| Vitamin C(Vc)is an essential vitamin for the human body and is widely used in various industries due to its free radical scavenging and antioxidant properties,and has a huge share in the global vitamin market.“The two-step fermentation method” is the main method for industrial production of vitamin C.The key step is to promote the growth of Ketogulonigenium vulgare and produce Vc precursor 2-keto-L-gulonic acid(2-KLG)by Bacillus spp.companion.Significant differences in the growth status and physiological metabolism of K.vulgare in the mixed bacterial system compared to its mono-bacterial fermentation.Studies have shown that quorum sensing of bacteria is involved in a variety of physiological processes and plays an important role in regulating bacterial growth.Therefore,the interaction between B.pumilus A9 and K.vulgare 25B-1 was explored by analyzing the effect of the quorum quenching lactonase Ytn P produced by B.pumilus A9 on K.vulgare 25B-1 quorum sensing.This study will provide some new clues to reveal the mechanism of massive growth and 2-KLG production of K.vulgare in the company of Bacillus spp.,and also provide some new ideas for the further research of Vc two step mixed bacterial fermentation.The main results of the study are as follows:(1)The 2-KLG production of K.vulgare 25B-1 in the mixed system was much higher than that in the monoculture system,and the growth status of both B.pumilus A9 and K.vulgare25B-1 was better than their individual growth.In addition,the concentration of the two bacteria was higher than that of the single bacteria system,but the expression of K.vulgare 25B-1quorum sensing system genes lux I and lux R in the mixed bacterial system was significantly lower than that in the mono-bacterial fermentation system,and the trend was negatively correlated with the concentration of K.vulgare 25B-1 and the amount of 2-KLG production.The quenching lactonase gene ytnp expression of B.pumilus A9 in the mixed bacterial system showed a significant increase during the period of massive accumulation of 2-KLG in K.vulgare 25B-1,and the production of B.pumilus A9 spores was more and earlier than that of monocultures,which was presumably related to the stress produced by K.vulgare 25B-1.The p H of the mixed bacterial system was maintained at about 6-7,which was favorable for B.pumilus A9 spore formation and the release of the quenching lactonase Ytn P.(2)The results of streak plate between K.vulgare 25B-1 and Agrobacterium tumefaciens KYC55 indicated that K.vulgare 25B-1 was able to produce AHL signaling molecules during the growth process.The gene ytnp encoding Ytn P in B.pumilus A9 was successfully cloned by homologous cloning.32.3 k Da Ytn P is a hydrophilic protein with no transmembrane structural domain,no signal peptide,a non-secretory protein,and contains a conserved structural domain of Lactamase＿B belonging to the metallo-beta-lactamase family and its conformation exists mainly as random coil.(3)The p ET-32a-ytnp expression vector was successfully constructed and transformed into E.coli BL21(DE3).The Ytn P recombinant protein was obtained by induction with IPTG.Ytn P was expressed as inclusion bodies,denatured with high concentration of urea and then purified and dialyzed for renaturation,and the final enzyme activity assay showed that the obtained Ytn P had AHL lactonase activity.(4)The 2-KLG production of K.vulgare 25B-1 increased by 11 % compared with that of the monoculture after the addition of Ytn P at a final concentration of 5 μg/m L,and by 14 %compared with that of the monoculture after the addition of Ytn P at final concentrations of 10μg/m L and 20 μg/m L.K.vulgare 25B-1 showed autolysis during monoculture growth,and the ORP was at a relatively high value in the first and middle growing stages,indicating that autolysis may be influenced by its own oxidative stress.The addition of Ytn P reduced the autolysis during the growth of K.vulgare 25B-1,and it is assumed that the AHL quorum sensing of K.vulgare 25B-1 is involved in regulation of autolysis process.(5)The expression levels of the genes encoding superoxide dismutase(SOD)and NADPH:quinone oxidoreductase s were significantly higher in K.vulgare 25B-1 monoculture system at the middle-growth stage after the addition of Ytn P,and it is assumed that K.vulgare25B-1 was unable to reduce the oxidative stress during growth through the autolysis process involved in AHL quorum sensing and increased the expression of antioxidant genes to cope with the oxidative stress.At the same time,K.vulgare 25B-1 increased the expression of isocitrate dehydrogenase gene after adding Ytn P,which may provide more reducing power to cope with oxidative stress. |
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