Efficient Biosynthesis of High-Value Succinic Acid and 5-Hydroxyleucine Using a Multienzyme Cascade and Whole-Cell Catalysis.

Succinic acid (SA) is applied in the food, chemical, and pharmaceutical industries. 5-Hydroxyleucine (5-HLeu) is a promising precursor for the biosynthesis of antituberculosis drugs. Here, we designed a promising synthetic route for the simultaneous production of SA and 5-HLeu by combining l-leucine dioxygenase (NpLDO), l-glutamate oxidase (LGOX), and catalase (CAT). Two bioconversion systems: "a multienzyme cascade catalysis in vitro" (MECCS) and "whole-cell catalysis system" (WCCS) were constructed. A high-activity NpLDO mutant was screened by error-prone polymerase chain reaction (PCR) and showed 6.1-fold improvement of catalytic activity. After optimization of reaction conditions, MECSS yielded 3.15 g/L SA and 3.92 g/L 5-HLeu, while the production of SA and 5-HLeu by the most effective WCSS reached 15.12 and 18.83 g/L, respectively. This is the first attempt to use ferrous iron/α-ketoglutarate-dependent dioxygenases for the simultaneous production of SA and hydroxy-amino-acid. This research provides a tool for industrial production of food of high-value products from low-cost raw materials.