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

基本信息

批准号：
7825252
负责人：
SHELLEY D. COPLEY
金额：
$ 28.9万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7825252
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 Pyruvates Reaction Serc Solutions Source Sugar Phosphates System Technology Threonine Transaminases Trioses Work 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

项目摘要

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）：5'Pyridoxal 5'磷酸（PLP）是一种必不可少的辅助因子，可催化涉及胺和氨基酸的广泛反应。大肠杆菌和其他3-蛋白杆菌合成的4-磷酸和1-脱氧-D-二硫代5-磷酸的PLP。缺乏PDXB的菌株（4-磷酸脱氢酶）无法在葡萄糖上生长，因为它不能产生PLP。我们发现，七种不同酶的过表达可以使该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。其余五个似乎有助于两种不同的潜在途径之一，这些途径允许绕过PDXB的台阶阻塞。这些途径中的第一个似乎使用通常提供其他功能的三种酶和一个未知功能的蛋白质对其进行了修补。尚未确定参与第二途径的酶。该项目将表征PDXA和AROB的可疑滥交以及这两种潜在途径所涉及的酶。我们将使用基因组改组来进化大肠杆菌的菌株，该大肠杆菌更有效地使用潜在的PLP合成途径。我们将通过基因组重新测序，转录分析和各种生化方法来表征进化的菌株，以识别菌株适应更有效地使用潜在途径的机制。该项目之所以新颖，是因为它涉及“未采取的道路”的进化潜力。虽然很明显，大自然并未探索合成关键代谢产物的所有可能解决方案，但我们很少有机会探索可能与现存生物体中发现的途径的潜力。我们对适应使用低效率潜在途径进行PLP合成所需的遗传变化的分析将为其他努力提供了将新型代谢模块纳入大肠杆菌的现有代谢网络和其他细菌用于工业目的。此外，该项目将通过将通常服务于细胞中其他功能的酶进行整合在一起，从而增强我们对组装新代谢途径的潜力的理解。这种途径可以允许降解人为化学物质，例如抗生素，农药和工业污染物。