Improved Whole-Cell Biocatalyst for the Synthesis of Vitamin E Precursor 2,3,5-Trimethylhydroquinone
Improved Whole-Cell Biocatalyst for the Synthesis of Vitamin E Precursor 2,3,5-Trimethylhydroquinone
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改进的全细胞生物催化剂用于合成维生素 E 前体 2,3,5-三甲基氢醌
DOI：
10.1021/acs.jafc.2c07768
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发表时间：
2023-01-09
影响因子：
6.1
通讯作者：
Yan，Xin
中科院分区：
文献类型：
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作者：
Ji，Junbin;Zeng，Caiting;Yan，Xin
2，3，5-三甲基对苯二酚(2，3，5-TMHQ)是合成维生素E的关键前体，如何在温和的反应条件下用化学法合成2，3，5-TMHQ仍是一大挑战。单加氧酶系统MpdAB能特异性催化2，3，6-三甲基苯酚(2，3，6-TMP)转化为2，3，5-TMHQ。然而，野生型MpdA催化能力较弱，底物的细胞毒性限制了2，3，5-TMHQ的生产效率。在此，通过同源模建和饱和突变来提高mpdA的催化活性。获得了两个对2，3，6-TMP具有较高活性的突变体L128A和L128K(1.86-1.87倍)。另一方面，通过实验室适应性进化获得了一株对2，3，6-TMP的抗性增强的进化菌株B5-4M(对1000μM2，3，6-TMP的抗性提高了8.15倍)。随后，携带L128K(或L128A)和MpDB的进化菌株B5-4M与野生型(表达MpdAB的菌株B5-4M)相比，2，3，5-TMHQ的产量提高了5.29倍(或4.87倍)。本研究为生产2，3，5-TMHQ提供了较好的遗传资源，并证明了通过酶修饰和实验室适应性进化可以提高2，3，5-TMHQ的合成效率。
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.