Tikh Ilya B, Quin Maureen B, Schmidt-Dannert Claudia
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota, United States of America.
PLoS One. 2014 Feb 21;9(2):e89734. doi: 10.1371/journal.pone.0089734. eCollection 2014.
The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in E. coli. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms Rhodobacter sphaeroides and Chlorobaculum tepidum. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg(2+) in the porphyrin ring, with no activity against non-Mg(2+) chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both in vitro, and in vivo as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in E. coli. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.
创建一种能够从阳光中获取能量以驱动其代谢过程的合成微生物，是实现目标化合物经济可行的生物合成生产的一种有吸引力的方法。我们的目标是设计和改造一种基因易处理的非光合微生物，以生产光捕获分子。此前，我们创建了一个模块化的多酶系统，用于在大肠杆菌中异源生产细菌叶绿素（BChl）途径的中间体。在本报告中，我们扩展了该途径，使其包括一种底物宽泛的8-乙烯基还原酶，该酶可以接受BChl生物合成的多种中间体。我们对来自模式光合生物球形红细菌和嗜热绿菌的8-乙烯基还原酶的同源物进行了信息丰富的比较分析。首次对这些酶进行纯化，从而对其进行详细的生化和生物物理表征。所获得的数据表明，这两种8-乙烯基还原酶底物宽泛，能够还原镁原卟啉IX、镁原卟啉IX甲酯和二乙烯基原叶绿素酸酯的C8-乙烯基。然而，活性取决于卟啉环中螯合的Mg(2+)的存在，未观察到对非Mg(2+)螯合中间体的活性。此外，圆二色性分析表明，这两种8-乙烯基还原酶似乎以不同的方式结合相同的底物。此外，我们发现，这两种8-乙烯基还原酶在体外以及作为我们工程系统一部分在体内的不同反应速率，导致只有一种同源物适用于我们在大肠杆菌中的BChl途径。我们的结果首次揭示了同源8-乙烯基还原酶的不同功能。这项研究也使我们朝着创建一种能够从阳光中获取能量以生物合成所需分子的非光合微生物又迈进了一步。
