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Metabolic control analysis of microbial fed-batch production of L-tryptophan

微生物补料分批生产L-色氨酸的代谢控制分析

基本信息

批准号：
191572767
负责人：
Professor Dr. Georg Sprenger
金额：
--
依托单位：
Lehrstuhl für Bioverfahrenstechnik
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/191572767?language=en
关键词：
Metabolic control analysis microbial fed

项目摘要

Microbial production processes are subject to regulatory mechanisms on the cellular level. They determine the maximum specific production rate as well as the overall process duration. Therefore, modern approaches for process optimization use biological (metabolic design) and engineering techniques (process design). By using microbial L-phenylalanine production from glycerol with E. coli as an example, the application of Metabolic Control Analysis (MCA) successfully revealed the limiting enzymatic reactions at various phases of a standardized fed-batch process. For this purpose, rapid media transition and short-time experiments (20 min) were established in parallelized small-scale stirred-tank reactors. This enabled the perturbation of the metabolism of cells from the production process in different directions by feeding various carbon sources. By expression of suitable phosphate-antiporter systems, also phosphorylated substrates like phosphoenolpyruvate could be applied during the short-time experiments. The combination of the stochastic methods from MCA and a thermodynamic analysis of the reaction network allowed the determination of control parameters in E. coli metabolism from metabolome and fluxome data that were generated from the short-time experiments. For the first time, it was thus possible to show changes in the metabolic control during L-phenylalanine production in the course of the production process. This new method for MCA of cells from production processes was established in a successful cooperation between the project partners (Georg Sprenger, University of Stuttgart and Dirk Weuster-Botz, Technische Universität München) and it shall now be extended and applied to microbial production of the amino acid L-tryptophan from glycerol with E. coli. The analysis of microbial L-tryptophan production in a production process highly increases the complexity of the metabolic network to be analyzed. The main reasons for the choice of this topic are the additional crosslinks between aromatic amino acid biosynthesis and central metabolism caused by the required synthesis of L-serine and 5-phospho-D-ribose-1-diphosphate. The aim of this research project is to identify the controlling metabolic reactions in the producing organism at specific process times during a fed-batch process for L-tryptophan production from glycerol with E. coli. Strains and process strategies for L-tryptophan production will be established. Furthermore, transporter systems for the uptake of important metabolic intermediates shall be implemented. This will enable the identification of controlling steps in E. coli metabolism during the production process by applying short-time experiments, fluxome, metabolome as well as transcriptome analyses and MCA. These results will be used for further strain improvement and validation in the fed-batch production process.

微生物生产过程受到细胞水平上的调节机制的影响。它们决定了最大比生产率以及整个工艺持续时间。因此，现代工艺优化方法使用生物（代谢设计）和工程技术（工艺设计）。以甘油为原料，利用E.以大肠杆菌为例，代谢控制分析（MCA）的应用成功地揭示了在标准化补料分批过程的各个阶段的限制性酶反应。为此，在平行的小型搅拌釜反应器中建立了快速介质过渡和短时间（20 min）实验。这使得通过供给各种碳源，能够在不同方向上干扰来自生产过程的细胞代谢。通过表达合适的磷酸盐-反向转运蛋白系统，也可以在短时间实验期间应用磷酸化底物如磷酸烯醇丙酮酸。结合MCA的随机方法和反应网络的热力学分析，可以确定E.大肠杆菌代谢的代谢组和通量组数据，从产生的短时间的实验。这是第一次，因此，可以显示在生产过程中L-苯丙氨酸生产过程中代谢控制的变化。这种用于生产过程中细胞MCA的新方法是在项目合作伙伴（斯图加特大学Georg Sprenger和慕尼黑工业大学Dirk Weuster-Botz）之间的成功合作中建立的，现在将其扩展并应用于用E.杆菌生产过程中微生物L-色氨酸生产的分析大大增加了待分析的代谢网络的复杂性。选择这一主题的主要原因是芳香族氨基酸的生物合成和所需的L-丝氨酸和5-磷酸-D-核糖-1-二磷酸的合成所引起的中心代谢之间的额外交联。本研究的目的是确定控制代谢反应的生产生物体在特定的工艺时间，在补料分批过程中的L-色氨酸生产甘油与E。杆菌将建立用于L-色氨酸生产的菌株和工艺策略。此外，还应实施用于摄取重要代谢中间体的转运系统。这将使得能够识别E.应用短时实验、通量组、代谢组学以及转录组学分析和MCA等方法对生产过程中的大肠杆菌代谢进行了研究。这些结果将用于补料分批生产工艺中的进一步菌株改进和验证。