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首页> 美国卫生研究院文献>The ISME Journal >A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity
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A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity

机译：一个基于功能的筛选器用于从元基因组中寻找RubisCO活性克隆：影响RubisCO活性的新型酶

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.
机译：1,5-双磷酸核糖羧化酶/加氧酶（RubisCO）是加尔文循环的关键酶，它负责地球的大部分初级生产。尽管对植物，蓝细菌和细菌中的RubisCO基因和酶的研究已经进行了多年，但对其在细菌中的调控和激活了解甚少。甚至更是如此，由于缺乏从环境中寻找活性RubisCO的可用的基于功能的筛选，关于宏基因组RubisCO的功能以及侧翼DNA上编码的酶的作用几乎没有任何信息。在这里，我们介绍了从宏基因组库中鉴定RubisCO活性fosmid克隆的第一个基于活性的方法。我们从热液排放液中构建了宏基因组文库，并筛选了1056个fosmid克隆。十二个克隆表现出RubisCO活性，宏基因组片段类似于Thiomicrospira crunogena的基因。这些克隆之一被进一步分析。它含有一个35.2 kb的宏基因组插入片段，带有RubisCO基因簇和侧翼DNA区域。该宏基因组片段上的12个基因和2个基因间区域的敲除表明RubisCO活性显着受损，并且归因于编码假定的转录调节子的基因的缺失以及被认为对RubisCO激活至关重要的基因。我们的新技术揭示了特征欠佳的基因与RubisCO活性之间的新颖联系。该屏幕打开了直接调查环境样品中的RubisCO基因和相应酶的大门。

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