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Metabolic pathway engineering of the toluene degradation pathway.

机译:甲苯降解途径的代谢途径工程。

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摘要

This thesis addresses the problem of how to examine a metabolic pathway and identify what are the key elements, specifically with respect to rate-limitation. The aim is to be able to analyze a pathway, identify the bottlenecks and implement genetic modifications to remove these bottlenecks. This is done by defining the system of interest and developing a predictive model using kinetic data. The model predictions can then be verified using fermentation data and genetic techniques to make the appropriate changes for improved performance. The test system chosen for this study was the TOL meta-cleavage pathway for the degradation of benzoate. This system was chosen on the basis of the application of pathway engineering principles to other systems. The modelling strategy and software was developed using principles from metabolic control theory and biochemical systems theory. By applying this to the TOL pathway using kinetic data, the control coefficients for the pathway were obtained as well as the system parameters required for the optimization of the pathway. The simulated results obtained from this model must be validated by experiment. Errors can arise both from incorrect assumptions in the model and from the fact that the kinetic data taken from individual in vitro experiments may not be applicable to the in vivo system. The effect of the presence of the TOL pathway on the behaviour of E.coli JM107 during fermentation was investigated and the transient concentration data necessary to identify the bottlenecks in the pathway measured. This data is then used to calculate the flux control coefficients for the TOL pathway. The predictive results were verified by the fermentation data which identified the first two enzymes in the pathway as having significant flux control coefficients. This final chapter also addresses the issue of flux analysis, that is, the calculation of the fluxes in the system to determine where fluxes to unwanted by-products occur and to indicate points of control. A graphical user interface is used to provide a user-friendly and intuitive means of building and customising metabolic pathways which can then be interfaced with instrumentation to provide on-line flux analysis.
机译:本论文解决了如何检查代谢途径并确定关键因素的问题,特别是在限速方面。目的是能够分析路径,识别瓶颈并实施基因改造以消除这些瓶颈。这是通过定义感兴趣的系统并使用动力学数据开发预测模型来完成的。然后可以使用发酵数据和遗传技术验证模型预测,以进行适当的更改以提高性能。此项研究选择的测试系统是苯甲酸降解的TOL元裂解途径。选择该系统是基于将路径工程原理应用于其他系统的基础。使用代谢控制理论和生化系统理论的原理开发了建模策略和软件。通过使用动力学数据将其应用于TOL途径,可以获得途径的控制系数以及途径优化所需的系统参数。从该模型获得的仿真结果必须通过实验进行验证。错误可能源于模型中的不正确假设,也可能来自以下事实:从单个体外实验获得的动力学数据可能不适用于体内系统。研究了TOL途径的存在对发酵过程中大肠杆菌JM107行为的影响,并测定了确定途径中瓶颈所需的瞬时浓度数据。然后,该数据用于计算TOL路径的磁通控制系数。通过发酵数据验证了预测结果,该数据确定了该途径中的前两种酶具有显着的通量控制系数。最后一章还讨论了通量分析的问题,即计算系统中的通量,以确定产生不希望的副产物的通量并指出控制点。图形用户界面用于提供建立和自定义代谢途径的用户友好和直观的方法,然后可以将其与仪器连接以提供在线通量分析。

著录项

  • 作者

    Regan, Lucy.;

  • 作者单位

    University of London, University College London (United Kingdom).;

  • 授予单位 University of London, University College London (United Kingdom).;
  • 学科 Biochemistry.
  • 学位 Ph.D.
  • 年度 1995
  • 页码 314 p.
  • 总页数 314
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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