Isolation, Oxygen-tolerant Domestication And Mechanisms For The Oxygen-tolerant Capability Of A 5-hydroxy-equol Producing Bacterium

Soy isoflavones, which are mainly composed of genistein and daidzein, are secondary metabolites produced during the growth of leguminous plants, including soybeans. Soy isoflavones, the structures of which are similar to that of estrogens, are capable of binding to estrogen receptors. Therefore, soy isoflavones are called phytoestrogens. Soy isoflavones have been reported to have numerous physiological functions, including anti-carcinogenic, anti-osteoporosis and positive role for treatment of cardio-vascular diseases. However, studies have shown that soy isoflavones are subjected to bioconversion by gut microflora after ingestion, the metabolites of which include dihydrodaidzein(DHD), tetrahydrodaidzein(THD), dihydrogenistein(DHG), equol, O-desmethylangolensin(O-Dma), 4-P-ethylphenol and so on.A newly isolated rod-shaped obligate anaerobic bacterium from fresh cock feces, which we named AUH-JLC257, was found to be capable of bioconverting isoflavone genistein to 5-hydroxy-equol under anaerobic conditions. The 16 S r DNA sequence(1,401bp) of the bacterium strain AUH-JLC257 showed the highest similarity(99.27%) to that of Slackia equolifaciens strain DZE.The metabolite 5-hydroxy-equol was identified by using UV spectrum, electrospray ionization mass spectrometry(ESI-MS), and 1H and 13 C nuclear magnetic resonance(NMR) analyses. Chiral stationary-phase high-performance liquid chromatography analysis and specific rotation examination demonstrated that the biosynthesized 5-hydroxy-equol was just(-)-5-hydroxy-equol. The maximal concentration of the substrate genistein that AUH-JLC257 could convert was 0.6mmol/L, the average bioconversion rate was 83.05%. In addition, strain AUH-JLC257 was also capable of bioconverting isoflavone daidzein to equol.An oxygen-tolerant domestication was carried out to improve the oxygen-tolerant ability of the obligate anaerobic bacterium AUH-JLC257. In the end, we successfully obtained an oxygen-tolerant mutant, which we named Aeroto-AUH-JLC2H-257.Our study revealed that strain Aeroto-AUH-JLC2H-257 differed from the native strain AUH-JLC257 in many aspects, including the cell morphology(from rod to filament), the improved capability to use different carbon sources, such as glucose, lactose, sucrose, maltose, salicina, cellose, mannose, raffinose, trehalose and esculin.High performance liquid chromatography(HPLC) detection showed that the obtained oxygen-tolerant strain Aeroto-AUH-JLC2H-257 could not bioconvert the substrate genistein or daidzein to 5-hydroxy-equol or equol anymore; however, this bacterial mutant strain could convert the substrate genistein or daidzein to DHG or DHD respectively. It is known that DHG and DHD are the precursors for production of 5-hydroxy-equol and equol respectively. In addition, in the presence of atmospheric oxygen, both the product DHG and DHD was converted to genistein and daidzein, the phenomenon of which was called reversal conversion. However, when we added proper kinds of reducing agents to the cultural medium, such kind of reversal conversion phenomenon disappeared. Furthermore, we observed that the bioconversion capacity of Aeroto-AUH-JLC2H-257 was significantly increased when we added reducing agents to the cultural medium. The maximum bioconversion capacity for genistein and daidzein was 1.6 mmol/L and 0.6 mmol/L respectively, and the average bioconversion rate of genistein was 79.37%, and that of daidzein was 83.67% when we added 0.10%(m/v) ascorbic acid to the cultural medium.Studies on mechanisms for the oxygen-tolerant mutant Aeroto-AUH-JLC2H-257 to grow in the presence of atmospheric oxygen found four possible pathways, including oxygen consumption by the formation of NO gas and the appearance of the NADH oxidase and NADH peroxidase system, driving out of the dissolved oxygen in cultural medium by producing of H2 gas, and oxygenproof by forming the “protective coat” on the bacterial cell surface.