Isolation, Oxygen-tolerant Domestication Of Isoflavone Biotransforming Bacteria And Oxygen-tolerant Mechanisms For The Obtained Oxygen-tolerant Mutant

Soy isoflavones, which are mainly composed by daidzein, genistein and glycitein, aresecondary metabolites, formed in the growing process of soy plants. Soy isoflavones are ofa wide range of physiological functions, such as antioxidant and anticancer activity, lipidlowering, prevention of osteoporosis and improvement of women’s menopausal syndromeand so on. After being absorbed by the gastrointestinal tracts of human beings or otheranimals, soy isoflavone will be degraded into different metabolites. Daidzein, for example,will be converted into dihydrodaidzein (DHD), equol and O-desmethylangolensin(O-Dma).A Gram-negative strictly anaerobic bacterium, which we named AUH-Julong365,capable of bioconverting isoflavone daidzein was isolated from human feces. Basic LocalAlignment Search Tool (BLAST) search on the GenBank revealed that the full-length of16S rDNA sequence for strain AUH-Julong365has the highest similarity to that ofEggerthella lenta ATCC25559(AF292375), the similarity of which was98.6%. Based onHPLC retention time, UV spectrum, ESI-MS and1H-NMR analyses, the metabolite ofdaidzein by strain AUH-Julong365was identified as equol. Furthermore, we found that thebiosynthesized equol from daidzein by strain AUH-JLC365was100%S-equol based onHPLC analysis by using a chiral column. The maximal concentration of the substrate thatbacterium strain AUH-Julong365may bioconvert was0.6mmol/L. In order to improve theanti-oxygen capability of the obligate anwerobic bacterium strain AUH-Julong365, wecarried out an oxygen-tolerant domestication process. However, in the end we did notobtain an oxygen-tolerant mutant which can not only grow but also show itsbiotransforming activity in the presence of atmospheric oxygen.Bacterium strain AUH-JLC140, which was isolated from the fresh coke feces, is astrictly anaerobic Gram-positive bacterium capable of bioconverting daidzein intoO-desmethylangolensin (O-Dma) under anaerobic conditions. Strain AUH-JLC140and itsapplication in biosynthesis of O-Dma were applied for national patent in November2011,the patent of which was issued by the state intellectual property office in October2012(patent number: ZL201110367963.1). An oxygen-tolerant domestication was carriedout to improve the oxygen-tolerant ability of Clostridium sp. AUH-JLC140. Then we gotan oxygen tolerant strain which we named Aeroto-AUH-JLC140, capable of anaerobicallybioconverting isoflavone daidzein to O-desmethylangolensin (O-Dma). In order to improvethe anti-oxygen capability of the obligate anwerobic bacterium strain AUH-JLC140, wecarried out an oxygen-tolerant domestication process. In the end, we successfully obtainedan oxygen-tolerant mutant, which we named Aeroto-AUH-JLC140, the mutant of whichnot only grew but also transformed the substrate daidzein to O-Dma.Strain Aeroto-AUH-JLC140differed from the original obligate anaerobic bacteriumAUH-JLC140by various characteristics, including a change in bacterial shape (frommiddle-to-long rods to short clumped rods; forming a “protective coat” on the surface ofthe bacterial cell), in biochemical traits (from indole negative to indole positive; fromamylohydrolysis positive to negative; H2S production from negative to positive). The average bioconversion rate of daidzein by strain Aeroto-AUH-JLC140grown aerobicallywas81.9%when the initial concentration of the substrate daidzein was or was lower than0.6mmol/L. When we added L-cysteine (0.05%, m/v) in the cultural medium, the averagebioconversion rate of daidzein was improved from81.9%to94.3%. The mechanisms thatthe oxygen-tolreant mutant Aeroto-AUH-JLC140can endure oxygen include: H2Sproduction capability; antioxidant “protective coat”; changing in bacterial shape andincreased bacterial growth rate etc.