Heterologous expression and characterization of a fumarate reductase from Lactobacillus panisapium with activity...

Lactobacillus panisapium, isolated from the gastrointestinal tract of a queen honeybee, converts trans-10-hydroxy-2-decenoic acid (10H2DA) into 10-hydroxydecanoic acid (10HDAA) in royal jelly. 10HDAA has shown significant anti-inflammatory potential. This study aimed to identify the reductase responsible for this bioconversion and characterize its properties.

An enzyme was partially purified from L. panisapium cell extracts based on its 10H2DA-reducing activity. Liquid chromatography-mass spectrometry and phylogenetic analyses identified the enzyme as a water-soluble fumarate reductase (~40% identity to reductases from Shewanella frigidimarina and Klebsiella pneumoniae). The reductase was heterologously expressed in Escherichia coli. The recombinant enzyme displayed activity toward fumarate and 10H2DA in the presence of flavin adenine dinucleotide and nicotinamide adenine dinucleotide hydride. The enzyme exhibited optimal stability at 50 °C and pH 6.0. An active-site glycine residue in the L. panisapium reductase likely facilitates the binding of large substrates such as 10H2DA. Substituting this residue with threonine decreased activity toward 10H2DA while maintaining activity toward fumarate. Structural modeling revealed that, compared with the homologous reductase from S. frigidimarina, the enzyme from L. panisapium lacks a heme-binding domain, again consistent with enhanced access for larger substrates. Whole cells of E. coli expressing the recombinant reductase effectively converted 10H2DA to 10HDAA, offering a scalable alternative to using purified recombinant enzyme.

This study provides insights into the structure-function relationship of a novel fumarate reductase-family enzyme from L. panisapium and demonstrates its potential for biocatalytic applications in producing 10HDAA-enriched royal jelly, contributing to the development of functional foods. © 2025 Society of Chemical Industry.