Developing plant chassis for heterologous expression of the pleuromutilin biosynthetic gene cluster

  • Konstantina Beritza

Student thesis: Master's ThesisMaster of Science (MSc)

Abstract

In an era of widespread antibiotic resistance, basidiomycete fungi are a useful source of possible antimicrobials, with pleuromutilin being one of them. Due to its mechanism of action which comprises the binding of the conserved peptidyl transferase centre, resistance to pleuromutilin is slow to be developed and has minimal cross-resistance with other antibiotics. The pleuromutilin biosynthetic gene cluster and pathway of chemical intermediates is known, and its heterologous expression has been achieved in fungi. This thesis reports attempts to express parts of the pleuromutilin pathway in different plant hosts, including Marchantia polymorpha and Nicotiana tabacum. Prior to genetic engineering of the hosts, this work shows that pleuromutilin, mutilin and a diverse selection of antibiotics impact on growth and morphology of Physcomitrella patens and M. polymorpha, when supplemented into growing media, indeed the pleuromutilin derivative, tiamulin led to inhibition of growth of P. patens and M. polymorpha.
The construction of multigene expression plasmids using Golden Gate technology is described. Transient expression was performed in N. tabacum and stable transformants obtained in M. polymorpha. Expression analysis using qPCR was carried out to validate the heterologous expression of the genes. The results from HPLC analysis were unclear and did not prove the presence of pleuromutilin metabolic intermediates, indicating that more work is needed to fine-tune the biosynthetic pathway in planta.
Date of Award26 Nov 2020
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorGary D Foster (Supervisor) & Andy M Bailey (Supervisor)

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