Function Identificationof a Lactonase Gene <i>lac1563</i> Involved in ProducingUrolithin A of Limosilactobacillus fermentum FUA033

This study aimed to identify the lactonase gene in Limosilactobacillus fermentum FUA033 that facilitates the conversion of ellagic acid into urolithin A. Lactonase gene candidates were identified genome analysis. A series of overexpression, knockout, and complementation strains of these candidate genes were subsequently generated. The urolithin A yield of the <i>lac1563</i> knockout strain was significantly reduced compared to the wild-type strain, whereas the overexpression strain exhibited a markedly higher yield. The complementation strain showed no significant difference in urolithin A yield relative to the wild-type strain. In contrast, alterations in the <i>lac329</i> gene did not significantly impact urolithin A production compared to the wild-type strain. Additionally, the candidate lactonase genes were cloned and heterologously expressed in Escherichia coli. The recombinant lactonase derived from the <i>lac1563</i> gene, unlike that from the <i>lac329</i> gene, demonstrated activity toward ellagic acid. Optimal activity of the <i>lac1563</i> lactonase occurred at 37 °C and pH 7.0. Enzyme activity was notably enhanced by Mn2+ and Co2+, while inhibited by Zn2+ and Cu2+. These findings contribute valuable insights into the metabolic pathway of ellagic acid conversion to urolithin A and establish the <i>lac1563</i> gene as a promising candidate for biotechnological applications aimed at urolithin A production.