In this work, the effects of PcaJ (beta-ketoadipate:succinyl-coenzyme A transferase)- and PcaD (beta-ketoadipate enollactone hydrolase)-inactivation on protocatechuic acid metabolism in Pseudomonas putida KT2440 were evaluated. Beta-ketoadipic add was produced from protocatechuic acid by the inactivation of PcaJ as expected; however, a portion of the produced beta-ketoadipic acid was converted to levulinic acid through a purification step consisting of extraction from the culture and recrystallization. On the other hand, muconolactone was purified from the culture of the PcaD-inactivated mutant of 172440, although beta-ketoadipate enol-lactone was supposed to be produced because it is the substrate of PcaD. Under aerobic conditions, it has been reported that lignin-related aromatics are metabolized through PCA 2,3- or 3,4- or 4,5-ring cleavage pathways, and muconolactone is an intermediate observed in the metabolism of catechol, not protocatechuic acid. Our results will provide a prospective route to produce muconolactone with a high yield through the protocatechuate-3,4-metabolic pathway. (C) 2015 The Society for Biotechnology, Japan. All rights reserved.