Culture-independent studies have indicated that there is significant diversity in the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzymes used by marine, freshwater, and terrestrial autotrophic bacteria. Surprisingly, little is known about the catalytic properties of many environmentally significant RubisCO enzymes. Because one of the goals of RubisCO research is to somehow modify or select for RubisCO molecules with improved kinetic properties, a facile means to isolate functional and novel RubisCO molecules directly from the environment was developed. In this report, we describe the first example of functional RubisCO proteins obtained from genes cloned and characterized from metagenomic libraries derived from DNA isolated from environmental samples. Two form IA marine RubisCO genes were cloned, and each gene supported both photoheterotrophic and photoautotrophic growth of a RubisCO deletion strain of Rhodobacter capsulatus, strain SBI/II−, indicating that catalytically active recombinant RubisCO was synthesized. The catalytic properties of the metagenomic RubisCO molecules were further characterized. These experiments demonstrated the feasibility of studying the functional diversity and enzymatic properties of RubisCO enzymes without first cultivating the host organisms. Further, this “proof of concept” experiment opens the way for development of a simple functional screen to examine the properties of diverse RubisCO genes isolated from any environment, and subsequent further bioselection may be possible if the growth conditions of complemented R. capsulatus strain SBI/II− are varied.
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机译:与文化无关的研究表明,海洋,淡水和陆生自养细菌使用的核糖1,5-双磷酸羧化酶/加氧酶(RubisCO)酶具有很大的多样性。令人惊讶地,对于许多对环境有意义的RubisCO酶的催化性质知之甚少。由于RubisCO研究的目标之一是以某种方式修饰或选择具有改进动力学性能的RubisCO分子,因此开发了一种从环境中直接分离功能性和新型RubisCO分子的简便方法。在本报告中,我们描述了从从环境样品中分离的DNA衍生的宏基因组文库中克隆和表征的基因获得的功能RubisCO蛋白的第一个例子。克隆了两种形式的IA海洋RubisCO基因,每个基因都支持荚膜红细菌RubisCO缺失菌株SBI / II - sup>的光异养和光自养生长,表明已合成了具有催化活性的重组RubisCO。宏基因组的RubisCO分子的催化特性得到了进一步的表征。这些实验证明了在不首先培养宿主生物的情况下研究RubisCO酶的功能多样性和酶学性质的可行性。此外,该“概念验证”实验为开发简单的功能筛选以检查从任何环境中分离的多种RubisCO基因的特性开辟了道路,并且如果互补荚膜荚膜梭菌菌株SBI的生长条件,则可能进行进一步的生物选择/ II - sup>有所不同。
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