POTENTIAL USE OF A BACTERIAL FLAVING-CONTAINING MONOOXYGENASE FOR THE REDUCTION OF TRIMETHYLAMINE OFF-ODOR

GUENDULAIN, TATIANA; BARRA, JOSÉ LUIS

Chapadmalal, Buenos Aires

Congreso; XVIII Reunión Anual de la Sociedad Argentina de Microbiología General (SAMIGE); 2023

Sociedad Argentina de Microbiología General (SAMIGE)

Trimethylamine (TMA) is a volatile tertiary amine that has a strong odor of decomposingfish, even in low concentrations. Protein hydrolysates made from marine byproducts arehighly nutritious, but they often contain TMA, giving them a fishy odor that makes themunappealing for consumption. Trimethylamine is also produced by the human intestinalmicrobiota from dietary precursors such as choline, carnitine, and trimethylamine oxide(TMAO). Normally, the trimethylamine generated in the intestine enters the bloodstreamand is transformed into odorless TMAO in the liver, through the action of the enzyme flavinmonooxygenase 3 (FMO3). Failure of the FMO3 enzyme results in a rare condition calledtrimethylaminuria (TMAU), in which abnormal amounts of TMA are secreted in urine, sweat,exhaled air, and other bodily secretions, giving the person an unpleasant body odorresembling that of decomposing fish. As a consequence, patients can suffer from severepsychosocial consequences.In this study, we analyze the potential use of a bacterial flavin-containingmonooxygenase (FMO) for the reduction of trimethylamine off-odor. The coding sequenceof a bacterial FMO was synthesized using codons optimized for expression in Escherichiacoli, with a His-tag at the C-terminal end. Overexpression of the FMO in E. coli BL21 λDE3allowed the production of large amounts of protein, which was then purified usingimmobilized metal affinity chromatography (IMAC). The activity of the purified FMO (in vitro)was evaluated using spectrophotometry, with trimethylamine as substrate. To assess its invivo activity, the FMO sequence was cloned into suitable vectors for expression inEscherichia coli Nissle 1917 (a probiotic strain) and a mutant strain with the gene encodinga TMAO reductase interrupted (E. coli Nissle 1917 ΔtorA). The activity of these geneticallymodified probiotic bacteria was analyzed in aerobic and anaerobic cultures, usingtrimethylamine as substrate.The purified FMO has activity with TMA as substrate, and its kinetic parameters weredetermined. Probiotic bacteria expressing the FMO have the ability to convert TMA intoTMAO under aerobic conditions. The potential use of these bacteria in the marine productderivatives industry and as complementary therapies for TMAU will be discussed.