Tefft Nicholas M, TerAvest Michaela A
Department of Biochemistry and Molecular Biology , Michigan State University , East Lansing , Michigan 48824 , United States.
ACS Synth Biol. 2019 Jul 19;8(7):1590-1600. doi: 10.1021/acssynbio.8b00498. Epub 2019 Jun 21.
Microbial electrosynthesis is an emerging technology with the potential to simultaneously store renewably generated energy, fix carbon dioxide, and produce high-value organic compounds. However, limited understanding of the route of electrons into the cell remains an obstacle to developing a robust microbial electrosynthesis platform. To address this challenge, we leveraged the native extracellular electron transfer pathway in MR-1 to connect an extracellular electrode with an intracellular reduction reaction. The system uses native Mtr proteins to transfer electrons from an electrode to the inner membrane quinone pool. Subsequently, electrons are transferred from quinones to NAD by native NADH dehydrogenases. This reverse functioning of NADH dehydrogenases is thermodynamically unfavorable; therefore, we added a light-driven proton pump (proteorhodopsin) to generate proton-motive force to drive this activity. Finally, we use reduction of acetoin to 2,3-butanediol via a heterologous butanediol dehydrogenase (Bdh) as an electron sink. Bdh is an NADH-dependent enzyme; therefore, observation of acetoin reduction supports our hypothesis that cathodic electrons are transferred to intracellular NAD. Multiple lines of evidence indicate proper functioning of the engineered electrosynthesis system: electron flux from the cathode is influenced by both light and acetoin availability, and 2,3-butanediol production is highest when both light and a poised electrode are present. Using a hydrogenase-deficient background strain resulted in a stronger correlation between electron transfer and 2,3-butanediol production, suggesting that hydrogen production is an off-target electron sink in the wild-type background. This system represents a promising step toward a genetically engineered microbial electrosynthesis platform and will enable a new focus on synthesis of specific compounds using electrical energy.
微生物电合成是一种新兴技术，具有同时储存可再生能源、固定二氧化碳和生产高价值有机化合物的潜力。然而，对电子进入细胞途径的了解有限，仍然是开发强大的微生物电合成平台的一个障碍。为了应对这一挑战，我们利用MR-1中天然的细胞外电子转移途径，将细胞外电极与细胞内还原反应连接起来。该系统利用天然的Mtr蛋白将电子从电极转移到内膜醌池。随后，电子通过天然的NADH脱氢酶从醌转移到NAD。NADH脱氢酶的这种反向功能在热力学上是不利的；因此，我们添加了一种光驱动质子泵（视紫红质）来产生质子动力以驱动这种活性。最后，我们利用异源丁二醇脱氢酶（Bdh）将乙偶姻还原为2,3-丁二醇作为电子受体。Bdh是一种依赖NADH的酶；因此，观察到乙偶姻的还原支持了我们的假设，即阴极电子被转移到细胞内的NAD。多条证据表明工程化电合成系统功能正常：来自阴极的电子通量受光和乙偶姻可用性的影响，并且当光和平衡电极都存在时，2,3-丁二醇的产量最高。使用缺乏氢化酶的背景菌株导致电子转移与2,3-丁二醇产量之间的相关性更强，这表明产氢是野生型背景中一个非目标电子受体。该系统代表了朝着基因工程微生物电合成平台迈出的有希望的一步，并将使人们能够重新聚焦于利用电能合成特定化合物。
