Synthetic Biology Applications of Single Domain Antibodies

Abstract

Synthetic biology is a powerful collection of tools that facilitates the design and construction of novel biological systems, with applications ranging from the development of synthetic life to the production of high value chemicals. The development of novel biological parts is a core principle of synthetic biology, as expanding the biological 'tool-kit' opens up new possibilities for the production of novel tools, pathways and compounds. Antibodies demonstrate high antigen affinity and specificity, making them ideal candidates for the development of novel synthetic biology tools. However full length antibodies require post-translational modifications that prevent their use in prokaryotic chassis, such as Escherichia coli, which are commonly used for synthetic biology applications. Single domain antibodies (sdAb), such as heavy chain variable domains (VHH) of camelid heavy chain only antibodies (HCAb), demonstrate improved stability and robustness when compared to conventional recombinant antibodies whilst retaining high antigen affinity and specificity. Furthermore, sdAb such as VHH are also capable of binding 'cryptic' epitopes on the target antigen, such as those that occur within clefts and cavities. As a result, these recombinant antibody fragments are ideally positioned for initial investigations into their use as tools for synthetic biology applications. This thesis describes the generation of, and subsequent investigations into, VHH with functionalities relating to the production of monoterpenoids in E. coli. First, a VHH phage display library was constructed and subsequently enriched against Streptomyces clavuligerus linalool synthase (bLinS). Next, Design of Experiments (DoE) was used to construct and optimise a platform that facilitated the production of VHH in the cytoplasm of E. coli, such that these recombinant antibody fragments may be utilised for synthetic biology applications. Finally, the ability of three anti-bLinS VHH to modulate the production of monoterpenoids in E. coli containing a heterologous linalool production pathway was investigated, revealing that these anti-bLinS VHH were capable of altering the flux within the pathway, thereby altering the profile of the terpenoid products obtained. As such, the platforms developed in this project, and the insights gained following their implementation, represent a foundation upon which further investigations and developments may build, so as to realise the full potential of VHH antibodies as powerful tools in the field of synthetic biology.

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nigel Scrutton (Supervisor) & Eriko Takano (Supervisor)