Identification and functional analysis of 2,3-dihydroxybenzoic acid and catechol catabolic pathway genes in...

Microorganisms catabolize many plant-derived aromatic compounds through the β-ketoadipate pathway. This pathway has two branches, beginning with protocatechuic acid and catechol, which converge on β-ketoadipate before further catalysis to produce tricarboxylic acid cycle intermediates. The protocatechuic acid branch of the pathway has been characterized in fungi.Here, we characterized the genes of the catechol branch of the β-ketoadipate pathway in the filamentous fungus Aspergillus niger. We predicted the genes involved using comparative transcriptomics and homology to characterized genes. We tested the predictions by constructing knockout mutants lacking candidate genes to observe the growth phenotype on 2,3dihydroxybenzoic acid (2,3-DHB), which is catabolized through the catechol branch, and by detection of metabolites accumulated in knockout mutants using mass spectrometry. Using these approaches to reconstruct the pathway, we confirmed the reported assignments of dhbA, crcA, kstA, and kctA as encoding 2,3-dihydroxybenzoate decarboxylase, catechol 1,2dioxygenase, β-ketoadipate:succinyl-CoA transferase, and β-ketoadipyl-CoA thiolase, respectively. We further assigned mciA, mliA, and kelA as encoding muconate cycloisomerase, muconolactone isomerase, and β-ketoadipate enol-lactonase, respectively. Additionally, we identified a transcription regulator involved with the β-ketoadipate pathway in fungi for the first time. This gene, pcaR, is upregulated on 2,3-DHB and deletion of the gene impaired growth on 2,3-DHB.