Project Grant R43GM145102

PRODUCTION OF NATURAL DEOXYSUGARS FOR CHEMICAL SYNTHESIS OF GLYCOSIDES - PROJECT SUMMARY THE ULTIMATE 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 ARE A NUMBER OF CHALLENGES WHICH NEED TO BE OVERCOME. FIRST, TITERS OF THE TDP-DEOXYSUGARS NEED 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 NEEDS TO BE DEVELOPED. FINALLY, THE DEOXYSUGARS NEED TO BE HYDROLYZED AND ISOLATED. SPECIFICALLY, IN PHASE I WE WILL DEMONSTRATE THE FEASIBILITY OF DEOXYSUGAR PRODUCTION USING D-FUCOSE DERIVED FROM DTDP-D-FUCOSE AS A MODEL. THIS WILL BE 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 CHEMICALLY DEFINED MEDIUM CAPABLE OF PRODUCING LARGER QUANTITIES OF DTDP-D-FUCOSE IN THE MEDIUM, 3) DEVELOPING A DTDP-D-FUCOSE PURIFICATION PROTOCOL FROM THE CHEMICALLY DEFINED 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 AT LEAST 18 ADDITIONAL DEOXYSUGARS FROM DTDP- DEOXYSUGARS. OPTIMIZATION OF THE DTDP-DEOXYSUGAR PRODUCTION PATHWAYS WILL BE CONDUCTED TO INCREASE THE AMOUNT IN THE MEDIUM TO AT LEAST 500 MG/L. THIS WILL ALLOW US TO PRODUCE UP TO A GRAM 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 PHASES I AND II WILL ALSO HELP SUPPORT THE SALE OF OUR DTDP-ACTIVATED DEOXYSUGARS.