Evolutionary ecology and multidisciplinary approaches to prospecting for monooxygenases as biocatalysts
Evolutionary ecology and multidisciplinary approaches to prospecting for monooxygenases as biocatalysts
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DOI：
10.1007/s10482-008-9227-1
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发表时间：
2008-06-01
影响因子：
2.6
通讯作者：
Coleman, Nicholas V.
中科院分区：
文献类型：
--
作者：
Holmes, Andrew J.;Coleman, Nicholas V.
探索微生物多样性的新技术使人们对勘探天然产品（生物勘探或生物发现）重新产生兴趣。虽然许多生物勘探项目乍一看可能没有什么共同之处，但绝大多数项目都面临着一个特殊的挑战。它们的目标是复杂的自然组合中的稀有到非常稀有的成员。尽管在培养新生物体和应用非培养技术分离新基因方面取得了进展，但对具有特定生长要求的相对罕见的生物体仍然存在系统性偏见。这些问题往往可以通过应用考虑到进化生态学原则的多学科方法来克服。我们的经验与勘探可溶性二铁单加氧酶（SDIMO）表明，传统的方法，生物体分离和宏基因组克隆系统下的样品在这个酶家族的多样性。这反映出含有空间数据集单胞菌的生物体通常是天然集合体中丰度相对较低的成员（因此不受直接克隆的影响），空间数据集单胞菌在每种生物体中具有离散的生理作用（因此不适合通用富集培养策略）。我们试图通过基于PCR的基因多样性调查来指导后续培养或克隆研究的评估，以克服这一点。这项调查的一个令人惊讶的结果是，使用简并引物的传统PCR方法也系统地欠采样的多样性，但巢式PCR策略揭示了前所未有的多样性。我们的结论是，许多基于PCR的基因勘探研究可能低估了目标：竞争对手的比例对他们的成功的影响。
New techniques to explore microbial diversity have led to resurgent interest in prospecting for natural products (bioprospecting or biodiscovery). Although many bioprospecting projects may share little in common at first glance, the vast majority share one particular challenge. Their targets are rare to very rare members of complex natural assemblages. Despite the advances made in bringing new organisms into cultivation and application of culture-independent techniques to isolation of novel genes there remain systematic biases against relatively rare organisms with specific growth requirements. These can frequently be overcome by application of multidisciplinary approaches that take into account principles of evolutionary ecology. Our experiences with prospecting for soluble di-iron monooxygenases (SDIMO) indicate that conventional approaches to organism isolation and metagenomic cloning systematically under-sample diversity in this enzyme family. This reflects that SDIMO-containing organisms are typically relatively low-abundance members of natural assemblages (thus biased against by direct cloning) and SDIMOs have discrete physiological roles in each organism (thus are not amenable to generic enrichment culture strategies). We have sought to overcome this by a PCR-based survey of gene diversity to guide evaluation of subsequent culture or cloning studies. A surprising outcome of this survey was that conventional PCR approaches using degenerate primers also systematically under-sampled diversity, but nested PCR strategies revealed unprecedented diversity. We conclude that many PCR-based gene-prospecting studies are likely to have under-estimated the impact of target:competitor ratios on their success.