Development of novel ELP-based transcriptional regulators for improved biomanufacturing

Abstract

Microbial chemical factories (MCFs) have become an attractive platform for producing valuable drugs, chemicals, and biofuels due to increasing environmental concerns, energy demands, and the difficulties associated with chemically synthesizing complex molecules. However, the potential of using microbes to produce many valuable products has not been fully realized due to low productivity and yields. Production may be enhanced through the dynamic control of metabolic pathways to alleviate metabolic imbalances and toxic product build-up, but very few tools are available to broadly implement this paradigm. I aim to expand this toolbox by developing tunable sensor-regulator devices that act as feedback controllers for toxic intermediate formation by responding to cues of cell health for improved production. Elastin-like polypeptide (ELP) will act as the sensing domain of the controller to indirectly sense toxic metabolite accumulation through changes in intracellular pH. ELPs make ideal sensors since they exhibit a sharp, inverse phase transition to indicators of cellular health such as pH and ionic strength, and external stimuli such as temperature. In this research, a library of ELPs that exhibit pH sensitivity and transition under various conditions was made and purified using a new organic solvent extraction method. It is hypothesized that fusion of ELP to orthogonal transcription factor will allow for the controlled expression of target genes in response to stimuli without disrupting native processes. As proof of concept, an ELP fusion to orthogonal sigma factor was designed to drive the expression of a fluorescent reporter protein. Initial designs successfully alter gene expression by 21% in response to temperature. To improve this response, an alternative feed-forward loop architecture was modelled, which predicted an improved response of 35% and increased ultrasensitivity. Refinement of this design and combinatorial construct libraries will generate various regulators with diverse outputs that may be integrated in bioproduction pathways for improved performance.