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

这个小企业创新研究第一阶段项目的更广泛影响/商业潜力可能是显著降低磷酸糖的制造成本，从而为用于治疗心脏病、癌症和退行性疾病的许多药物及其衍生物的低成本生产创造机会。例如，果糖1,6-二磷酸（FBP）是一种非常重要的治疗心脏疾病的药物。此外，成本较低的FBP，以及两种耐热酶（即醛缩酶和三磷酸异构酶），将为制药行业和学术界提供价格合理的甘油醛3-磷酸（G3P）和磷酸二羟丙酮（DHAP）。G3P和DHAP都是合成许多用于药物生产的碳-碳手性化合物的重要前体。在这个项目中开发的体外合成生物系统平台可能有助于释放修饰磷酸糖在药物发现和药物合成中的全部潜力。此外，该平台比典型的化学合成更环保：反应条件适中，环境足迹显著减少。通过为目前使用化学合成的制药工厂建立一种可行的、环保的替代方案，该平台也有可能引领生物制造的下一个范式转变。本一期研究项目的技术目标是验证通过非天然无atp酶途径生物合成果糖1,6-二磷酸、甘油醛3-磷酸和磷酸二羟基丙酮的技术可行性。与糖酵解不同，这种新途径既不涉及昂贵的辅酶（如NAD+、ATP），也不需要ATP再生。此外，来自超嗜热微生物的热稳定酶将用于在水溶液中进行50-60℃的反应。嗜热酶比中温酶具有更长的寿命，并且相对较高的反应温度消除了可能的微生物污染。该项目的目标是通过一种新的无atp酶途径，从低成本底物（即淀粉和焦磷酸盐）中生物合成果糖1,6-二磷酸、甘油醛3-磷酸和二羟基丙酮磷酸。技术任务是：(1)从各种嗜热微生物中克隆和表达几种重组焦磷酸磷酸果糖激酶；(2)验证生物制造磷酸糖合成途径的可行性；(3)优化合成途径，实现高性价比生产；(4)通过一系列纯化步骤得到克高纯度的果糖1,6-二磷酸晶体。最重要的任务是高效表达重组，高活性焦磷酸磷酸果糖激酶，这使得省略糖酵解？atp依赖性磷酸果糖激酶

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