Project Grant R44GM145102

PRODUCTION OF NATURAL DEOXYSUGARS FOR USE IN CHEMICAL SYNTHESIS OF GLYCOSIDES - PROJECT SUMMARY THE GOAL OF THE PROPOSAL PRESENTED HEREIN IS TO PRODUCE NATURALLY OCCURRING 6-DEOXYSUGARS, INCLUDING DI- AND TRI-DEOXYSUGARS, AMINO SUGARS AND BRANCHED-CHAIN SUGARS. THESE SPECIALIZED HEXOSES ARE FOUND AS IMPORTANT STRUCTURAL COMPONENTS THROUGHOUT PLANT AND MICROBIAL SECONDARY METABOLITES, OFTEN PLAYING A CRUCIAL ROLE IN CONFERRING ACTIVITY IN BIOACTIVE NATURAL PRODUCTS, SUCH AS ANTIBIOTICS AND ANTICANCER THERAPEUTICS. THEY ARE OF INCREASING INTEREST IN GLYCOENGINEERING EFFORTS, WHICH AIM TO ALTER SUGAR SUBSTITUENTS FOUND ON GLYCOSYLATED NATURAL PRODUCTS OR APPEND SUGAR MOIETIES TO NON-GLYCOSYLATED NATURAL PRODUCTS. IN NATURE, 6-DEOXYSUGARS ARE FREQUENTLY BUILT UP AND MODIFIED AS PART OF ACTIVATED DTDP-NUCLEOSIDE SUGARS INSTEAD OF INDEPENDENT MOLECULES. WE PROPOSE TO INVESTIGATE A NOVEL APPROACH TO PRODUCE THESE RARE DEOXYSUGARS USING A PROPRIETARY E. COLI SYSTEM WE FIRST DEVELOPED TO PRODUCE ACTIVATED TDP-DEOXYSUGARS. TO DO THIS, THERE WERE SEVERAL CHALLENGES TO ADDRESS IN THE PHASE I FEASIBILITY STUDY. FIRST, TITERS OF THE TDP- DEOXYSUGARS NEEDED TO BE INCREASED, PREFERENTIALLY ACCUMULATING MORE MATERIAL IN THE GROWTH MEDIUM. SECOND, AN EFFECTIVE WAY TO SEPARATE AND PURIFY THE MOLECULES FROM THE GROWTH MEDIUM NEEDED TO BE DEVELOPED. FINALLY, THE DEOXYSUGARS NEEDED TO BE HYDROLYZED AND ISOLATED. IN PHASE I WE DEMONSTRATED THE FEASIBILITY OF DEOXYSUGAR PRODUCTION USING D-FUCOSE DERIVED FROM DTDP- D-FUCOSE AS A MODEL. THIS WAS ACCOMPLISHED BY 1) INCREASING PRODUCTION OF DTDP-D-FUCOSE BY ENHANCING EXPRESSION AND SOLUBILITY OF FCF1, THE ENZYME USED TO MAKE DTDP-D-FUCOSE FROM TKDG, 2) DETERMINING A MEDIUM CAPABLE OF PRODUCING LARGER QUANTITIES OF DTDP-D-FUCOSE IN THE MEDIUM, 3) DEVELOPING A DTDP-D-FUCOSE PURIFICATION PROTOCOL FROM THE MEDIUM, 4) DEVELOPING A METHOD FOR HYDROLYSIS OF DTDP-D-FUCOSE AND THE SUBSEQUENT PURIFICATION OF D-FUCOSE. IN PHASE II, WE WILL EXPAND THE PROCESS TO MANUFACTURE 18 ADDITIONAL DEOXYSUGARS FROM DTDP- DEOXYSUGARS. FURTHER OPTIMIZATION OF THE DTDP-DEOXYSUGAR PRODUCTION PATHWAYS WILL BE CONDUCTED TO INCREASE THE AMOUNT IN THE MEDIUM TO 0.5-1.0 G/L. THIS WILL ALLOW US TO PRODUCE UP TO A 2.5 G OF EACH DEOXYSUGAR FROM A SINGLE 10 L FERMENTER. IN PHASE III, WE WILL COMMERCIALIZE THE DEOXYSUGARS, INDIVIDUALLY AND AS A KIT, TO RESEARCHERS IN ACADEMIA AND INDUSTRY. THROUGH EXPANDED PRODUCTION, THE WORK DONE IN PHASE II WILL ALSO HELP INCREASE PRODUCTION OF THE DTDP-ACTIVATED DEOXYSUGARS AS IMPORTANT SECONDARY PRODUCTS.