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

在这个合成生物学时代，有可能重新设计和构建新的生物系统，以实现有用的新功能，但人们往往忽视了这种方法本身就有缺陷，因为有毒物质的积累。在将细胞用作工厂时，原料的采购和中间体/产品的积累往往会证明对细菌有害。这就是通过工程将钴胺(维生素B12)转化为大肠杆菌的方法。在这里，是钴的供应，这是维生素的金属中心，细菌很难处理。由于细胞没有特定的钴输入系统，必须将高水平的外源金属添加到生长介质中，以便将金属运输到维生素合成部位，并允许生产营养物质。然而，高水平的钴会影响细胞的活力，因为钴会干扰氧化还原蛋白中铁-S中心的形成。为了克服这个金属毒性问题，我们概述了一个研究计划，以增强钴的摄取，增加其内部细胞浓度，并调节其输出。这将通过开发系统来实现，以允许更好地吸收金属，增加内部结合能力，并加强将金属输送到代谢途径中需要的地方。我们将通过在单元内使用内部记者来监测这些程序的有效性，这些记者将提供钴水平的读数以及如何在内部使用钴。这项研究还将允许对一些金属运输和结合蛋白进行表征。这些分析的结果将告知如何进一步设计细胞，以便在较低的金属浓度下增强钴胺的合成。根据这些信息，我们将开发一种独立的、完全布线的金属电路，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.