Biological production of muconic acid via a prokaryotic 2,3-dihydroxybenzoic acid decarboxylase.

Non-oxidative decarboxylases belong to a unique enzyme family that does not require any cofactors. Here we report the characterization of a 2,3-dihydroxybenzoic acid (2,3-DHBA) decarboxylase (BDC) from Klebsiella pneumoniae and explore its application on the production of muconic acid. The enzyme properties were systematically studied, including the optimal temperature and pH, kinetic parameters, and substrate specificity. On this basis, we designed an artificial pathway for muconic acid production by connecting 2,3-DHBA biosynthesis with its degradation pathway. Over-expression of entCBA and the key enzymes in the shikimate pathway led to the production of 900 mg L(-1) of 2,3-DHBA. Further, expression of the BDC coupled with catechol 1,2-dioxygenase achieved the conversion of 2,3-DHBA into muconic acid. Finally, assembly of the total pathway resulted in the de novo production of muconic acid up to 480 mg L(-1).