Samenvatting
Whole-cell biosensors are a key enabling technology in metabolic engineering. Biosensors based on metabolite-responsive transcription factors (MRTFs) have proven their use in a range of applications, of which the dynamic regulation of metabolic fluxes has received most attention by the research community. However, limited understanding of the tunability of biosensors has made their development labour-intensive and delayed their en masse industrial application. Mathematical modelling efforts have addressed this bottleneck, and one notable study has revealed theoretical constraints for biosensor design and exposed tunable parameters that allow orthogonal control of the biosensor dynamic range and sensing threshold. However, it was based on the well-characterised lac repressor and has never been applied to new biosensors, that could possess more complex mechanisms not covered by the model.The first part of this thesis aimed to obtain further insight in the contribution of the different tunable parameters on the biosensor response curve by investigating this phenomenological model. Simulations revealed interesting behaviour in response to perturbations in the a1 parameter (representing the maximum increase in transcription factor activity) that did not fully correspond to the effect of tuning the expression level of repressed-repressors described in literature, potentially implying that this parameter alone cannot capture that effect. In addition, the proposed orthogonal control of the biosensor dynamic range was nuanced by identifying limiting conditions that moderate this effect. A parameter estimation method was implemented in Python to fit biosensor characterisation data to this model, which will allow to study the effect of these tunable parameters more quantitatively and provide further insight in the validity of the model.
For the second part of this thesis, biosensors responsive to the industrially important intermediate compound β-alanine were studied in Escherichia coli. Six mutated biosensors were constructed by introducing mutations in the predicted DNA binding sites of Cupriavidus necator OapR, and their response to β-alanine was quantified in vivo. A range of dose-response characteristics emerged with affected dynamic ranges, thresholds and sensitivies across the mutant strains. Notably, a single-nucleotide substitution in the I3 site reduced the dynamic range 100-fold, and the resulting biosensor appeared to have lost the ability to activate the expression from the oapTD promoter and thus function solely as a repressor. On the other hand, four biosensors based on the archaeal MRTF AHOS_RS02205 from ”Acidianus hospitalis” were parameterised. All biosensors were functional in E. coli, either as repressed-repressors or repressed-activators, which is exceptional due to the evolutionary distance separating Bacteria and Archaea.
In conclusion, this thesis project has extracted additional information from a phenomenological model that could aid biosensor response curve engineering and provided the first steps for its practical application to new biosensors. On the other hand, β-alanine-responsive biosensors were engineered, resulting in a set of novel biosensors with potential applications for the production of β-alanine-derived products, such as the promising platform molecule 3-hydroxypropionic acid.
| Datum prijs | 14 sep. 2022 |
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| Originele taal | English |
| Prijsuitreikende instantie |
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| Begeleider | Eveline Peeters (Promotor), Sophie de Buyl (Co-promotor), Indra Bervoets (Advisor), Amber Bernauw (Advisor), Remy Loris (Jury) & Wim Vranken (Jury) |