Macrolides are a class of antibiotics which are in high demand for clinical applications. They are biosynthesized via large assembly line Polyketide Synthases (PKS) arranged in a modular fashion. There is an urgent need for novel macrolides due to the growing resistance of bacteria towards them. Combinatorial biosynthesis is an approach to reprogram the PKS modules in order to diversify macrolides. However due to the size and the complexity of PKS combinatorial biosynthesis approach is challenging. Directed evolution is an alternative approach where large libraries of enzyme variants which can diversify the macrolides can be screened and it is important to develop high throughput screening methods. MphR is a transcriptional regulator protein which represses the transcription upon binding to a DNA promoter sequence. It can bind to macrolides promiscuously and release from the DNA promoter de-repressing the transcription. In this research a gene expressing the Green Fluorescence Protein (GFP) is incorporated to the biosensor system. Macrolides will bind to MphR, that will de-repress a gene cassette with gfp gene leading to green fluorescence in the presence of macrolides. In this research the sensitivity of macrolide MphR biosensor is improved by a gene knockout approach where a gene expressing a protein related to antibiotic resistance is knocked out from the <i>E.coli</i> chromosome to obtain higher sensitivity of the biosensor. Furthermore it focuses on developing a ratio metric biosensor for the simultaneous detection of erythromycin and clarithromycin which is a semi-synthetic derivative of erythromycin.

Developing transcriptional repressor protein, MphR based macrolide antibiotic biosensors for high throughput screening of enzyme variant libraries

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.