Studies on the late-stage biosynthesis of the antibiotic Mupirocin

Connolly, Jack Andrew (2018). Studies on the late-stage biosynthesis of the antibiotic Mupirocin. University of Birmingham. Ph.D.

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Abstract

There is a clear and well-established need for new antibiotics. The biosynthesis of the antibiotic mupirocin is an important model trans-AT polyketide synthase pathway. A key tailoring step is the removal of a hydroxyl from the intermediate pseudomonic acid B (PA-B), which generates active pseudomonic acid A (PA-A). This is proposed to occur on discrete proteins after release from enzyme MmpB.
In this work, a systematic screen for genes required for this key step was developed, which implicated ten genes. On a multi-gene expression plasmid, these ten genes encoded all functions necessary for conversion. This re-identified seven genes from the existing proposed model. A novel requirement for MupM, MupN, and the DNA sequence but not protein function ofMupL was uncovered. This represents the first identification of all genes required for this surprisingly convoluted pathway.
With the aim of re-engineering the location of these tailoring steps, domains from thiomarinol enzyme TmpB were inserted into MmpB. Low-level conversion to PA-A was achieved using thiomarinol tailoring enzymes. The TmpB KS0 domain was used to load the normally free-standing MacpE, and high-level conversion was achieved on the hybrid enzyme. This success opens the future path to further MmpB modifications to generate mupirocin derivatives.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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