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Systematic Analysis of Bottlenecks in a Multibranched and Multilevel Regulated Pathway: The Molecular Fundamentals of l-Methionine Biosynthesis in Escherichia coli
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-10-01 00:00:00 , DOI: 10.1021/acssynbio.8b00249 Jian-Feng Huang 1 , Zhen-Yang Shen 1 , Qiao-Li Mao 1 , Xiao-Ming Zhang 1 , Bo Zhang 1 , Jia-Shu Wu 1 , Zhi-Qiang Liu 1 , Yu-Guo Zheng 1
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-10-01 00:00:00 , DOI: 10.1021/acssynbio.8b00249 Jian-Feng Huang 1 , Zhen-Yang Shen 1 , Qiao-Li Mao 1 , Xiao-Ming Zhang 1 , Bo Zhang 1 , Jia-Shu Wu 1 , Zhi-Qiang Liu 1 , Yu-Guo Zheng 1
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To produce chemicals and fuels from renewable resources, various strategies and genetic tools have been developed to redesign pathways and optimize the metabolic flux in microorganisms. However, in most successful cases, the target chemicals are synthesized through a linear pathway, and regular methodologies for the identification of bottlenecks and metabolic flux optimization in multibranched and multilevel regulated pathways, such as the l-methionine biosynthetic pathway, have rarely been reported. In the present study, a systematic analysis strategy was employed to gradually reveal and remove the potential bottlenecks limiting the l-methionine biosynthesis in E. coli. 80 genes in central metabolism and selected amino acids biosynthetic pathways were first repressed or upregulated to probe their effects on l-methionine accumulation. The l-methionine biosynthetic pathway was then modularized and iteratively genetic modifications were performed to uncover the multiple layers of limitations and stepwise improve the l-methionine titer. The metabolomics data further revealed a more evenly distributed metabolic flux in l-methionine biosynthesis pathway of the optimal strain and provided valuable suggestions for further optimization. The optimal strain produced 16.86 g/L of l-methionine in 48 h by fed-batch fermentation. This work is the first to our knowledge to systematically elucidate the molecular fundamentals of multilevel regulation of l-methionine biosynthesis. It also demonstrated that the systematic analysis strategy can boost our ability to identify the potential bottlenecks and optimize the metabolic flux in multibranched and multilevel regulated pathways for the production of corresponding chemicals.
中文翻译：
多分支和多水平调控途径中瓶颈的系统分析：大肠杆菌中L-蛋氨酸生物合成的分子基础
为了从可再生资源生产化学品和燃料，已经开发了各种策略和遗传工具来重新设计途径并优化微生物的代谢通量。然而，在最成功的情况下，目标化学品通过线性途径合成，并定期方法为瓶颈的鉴定和代谢通量优化在多支链和多级调节的途径，如升-甲硫氨酸生物合成途径，很少有报道。在本研究中，系统的分析策略用来逐渐显露并除去潜在的瓶颈限制升在蛋氨酸生物合成大肠杆菌。首先抑制或上调中央代谢和选定氨基酸生物合成途径中的80个基因，以探究它们对1-甲硫氨酸积累的影响。然后将1-甲硫氨酸生物合成途径模块化，并进行迭代遗传修饰以揭示多层限制并逐步提高1-甲硫氨酸滴度。代谢组学数据进一步显示在一个更均匀分布的代谢通量升-甲硫氨酸生物合成途径的最佳菌株的，并为进一步优化提供了有价值的建议。最佳菌株产生16.86 g / L的l分批补料发酵48小时制得蛋氨酸。这项工作是我们所了解的第一个系统阐明1-甲硫氨酸生物合成多级调节分子基础的知识。它还表明，系统的分析策略可以提高我们识别潜在瓶颈的能力，并优化多支链和多级调节途径中的代谢通量，以生产相应的化学物质。
更新日期：2018-10-01
中文翻译：
多分支和多水平调控途径中瓶颈的系统分析：大肠杆菌中L-蛋氨酸生物合成的分子基础
为了从可再生资源生产化学品和燃料，已经开发了各种策略和遗传工具来重新设计途径并优化微生物的代谢通量。然而，在最成功的情况下，目标化学品通过线性途径合成，并定期方法为瓶颈的鉴定和代谢通量优化在多支链和多级调节的途径，如升-甲硫氨酸生物合成途径，很少有报道。在本研究中，系统的分析策略用来逐渐显露并除去潜在的瓶颈限制升在蛋氨酸生物合成大肠杆菌。首先抑制或上调中央代谢和选定氨基酸生物合成途径中的80个基因，以探究它们对1-甲硫氨酸积累的影响。然后将1-甲硫氨酸生物合成途径模块化，并进行迭代遗传修饰以揭示多层限制并逐步提高1-甲硫氨酸滴度。代谢组学数据进一步显示在一个更均匀分布的代谢通量升-甲硫氨酸生物合成途径的最佳菌株的，并为进一步优化提供了有价值的建议。最佳菌株产生16.86 g / L的l分批补料发酵48小时制得蛋氨酸。这项工作是我们所了解的第一个系统阐明1-甲硫氨酸生物合成多级调节分子基础的知识。它还表明，系统的分析策略可以提高我们识别潜在瓶颈的能力，并优化多支链和多级调节途径中的代谢通量，以生产相应的化学物质。
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