S34: Functional metagenomics for antimicrobial discovery from large-insert soil libraries

Many clinically-used antibiotics are derived from cultured microorganisms; however, the majority of soil microbes are recalcitrant to cultivation. We constructed soil metagenomic libraries with over 120,000 clones and average insert sizes greater than 100 kb in a shuttle bacterial artificial chromosome vector. An analysis of 16S rRNA gene sequences present within the library indicated a very diverse assemblage of microbial genomes, representing nine bacterial phyla. The library also contained diverse Type I polyketide synthase (PKS) pathways with a broad range of homology (35-85%) with known PKS domains. We screened 19,200 E. coli clones for inhibition of growth of methicillin-resistant Staphyloccocus aureus (MRSA) using a 96-well microtitre plate format.  In situ lysis of the E. coli host enabled detection of both intra- and extracellular compounds, yielding a total of 32 anti-MRSA clones. Next-gen sequencing and annotation of these clones revealed genes predicted to be involved in various biosynthetic pathways as well as many with no significant GenBank similarity. Interestingly, multiple clones were capable of modifying the chloramphenicol added to the culture medium, thereby resulting in modification of an existing antimicrobial scaffold.  The metagenomic library was also conjugally transferred into Sinorhizobium meliloti and screened for activity against multiple bacterial pathogens. The genetic and biochemical characterization of antimicrobial expressing S. meliloti clones is ongoing. These results illustrate that large-insert soil metagenomic libraries can be screened using sequence-based and innovative functional screening methods to access previously undescribed genomic and biochemical diversity.