Unravelling the regulatory network of antibiotic production

  • Marc Biarnes Carrera

Student thesis: Phd


Chemically inducible signalling circuits based on the acyl homoserine lactone signalling molecules are popular tools for regulating expression of synthetic gene circuits. However, these systems, derived from quorum sensing mechanisms, have several limitations: they have been found to crosstalk, both at signal and host level, and transcription of their promoters is leaky. In this thesis, I explore whether the cell-to-cell communication mechanism governing antibiotic production in Streptomyces, the gamma-butyrolactone system, would be a suitable candidate to overcome these limitations. First, I introduce the gamma-butyrolactone systems from different Streptomyces species, highlighting the natural diversity of building blocks that could be used for synthetic biology purposes. Then, I present a key method that allowed seamless extraction, detection and relative quantification of gamma-butyrolactones; focusing on the gamma-butyrolactone system from S. coelicolor A3(2). Second, all the genes involved in the biosynthesis of S. coelicolor A3(2) gamma-butyrolactones, SCB1 3, were identified in its native environment and I introduced the whole biosynthetic pathway into E. coli BL21. This resulted in production of gamma-butyrolactone SCB2 under various experimental conditions. Next, I tested an optimised gamma-butyrolactone receptor system for orthogonality against the acyl homoserine lactone systems from Vibrio fischeri and Pseudomonas aeruginosa, showing that the gamma-butyrolactone and acyl-homoserine lactone systems are signal and promoter orthogonal. Alongside, I evaluated the regulatory role of a characteristic promoter overlap between the gamma-butyrolactone key synthase scbA and receptor scbR, a unique feature from the gamma-butyrolactone system of S. coelicolor A3(2), by uncoupling the scbR and scbA promoters. I then compared the phenotype of the cells with uncoupled promoter and the coupled promoter configuration, both at RNA and protein levels, using a systematic approach, where different layers of regulation are sequentially added to the complex system. Finally, I attempted expanding the number of gamma-butyrolactone-like signals to produce in E. coli, such as butenolides, furans, or avenolides. Then, given that biosynthesis of gamma-butyrolactones and gamma-butyrolactone-like compounds is modular, I envisaged a device that could be employed to rationally design novel gamma-butyrolactones by manipulating the expression of these modules. Consequently, more information about how natural gamma-butyrolactones interact with their cognate receptors is required. This would allow engineering receptors capable of binding synthetic signals. Towards this goal, I propose a framework for the overexpression and purification of gamma-butyrolactone receptors, in order to attempt their crystallisation. Overall, the work here presented shows the first steps towards gamma-butyrolactone-based signalling systems for synthetic biology in E. coli. The gamma-butyrolactone system from S. coelicolor A3(2) was shown to be a robust tool that can be used to generate synthetic circuits for synthetic biology, either as a standalone tool or in parallel to acyl homoserine lactone systems. Furthermore, production of other gamma-butyrolactones and gamma-butyrolactone-like compounds has been achieved, and the first steps towards the production of soluble gamma-butyrolactone receptor are reported. This would enable the future development of a platform where signalling molecules are rationally designed, by expression of biosynthetic genes in a plug-and-play fashion, to program synthetic cells to generate complex functions.
Date of Award1 Aug 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDavid Leys (Supervisor), Rainer Breitling (Supervisor) & Eriko Takano (Supervisor)


  • signalling molecules
  • orthogonal regulation
  • regulatory circuits
  • GBL
  • butyrolactone
  • biotechnology
  • synthetic biology
  • Streptomyces

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