21ENGBIO: In Cell Assembly of Artificial Imine Reductases for Whole-Cell Catalysis

该项目旨在组装人造酶，这种人造酶由合成催化剂组成，通过锚基连接到活细菌细胞内的蛋白质支架上。通过将合成催化剂的反应性与蛋白质的选择性和生物相容性相结合，所产生的人造金属酶旨在工程细胞的细胞环境中执行有效的催化反应。为了实现细菌细胞对催化剂的摄取，我们建议利用活跃的细菌铁运输途径。使用参与必需铁运输的细菌蛋白是有利的，因为它们是由细菌细胞产生并分泌到周质中，这是催化发生的合适间隔。在那里，蛋白质准备捕获锚定的催化剂，该催化剂被伪装成含铁的“特洛伊木马”活跃地通过外膜运输。小底物可以通过孔道进入，形成的产物可以释放或进一步反应。因此，这种方法最终可能利用细菌细胞的新陈代谢在活细胞中生产有价值的靶分子。接下来将研究催化、从蛋白质支架中释放基于Ir基的催化剂、它的回收和再利用。

The project aims at the assembly of artificial enzymes that consist of a synthetic catalyst that is attached via an anchor group to a protein scaffold inside living bacterial cells. By combining the reactivity of the synthetic catalyst with the selectivity and biocompatibility of the protein, the resulting artificial metalloenzymes aim to perform efficient catalytic reactions within the cellular environment of engineered cells. To achieve the uptake of the catalysts by bacterial cells we propose to take advantage of active bacterial iron-transport pathways. Using bacterial proteins that are involved in the transport of essential iron is advantageous since it they are produced by the bacterial cell and secreted into the periplasm, a suitable compartment for catalysis to take place. There the proteins are ready for the capture of the anchored catalyst that is actively transported through the outer membrane disguised as an iron-containing 'Trojan horse'. Small substrates can enter through porins and the products formed can either be released or react further. Consequently, this approach could eventually utilise the metabolism of the bacterial cell for the production of valuable target molecules in living cells.Following catalysis, the release of the iridium-based catalyst from the protein scaffold, its recovery and re-use will be investigated.