Studies on key steps controlling biosynthesis of antibiotics thiomarinol and mupirocin

Omer-Bali, Ahmed Mohammed (2013). Studies on key steps controlling biosynthesis of antibiotics thiomarinol and mupirocin. University of Birmingham. Ph.D.

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Abstract

The modular polyketide synthase responsible for biosynthesis of the antibiotic mupirocin occupies 75 kb of Pseudomonas fluorescens NCIMB 10586, while a hybrid of PKS/NRPS is responsible for biosynthesis of the antibiotic thiomarinol located on a 97 kb plasmid pTML1 in Pseudoalteromonas spp SANK 73390. Biosynthesis of the acyl side chains in mupirocin and thiomarinol are thought to be either through esterification of the fully synthesised fatty acid (C\(_9\) or C\(_8\)) or through extension of the PK derived ester starter unit which is predicted to be carried out on MmpB and TmpB. mupU/O/V/C/F and macpE are proposed to be sufficient for the conversion of pseudomonic acid B to pseudomonic acid A. Mupirocin is regulated via quorum sensing, while regulation of thiomarinol was not identified.

Production of thiomarinol was determined to occur after 8 hours of growth, while acidic conditions and use of acetone with ethyl acetate improved the extraction. TmlU, the thiomarinol amide ligase, did not complement a mupU mutant in mupirocin, and was found to block the biosynthesis of 9-hydroxynonanoic acid, causing truncation of 9-HN. This suggests that MupU, prevents MmpB from being an iterative PKS. KS-B2/ACP-B2 was shown to be involved in the removal of C8-OH from thiomarinol. Genetically manipulated mupU increased the production of mupirocin to 3 to 4 fold without abolishing PA-B production. Fused mupU-macpE complemented the NCIMB10586ΔmupUΔmacpE double mutant. However, insertion of this fusion into MmpB blocked the biosynthesis of mupirocin, while insertion after MmpA did not changed the pathway. Attempts to mobilise pTML1 revealed that a hybrid plasmid of RK2-R6K γ-ori was integrated into pTML1 but recovery of this cointegrate has not yet been recovered in E. coli.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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