Next-Generation Functional and Structural Soil Metagenomics.

Recent progress in molecular microbial ecology has revealed that soil is one of the most diverse microbial communities, but < 1% of viable microorganisms in soil can be recovered by traditional culturing techniques. Culture-independent metagenomic methods are limited by the challenges of constructing unbiased large inserts > 50kb for screening and next-gen sequencing (NGS). We have succeeded in preparing and cloning high quality high molecular weight (HMW, >100kb) metagenomic DNA from soil using bacterial artificial chromosome (BAC) shuttle vectors for expression in gram-positive and gram-negative hosts. To access the genetic diversity and biochemical potential of soil microorganisms, we have constructed the first-ever Random Shear shuttle BAC library of a soil sample with average inserts of 110 kb. Heterologous expression of this BAC library containing 19,200 BAC clones in Escherichia coli for functional screening identified 28 clones inhibiting the growth of Methicillin-resistant Staphyloccocus aureus (MRSA), three BACs showed cellulase-related activity, and three clones produced pigments. Sequence analysis of the anti-MRSA clones by multiplex 454 or Ion Torrent platforms revealed novel secondary metabolic pathways, unique sequences compared to the GenBank database, and their potential origins of the soil microorganisms in the community. These new metagenomic technologies have significant potential for discovering novel natural products and industrial enzymes as well as revealing large tracts of metagenomes, secondary metabolic pathways, and gene clusters including regulatory regions of the microbial communities.