Recent progress in molecular microbial ecology has revealed that soil is one of the most diverse microbial communities, but < 1% of viable microorganisms 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). NGS of metagenomes provides valuable genetic information but the daunting amount of short read sequence information compromises functional genomics and assembly. Large-insert bacterial artificial chromosome (BAC) libraries of metagenomes could be an excellent tool to bridge this gap. The research objectives are to prepare ultrapure mega-base-size DNA from soil, construct Random Shear BAC libraries with average insert size at least 100kb in a shuttle BAC vector, and express soil metagenomic DNA and secondary metabolic pathways of un-cultivatable soil bacterial communities. High molecular weight (HMW) genomic DNA was purified from soil, the DNA was mechanically sheared, Random Shear BAC libraries were constructed, library screening was performed in 96-well and 384-well formats, and positive clones were sequenced by next-gen platforms.
Results/Conclusions
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 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. The take-home message is that 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.