Crotonase Catalysis Enables Flexible Production of Functionalized Prolines and Carbapenams

The biocatalytic versatility of wildtype and engineered carboxymethylproline synthases (CMPSs) is demonstrated by the preparation of functionalized 5-carboxymethylproline derivatives methylated at C-2, C-3, C-4, or C-5 of the proline ring from appropriately substituted amino acid aldehydes and malonyl-coenzyme A. Notably, compounds with a quaternary center (at C-2 or C-5) were prepared in a stereoselective fashion by engineered CMPSs. The substituted-5-carboxymethyl-prolines were converted into the corresponding bicyclic β-lactams using a carbapenam synthetase. The results demonstrate the utility of the crotonase superfamily enzymes for stereoselective biocatalysis, the amenability of carbapenem biosynthesis pathways to engineering for the production of new bicyclic β-lactam derivatives, and the potential of engineered biocatalysts for the production of quaternary centers.

本研究以适当取代的氨基酸醛与丙二酰辅酶A（malonyl-coenzyme A）为底物，成功制备了脯氨酸环C-2、C-3、C-4或C-5位甲基化修饰的功能化5-羧甲基脯氨酸衍生物，由此证明了野生型及工程化羧甲基脯氨酸合酶（carboxymethylproline synthases, CMPSs）的生物催化通用性。值得注意的是，工程化CMPSs可通过立体选择性（stereoselective）方式，合成在C-2或C-5位带有季碳中心（quaternary center）的目标化合物。将取代后的5-羧甲基脯氨酸经碳青霉烷合酶（carbapenam synthetase）催化，可转化为对应的双环β-内酰胺类化合物（bicyclic β-lactams）。本研究结果证实了巴豆酸酶超家族（crotonase superfamily）酶在立体选择性生物催化中的应用价值，证明碳青霉烯生物合成途径（carbapenem biosynthesis pathways）可经工程化改造用于制备新型双环β-内酰胺衍生物，同时也展现了工程化生物催化剂在合成季碳中心方面的应用潜力。