(686d) Connecting Anthranilate Catabolism and Anabolism Leads to a Novel Muconic Acid Biosynthetic Approach

Adipic acid is an important platform chemical that can be used for plastic and nylon production. The global demand for adipic acid is over 2 billion kilograms annually. Currently, the major approach to adipic acid production relies on chemical synthesis using benzene as the starting material. However, this approach is generally considered nonrenewable and environmentally incompatible due to the toxicity of the starting material and intermediates and the release of greenhouse gas N₂O. Microbial synthesis from renewable carbon sources provides a feasible and promising alternative in the circumstance of environment deterioration and petroleum depletion. Indeed, adipic acid can be easily generated by hydrogenation of muconic acid, a naturally existing intermediate in aromatic compounds degradation by some soil bacteria. Draths and Frost first reported the constitution of an artificial pathway in Escherichia coli by shunting 3-dehydroshikimate from shikimate pathway, affording the production of muconic acid from glucose. Here we designed a novel muconic acid biosynthetic pathway by connecting anthranilate catabolism with anabolism. Specifically, anthranilate, an E. coli endogenous intermediate in the tryptophan biosynthetic branch was sequentially converted into catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO). First of all, screening for efficient ADO and CDO from different microorganisms led to the convertion of anthranilate to gram per liter-level of muconic acid. Further, to achieve the de novo muconic acid biosynthesis, anthranilate overproducing strains were constructed by blocking tryptophan biosynthesis and over-expressing the key enzymes in shikimate pathway. Interestingly, introduction of a strengthened glutamine regeneration system by over-expressing glutamine synthase dramatically enhanced anthranilate production. Finally, the engineered strain carrying the full pathway produced 390 mg/L of muconic acid from simple carbon sources in shake flask. This approach will demonstrate scale-up potential for microbial production of muconic acid with further condition and pathway optimizations.