Pseudomonic acid (mupirocin) produced by is a polyketide antibiotic which blocks isoleucyl-tRNA synthetase. Knowledge of the biosynthetic pathways leading to pseudomonic acid production may help in engineering related antibiotics with other useful properties. To help define these pathways, we have isolated 13 non-producing mutants using Tn5 and Tn1725 mutagenesis. Seven of the Tn5 insertions mapped within a 55 kb region. The remaining insertions, and one gene encoding resistance to pseudomonic acid, mapped at separate locations. DNA that overlapped the seven clustered Tn5 insertions was isolated on a series of clones, extending over a region in excess of 60 kb. Non-producing mutants generated via gene disruption suggest that the biosynthetic cluster extends throughout this region.


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