Efficient synthesis 1,4-cyclohexanedicarboxaldehyde by an engineered alcohol oxidase.

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.