Session: Current Topics in Biological Chemistry:

Macrolide antibiotics, synthesized via giant polyketide synthases (PKS) are in high demand for clinical applications. Due to the growing resistance of bacteria towards antibiotics there is an urgent need of novel macrolide antibiotics. Combinatorial biosynthesis is an approach to reprogram the PKSs, however it is challenging due to their size and complexity. Directed evolution is an alternative approach where large libraries of enzyme variants that lead to functional group modifications in macrolides are evolved. Hence it is important to develop high throughput screening methods to screen these enzyme libraries. MphR is a transcriptional repressor protein which undergoes transcriptional de-repression upon binding to macrolides such as erythromycin. The biosensor in this study consists of a two plasmid system, in which upon binding of macrolide to MphR a gene cassette is transcribed expressing green fluorescent protein (GFP). A gene knockout approach where genes expressing proteins that are involved in antibiotic transport were disrupted in the E.coli chromosome was used in order to improve the sensitivity of the biosensor. The tolCΔ single gene knockout strain and bamBΔtolCΔ double knockout strain along with the two plasmid system showed improved sensitivity due to high influx and retaining more macrolide in the cell. Furthermore, these sensor systems were used to create an orthogonal biosensor pair, capable of reporting differential concentrations of erythromycin and its semi synthetic derivative clarithromycin.

Jayani Angela Christopher

Graduate Research Assistant, Virginia Commonwealth University

Appearances