A GENOME-MINING STRATEGY FOR IDENTIFICATION OF NEW ANTIBIOTICS PRODUCED BY SHORT LANB ENZYMES

Project Summary/Abstract Development of antibiotic resistance by bacterial pathogens poses an inheritneed for the identification of new chemical entities that will serve as new classes ofantibiotics. Lanthipeptides are a family of ribosomally synthesized post-translationallymodified peptides (RiPPs) consisting of structurally diverse natural products many ofwhich are antibiotics. In this proposal, a genome mining strategy for the identification ofnew natural product RiPPs will be explored. The recent discovery of a new class oftruncated LanB enzymes, herein termed short LanBs (sLanB), has promptedinvestigation of the natural products they produce. Given the rich history oflanthipeptides, formerly known as lantibiotics, as antimicrobial compounds, the naturalproducts derived from sLanB enzymes are expected to possess similar therapeutic valueand structural diversity. The first aim of this proposal involves the isolation of a natural product from amodel gene cluster in Pseudomonas syringae containing a single sLanB gene. Theenzymes encoded by the gene cluster will be studied by heterologous expression in E.coli. After determining the natural product structure, isolation from the native organismwill be attempted by chemical derivatization. Chromosomal disruption by homologousrecombinations represents an alternative method for natural product determination byisolation of advanced biosynthetic intermediates. Building upon our initial studies, the second aim of this proposal involvesidentification of a natural product from a complex gene cluster in Desmospora sp. 8437,containing a total of seven sLanB enzymes. The enzymes encoded by the gene clusterwill be heterologous expressed in E. coli. In vivo co-expression of all seven sLanBs withtagged precursor peptide will allow for rapid determination of enzyme activity. Themodifications by the sLanB enzymes will be analyzed by mass spectrometry. Thisoverall strategy is expected to allow for rapid isolation of natural products derived frommultiple sLanB enzymes. Natural products isolated from these two gene clusters will beevaluated for their antimicrobial activity as potential therapeutics.