Mining the cryptic nonribosomal peptide synthetase systems of streptomyces laurentii

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Suidan, Tala Mubadda
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In bacteria and fungi, nonribosomal peptide synthetase (NRPS) systems produce secondary metabolites that often possess anti-microbial, anti-fungal, anti-cancer, or other biological activities. Since microorganisms continue to develop resistance against known pharmaceutical treatments, the need for new drugs remains critical. Analyses of bacterial genomes reveal many more biosynthetic gene clusters than the number of known metabolites produced in a given species. Previous investigations of such cryptic clusters resulted in the discovery of novel secondary metabolites. This project evaluated two cryptic NRPS biosynthetic gene clusters encoded in the Streptomyces laurentii ATCC 31255 (S. laurentii) genome: nrps2 and nrps3. Bioinformatic analyses of the S. laurentii genomic data allowed for the identification of these two cryptic NRPS biosynthetic gene clusters and a proposal of the core structures for the associated products. Determination of the loci of the nrps2 and nrps3 clusters involved the polymerase chain reaction (PCR), cloning, sequencing, and bioinformatic analysis. The bioinformatic analyses predict that the NRPS3 system produces a novel nonribosomal peptide. On the other hand, NRPS2 is predicted to produce a blue pigment, indigoidine, a metabolite previously isolated from Streptomyces virginiae MAFF 6014, Streptomyces lavendulae ATCC 11924, and Erwinia chrysanthemi. Thus, S. laurentii is established as an alternate producer of that compound (6-8). For NRPS2, characterizing the protein and identifying the metabolite produced also required PCR and cloning in addition to metal-chelate affinity chromatography of the heterologously-expressed protein, fast protein liquid chromatography (FPLC), in vitro assays, and high performance liquid chromatography (HPLC). Inducing production of the new compound in vivo was attempted, and future genetic disruption studies in conjunction with genetic and chemical complementation assays will confirm that the proteins encoded within the genetic cluster are responsible for blue pigment biosynthesis. Study of NRPS2 as an additional indigoidine-producing enzyme illuminates critical residues and motifs that are responsible for the unusual chemistry performed by these enzymes. Overall, the results from both projects aid in the continued battle against microbes, cancer, and other human disease states by adding either a new source for a known metabolite or a new peptide product, which adds to nature s arsenal of biologically active compounds.
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