Integrated System for Rapid Production of Activated Sugars and Glycosylated Molecules

Glycan modifications of natural products are increasingly important in the discovery of new pharmaceuticalsWhile glycodiversity gives rise to important therapeutic properties of bioactive moleculesexploring glycodiversity has challengesIsolating glycosylated compounds from nature is often difficult due to low concentrations and the presence of multiple related molecules with similar structures and propertiesChemical synthesis is challenging task and may only produce small milligram amountsThe enzymatic synthesis of glycans has many potential advantages for producing both natural and novel compounds is significantly limited by the availability of the activated NDP sugar substrates required by glycosyltransferases involved in the glycosylation reactionsAt zuChem we have engineered proprietary enzymes involved in activated sugar synthesis including sugar kinases and nucleotidyltransferase with broadened substrate specificities producing a wide range of sugarphosphates and activated sugarsThe enzymes are stable and carry out reactions quickly and at high titerDespite thishoweverchallenges remain with their use on a rapid and broad scaleThe enzymes exhibit product and substrate inhibition and run under different physiological reaction conditionsThis prevents a simple one pot method for activated sugars to be developedintermediates need complex purification before use in the next reaction stepAs a resultthroughput and the ability to produce larger volumes of activated sugars is limitedFinallythe cost of nucleotides remains expensive for large scale production of compoundsHerewe propose to develop an integrated system for simple production of a large library of natural and novel activated NDP sugarsThe system will contain enzymes engineered to eliminate substrate product inhibition and work under similar physiological conditionsThey will be utilized in a one pot or immobilized setup with a nucleotide recycling systemStarting from the proprietary enzymes we have developed we will use a powerful screen we have designed to rapidly identify mutants with improved enzyme propertiesIn Phase I we will demonstrate the feasibility of developing the systemWe will show that a polyphosphate kinase can be engineered to tolerate higher levels of the ADP co productand a nucleotidyltransferase can be engineered with decreased sensitivity to nucleotidesIn Phase II we will further improve the enzymesoptimize mutation combinations and optimize the cofactor recycling systemWe will also develop one pot and or enzyme immobilization methods to create an integrated system for the rapid production of activated sugarsThe system will be able to produce not only activated sugarsbut custom glycosylated products when fed only sugarcatalytic amounts of nucleotide triphosphatesNTPpolyphosphateand the customer s desired glycosyltransferase and substrateAt preparative and industrial scales the system will be adapted for the production of important oligosaccharidesIn Phase III we will commercialize the system by selling or licensing it for use to researchersand selling custom activated sugarsoligosaccharides and other glycosylated compounds Project Narrative We will develop a novel integrated system for the enzymatic synthesis of activated sugars with in situ nucleotide regenerationThis technology will simplify production of a large library of both natural and rare activated sugars nucleotidesmake them more readily available at research scale for glycodiversity experiments and at large scale for production of glycosylated small moleculesproteinsand oligosaccharidesThe proposed research has the potential to open up several multi billion dollar markets in anti infective and anticancer therapeutics