1,4-环己烷二甲胺(1,4-BAC)是生物基材料的重要单体,它在包括有机合成在内的各个领域得到了广泛的应用,医学,化学工业,和材料。目前,它的合成主要依靠化学方法,受到诸如昂贵的金属催化剂等问题的困扰,苛刻的反应条件,和安全风险。因此,有必要为其合成探索更绿色的替代品。在这项研究中,成功开发了一种双细菌三酶级联转化途径,将1,4-环己烷二甲醛转化为1,4-环己烷二甲胺。该途径使用大肠杆菌衍生的氨基转移酶(EcTA),酿酒酵母谷氨酸脱氢酶(ScGlu-DH),和博伊丁念珠菌衍生的甲酸脱氢酶(CbFDH)。通过结构引导的蛋白质工程,一个有益的突变体,EcTAF91Y,获得了,与野生型相比,比活性增加2.2倍,kcat/Km增加1.9倍。通过构建重组菌株和优化反应条件,发现在最优条件下,底物浓度为40g/L可产生(27.4±0.9)g/L的产物,对应于67.5%±2.1%的摩尔转化率。
1,4-cyclohexanedimethylamine (1,4-BAC) is an important monomer for bio-based materials, it finds wide applications in various fields including organic synthesis, medicine, chemical industry, and materials. At present, its synthesis primarily relies on chemical method, which suffer from issues such as expensive metal catalyst, harsh reaction conditions, and safety risks. Therefore, it is necessary to explore greener alternatives for its synthesis. In this study, a two-bacterium three-enzyme cascade conversion pathway was successfully developed to convert 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine. This pathway used Escherichia coli derived aminotransferase (EcTA), Saccharomyces cerevisiae derived glutamate dehydrogenase (ScGlu-DH), and Candida boidinii derived formate dehydrogenase (CbFDH). Through structure-guided protein engineering, a beneficial mutant, EcTAF91Y, was obtained, exhibiting a 2.2-fold increase in specific activity and a 1.9-fold increase in kcat/Km compared to that of the wild type. By constructing recombinant strains and optimizing reaction conditions, it was found that under the optimal conditions, a substrate concentration of 40 g/L could produce (27.4±0.9) g/L of the product, corresponding to a molar conversion rate of 67.5%±2.1%.