Biosynthetic and self-resistance investigations of kibdelomycin.

The need for new antibiotic drug leads is pressing given the appearance of multidrug resistant (MDR) and extremely drug-resistant (XDR) strains of pathogenic bacteria. Kibdelomycin is a natural product produced by Kibdelosporangium sp. (MA7385) and Amycolatopsis sp. FERM BP-11411 and was previously identified to have a new binding motif to DNA gyrase, in part due to interesting structural features found in the molecule, and a low frequency of resistance making it an excellent lead compound for the development of Gram-negative antibacterial agents. Kibdelomycin represents an exciting platform that can be used for the discovery of lead compounds for the treatment of antibiotic resistant bacterial infections. In this R21 application, we propose a two-pronged approach to investigate the biosynthesis of kibdelomycin with the ultimate goal of creating kibdelomycin variants that have a greater activity against Acinetobacter baumannii and Pseudomonas aeruginosa, two bacterial pathogens that the Center for Disease Control classifies as urgent and serious threats, respectively. In Aim 1.1, we propose to develop a genetic system for Amycolatopsis sp. FERM BP-11411 in analogy to our team’s previous success in Amycolatopsis mediterranei S699. This will provide access to kibdelomycin variants that are difficult to access via synthesis. In Aim 1.2, we propose to investigate the early biosynthetic steps of how kibdelomycin is assembled by a bottom-up synthetic biology approach synthesizing constructs to produce early intermediates that can be used in analog synthesis. In Aim 2, we engage in a complementary approach to study the natural resistance of the producing strain Kibdelosporangium sp. (MA7385). These self- resistance studies will help dissect the amino acid substitutions present in the kibdelomycin-resistant DNA gyrase to preemptively understand how resistance might evolve in clinical bacterial strains and to guide future synthetic investigations into analogs that can avert this outcome.