Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
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DOI：
10.1128/mbio.00190-10
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发表时间：
2010-11-01
期刊：
影响因子：
6.4
通讯作者：
Gralnick, Jeffrey A.
中科院分区：
文献类型：
--
作者：
Flynn, Jeffrey M.;Ross, Daniel E.;Gralnick, Jeffrey A.
细胞新陈代谢是一系列紧密相连的氧化和还原，必须达到平衡。发酵过程中细胞内电子载体的循环通常需要底物转化为不需要的产物，而呼吸作用则需要不断增加电子受体。使用基于电极的电子受体来平衡生物转化可能会克服这些限制。为了验证这一假设，对金属还原细菌Shewanella onedensis进行了改造，使其按化学计量比将甘油转化为乙醇，这种生物转化不会发生，除非通过外部反应(如电极还原)移除两个电子。将多个模块组合成一个改变单胞菌代谢的质粒：一个甘油模块，由来自大肠杆菌的glpF、glpK、glpD和tpiA组成；一个乙醇模块，包含来自运动发酵单胞菌的pDC和adh。产品产量的进一步增加是通过敲除编码磷酸乙酰转移酶的PTA，将通量转移到乙醇而不是生产醋酸酯来实现的。在这第一代演示中，甘油转化为乙醇需要存在电极来平衡反应，而电极连接的速率与在工程大肠杆菌中观察到的体积转化率相同。将微生物生物催化与当前的生产联系起来，可以通过转移其他不平衡的反应来产生纯产品，从而消除氧化还原限制，并为下一代生物生产策略提供新的平台。重要的是，所有由全细胞或酶催化的反应都必须达到氧化还原平衡。在极少数情况下，可以通过完全平衡的发酵实现转化，允许在单个产品中回收所有电子等价物。在大多数生物转化中，有机体必须产生酸、气体和/或酒精的混合物，再多的酶或菌株工程也无法克服这一基本要求。由于甘油的还原状态比乙醇和二氧化碳更低，生物柴油酯交换过程中产生的甘油按化学计量转化为乙醇和二氧化碳，没有副产品，这是一种不可能的发酵。甘油到乙醇的不平衡转化一直被认为只有两种解决方案：将甘油发酵成乙醇和潜在有用的副产品，或者通过小心地引入氧气来“燃烧”多余的电子。在这里，我们使用甘油到乙醇的例子来演示第三种策略，使用直接连接到电极的细菌。
Cellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. The use of electrode-based electron acceptors to balance biotransformations may overcome these constraints. To test this hypothesis, the metal-reducing bacterium Shewanella oneidensis was engineered to stoichiometrically convert glycerol into ethanol, a biotransformation that will not occur unless two electrons are removed via an external reaction, such as electrode reduction. Multiple modules were combined into a single plasmid to alter S. oneidensis metabolism: a glycerol module, consisting of glpF, glpK, glpD, and tpiA from Escherichia coli, and an ethanol module containing pdc and adh from Zymomonas mobilis. A further increase in product yields was accomplished through knockout of pta, encoding phosphate acetyltransferase, shifting flux toward ethanol and away from acetate production. In this first-generation demonstration, conversion of glycerol to ethanol required the presence of an electrode to balance the reaction, and electrode-linked rates were on par with volumetric conversion rates observed in engineered E. coli. Linking microbial biocatalysis to current production can eliminate redox constraints by shifting other unbalanced reactions to yield pure products and serve as a new platform for next-generation bioproduction strategies.IMPORTANCE All reactions catalyzed by whole cells or enzymes must achieve redox balance. In rare cases, conversion can be achieved via perfectly balanced fermentations, allowing all electron equivalents to be recovered in a single product. In most biotransformations, organisms must produce a mixture of acids, gasses, and/or alcohols, and no amount of enzyme or strain engineering can overcome this fundamental requirement. Stoichiometric conversion of glycerol, a waste product from biodiesel transesterification, into ethanol and CO2 with no side products represents such an impossible fermentation, due to the more reduced state of glycerol than of ethanol and CO2. The unbalanced conversion of glycerol to ethanol has been viewed as having only two solutions: fermenting glycerol to ethanol and potentially useful coproducts or "burning off" excess electrons via careful introduction of oxygen. Here, we use the glycerol-to-ethanol example to demonstrate a third strategy, using bacteria directly interfaced to electrodes.