Varaljay Vanessa A, Satagopan Sriram, North Justin A, Witte Brian, Dourado Manuella N, Anantharaman Karthik, Arbing Mark A, Hoeft McCann Shelley, Oremland Ronald S, Banfield Jillian F, Wrighton Kelly C, Tabita F Robert
Department of Microbiology, The Ohio State University, Columbus, OH, 43210, USA.
The Botanical Research Institute of Texas, Fort Worth, TX, 76107, USA.
Environ Microbiol. 2016 Apr;18(4):1187-99. doi: 10.1111/1462-2920.13138. Epub 2016 Jan 21.
Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO2 /O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2 -fixing enzymes not previously characterized.
1,5-二磷酸核酮糖羧化酶/加氧酶（RubisCO）是一种关键但效率极低的酶，它催化地球上几乎所有碳的固定。在此，我们报告了一种功能宏基因组筛选方法，可直接从自然微生物群落中未培养且大多未知的成员中获得具有生理活性的RubisCO分子。筛选基于在能够表达环境脱氧核糖核酸（DNA）的宿主菌株中依赖二氧化碳的生长，无需纯培养或筛选重组克隆的酶活性。使用从土壤、河流自养富集物、光合生物膜和地下含水层提取的DNA，筛选出了17个编码功能性RubisCO的序列。值得注意的是，回收了三种相关的II型RubisCO，它们与未培养的Gallionellaceae科成员的宏基因组支架具有高度序列相似性。其中一种Gallionellaceae RubisCO被纯化，并显示具有典型II型酶的二氧化碳/氧气特异性。X射线晶体学确定该酶是六聚体，这是第二个解析出的II型多聚体，也是从未培养细菌中获得的第一个RubisCO结构。功能宏基因组筛选利用了自然生物多样性和环境群落中固有的数十亿年进化，为发现以前未表征的二氧化碳固定酶提供了一个新窗口。
