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Title: Molecular biological studies on the genes of Pseudomonas fluorescens NC1B10586 which encode biosynthesis of the polyketide antibiotic mupirocin
Author: El-Sayed, Ahmed Kassem Abd El-Samad
ISNI:       0000 0004 2678 0893
Awarding Body: The University of Birmingham
Current Institution: University of Birmingham
Date of Award: 2001
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This study describes the organisation and characterisation of the genes involved in the polyketide antibiotic pseudomonic acid (mupirocin) biosynthesis by Pseudomonas fluorescens NCIB10586. Suicide vector and PCR strategies were used to isolate and clone chromosomal DNA segments flanking a 17.1 kb fragment which was previously cloned. The sequence analysis of the 83.993 kb from the whole DNA fragments obtained by these strategies revealed the involvement of approximately 74.5 kb genomic DNA in the mupirocin biosynthesis. The study predicted that this region encodes the whole biosynthetic pathway, since the disturbance of the upstream and downstream region of the 74.5 kb does not affect mupirocin production where many suicide plasmid insertions within the 74.5 kb region disturbed the biosynthesis. The sequence analysis revealed two sets of tRNA genes immediately upstream of the mup operon indicating that the mupirocin gene cluster might be integrated within the P. fluorescens genome like a pathogenicity island. The translated amino acid sequence revealed a unique order of biosynthetic genes comprising a mixture of (PKS) typel and typell systems. Four main large open reading frames designated orfl, orflI, orflII and orflV encoded multifunctional enzyme complexes resembling type I systems of polyketide synthases (PKS) and fatty acid synthases (FAS). Additional individual genes were identified within the mup cluster as type 11 systems. Some other genes were also identified as tailoring genes probably involved in modification functions such as oxidation, reduction, hydration and ligation. Also a putative mupirocin resistance gene which encodes isoleucyltRNA synthetase activity was identified within the cluster. In an attempt to accumulate intermediate products during mupirocin biosynthesis, in-frame deletions within some domains and genes involved in mupirocin biosynthesis were performed using the suicide vector strategy. This would help in studying the catalytic activity of these genes and subsequently understanding how mupirocin could be assembled. The regulation of mupirocin biosynthesis was first described in this study by the discovery of the mupirocin regulatory genes mupR and mupI. The regulation of mupirocin is controlled via a quorum-sensing system similar to LasR/LasI and LuxR/LuxI systems. A reporter gene fusion and deletion mutation studies were performed in order to determine the effect of mupR and mupI on the putative promoter region of mup operon and consequently on mupirocin productivity. According to information extracted from the putative activities of the identified domains and genes, the predicted pathway of mupirocin assembly was postulated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available