Binzhou Medical University, Yantai, China.
Binzhou Medical University, Yantai, China; Shandong Provincial Key Laboratory of Biomass Gasification, Energy Research Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China.
Enzyme Microb Technol. 2018 Dec;119:52-57. doi: 10.1016/j.enzmictec.2018.09.002. Epub 2018 Sep 5.
A sufficient supply of reducing equivalents is essential for obtaining the maximum yield of target products in anaerobic fermentation. The pyruvate dehydrogenase (PDH) complex controls the critical step in pyruvate conversion to acetyl-CoA and NADH. However, in anaerobic Escherichia coli, PDH residing in the dihydrolipoamide dehydrogenase (LPD) component is normally inactive due to inhibition by NADH. In this study, the protein engineering of LPD by structural analysis was explored to eliminate this inhibition. A novel IAA350/351/358VVV triple mutant was successfully verified to be more effective than other LPD mutants reported till date. Notably, PDH activity with the triple mutant at an [NADH]/[NAD] ratio of 0.15 was still higher than that of the wild-type without NADH addition. The altered enzyme of the PDH complex was also active in the presence of such high NADH levels. This is the first study concerning protein engineering of PDH by structure-guided design. The presence and functional activity of such an NADH-insensitive PDH complex provides a useful metabolic element for fermentation products and has potential for biotechnological application.
在厌氧发酵中，获得目标产物的最大产量，需要有足够的还原当量供应。丙酮酸脱氢酶（PDH）复合物控制着丙酮酸转化为乙酰辅酶 A 和 NADH 的关键步骤。然而，在厌氧大肠杆菌中，由于 NADH 的抑制作用，位于二氢硫辛酸脱氢酶（LPD）成分中的 PDH 通常是无活性的。在这项研究中，通过结构分析对 LPD 进行了蛋白质工程改造，以消除这种抑制。一种新型的 IAA350/351/358VVV 三突变体被成功验证，比迄今为止报道的其他 LPD 突变体更有效。值得注意的是，在 [NADH]/[NAD] 比值为 0.15 的情况下，该三重突变体的 PDH 活性仍高于不添加 NADH 的野生型。在如此高的 NADH 水平下，PDH 复合物的改变酶也具有活性。这是首次通过结构导向设计对 PDH 进行蛋白质工程研究。这种存在且具有功能活性的 NADH 不敏感 PDH 复合物为发酵产物提供了有用的代谢元件，具有生物技术应用的潜力。
