STTR Phase I: Low-Cost Biosynthesis of Sugar Phosphates via ATP-Free Enzyme Cocktails

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The broader impact/commercial potential of this Small Business Innovation Research Phase I project could be a significant decrease of sugar phosphate manufacturing costs, thereby creating opportunities for the low-cost production of numerous drugs and their derivatives used to treat cardiac diseases, cancers, and degenerative diseases. For example, fructose 1,6-bisphosphate (FBP) is a very important drug for treating cardiac diseases. Furthermore, less costly FBP, along with two thermostable enzymes (i.e., aldolase and triosephosphate isomerase), will provide affordable glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to both the pharmaceutical industry and academia. Both G3P and DHAP are important precursors for the synthesis of numerous carbon-carbon chiral compounds used for pharmaceutical production. The in-vitro synthetic biosystems platform to be developed in this project could be instrumental in unlocking the full potential of modified sugar phosphates in drug discovery and drug synthesis. Furthermore, this platform is much more environmentally friendly than typical chemical synthesis: modest reaction conditions and significant decreases in environmental footprint. This platform also has the potential to lead the next paradigm shift in biomanufacturing, by establishing a viable, environmentally friendly alternative for pharmaceutical manufacturing plants currently using chemical synthesis. The technical objectives in this Phase I research project are to validate the technological feasibility of the biosynthesis of fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate via a non-natural ATP-free enzymatic pathway. In contrast to glycolysis, this novel pathway involves neither costly coenzymes (e.g., NAD+, ATP) nor requires ATP regeneration. Also, thermostable enzymes from hyperthermophilic microorganisms will be used to carry out reactions at 50-60 deg C in aqueous solution. Thermophilic enzymes have a longer lifetime than mesophilic enzymes, and the relatively high reaction temperature eliminates possible microbial contamination. The goal of this project is to demonstrate the biosynthesis of fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate from low-cost substrates, that is, starch and pyrophosphate, via a novel ATP-free enzymatic pathway. The technical tasks are to (1) clone and express several recombinant pyrophosphate phosphofructokinases from various thermophilic microorganisms; (2) validate the feasibility of the synthetic pathway for biomanufacturing sugar phosphates; (3) optimize the synthetic pathway for cost-effective production; and (4) obtain grams of high-purity fructose 1,6-bisphosphate crystals via a series of purification steps. The most essential task is the efficient expression of a recombinant, high-activity pyrophosphate phosphofructokinase, which enables the omission of glycolysis? ATP-dependent phosphofructokinase from our pathway