Promiscuity, serendipity, and metabolic innovation

生物信息学和结构/功能研究表明，大多数现存的代谢途径 是从以前存在的混杂的酶活性拼凑起来的，即使 效率低下，提供了一定程度的催化作用。然而，我们无法解释为什么某些途径 而不是其他成千上万的可能性。此外，我们对 新的途径拼凑在一起，通过突变改善通量的过程。 本项目将开发一个模型系统，其中吡哆醛5- 磷酸盐（PLP）被破坏，以确定恢复PLP的“偶然途径”（SP 通过将通常服务于其他生物的酶的混杂活性拼凑在一起来合成。 功能协调发展的将表征在SP中催化步骤的酶。的机制 将鉴定哪些突变将通过SP的通量增加到生理学上显著的水平。 最后，进化结果对环境条件和基因组的依赖性 内容将被定义。 该项目的可行性得到了初步结果的支持， E. p. pdxB的进化大肠杆菌在不同的条件和证据表明，PLP的合成可以恢复 在其他条件下进化后，也是如此。生物化学、遗传学和代谢组学方法 揭示了突变使这些SP之一能够组装的机制。 E. PLP合成所需的基因已被删除的大肠杆菌和其它细菌， 进行实验室进化。如果生长能够恢复，负责的突变将是 鉴定这些突变促进招募一种 混杂的酶来取代缺失的酶，或者，更有趣的是，SP的出现 将使用一系列生物化学、遗传学和组学方法来定义。 该项目将为代谢途径的组装提供新的见解， 内容和生长条件影响新途径的演变，以及最初如何通过 由于突变，低效途径可能会增加。这些见解将帮助我们理解 细菌如何在地球生命的历史中进化，以及它们将如何继续 由于人类引入人为化学物质而施加新的选择压力， 例如杀虫剂和药品。降解途径的演变 人为化合物对于最大限度地减少对生态系统健康的不利影响至关重要， 人类另一方面，新途径的进化可能是一种未被认识的机制 对用于治疗传染病和癌症的抗代谢药物的耐药性。

Bioinformatic and structure/function studies suggest that most extant metabolic pathways were patched together from previously existing promiscuous enzyme activities that, even if inefficient, provided some degree of catalysis. However, we cannot explain why certain pathways arose rather than the thousands of other possibilities. Further, we have little insight into the process by which novel pathways were patched together and flux was improved via mutations. This project will exploit a model system in which the pathway for synthesis of pyridoxal 5- phosphate (PLP) has been disrupted to identify “serendipitous pathways” (SPs) that restore PLP synthesis by patching together promiscuous activities of enzymes that normally serve other functions. The enzymes that catalyze steps in SPs will be characterized. The mechanisms by which mutations increase flux through a SP to a physiologically significant level will be identified. Finally, the dependence of the evolutionary outcome on environmental conditions and genome content will be defined. The feasibility of this project is supported by preliminary results identifying two SPs after evolution of ∆pdxB E. coli in different conditions and evidence that PLP synthesis can be restored after evolution in other conditions, as well. Biochemical, genetic, and metabolomics approaches have revealed the mechanisms by which mutations enabled assembly of one of these SPs. E. coli and other bacteria in which a gene required for PLP synthesis has been deleted will be subjected to laboratory evolution. If growth can be restored, the responsible mutations will be identified. The mechanisms by which these mutations have facilitated recruitment of a promiscuous enzyme to replace the missing enzyme, or, more interestingly, emergence of a SP will be defined using a battery of biochemical, genetic and ‘omics approaches. This project will provide new insights into the assembly of metabolic pathways, how genome content and growth conditions affect the evolution of novel pathways, and how flux through initially inefficient pathways can be elevated due to mutations. These insights will help us understand how bacteria have evolved throughout the history of life on earth, and how they will continue to evolve as humans exert new selective pressures due to introduction of anthropogenic chemicals such as pesticides and pharmaceuticals. Evolution of pathways capable for degradation of anthropogenic compounds is critical to minimize adverse effects on the health of ecosystems and humans. On the other hand, evolution of novel pathways might be an unrecognized mechanism for resistance to antimetabolite drugs used to treat infectious diseases and cancer.