Biosynthetic Studies and Combinatorial Biosynthesis of Pleuromutilin Antibiotics

  • Khairunisa Khairudin

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Pleuromutilin has potential as a next-generation antibiotic, and many semi-synthetic pleuromutilin derivatives have been developed. Recently, characterization of individual enzymatic steps involved in the production of pleuromutilin has been carried out. A linear pathway of pleuromutilin biosynthesis was established; however, there is a possibility of alternative or shunt pathways.

Thus, the first part of this thesis aimed to investigate if any other possible routes could lead to the biosynthesis of pleuromutilin. Two alternative pathways were identified from the expression of various combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. However, neither route led to mature pleuromutilin due to the lack of activity of P450-3. It was shown that most of the tailoring enzymes except for P450- 3 exhibited a more relaxed substrate specificity as they were able to catalyze reactions in different premutilin intermediates. The structure-activity of pleuromutilin and its derivatives, including one synthesized by a semi-synthetic approach, was also investigated through antibacterial assay against Bacillus subtilis. It was observed that missing the substituents, 3-ketone, 11-OH or 14-acetyl from the pleuromutilin core affected the antibacterial activity of the pleuromutilin. Thus, it was important to retain the three side groups on the pleuromutilin core to maintain the bioactivity of the pleuromutilin, although further modification can be done on the C-14 to improve its activity.

The second part of this study evaluated the potential of using the A. oryzae secondary host as a platform for further in vivo derivatization of the pleuromutilin core through the expression of foreign genes. However, no accumulation of new pleuromutilin analogs could be detected, which suggested the difficulties in achieving pleuromutilin hybrid products through combinatorial biosynthesis. Further knowledge of the interaction between enzymes and their substrates may be required to find suitable hybrid enzyme combinations that could lead to the biosynthesis of compounds with diverse chemical structures and possibly improve the antimicrobial activities.
Date of Award25 Sep 2018
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorAndy M Bailey (Supervisor) & Gary D Foster (Supervisor)

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