biosciences School studentship.

⁴ To whom correspondence should be addressed: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Tel.: 44-121-414-5903; Fax: 44-121-414-5925; E-mail: c.m.thomas{at}bham.ac.uk.

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