A novel in vitro multi-enzymatic system was constructed for the conversion of CO₂ into optically pure N-carbamoyl-L-aspartate and orotate. Four cascade enzymes, carbamoyl phosphate synthase (CPS), aspartate carbamoyl-transferase (ATCase), dihydroorotase (DHOase), and dihydroorotate dehydrogenase (DHOD), were used for the biotransformation of feedstocks into products in one pot while polyphosphate kinase (PPK) was coupled for ATP regeneration. Under the optimal reaction conditions and substrate concentrations, 19.2 mM N-carbamoyl-L-aspartate was produced within 3.0 h, which enabled the synthesis of 15.5 mM orotate. The yields of N-carbamoyl-L-aspartate and orotate based on the added L-aspartic acid reached 96.0% and 93.3%, respectively. This work highlights the ease of producing optically pure N-carbamoyl-L-aspartate and orotate via an in vitro biomanufacturing platform and provides a novel pathway for the utilization of renewable feedstock for the synthesis of value-added commodity chemicals.

中文翻译：

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通过体外多酶级联将二氧化碳转化为光学纯的N-氨基甲酰基-L-天冬氨酸和乳清酸酯

构建了一种新颖的体外多酶系统，用于将CO ₂转化为光学纯的N-氨基甲酰基-L-天冬氨酸和乳清酸酯。一锅中使用四种级联酶将氨基甲酸酯磷酸合酶（CPS），天冬氨酸氨基甲酰转移酶（ATCase），二氢乳清酶（DHOase）和二氢乳清酸脱氢酶（DHOD）用于将原料生物转化为产品，而聚磷酸激酶（PPK）耦合用于ATP再生。在最佳反应条件和底物浓度下，在3.0小时内生成了19.2 mM N-氨基甲酰基-L-天门冬氨酸，这使得能够合成15.5 mM乳清酸酯。N-氨基甲酰基-L的产量-基于添加的L-天冬氨酸的-天冬氨酸和乳清酸酯分别达到96.0％和93.3％。这项工作突出了易于制备光学纯的Ñ氨基甲酰基大号天冬氨酸和乳清酸通过一个体外生物制造平台，并提供了可再生原料的利用率为增值通用化学品合成中的新途径。