Functional Metagenomics of Soil Microbial Secondary Metabolite Pathways

Soil microorganisms express diverse bioactive natural products, the majority of which are recalcitrant to cultivation. A novel method was developed to capture large DNA fragments from soil (up to 180 kb), enrich large BAC clones to identify complete natural product synthesis pathways, and express these pathways in an E. coli strain engineered to support small molecule synthesis. Screening a 19,200 BAC clone library from agricultural soil for antimicrobial activity produced 38 hits, none of which contained a recognizable natural product pathway. An alternative approach is to sequence each BAC clone and analyse the contents by homology comparison to GenBank. Sequencing 19,200 individual clones is not feasible, thus an alternative strategy using a multiplex pooling method termed BAC Sudoku sequencing was developed. Contigs were assembled for each pool and screened for secondary metabolite gene clusters using antiSMASH 2.0, resulting in hundreds of novel PKS/NRPS pathway containing clones. The cloned pathways are very divergent from known pathways, with the GC content varying from 41 to 76% and the amino acid identity of the KS domains ranging from 32 to 83% to the best matching BLAST hit. Expression of these PKS pathway-containing clones in an E. coli strain engineered for polyketide metabolite support resulted in multiple cases of heterologous expression as determined by anti-infective screening and structural analysis. These results indicate a high degree of unique sequence space has been recovered on large-insert metagenomic clones, some of which are capable of being expressed, thereby expanding our available resources for natural product discovery.