Biosensor Engineering for the Development of Yeast Cell Factories

Allosterically regulated transcription factor-based biosensors can play a key role in engineering yeast cell factories by providing new tools for the selection of designs with improved characteristics. The recent drop in the cost of DNA synthesis, combined with the advent of CRISPR-based technologies allow for fast and efficient construction of very large combinatorial libraries. The research performed during my PhD was therefore motivated by the necessity of accelerating the speed at which we are able to develop new screening systems to match the pace at which we are currently able to construct such libraries and to improve the performance of already established engineered strains. The first manuscript reviews the recent advances in biosensor engineering efforts and its applications in developing improved yeast cell factories. In this review the parameters characterizing the biosensors’ dose-response curve are described, as well as the common engineering strategies applied to improve them. The second manuscript in this thesis focuses on gaining deeper understanding of the rules regulating ligand specificity and promiscuity. In this study VanR from Caulobacter rescentus was engineered to lose the specificity towards vanillic acid, its main ligand, without hindering the ability to detect vanillin, a secondary ligand recognized at higher concentrations and with lower affinity. The third manuscript explores the effect of the position of the transcription factor binding site, also referred to as operator site, in biosensor reporter promoters. Although biosensors founded on allosterically regulated transcription factors are well-established tools for sensing small molecules in yeast, we noticed a lack of information regarding reporter promoter engineering, even though it has been previously shown it can dramatically influence the final biosensor output. To investigate this topic, we constructed libraries for two activators and one repressor covering more than 300 operator designs. After screening by flow cytometry we were able to observe promoter regions with high and low dynamic range areas for the operator positioning of the transcriptional repressor and, more importantly, we observed that transcriptional activators require specific operator positions in order to function. From this study we were able to provide a platform reporter promoter and design guidelines on how to introduce allosterically regulated transcriptional repressors and activators from prokaryotes into Saccharomyces cerevisiae. The fourth study tackles the issue of the loss of productivity in prolonged cultivations by enabling a biosensor-mediated stabilization of product formation in S. cerevisiae. The introduction of a heterologous biosynthetic pathway for the production of valuable chemicals often comes with a fitness cost which can compromise the growth rate of engineered strains. Eventually, the evolutionary pressure will select for mutations that increase the growth rate at the expenses of product formation, which in turn lowers the final yield of a fermentation process. To tackle this issue, we coupled allosterically regulated transcription factors to the expression of essential genes in order to try to avoid the evolution- driven escape mechanism. By doing so, we were able to construct a stabilized strain that in a fedbatch
setup was able to produce 6 times higher amounts of vanillin related metabolites compared to a non-stabilized strain.