Bioprocess engineering through dynamic control of metabolic pathways

代谢工程中的常见做法是在没有反馈控制的情况下，在生产过程中强烈过度表达酶。当单个细胞发现自己处于需要较少合成和重新分配化学前体的次优条件时，缺乏动态控制就成了问题。尽管像变构反馈调节这样的必要机制早在几十年前就已为人所知，但我们对它们在活细胞中的功能的不完全理解阻碍了这种类型的调节的实施。在我最近的工作中，我开发了一种方法，利用代谢组学和计算模型来研究细菌中变构反馈调节的功能。这个项目的目标是应用这些工具来寻找和实施结构原理，这些原理在生产宿主中协调自然代谢和化学物质的高过剩生产。值得注意的是，我将考虑工业规模的生物过程的条件，这些过程遭受产量过早下降的影响，并且不是理想的搅拌区，可能对生产主机不利。具体地说，我计划使用计算建模来寻找反馈调节，以实现生产途径与生产菌株在生物反应器环境中的新陈代谢的最佳协调。为了在细胞中实现控制电路，我计划使用现有的酶设计方法来构建合成变构酶。因此，将对已建立的蛋白质片段互补、结构域插入和模块重组程序进行评估。合成变构酶在生产菌株中的活性和功能是用我们结合代谢组学和计算模型的新方法来确定的。通过这种方法，我计划实现对大肠杆菌中氨基酸过量生产的自主调节。本研究的长期目标是了解代谢控制电路的基本设计原理，并将其转移到需要自主调节的生物技术相关系统中。动态控制可以提高生产菌株的整体适合度，从而保持生物过程的高生产率。一种机会是设计对大型生物反应器中存在的不理想搅动区域做出反应的细胞，每当细胞处于碳源或氧气供应不足的区域时，代谢信号可能会切断生产途径，直到细胞恢复。

The common practice in metabolic engineering is strong overexpression of enzymes in the production pathway without feedback control. The lack of dynamic control becomes problematic when an individual cell finds itself in suboptimal conditions that would require lower synthesis and redistribution of chemical precursors. Although the required mechanisms like allosteric feedback regulation are known since decades, our incomplete understanding of their function in a living cell stands in the way to implement this type of regulation. In my recent work I developed a method to investigate the function of allosteric feedback regulation in bacteria using metabolomics and computational modeling.The goal of this project is to apply these tools to search and implement structural principles that coordinate natural metabolism with high overproduction of a chemical in a production host. Notably, I will consider conditions of industrial scale bioprocesses, that suffer from premature decrease of yields and not ideally stirred zones that might be detrimental to the production host. Specifically, I plan to use computational modeling in order to search for feedback regulation that achieves optimal coordination of the production pathway with metabolism of the production strain in the environment of a bioreactor. In order to implement the control circuits in a cell, I plan to construct synthetic allosteric enzymes using existing approaches of enzyme design. Therefore established procedures of protein fragment complementation, domain insertion and modular recombination will be evaluated. The activity and function of the synthetic allosteric enzymes in the production strain is determined with our novel method that combines metabolomics and computational modeling. With this approach I plan to achieve autonomous regulation of amino acid overproduction in the bacterium Escherichia coli.The long-term goal of this research is that we understand fundamental design principles of metabolic control circuits and transfer them to biotechnological relevant systems that require autonomous regulation. Dynamic control can improve overall fitness of production strains and thereby maintains high productivity of bioprocesses. One opportunity is to engineer cells that respond to not ideally stirred zones present in large-scale bioreactors, whenever a cell is in a zone with low supply of carbon source or oxygen, a metabolic signal could switch off the production pathway until the cell has recovered.