Groningen Research Institute of Pharmacy (GRIP) Department of Chemical and Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
Chemistry. 2023 Jun 2;29(31):e202300697. doi: 10.1002/chem.202300697. Epub 2023 Apr 12.
Multi-enzymatic cascades exploiting engineered enzymes are a powerful tool for the tailor-made synthesis of complex molecules from simple inexpensive building blocks. In this work, we engineered the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) into an effective aldolase with 160-fold increased activity compared to 4-OT wild type. Subsequently, we applied the evolved 4-OT variant to perform an aldol condensation, followed by an epoxidation reaction catalyzed by a previously engineered 4-OT mutant, in a one-pot two-step cascade for the synthesis of enantioenriched epoxides (up to 98 % ee) from biomass-derived starting materials. For three chosen substrates, the reaction was performed at milligram scale with product yields up to 68 % and remarkably high enantioselectivity. Furthermore, we developed a three-step enzymatic cascade involving an epoxide hydrolase for the production of chiral aromatic 1,2,3-prim,sec,sec-triols with high enantiopurity and good isolated yields. The reported one-pot, three-step cascade, with no intermediate isolation and being completely cofactor-less, provides an attractive route for the synthesis of chiral aromatic triols from biomass-based synthons.
利用工程酶的多酶级联反应是一种从简单廉价的起始原料定制合成复杂分子的有力工具。在这项工作中，我们将广谱酶 4-氧代戊烯二酸互变异构酶（4-OT）工程化为一种有效的醛缩酶，其活性比 4-OT 野生型提高了 160 倍。随后，我们应用进化后的 4-OT 变体进行醛缩合反应，然后由先前工程化的 4-OT 突变体催化环氧化反应，在一锅两步级联反应中，从生物质衍生的起始原料合成对映体富集的环氧化物（高达 98%ee）。对于三个选择的底物，在毫克规模下进行反应，产物收率高达 68%，对映选择性非常高。此外，我们开发了一种三步酶级联反应，涉及环氧化物水解酶，用于生产具有高对映纯度和良好分离收率的手性芳香 1,2,3-原、仲、仲三醇。所报道的一锅三步级联反应无需中间分离且完全无辅因子，为从生物质合成子合成手性芳香三醇提供了一条有吸引力的途径。
