Cell circuitry for metals: Integrative metabolism for cobalt uptake and cobalamin production

在这个合成生物学的时代，有可能重新设计和构建新的生物系统来执行有用的新功能，但由于有毒物质的积累，这种方法本身就存在缺陷，这一点经常被忽视。在使用细胞作为工厂时，原料的采购和中间体/产物的积累往往对细菌有害。这就是将钴胺素（维生素B12）途径引入大肠杆菌的情况。在这里，它提供了钴，维生素的金属中心，这是细菌难以应对的。由于细胞没有特定的钴输入系统，因此必须向生长培养基中添加高外源水平的金属，以允许金属运输到维生素合成部位并允许营养物质的生产。然而，高水平的钴会干扰氧化还原蛋白中Fe-S中心的形成，从而损害细胞的生存能力。为了克服这种金属毒性问题，我们概述了一项研究计划，以提高钴的吸收，增加其内部细胞浓度，并调节其出口。这将通过开发系统来实现，以改善金属的吸收，增加内部结合能力，并增强金属在代谢途径中被输送到需要的地方。我们将通过在电解槽内使用内部报告器来监测这些程序的有效性，这些报告器将提供钴水平及其内部使用情况的读数。这项研究还将使许多金属运输和结合蛋白的特征化成为可能。这些分析的结果将告诉我们如何进一步改造细胞，以便在较低的金属浓度下增强钴胺素的合成。根据这些信息，我们将开发一个独立的、完全有线的金属电路，它将能够控制和调节细胞钴的供应。

In this era of synthetic biology, where it is possible to redesign and construct novel biological systems to perform new functions for useful purposes, it is often overlooked that such approaches are inherently flawed because of the accumulation of toxic materials. In using cells as factories the procurement of starting materials and the accumulation of intermediates/products can often prove detrimental to the bacterium. Such is the case with engineering the cobalamin (vitamin B12) pathway into E. coli. Here, it is the provision of cobalt, the metal centre of the vitamin, which is difficult for the bacterium to cope with. As the cell does not have a specific cobalt import system, high exogenous levels of the metal have to be added to the growth medium to permit transport of the metal to the site of vitamin synthesis and to allow production of the nutrient. However, the high level of cobalt compromises the viability of the cell as the cobalt interferes with Fe-S centre formation in redox proteins.To overcome this metal toxicity issue we have outlined a research plan to enhance cobalt uptake, increase its internal cellular concentration, and modulate its export. This will be accomplished by developing systems to allow improved uptake of the metal, increased internal binding capacity and enhanced delivery of the metal to where it is required within the metabolic pathway. We will monitor the effectiveness of these procedures by using internal reporters within the cell that will provide readouts on the level of cobalt and how it is being used internally. The research will also allow the characterisation of a number of metal transport and binding proteins. The results of these analyses will inform on how the cell can be engineered further so that cobalamin synthesis can be enhanced at lower metal concentrations. From this information we will develop a self contained, fully wired, metal circuit that will be able to control and regulate cellular cobalt supply.