Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.
Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.
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DOI：
10.1038/ncomms10008
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发表时间：
2015-12-07
影响因子：
16.6
通讯作者：
Hollfelder F
中科院分区：
文献类型：
--
作者：
Colin PY;Kintses B;Gielen F;Miton CM;Fischer G;Mohamed MF;Hyvönen M;Morgavi DP;Janssen DB;Hollfelder F
不可培养的细菌群落提供了丰富的生物催化剂来源，但通过功能宏基因组学进行实验发现它们是困难的，因为实验者的机会很小。在这里，我们展示了在微流控微滴区室中的百万元宏基因组文库的功能筛选。使用诱饵底物，新的水解酶的硫酸单酯和磷酸三酯进行了鉴定，主要是基于混杂的活动，推测不会受到选择压力。跨越三个蛋白质超家族，它们在序列空间中开辟了新天地：混杂现在将酶与仅远相关的序列连接起来。大多数命中无法通过序列分析预测，因为所需的活性从未归因于相似的序列，这表明这种方法如何补充宏基因组测序数据的生物信息学收获。功能筛选库的前所未有的规模与出色的检测灵敏度已在鉴定罕见的基因构成催化通用枢纽序列空间作为潜在的起点收购新的功能。 来自不可培养微生物的环境DNA包含具有有用功能的基因，这些基因仍然难以识别和分离。在这里，Colin，Kintses等人展示了以pL体积筛选数百万个样品以直接鉴定新的酶活性并补充基于序列的方法。
Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions. Environmental DNA from unculturable microorganisms contains genes with useful functions that remain difficult to identify and isolate. Here Colin, Kintses et al. demonstrate the screening of millions of samples in pL volumes to directly identify new enzymatic activities and complements sequence-based approaches.