An enzyme module system for the synthesis of dTDP-activated deoxysugars from dTMP and sucrose

A flexible enzyme module system is presented that allows preporative access to important dTDP-activated deoxyhexoses from dTMP and sucrose. The strategic combination of the recombinant enzymes dTMP-kinase and sucrose synthase (SuSy), and the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmID (4-ketoreductose) from the biosynthetic pathway of dTDP-beta-(L)-rhamnose was optimized. The SuSy module (dTMP-kinase, SuSy, +/- RmIB) yielded the precursor dTDP-alpha-(D)-glucose (2) or the biosynthetic intermediate dTDP-6-deoxy-4-keto-alpha-(D)-glucose (3) on a 0.2-0.6 g scale with overall yields of 62 % and 72 % respectively. A two-step strategy in which the SuSy module was followed by the deoxysugar module (RmIC and RmID) resulted in the synthesis of dTDP-beta-(L)-rhomnose (4; 24.1 mu mol, overall yield: 35.9%). Substitution of RmIC by DnmU from the dTDP-beta-(L)-daunosamine pathway of Streptomyces peucetius in this module demonstrated that DnmU acts in vitro as a 3,5-epimerose with 3 as substrate to yield 4 (32.2 mu mol, overall yield: 44.7%). Chemical reduction of 3 with NaBH4 gave a mixture of the C-4 epimers dTDP-a-D-quinovose (6) and MP-a-D-fucose (7) in a ratio of 2:1. In summary, the modular character of the presented enzyme system provides valuable compounds for the biochemical characterization of deoxysugar pathways playing a major role in microbial producers of antibiotic and antitumour agents.