Santana-Pereira Alinne L R, Sandoval-Powers Megan, Monsma Scott, Zhou Jinglie, Santos Scott R, Mead David A, Liles Mark R
Department of Biological Sciences, Auburn University, Auburn, AL, United States.
Lucigen Corporation, Middleton, WI, United States.
Front Microbiol. 2020 Dec 7;11:585398. doi: 10.3389/fmicb.2020.585398. eCollection 2020.
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. To identify the biosynthetic potential of soil microorganisms using a culture-independent approach, we constructed a large-insert metagenomic library in from a topsoil sampled from the Cullars Rotation (Auburn, AL, United States), a long-term crop rotation experiment. 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. 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. The NRPS and PKS clusters identified by NGS were distinct from known BGCs in the MIBiG database or those PKS clusters identified by PCR. 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. 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.
从历史上看，土壤微生物一直是天然产物发现的丰富资源，但这些微生物中的大多数仍未被培养，其生物合成能力也未得到充分探索。为了使用一种不依赖培养的方法来鉴定土壤微生物的生物合成潜力，我们从美国阿拉巴马州奥本的卡拉斯轮作（Cullars Rotation）的表层土壤中构建了一个大插入片段宏基因组文库，这是一个长期的作物轮作实验。使用PCR或NGS（下一代测序）多重混合策略对文库克隆进行生物合成基因簇（BGC）筛选，并结合生物信息学分析来鉴定与每个宏基因组克隆相关的重叠群。从19200个克隆中总共检测到1015个BGC，鉴定出223个携带聚酮合酶（PKS）和/或非核糖体肽合成酶（NRPS）簇的克隆（1.2%），与针对I型聚酮合酶（KS）结构域的PCR筛选相比，命中率有了显著提高。通过NGS鉴定的NRPS和PKS簇与MIBiG数据库中已知的BGC或通过PCR鉴定的那些PKS簇不同。同样，通过文库的NGS获得的16S rRNA基因序列包括许多未通过PCR回收的代表序列，这与在KS扩增子筛选中观察到的相同偏差一致。这项研究为天然产物发现提供了新的资源，并规避了扩增偏差，以便对土壤宏基因组文库进行注释，从而更全面地了解其功能和系统发育多样性。
