Improved Whole-Cell Biocatalyst for the Synthesis of Vitamin E Precursor 2,3,5-Trimethylhydroquinone

• Published in: Journal of agricultural and food chemistry Vol. 71; no. 2; pp. 1162 - 1169
• Main Authors: Ji, Junbin, Zeng, Caiting, Wu, Panpan, Wang, Yuying, Chen, Xueting, Yan, Xin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.01.2023
• Subjects: Biotechnology and Biological Transformations; Diazonium Compounds; Pyridines; Vitamin E; site-directed mutagenesis; whole-cell catalyst; 2,3,5-trimethylhydroquinone; adaptive laboratory evolution
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.2c07768

2,3,5-Trimethylhydroquinone (2,3,5-TMHQ) is the key precursor in the synthesis of vitamin E. It is still a major challenge to produce 2,3,5-TMHQ under mild reaction conditions by chemical methods. The monooxygenase system MpdAB can specifically catalyze the conversion of 2,3,6-trimethylphenol (2,3,6-TMP) to 2,3,5-TMHQ. However, the weak catalytic capacity of wild-type MpdA and the cytotoxicity of the substrate limited the production efficiency of 2,3,5-TMHQ. Here, homologous modeling and saturation mutation were performed to increase the catalytic activity of MpdA. Two variants, L128A and L128K, with higher activity toward 2,3,6-TMP (1.86–1.87-fold) were obtained. On the other hand, an evolved strain B5-4M-evolved with enhanced resistance to 2,3,6-TMP (8.15-fold higher for 1000 μM 2,3,6-TMP) was obtained through adaptive laboratory evolution. Subsequently, a 5.29-fold (or 4.87-fold) improvement in 2,3,5-TMHQ production was achieved by a strain B5-4M-evolved harboring L128K (or L128A) and MpdB, in comparison with that of the wild type (strain B5-4M expressing MpdAB). This study provides better genetic resources for producing 2,3,5-TMHQ and proves that the synthesis efficiency of 2,3,5-TMHQ can be improved through enzyme modification and adaptive laboratory evolution.