Direct Stimulation of Bacterial Metabolism with Applied Electrical Current

描述（由申请人提供）：活细胞通过在催化剂产物和合适的电子受体之间转移电子来获得生长和存活所需的能量。最有利的末端电子受体是氧，但在缺氧环境中，大多数细胞使用其他可溶性因子来接受代谢产生的电子。希瓦氏菌属中的细菌实际上可以使用固体胞外金属作为呼吸的底物，并且它们已经进化出一系列周质和外膜蛋白来作为与这些金属的直接电连接。希旺氏菌的几个成员作为机会致病菌具有临床相关性，因此其独特的呼吸能力的基础值得进一步研究。此外，在制造的电路和活细胞的中心代谢之间存在遗传编码的途径，为研究和工程化微生物开辟了新的前景。最近的结果表明，这些管道也可以反向传递电流，允许细胞外电子进入细胞并驱动合成过程。如果与碳固定相结合，施加的电流可以为高价值化学品的生产提供多方面的投入，包括药品。该提案概述了表征和提高希瓦氏菌细胞接受电流的能力的研究，使用电活性生物反应器结合表达荧光蛋白传感器的细胞显微镜，转录组测序和反向遗传学测定。最后，对E.将构建表达希瓦氏菌基因的大肠杆菌，以确定赋予电子摄取的有效遗传电路。

DESCRIPTION (provided by applicant): Living cells gain energy for growth and survival by transferring electrons between the products of catabolism and a suitable electron acceptor. The most favorable terminal electron acceptor is oxygen, but in oxygen-poor environments most cells use other soluble factors to accept the electrons generated by metabolism. Bacteria in the genus Shewanella can actually use solid extracellular metals as substrates for respiration, and they have evolved a series of periplasmic and outer-membrane proteins to serve as a direct electrical junction with these metals. Several members of the shewanellae have clinical relevance as opportunistic pathogens, and the basis of their unique respiratory abilities thus warrants further investigation. Furthermore, the existence of a genetically-encoded pathway between manufactured circuits and the central metabolism of living cells opens new vistas in studying and engineering microorganisms. Recent results suggest that these conduits can also pass current in reverse, allowing extracellular electrons to enter the cell and drive synthetic processes. If combined with carbon fixation, applied current could make a versatile input for the production of high-value chemicals, including pharmaceuticals. This proposal outlines research to characterize and improve the capacity of Shewanella cells to accept electrical current, using electrically active bioreactors in combination with microscopy of cells expressing fluorescent protein sensors, transcriptome sequencing, and reverse genetics assays. Finally, a strain of E. coli expressing genes from Shewanella will be constructed in order to define an efficient genetic circuit conferring electron uptake.