Muconic acid is a promising platform biochemical and precursor to adipic acid, which can be used to synthesize various plastics and polymers. In this study, the systematic construction and comparative evaluation of a modular network of non-natural pathways for muconic acid biosynthesis was investigated in Escherichia coli, including via three distinct and novel pathways proceeding via phenol as a common intermediate. However, poor recombinant activity and high promiscuity of phenol hydroxylase ultimately limited “phenol-dependent” muconic acid production. A fourth pathway proceeding viap-hydroxybenzoate, protocatechuate, and catechol was accordingly developed, though with muconic acid titers by this route reaching just 819 mg/L, its performance lagged behind that of the established, “3-dehydroshikimiate-derived” route. Finally, these two most promising pathways were coexpressed in parallel to create a synthetic “metabolic funnel” that, by enabling maximal net precursor assimilation and flux while preserving native chorismate biosynthesis, nearly doubled muconic acid production to up to >3.1 g/L at a glucose yield of 158 mg/g while introducing only a single auxotrophy. This generalizable, “funneling” strategy is expected to have broad applications in metabolic engineering for further enhancing production of muconic acid, as well as other important bioproducts of interest.

中文翻译：

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通过替代途径和合成的“代谢漏斗”生产粘康酸

粘康酸是有前途的平台生化剂和己二酸的前体，可用于合成各种塑料和聚合物。在这项研究中，在大肠杆菌中研究了粘康酸生物合成的非天然途径的模块化网络的系统构建和比较评估，包括通过三种不同的新颖途径，通过酚作为常见中间体进行。然而，不良的重组活性和酚羟化酶的滥交最终限制了“酚依赖性”粘康酸的生产。通过p进行的第四条途径相应地开发了-羟基苯甲酸酯，原儿茶酸酯和邻苯二酚，尽管通过该途径的粘康酸滴度仅为819 mg / L，但其性能落后于已确立的“ 3-脱氢ki草酸酯衍生”途径。最后，将这两个最有希望的途径并行共表达，以创建合成的“代谢漏斗”，该漏斗通过最大程度地吸收净前体并保持天然分支酸生物合成，同时使最大的净前体同化和通量，在一天的时间里将粘康酸的产量提高了近一倍，达到了> 3.1 g / L。葡萄糖产量为158 mg / g，而仅引入单个营养缺陷型。预期这种普遍的“漏斗”策略将在代谢工程中得到广泛应用，以进一步提高粘康酸以及其他重要的重要生物产物的产量。