Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
J Biol Eng. 2010 Feb 25;4:3. doi: 10.1186/1754-1611-4-3.
The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued.
Here we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds.
Through the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.
代谢工程在生产理想代谢物方面具有巨大的潜力，特别是替代燃料和其他高度还原的分子。工程方法必须重新引导化学还原当量的转移，防止这些电子流失到一般的细胞代谢中。对于储存在蛋白质中铁硫簇中的高能电子来说尤其如此，当两个这样的簇被紧密接近时，这些电子很容易被转移。因此，铁硫蛋白需要确保正确的伙伴之间相互作用的机制，类似于许多信号转导蛋白。虽然近年来在分离工程代谢途径方面取得了进展，但在体内设计隔离电子代谢电路的工作尚未开展。
在这里，我们展示了一种在大肠杆菌中合成的产氢电子转移电路可以通过多种方法与现有的细胞代谢隔离，在许多情况下可以改善该途径的功能。我们的电路由异源表达的[Fe-Fe]-氢化酶、铁氧还蛋白和丙酮酸-铁氧还蛋白氧化还原酶（PFOR）组成，允许氢气的产生与葡萄糖的分解偶联。我们表明，通过删除竞争反应、合理设计蛋白质相互作用表面、直接融合相互作用的伴侣以及将途径组件共定位在异源蛋白支架上，可以隔离这种合成途径。
通过在体内构建和表征一种合成代谢电路，我们展示了一种新的系统，允许对隔离的电子转移途径进行可预测的工程设计。该系统的开发为替代能源生产中工程途径的优化以及理解蛋白质之间的电子转移如何被控制提供了工作原理。
