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Deoxythymidine diphospho- l -rhamnose (dTDP- l -rhamnose) is used by prokaryotic rhamnosyltransferases as the glycosyl donor for the synthesis of rhamnose-containing polysaccharides and compounds that have potential in pharmaceutical development, so its efficient synthesis has attracted much attention. In this study, we successfully cloned four putative dTDP- l -rhamnose synthesis genes  Ss-rmlABCD  from  Saccharothrix syringae  CGMCC 4.1716 and expressed them in  Escherichia coli . The recombinant enzymes, Ss-RmlA (glucose-1-phosphate thymidylyltransferase), Ss-RmlB (dTDP- d -glucose 4,6-dehydratase), Ss-RmlC (dTDP-4-keto-6-deoxy-glucose 3,5-epimerase), and Ss-RmlD (dTDP-4-keto-rhamnose reductase), were confirmed to catalyze the sequential formation of dTDP- l -rhamnose from deoxythymidine triphosphate (dTTP) and glucose-1-phosphate (Glc-1-P). Ss-RmlA showed maximal enzyme activity at 37°C and pH 9.0 with 2.5mMMg 2+ , and the  K  m  and  k  cat  values for dTTP and Glc-1-P were 49.56μM and 5.39s −1 , and 117.30μM and 3.46s −1 , respectively. Ss-RmlA was promiscuous in the substrate choice and it could use three nucleoside triphosphates (dTTP, dUTP, and UTP) and three sugar-1-Ps (Glc-1-P, GlcNH 2 -1-P, and GlcN 3 -1-P) to form nine sugar nucleotides (dTDP-GlcNH 2 , dTDP-GlcN 3 , UDP-Glc, UDP-GlcNH 2 , UDP-GlcN 3 , dUDP-Glc, dUDP-GlcNH 2 , and dUDP-GlcN 3 ). Ss-RmlB showed maximal enzyme activity at 50°C and pH 7.5 with 0.02mM NAD + , and the  K  m  and  k  cat  values for dTDP-glucose were 98.60μM and 11.2s −1 , respectively. A one-pot four-enzyme reaction system was developed by simultaneously mixing all of the substrates, reagents, and four enzymes Ss-RmlABCD in one pot for the synthesis of dTDP- l -rhamnose and dUDP- l -rhamnose with the maximal yield of 65% and 46%, respectively, under the optimal conditions. dUDP- l -rhamnose was a novel nucleotide-activated rhamnose reported for the first time.

Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose