Elucidation and Evolutionary Potential of a Latent Pathway for PLP Synthesis

PLP 合成潜在途径的阐明和进化潜力

• 批准号: 8072108
• 负责人: SHELLEY D. COPLEY
• 金额: $ 28.57万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072108
• 关键词: 1-deoxy-2-pentulose; 3-hydroxybutanal; Acids; Active Sites; Address; Amines; Amino Acids; Ammonia; Anabolism; Antibiotics; Archaea; Bacteria; Biochemical; Biochemical Pathway; Bypass; Carbon; Carboxy-Lyases; Catalysis; Cells; Chemicals; Complex; D-xylulose-5-phosphate; Decarboxylation; Dihydroxyacetone Phosphate; Enzymes; Escherichia coli; Evolution; Genes; Genetic; Genome; Genomics; Glucose; Glutamine; Glyceraldehyde 3-Phosphate; Growth; Hydrolase; Industrial Waste; Ions; Lesion; Metabolic; Metabolic Pathway; Metabolism; Metals; Microbe; Molecular; Mutation; Nature; Organism; Oxidoreductase; Pathway interactions; Pesticides; Pheromone; Phosphoric Monoester Hydrolases; Phosphotransferases; Process; Proteins; Proteobacteria; Pyridoxal; Pyridoxal Phosphate; Pyridoxamine; Pyridoxine 5 Phosphate Oxidase; Pyruvate; Reaction; Serc; Solutions; Source; Sugar Phosphates; System; Technology; Threonine; Transaminases; Trioses; Work; active control; anthropogenesis; base; catalyst; cell type; chemical reaction; cofactor; erythrose 4-phosphate; fitness; genetic analysis; inorganic phosphate; lysine 2,3-aminomutase; novel; overexpression; plant fungi; pyridoxine; pyridoxine 5-phosphate; quorum sensing; racemization; ribose-5-phosphate; ribulose 5-phosphate; transamination

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.

描述（由申请人提供）：吡哆醛5 '磷酸（PLP）是一种必需的辅因子，可催化涉及胺和氨基酸的广泛反应。E.大肠杆菌和其它3-变形菌从赤藓糖4-磷酸和1-脱氧-D-木酮糖5-磷酸合成PLP。缺乏PdxB（4-磷酸二磷酸脱氢酶）的菌株不能在葡萄糖上生长，因为它不能产生PLP。我们已经发现，七种不同酶的过表达允许这种菌株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。剩下的五个似乎促进了两种不同的潜在途径之一，允许绕过PdxB缺乏所阻断的步骤。第一条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。参与第二途径的酶尚未确定。该项目将表征PdxA和AroB的可疑混杂活性以及参与这两种潜在途径的酶。我们将使用基因组改组来进化E.大肠杆菌，使用潜在的PLP合成途径更有效。我们将通过基因组重测序、转录谱分析和各种生化方法来表征进化菌株，以确定菌株适应更有效地使用潜伏途径的机制。 这个项目是新颖的，因为它解决了“未采取的道路”的进化潜力。虽然很明显，大自然并没有探索所有可能的解决方案来合成关键代谢物，但我们很少有机会探索可能与现存生物体中发现的途径一样有效的途径的潜力。 我们对适应PLP合成的低效潜在途径所需的遗传变化的分析将为其他努力提供信息，以将新的代谢模块纳入E.大肠杆菌和其他细菌用于工业用途。此外，该项目将增强我们对通过将通常在细胞中发挥其他功能的酶拼凑在一起来组装新代谢途径的潜力的理解。这些途径可以使抗生素、杀虫剂和工业污染物等人为化学物质降解。