Enhanced Oxygen-tolerant Capacity And Aerobic Bioconversion Efficiency Of Oxygen-tolerant Mutant Derived From Isoflavone Biotransforming Bacterium By Protoplast Fusion

To date, all the reported isoflavone biotransforming bacteria are strictlyanaerobes which can only exhibit their isoflavone biotransforming activity understrictly anaerobic conditions. In order to improve the oxygen-tolerant capacity ofisoflavone biotransforming bacteria, an oxygen-tolerant mutant designatedAeroto-Niu-O16was first obtained by our lab from an obligate anaerobic bovinerumen bacterium Niu-O16after a long-term oxygen-tolerant domestication process.The oxygen-tolerant mutant strain Aeroto-Niu-O16not only grew but also convertedisoflavones daidzein and genistein to dihydrodaidzein (DHD) and dihyrogenistein(DHG) in the presence of atmospheric oxygen. Compared with the original obligateanaerobic bovine rumen bacterium strain Niu-O16, the oxygen-tolerance of themutant strain Aeroto-Niu-O16was obviously improved. However, on the other hand,the bioconversion efficiency of strain Aeroto-Niu-O16was significantly lower thanthat of strain Niu-O16under obligate anaerobic conditions. In this study, in order tofurther improve both the oxygen-tolerant capacity and the isoflavone bioconversionefficiency of the oxygen-tolerant mutant strain Aeroto-Niu-O16, protoplast fusion wascarried out between the strain Aeroto-Niu-O16and a predominant intestinal aerobicbacterium isolated from animal feces.Among all the previously isolated20predominant intestinal aerobic bacteria,only five bacterial strains including strain M3, M4, M5and M6isolated from ICRmice feces and strain Z5isolated from pig feces were sensitive to lysozyme. Furtherstudy showed that among all the5lysozyme sensitive strains mentioned above, onlystrain Escherichia fergusonii M6isolated from ICR mice had both the highestprotoplast preparation rate and the highest regeneration rate. Therefore, we chosestrain M6as the predominant aerobic bacterium for protoplast fusion study. Weinvestigated different factors that influence the protoplast preparation andregeneration rate, the factors of which include bacterial age of strain M6, enzymeconcentration, enzymolysis time, osmotic pressure stabilizer and addition of differentchemical compounds. Finally we determined the optimal conditions that suitable for protoplast preparation and regeneration of strain M6. The protoplasts of strain M6washeat-inactivated before protoplast fusion. Polyethylene glycol (PEG)-assistedprotoplast fusion was carried out between the heat-inactivated protoplasts of strain M6and non-heat-treated normal protoplasts of strain Aeroto-Niu-O16. One fusant, whichwe named AUH-RHZ-1, was obtained. We found that the fusant strain AUH-RHZ-1not only grew but also converted isoflavones daidzein and genistein to DHD and DHG,respectively, in the presence of atmospheric oxygen.The fusant strain AUH-RHZ-1inherited two characteristics, i.e. the positiveamylohydrolysis activity and the capability to produce hydrogen gas, from theparental strain M6, However, the fusant was identical or similar to the other parentalstrain Aeroto-Niu-O16by various characteristics, including cell morphology,biochemical traits,16S rDNA sequences, patterns of random amplified polymorphicDNA (RAPD) and esterase isoenzyme, etc. It is worthy to noticing here that ananti-oxidant “protective coat” forms during the growth of the parental strainAeroto-Niu-O16in the presence of atmospheric oxygen, however, such a similarstructure was not detected on the fusant strain AUH-RHZ-1when it was grown underexactly the same conditions. Further more, we found that the tolerance to certainantibiotics, including metronidazole, azithromycin and gentamicin, of the fusant strainAUH-RHZ-1was obviously enhanced compared with its parental strains. Study onthe oxygen-tolerant capacity, including the kinetics of the oxidation-reductionpotential (ORP), the influence of the inoculum concentration and the culture depth ofthe medium in cultural tube, demonstrated that the oxygen-tolerance of the fusantstrain AUH-RHZ-1was obviously improved. On the other hand, compared with thatof the parental strain Aeroto-Niu-O16, the aerobic bioconverting efficiency ofisoflavone daidzein at different concentrations was obviously increased, and that ofisoflavone genistein at different concentrations was also increased to different extent.This is the first report demonstrating that protoplast fusion is an efficient way toimprove both the oxygen-tolerant capacity and the bioconverting efficiency of anoxygen-tolerant isoflavone biotransforming bacterial mutant.