FluxProbe - Using biocatalysis for probing the functional space of single microbial catalysts

全细胞生物催化剂由于其无与伦比的手性和位置选择性而成为有机合成的有力工具。但是，最小的催化单位，即单细胞，对生物催化有什么贡献呢？由于缺乏合适的分析，单个细胞反应性的离散速率以及控制细胞功能的机制在很大程度上仍然未知。项目7将建立一个基于芯片的分析框架，用于在单细胞水平上量化生物催化剂的性能。通过整合微流控生物反应器技术和用于生物催化产物质谱分析的小型化分析，将首次定量评估单细胞的条件依赖性催化性能。然而，催化产物不仅将被定量，而且还将用稳定同位素进行代谢富集以用作代谢探针（“通量探针”）。借助质谱的力量，产物分子中的同位素标记将揭示细胞的催化网络操作，并最终将代谢状态与反应性和细胞外反应条件联系起来。与WG Belder合作进行的关键实验已经验证了这一概念的可行性。

Whole-cell biocatalysts are powerful tools for organic synthesis due to their unmatched chiral and positional selectivity. But what is the contribution of the minimal catalytic unit, the single cell, to biocatalysis? Discrete rates for reactivity of individual cells, as well as mechanisms that control cellular functionality, are still largely unknown due to a lack of suitable analytics. Project 7 will establish a chip-based analytical framework for quantifying biocatalyst performance at the single cell level. By integrating microfluidic bioreactor technologies and miniaturized analytics for mass spectrometry of biocatalytic products, the conditions-dependent catalytic performance of single cells in terms will be evaluated quantitatively for the first time. However, catalytic products will not only be quantified, but also metabolically enriched with stable isotopes to serve as a metabolic probe (“flux probe”). With the power of mass spectrometry, the isotope labelling in the product molecules will reveal the catalytic network operation of the cell and finally enable connecting metabolic status with reactivity and extracellular reaction conditions. Key experiments performed in collaboration with the WG Belder already validated the feasibility of this concept.