Biswas Pilot Project Summary
Lantibiotics are ribosomally synthesized peptides with antimicrobial activities that inhibit a comparatively broad but defined range of target pathogens that include streptococci and staphylococci. Lantibiotics are post-translationally modified and contain unusual amino acids, such as dehydrated and lanthionine residues. MU1140 is such a lantibiotic that is produced by Streptococcus mutans strain JH1140, which is a clinical isolate. The production and secretion of MU1140 by JH1140 is relatively low and the biosynthetic operon is not well-characterized. The JH1140 strain is genetically poorly manipulatable. In Aim 1, to increase the production of MU1140, we propose to use a genetically well-studied S. mutans strain, UA159. Since the biosynthetic operon for MU1140 is large, we will use a recently developed novel gene cloning technique called NabLC to insert the operon from JH1140 to UA159 to generate a MU1140 producer strain, UA149-MU. This will facilitate genetic manipulation in the new strain for increased production of MU1140. In Aim 2, once the strain is established, we will then modify the structural gene for the MU1140 lantibiotic based on the homologous sequence analysis from the GenBank to increase the efficiency of inhibition and to increase the inhibitory spectra. We will also use a novel approach where we will use a well-studied lantibiotic modification system from nisin biosynthesis to modify MU1140 to obtain novel variants that might display new inhibitory properties. Finally, we will also attempt to modify MU1140 in vitro using a cell-free protein synthesis (CFPS) system that is supplemented with purified modification enzymes from nisin. In the future, bioactive products generated from this study will be tested for commercial use to inhibit various multidrug resistant gram-positive pathogens.