Thompson Brian, Pugh Shawn, Machas Michael, Nielsen David R
Chemical Engineering, School for Engineering of Matter, Transport, and Energy, Arizona State University , Tempe, Arizona 85287, United States.
ACS Synth Biol. 2018 Feb 16;7(2):565-575. doi: 10.1021/acssynbio.7b00331. Epub 2017 Nov 2.
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 via p-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.
粘康酸是一种很有前景的平台生物化学物质和己二酸的前体，可用于合成各种塑料和聚合物。在本研究中，对大肠杆菌中粘康酸生物合成非天然途径的模块化网络进行了系统构建和比较评估，包括通过以苯酚为共同中间体的三种不同的新途径。然而，苯酚羟化酶的重组活性差和高混杂性最终限制了“依赖苯酚”的粘康酸生产。因此，开发了第四条通过对羟基苯甲酸、原儿茶酸和儿茶酚的途径，尽管通过该途径的粘康酸滴度仅达到819 mg/L，但其性能落后于已确立的“3-脱氢莽草酸衍生”途径。最后，将这两条最有前景的途径平行共表达，以创建一个合成的“代谢漏斗”，通过在保留天然分支酸生物合成的同时实现最大净前体同化和通量，在葡萄糖产率为158 mg/g的情况下，将粘康酸产量提高了近一倍，达到>3.1 g/L，同时仅引入一种营养缺陷型。这种可推广的“漏斗式”策略有望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目标生物产品的产量。
