Assessment of the Fluorescent Activities of Different Fluorescent Proteins for Potential Use as Biosensors in Saccharomyces cerevisiae

Sammanfattning: Development of genetically modified microorganisms such as Saccharomyces cerevisiae for use as microbial cell factories to produce fine and bulk chemicals has lately proceeded very rapidly, much thanks to new gene editing methods based on a CRISPR/Cas9 system adapted from bacteria which increases the rate of genetic engineering. However, development of methods which enable fast and simple screening of the best performing strains with specific sought-after characteristics is not advancing at the same pace. Therefore, screening can be a bottleneck in the cell factory engineering process, slowing down the overall development time. In this context, biosensors where inducible promoters are coupled to reporter molecules are of interest because they allow for high throughput screening. As reporter molecules, fluorescent proteins are good candidates because of their non- invasiveness and easy detection through, for example, fluorescence imaging or flow cytometry. Biosensors for analyzing multiple properties in parallel require bright fluorescent proteins with several different colors. In this project, the fluorescent activities of fluorescent protein candidates were investigated by integrating the genes encoding the proteins into the genome of S. cerevisiae and analyzing the fluorescent activities using flow cytometry. The candidates yEGFP, mEGFP, CyOFP1, yeast codon optimized CyOFP1 and yeast codon optimized mBeRFP were bright enough to be distinguished from the background fluorescence, whereas the strain with the gene encoding smURFP could not be distinguished from the background fluorescence, not even after attempting to increase the fluorescence signals by supplying the strain with a cofactor necessary for smURFP fluorescence. Not all proteins were suitable to be combined due to fluorescence spillover. Two strains were generated, one with codon optimized CyOFP1 and mEGFP and one with codon optimized mBeRFP and mEGFP. The data for these strains suggested that bright signals derived from each fluorescent protein could be detected, showing promise for use as dual biosensors. More work is required to find suitable combinations of three or more fluorescent proteins for biosensor applications, and it is recommended that the method of color compensation is investigated to handle unavoidable spillover fluorescence.