![]() ![]() Culture-independent, amplification-based surveys have resulted in significant insights into microbial ecology and population dynamics ( Schöler et al., 2017), while having their own inherent biases that obscure and distort our view of microbial diversity due to PCR amplification biases ( Eloe-Fadrosh et al., 2016). Natural products from soil microorganisms have historically been a rich resource for antibiotics ( Schatz et al., 1944 Ling et al., 2015 Hover et al., 2018) however, the vast majority of encoded BGCs have yet to be characterized since most soil microbes are recalcitrant to cultivation ( Janssen, 2006 Howe et al., 2014 Soliman et al., 2017 Orellana et al., 2018). Interactions between microorganisms are, to a large extent, mediated by bioactive natural products that are encoded by BGCs ( Reddy et al., 2012 Hill et al., 2014 Wang et al., 2014). There is a tremendous degree of microbial diversity in soils ( Torsvik and Ovreas, 2002), and soilborne microorganisms are key to biogeochemical and ecological processes. Its application can be extended to the detection of any genes of interest, thereby improving the power of functional metagenomic studies. We conclude that a more complete exploration of environmental metagenomes for their biosynthetic potential can be achieved using this approach. This approach uncovered heretofore undiscovered BGC and phylogenetic diversity revealing that much of our current knowledge has been obscured by traditional culturing or targeted amplification strategies. The approach developed in this study used next-generation sequencing in a pooled format, enabling bioinformatic identification of gene content associated with individual metagenomic clones. Direct cloning of soil metagenomic DNA can circumvent these limitations, yet identification of BGCs from metagenomic sequences can also be biased. Soil microorganisms have been an important reservoir of antibiotic and other bioactive compounds, yet our knowledge of their biosynthetic gene clusters (BGCs) has been limited by culture- and PCR-biases. This study provides novel resources for natural product discovery and circumvents amplification bias to allow annotation of a soil metagenomic library for a more complete picture of its functional and phylogenetic diversity. Likewise, 16S rRNA gene sequences obtained by NGS of the library included many representatives that were not recovered by PCR, in concordance with the same bias observed in KS amplicon screening. The NRPS and PKS clusters identified by NGS were distinct from known BGCs in the MIBiG database or those PKS clusters identified by PCR. A total of 1,015 BGCs were detected from 19,200 clones, identifying 223 clones (1.2%) that carry a polyketide synthase (PKS) and/or a non-ribosomal peptide synthetase (NRPS) cluster, a dramatically improved hit rate compared to PCR screening that targeted type I polyketide ketosynthase (KS) domains. Library clones were screened for biosynthetic gene clusters (BGCs) using either PCR or a NGS (next generation sequencing) multiplexed pooling strategy, coupled with bioinformatic analysis to identify contigs associated with each metagenomic clone. To identify the biosynthetic potential of soil microorganisms using a culture-independent approach, we constructed a large-insert metagenomic library in Escherichia coli from a topsoil sampled from the Cullars Rotation (Auburn, AL, United States), a long-term crop rotation experiment. Soil microorganisms historically have been a rich resource for natural product discovery, yet the majority of these microbes remain uncultivated and their biosynthetic capacity is left underexplored. 3Varigen Biosciences Corporation, Madison, WI, United States.2Lucigen Corporation, Middleton, WI, United States.1Department of Biological Sciences, Auburn University, Auburn, AL, United States.Santana-Pereira 1, Megan Sandoval-Powers 1, Scott Monsma 2, Jinglie Zhou 1, Scott R. ![]()
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