Elucidation and Evolutionary Potential of a Latent Pathway for PLP Synthesis

描述(由申请人提供)：吡哆醛5‘磷酸(PLP)是一种重要的辅因子，催化涉及胺和氨基酸的广泛反应。大肠埃希菌和其他3-蛋白细菌从红素4-磷酸和1-脱氧-D-木糖-5-磷酸合成PLP。缺乏PdxB(赤磷酸脱氢酶)的菌株不能在葡萄糖上生长，因为它不能产生PLP。我们发现，七种不同酶的过度表达使该菌株能够在葡萄糖上缓慢生长。其中两个(PdxA和AROB)可能具有混杂的PdxB活性。剩下的五条似乎促进了两条不同的潜在途径中的一条，这两条不同的潜在途径允许绕过因缺乏PdxB而被阻止的步骤。这些途径中的第一条似乎是使用三种通常具有其他功能的酶和一种未知功能的蛋白质拼接在一起的。参与第二条途径的酶还没有确定。这个项目将描述可疑的PdxA和AROB的混杂活性以及这两个潜在途径中涉及的酶。我们将使用基因组改组来进化出更有效地使用潜在PLP合成途径的大肠杆菌菌株。我们将通过基因组重新测序、转录图谱和各种生化方法来表征进化的菌株，以确定菌株适应更有效地使用潜在途径的机制。 这个项目是新颖的，因为它解决了“未被选择的道路”的进化潜力。虽然很明显，大自然并没有探索合成关键代谢物的所有可能的解决方案，但我们很少有机会探索一条可能与现有生物体中发现的途径一样的途径的潜力。 我们对适应使用低效的PLP合成潜在途径所需的遗传变化的分析，将为其他努力将新的代谢模块整合到现有的大肠杆菌和其他细菌的代谢网络中用于工业目的提供信息。此外，这个项目将加强我们对组装新的代谢途径的潜力的理解，方法是将通常在细胞中发挥其他功能的酶拼接在一起。这种途径可以降解抗生素、杀虫剂和工业污染物等人为化学物质。

DESCRIPTION (provided by applicant): Pyridoxal 5'phosphate (PLP) is an essential cofactor that catalyzes a wide range of reactions involving amines and amino acids. E. coli and other 3- proteobacteria synthesize PLP from erythrose 4-phosphate and 1-deoxy-D- xylulose 5-phosphate. A strain lacking PdxB (erythronate 4-phosphate dehydrogenase) cannot grow on glucose because it cannot make PLP. We have found that overexpression of seven different enzymes allows this strain to grow slowly on glucose. Two of these (PdxA and AroB) probably have promiscuous PdxB activity. The remaining five appear to facilitate one of two different latent pathways that allow the step blocked by the absence of PdxB to be bypassed. The first of these pathways appears to be patched together using three enzymes that normally serve other functions and a protein of unknown function. The enzymes involved in the second pathway have not yet been identified. This project will characterize the suspected promiscuous activities of PdxA and AroB and the enzymes involved in both latent pathways. We will use genome shuffling to evolve strains of E. coli that use the latent PLP synthesis pathways more efficiently. We will characterize the evolved strains by genome re-sequencing, transcriptional profiling, and various biochemical approaches to identify the mechanisms by which the strains have adapted to use a latent pathway more efficiently. This project is novel because it addresses the evolutionary potential of "roads not taken". While it is obvious that nature has not explored all possible solutions to the synthesis of critical metabolites, we rarely have an opportunity to explore the potential of a pathway that might serve as well as those found in extant organisms. Our analysis of the genetic changes required for adaptation to the use of the inefficient latent pathways for PLP synthesis will inform other efforts to incorporate novel metabolic modules into the pre-existing metabolic network of E. coli and other bacteria for industrial purposes. In addition, this project will enhance our understanding of the potential for assembling novel metabolic pathways by patching together enzymes that normally serve other functions in the cell. Such pathways could allow degradation of anthropogenic chemicals such as antibiotics, pesticides, and industrial pollutants.