Mapping and Redirecting Metabolic Fluxes to Attain Significantly Lessen Oxygen Requirements, Acid Production, and Enhanced Recombinant Culture Productivity

9622821这项建议正在进行研究，以解决重组大肠杆菌经常报道的蛋白质生产瓶颈之一，即半氧化糖和碳水化合物(例如醋酸盐)的过量生产，这会降低总碳产率，并表明代谢生物合成/分解代谢通量效率低下。本研究的具体目标是：(1)在研究人员对芽孢杆菌的研究的基础上，通过实验验证(使用核磁共振)以丙酮酸激酶(PYK)为中心的优化通量分配的可行性；(2)通过删除PYK同工酶将增产策略转移到大肠杆菌中，并通过代谢通量分析进行分析，再次与核磁共振相结合；(3)根据突变大肠杆菌的浓度、重组蛋白的生产和氧气消耗来确定放大潜力，以及(4)评估是否如高糖饲料大肠杆菌培养中所显示的那样，在耗能的蛋白分解和ATP的产生之间存在联系。***

9622821 Ataai This proposal is on research which addresses one of the bottlenecks in protein production frequently reported for recombinant E. coli, namely overflow production of semi-oxidized sugars and carbohydrates (e.g. acetate) which reduces the total carbon yield and also indicates inefficient metabolic biosynthetic/catabolic fluxes. The specific goals of this research are to: (1) build on the investigators' Bacillus research to experimentally validate (using NMR) the feasibility of optimized flux distribution centered around pyruvate kinase (PYK), (2) transfer the yield-increasing strategy to E. coli by deleting PYK isozymes and analyze via metabolic flux analysis, again coupled with NMR, (3) determine scale-up potential in terms of mutant E. coli concentration, recombinant protein production, and oxygen consumption, and, (4) evaluate whether there is a link between energy-consuming proteolysis and ATP-generation as shown in high glucose feed E. coli cultures. ***