S71: Proteomics-based discovery of new natural product gene clusters in Bacillus and Streptomyces

Natural products are a diverse collection of medically relevant compounds. Two very important families of natural products are the nonribosomal peptides (NRPs) and polyketides (PKs) due to the great diversity in structure and activity they exhibit. Their biosyntheses are carried out by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) that have enormous size (> 200 kDa) and multimodular architecture. Genome sequencing projects have revealed immense bacterial biosynthetic capacity. However, discovery of new natural products and association to their biosynthetic pathways remain challenging. We have developed a new proteomics-based method to discover and link NRPS/PKS enzymes with their small molecule products. Combining a gel-based method for selecting high molecular weight proteins, in-gel trypsin digestion and Fourier-transform mass spectrometry (FTMS), we have detected natively expressed NRPS and PKS proteins from Bacillus and Streptomyces species, with or without sequenced genomes. Peptide sequences of the identified biosynthetic proteins were used to design degenerate nucleotide primers to amplify and sequence portions of the expressed biosynthetic gene cluster. Bioinformatic analysis of the newly discovered clusters is currently guiding our efforts to isolate novel polyketides and/or non-ribosomal peptides.