Toward Synthetic Biology Strategies for Adipic Acid Production: An in Silico Tool for Combined Thermodynamics and Stoichiometric Analysis of Metabolic Networks

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.

己二酸（adipic acid）作为尼龙-6,6的前体物质，近年来在合成生物学领域广受关注。本研究利用一款用于基元通量模式（elementary flux modes）网络嵌入热力学分析的新型工具，对16种不同的己二酸合成途径开展评估。该工具可在确定的代谢物浓度条件下，区分热力学可行与不可行的通量模式，从而实现理论产率的热力学可行性判定。此外，研究还识别出了始终导致通量分布不可行的模式，这将助力定制化菌株设计的开发。对细胞外排机制的综述研究表明，唯有与ATP水解偶联时，胞外产物方可实现显著积累。化学计量分析显示，最大理论产物碳产率高度依赖代谢途径类型：在大肠杆菌（Escherichia coli）与酿酒酵母（Saccharomyces cerevisiae）的代谢模型中，以葡萄糖和/或棕榈酸为底物时，产物碳产率范围为32%至99%。同样值得关注的是，多条代谢途径中的代谢物浓度似乎受到热力学限制。因此，热力学可行的通量分布数量有所缩减，部分场景下甚至导致整条途径丧失可行性，凸显了途径选择的关键意义。唯有基于莽草酸途径（shikimate pathway）的合成途径，在较宽的浓度与pH范围内均具备热力学优势。酿酒酵母的低pH耐受特性，可使部分途径的热力学平衡向产物生成方向偏移。研究识别出的一种不可行模式显示，线粒体苹果酸脱氢酶的可逆性与当前学术认知相悖，这对酿酒酵母的代谢构成了重大限制。最后，通过对工业相关约束条件的评估发现，大肠杆菌中两条基于莽草酸途径的合成途径具备最优的稳定性。