ENGINEERING TUNABLE THERMOSENSORS IN BACTERIA FOR NEXT GENERATION BIOTECHNOLOGY APPLICATIONS

Abstract

Temperature is a ubiquitous physical cue that is easy to apply, allowing cellular behaviours to be controlled in a contactless fashion via heat-inducible/repressible systems. However, existing heat-repressible systems are limited in numbers and rely on either thermal-sensitive mRNA or transcription factors which suffer delays, have complex designs or function at low temperatures. To provide an alternative mode of thermal regulation and overcome these shortcomings, the research presented in this thesis developed a library of compact and fast-switching thermal-repressible split-T7 RNA polymerases through fusion with temperature-sensitive Tlpa’s domains to enable direct thermal control of polymerase activity. Leveraging on developed screening platforms, the large mutant library extended temperature tunability for different microbial applications. Consequentially, for bioproduction, the two innovative thermal control strategies achieved active thermal control of cell proportions within co-cultures and the direct thermal induction of vanillin bioconversion. Overall, the studies presented as facilitated by innovations in thermosensor designs, screening and platform technologies has expanded avenues for thermal control in next generation biotechnology applications.