Reduction of ferulic acid as an electron acceptor under anaerobic conditions by the heterofermentative lactic acid bacterium <i>Weissella cibaria</i>

When the heterofermentative lactic acid bacterium <i>Weissella cibaria</i> JCM12495 was cultivated anaerobically in medium supplemented with 3-hydroxy-4-methoxycinnamic acid (ferulic acid), all the added ferulic acid was converted to 3-(4-hydroxy-3-methoxyphenyl)propionic acid (HMPA, dihydroferulic acid) via reduction of the propenoic acid side chain. Complete conversion to HMPA was achieved within 16 h, even at an initial ferulic acid concentration of 10 mM. Concurrently, the production of typical heterolactic fermentation products, such as lactic acid and ethanol, was suppressed, while acetic acid formation was increased. Transcriptomic analysis revealed that only two genes were significantly induced by ferulic acid addition, and these genes form an operon in the genome. We individually expressed these two genes in <i>Escherichia coli</i> and assessed their enzymatic activities <i>in vitro</i> against ferulic acid by identifying reaction products using liquid chromatography quadrupole time-of-flight mass spectrometry. When ferulic acid and acetyl-CoA were added to a reaction mixture containing the first expressed protein, feruloyl-CoA was detected as the product. Subsequently, when the generated feruloyl-CoA was incubated with the second expressed protein in the presence of FAD and NADH, the formation of HMPA was confirmed. These results demonstrate that the two genes encode a ferulate CoA-transferase (FarA), which catalyzes the CoA ligation of ferulic acid, and a feruloyl-CoA reductase (FarB), which reduces the propenoic acid side chain of feruloyl-CoA to produce HMPA. Our findings reveal an anaerobic respiratory pathway coupled to heterolactic fermentation, in which ferulic acid acts as an electron acceptor, thereby influencing carbohydrate utilization during heterolactic fermentation.IMPORTANCEProbiotics and lactic acid bacteria (LAB) in fermented foods are of significant interest due to their health-promoting effects. This study shows that <i>Weissella cibaria</i>, a common LAB found in plant-derived fermented foods, uses ferulic acid, a compound bound to dietary fibers such as arabinoxylans, as an external electron acceptor for anaerobic redox balancing. This metabolic strategy is associated with redox regulation during heterolactic fermentation and leads to the production of 3-(4-hydroxy-3-methoxyphenyl)propionic acid, a metabolite that has been reported to have health benefits in humans. These findings reveal an unrecognized link between LAB metabolism, dietary polyphenol conversion, and potential probiotic functions, providing new insight into how gut-associated microbes contribute to host health.