ACYL carrier proteins in the biosynthesis of mupirocin by Pseudomonas fluorescens NCIMB 10586

Mupirocin or Pseudomonic acid A is a polyketide antibiotic produced by Pseudomonas fluorescens NCIMB10586. The presence of 16 acyl carrier proteins (mACPs) distinguishes the mupirocin biosynthetic cluster from other known systems. This study investigated whether all the ACPs encoded in the tailoring region (macps12-16) are essential, why there are tandem copies (macp3/4 & macp5/6/7) in the modular steps and at what stage the essential ACPs act. For the tandem clusters domain deletions showed that a single mACP from the cluster can provide basic activity to the pathway though the overall rate of mupirocin production is significantly reduced. Inactivation of ACPs by amino acid substitution in the double cluster, suggests that substrates may be held by either of the ACPs which might be arranged "in parallel". In the triplet cluster, amino acid substitution showed that an inactive ACP5 can form a roadblock and that it might be arranged "in series" to ACP6 and 7. Thus while both the doublet and the triplet clusters serve to increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. Deletion of discrete macps (12-15) in the tailoring region resulted in the complete loss of antibacterial activity except Δmacp16 which produced an intermediate with a mass of 516.2 Da and a structure corresponding to PA-B. Specificity studies have suggested that most of the tailoring mACPs are specific in action and the C-terminus might be responsible for specificity. Thus there may be a single ACP for each step of the biosynthetic pathway and the use of appropriate ACPs will be necessary when modifying the pathway.EThOS - Electronic Theses Online ServiceGBUnited Kingdo