Adipic acid, a nylon-6,6 precursor, has recently gained popularity in synthetic biology. Here, 16 different production routes to adipic acid were evaluated using a novel tool for network-embedded thermodynamic analysis of elementary flux modes. The tool distinguishes between thermodynamically feasible and infeasible modes under determined metabolite concentrations, allowing the thermodynamic feasibility of theoretical yields to be assessed. Further, patterns that always caused infeasible flux distributions were identified, which will aid the development of tailored strain design. A review of cellular efflux mechanisms revealed that significant accumulation of extracellular product is only possible if coupled with ATP hydrolysis. A stoichiometric analysis demonstrated that the maximum theoretical product carbon yield heavily depends on the metabolic route, ranging from 32 to 99% on glucose and/or palmitate in Escherichia coli and Saccharomyces cerevisiae metabolic models. Equally important, metabolite concentrations appeared to be thermodynamically restricted in several pathways. Consequently, the number of thermodynamically feasible flux distributions was reduced, in some cases even rendering whole pathways infeasible, highlighting the importance of pathway choice. Only routes based on the shikimate pathway were thermodynamically favorable over a large concentration and pH range. The low pH capability of S. cerevisiae shifted the thermodynamic equilibrium of some pathways toward product formation. One identified infeasible-pattern revealed that the reversibility of the mitochondrial malate dehydrogenase contradicted the current state of knowledge, which imposes a major restriction on the metabolism of S. cerevisiae. Finally, the evaluation of industrially relevant constraints revealed that two shikimate pathway-based routes in E. coli were the most robust.

中文翻译：

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己二酸生产的合成生物学策略：代谢网络的热力学和化学计量分析相结合的 in silico工具

己二酸，一种尼龙6,6的前体，最近在合成生物学中得到普及。在这里，使用新型工具对元素通量模式进行网络嵌入式热力学分析，评估了十六种生产己二酸的生产路线。该工具在确定的代谢物浓度下区分热力学可行模式和不可行模式，从而可以评估理论产率的热力学可行性。此外，确定了总是导致通量分布不可行的模式，这将有助于定制应变设计的发展。审查细胞外排机制表明，只有与ATP水解结合，细胞外产物的大量积累才是可能的。大肠杆菌和酿酒酵母代谢模型。同样重要的是，代谢物的浓度似乎在热力学上受到多种途径的限制。因此，减少了热力学上可行的通量分布，在某些情况下甚至使整个路径变得不可行，从而突出了选择路径的重要性。仅基于the草酸酯途径的途径在大浓度和pH范围内在热力学上是有利的。酿酒酵母的低pH值能力使某些途径的热力学平衡向产物形成转移。一种确定的不可行模式揭示线粒体苹果酸脱氢酶的可逆性与目前的知识状态相矛盾，这对酿酒酵母的代谢构成了重大限制。最后，对与工业相关的限制条件的评估表明，大肠杆菌中两种基于sh草酸途径的途径最为稳健。