A novel phthalic acid degradation pathway in Klebsiella variicola SY1 and engineering the strain for degradation of multiple phthalate esters.

Phthalic acid (PA) is a key intermediate in the biodegradation of phthalate esters (PAEs), and its aerobic degradation pathways exhibit considerable diversity. In this study, a novel aerobic PA degradation bypass was discovered in Klebsiella variicola SY1. After blocking the classical 4,5-dioxygenase pathway, the strain retained the ability to utilize PA, and gene 5193 encoding a UbiD family decarboxylase was significantly upregulated. Further knockout of gene 5193 caused the loss of PA utilization ability. Metabolomic and LC-MS analysis revealed that this bypass is initiated by 4-monooxygenation of PA to 4-hydroxyphthalic acid, followed by decarboxylation to 4-hydroxybenzoic acid, which then enters protocatechuic acid or phenol metabolism. Heterologous expression of gene 5193 did not confer the ability to convert PA or 4-hydroxyphthalic acid (4-HP), indicating that its product is not directly responsible for substrate conversion. Furthermore, by constructing a constitutive expression vector and introducing the esterase gene pehA, the recombinant strain achieved degradation rates exceeding 90% for dimethyl phthalate, diethyl phthalate, and dibutyl phthalate, effectively broadening the substrate spectrum. This finding enriches the understanding of the diversity of aerobic PA metabolism and provides both a strain resource and theoretical support for the bioremediation of phthalate ester pollution.