Bioprocess engineering through dynamic control of metabolic pathways

代谢工程中常见的做法是在没有反馈控制的情况下，生产途径中酶的强烈过表达。当单个细胞发现自己处于不理想的条件下，需要较少的化学前体合成和重新分配时，缺乏动态控制就会成为问题。虽然像变构反馈调节这样的必要机制在几十年前就已经为人所知，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调节的实现。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变构反馈调节的功能。该项目的目标是应用这些工具来搜索和实施结构原则，以协调生产宿主中自然代谢与高过量生产的化学物质。值得注意的是，我将考虑工业规模生物工艺的条件，这些条件会导致产量过早下降，并且没有理想的搅拌区，这可能对生产宿主有害。具体而言，我打算利用计算建模的方法，在生物反应器环境中寻找生产途径与生产菌株代谢的最优协调的反馈调节。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构建合成变构酶。因此，将评估已建立的蛋白质片段互补、结构域插入和模块重组程序。利用代谢组学和计算模型相结合的新方法测定了生产菌株合成变构酶的活性和功能。通过这种方法，我计划在大肠杆菌中实现氨基酸过量生产的自主调节。这项研究的长期目标是，我们了解代谢控制电路的基本设计原则，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应度，从而保持生物过程的高生产率。一个机会是设计细胞，使其对大规模生物反应器中存在的不理想搅拌区域做出反应，每当细胞处于碳源或氧气供应不足的区域时，代谢信号就会关闭生产途径，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.