Development and application of mass spectrometry-based methods for systems biology: regulation of central carbon metabolism of Escherichia coli

• Main Author: Shen, Yang
• Format: Others
• Language: English
• Published: HKBU Institutional Repository 2015
• Subjects: Biological systems;Carbon;Escherichia coli;Mass spectrometry;Metabolism;Methodology.
• Online Access: https://repository.hkbu.edu.hk/etd_oa/209; https://repository.hkbu.edu.hk/cgi/viewcontent.cgi?article=1208&context=etd_oa

As an emerging area, systems biology provides a new paradigm not only for studying the cellular organization and regulation, but also for investigating how the systemic behaviors emerge in biological systems. One of the main objectives of systems biology is to understand mechanism and principle of the strategy that is applied in the metabolic network response to the environment and resources availability. Mass spectrometry (MS) was employed as the high-throughput technique tools to collect reliable data to obtain a quantitative understanding of the regulation of metabolic network in different cases. A LC-MS based method was developed and optimized for the measurement of central carbon metabolites in Escherichia coli. It could avoid the leakage problem and the "false high level" caused by the metabolites excreted to the medium, and provides better coverage as well as more accurate quantitative results of intracellular metabolites from different conditions. The developed method was employed to investigate the metabolic response to the nutrition stress. Intracellular concentrations of central carbon metabolites were measured under different nutrition conditions. The FBP concentration revealed the carbon influx because it served as a sensor of glycolytic flux and the α-ketoglutarate served as a coordinator of carbon and nitrogen flux response to the nutrient availabilities. A scenario was made that cell coordinated the catabolic and anabolic metabolism under different conditions by α-ketoglutarate and cAMP signaling. The overflow metabolism of E. coli was studied. A robust linear relation between acetate excretion rate and growth rate was observed. Gene expression level and quantitative proteomics approach were employed under perturbations such as mutants and increased energy demand (drained the energy by DNP). Acetate overflow in E. coli results from the tradeoff between efficient utilization of carbon resources by respiration and efficient utilization of proteome resources by fermentation. The physiology-driven approach was employed to investigate the potential targets of sRNA RyhB and the function of chaperon Hfq. Construction of a truncated RyhB mutant (RyhBt) was performed to confirm the necessity of hfq to RyhB. The expression of RyhB and RyhBt can both slow the growth rate. However, after the deletion of hfq, the growth defects induced by RyhB disappeared but still existed in the RyhBt strain without Hfq. It indicated that RyhB played its function by binding with Hfq at the special regions. Proteomics approach discovered some target genes of RyhB and RyhBt in both TCA cycle and nitrogen assimilation pathway. The relative abundance of proteins reflected that the RyhB and RyhBt affected the target differently because they had different binding sites with chaperons or targets. It will provided valuable information for revealing the inner mechanism of the physiology changes caused by sRNAs. GC-MS method was developed to identify and quantify the metabolites in E.coli cells. Three different derivatization methods for GC-MS were compared and optimized. The pool size of glutamine and glutamate was stable in the wild type strain at certain conditions but changed significantly in the GOGAT- strain especially the nitrogen was limited.