Averesch Nils J H, Martínez Verónica S, Nielsen Lars K, Krömer Jens O
Centre for Microbial Electrochemical Systems (CEMES), Advanced Water Management Centre (AWMC), The University of Queensland , Brisbane 4072, Australia.
Universities Space Research Association at NASA Ames Research Center , Moffett Field, California 94035, United States.
ACS Synth Biol. 2018 Feb 16;7(2):490-509. doi: 10.1021/acssynbio.7b00304. Epub 2017 Dec 26.
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.
己二酸作为尼龙-6,6的前体，最近在合成生物学领域受到了广泛关注。在此，我们使用一种用于基本通量模式网络嵌入热力学分析的新型工具，评估了16种不同的己二酸生产途径。该工具能够在确定的代谢物浓度下区分热力学可行和不可行的模式，从而可以评估理论产率的热力学可行性。此外，还确定了总是导致不可行通量分布的模式，这将有助于定制菌株设计的开发。对细胞外排机制的综述表明，只有与ATP水解偶联，细胞外产物才可能大量积累。化学计量分析表明，最大理论产物碳产率在很大程度上取决于代谢途径，在大肠杆菌和酿酒酵母的代谢模型中，以葡萄糖和/或棕榈酸酯为底物时，该产率范围为32%至99%。同样重要的是，在几种途径中，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减少，在某些情况下甚至使整个途径变得不可行，这突出了途径选择的重要性。只有基于莽草酸途径的路线在较大的浓度和pH范围内在热力学上是有利的。酿酒酵母的低pH能力使一些途径的热力学平衡向产物形成方向移动。一种确定的不可行模式表明，线粒体苹果酸脱氢酶的可逆性与当前的知识状态相矛盾，这对酿酒酵母的代谢施加了重大限制。最后，对工业相关约束的评估表明，大肠杆菌中两条基于莽草酸途径的路线最为稳健。
