Ryoji Mitsui, Hikaru Maruko, Riyo Awa, Fusako Kawamoto, Daigo Iwasaki, Yosuke Nishitani, Hiroshige Kuwahara, Takanori Yano
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When the heterofermentative lactic acid bacterium Weissella cibaria 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 Escherichia coli and assessed their enzymatic activities in vitro 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.
Research papers (academic journals)
