A highly selective cell-based fluorescent biosensor for genistein detection

Abstract

Genistein, an isoflavone found mainly in legumes, has been shown to have numerous health benefits for humans. Therefore, there is substantial interest in producing it using microbial cell factories. To aid in screening for high genistein producing microbial strains, a cell-based biosensor for genistein was developed by repurposing the Gal4DBD-ERα-VP16 (GEV) transcriptional activator in Saccharomyces cerevisiae. In the presence of genistein, the GEV sensor protein binds to the GAL1 promoter and activates transcription of a downstream GFP reporter. The performance of the biosensor, as measured by fold difference in GFP signal intensity after external genistein induction, was improved by engineering the sensor protein, its promoter and the reporter promoter. Biosensor performance increased when the weak promoter REV1p was used to drive GEV sensor gene expression and the VP16 transactivating domain on GEV was replaced with the tripartite VPR transactivator that had its NLS removed. The biosensor performance further improved when the binding sites for the inhibitor Mig1 were removed from and two additional Gal4p binding sites were added to the reporter promoter. After genistein induction, our improved biosensor output a GFP signal that was 20 times higher compared to the uninduced state. Out of the 8 flavonoids tested, the improved biosensor responded only to genistein and in a somewhat linear manner. The improved biosensor also responded to genistein produced in vivo, with the GFP reporter intensity directly proportional to intracellular genistein concentration. When combined with fluorescence-based cell sorting technology, this biosensor could facilitate high-throughput screening of a genistein-producing yeast cell factory.