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）是一种重要的辅助因子，催化涉及胺和氨基酸的广泛反应。大肠杆菌和其他3-变形菌由4-磷酸红酶和1-脱氧-d - 5-磷酸木糖合成PLP。缺乏PdxB（赤藓酸4-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，7种不同酶的过度表达使这种菌株在葡萄糖上生长缓慢。其中两种（PdxA和AroB）可能具有混杂的PdxB活性。剩下的五种似乎促进了两种不同潜在途径中的一种，这些潜在途径允许绕过PdxB缺失所阻断的步骤。第一种途径似乎是用三种通常具有其他功能的酶和一种功能未知的蛋白质拼凑在一起的。参与第二种途径的酶尚未被确定。该项目将表征PdxA和AroB以及参与这两种潜在途径的酶的可疑混杂活性。我们将使用基因组洗牌来进化更有效地利用潜在PLP合成途径的大肠杆菌菌株。我们将通过基因组重测序、转录谱分析和各种生化方法来描述进化菌株的特征，以确定菌株适应更有效地使用潜伏途径的机制。

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.