Cheng Yaqi, Song Wei, Chen Xiulai, Gao Cong, Liu Jia, Guo Liang, Zhu Meng, Liu Liming, Wu Jing
School of Life Sciences and Health Engineering, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Bioresour Bioprocess. 2022 Aug 13;9(1):80. doi: 10.1186/s40643-022-00570-y.
In this study, we selected and engineered a flavin adenine dinucleotide (FAD)-dependent alcohol oxidase (AOX) to produce 1,4-cyclohexanedicarboxaldehyde (CHDA), an initial raw material for spiral compounds, from 1,4-cyclohexanedimethanol (CHDM). First, the structure of alcohol oxidase from Arthrobacter cholorphenolicus (AcCO) was analyzed, and the mechanism of AcCO-catalyzed primary alcohol oxidation was elucidated, demonstrating that the energy barrier of the hydride (H) transfer (13.4 kcal·mol and 20.4 kcal·mol) decreases the catalytic efficiency of the primary alcohol oxidation reaction. Therefore, we designed a protein engineering strategy to adjust the catalytically active conformation to shorten the distance of hydride (H) transfer and further decreased the core energy barrier. Following this strategy, variant W4 (S101A/H351V/N378S/Q329N) was obtained with 112.5-fold increased catalytic efficiency to produce CHDA compared to that of the wild-type strain. The 3 L scale preparation of CHDA reached a titer up to 29.6 g·L with a 42.2% yield by an Escherichia coli whole-cell catalyst, which demonstrates the potential of this system for industrial application.
在本研究中，我们筛选并改造了一种黄素腺嘌呤二核苷酸（FAD）依赖性醇氧化酶（AOX），以从1,4 - 环己烷二甲醇（CHDM）生产1,4 - 环己烷二甲醛（CHDA），后者是螺旋化合物的起始原料。首先，分析了嗜氯节杆菌醇氧化酶（AcCO）的结构，阐明了AcCO催化伯醇氧化的机制，结果表明氢化物（H）转移的能垒（13.4 kcal·mol和20.4 kcal·mol）降低了伯醇氧化反应的催化效率。因此，我们设计了一种蛋白质工程策略来调整催化活性构象，以缩短氢化物（H）转移的距离，并进一步降低核心能垒。按照该策略，获得了变体W4（S101A/H351V/N378S/Q329N），与野生型菌株相比，其生产CHDA的催化效率提高了112.5倍。利用大肠杆菌全细胞催化剂进行3 L规模的CHDA制备，效价高达29.6 g·L，产率为42.2%，这证明了该系统在工业应用中的潜力。
