| Recently, Bacillus subtilis spore preparations are promising probiotics and biocontrol agents, which can be used in plants, animals and humans. However, higher production cost limitited its wide application in agriculture. How to increase the production of B.subtilis, especially the spore yields of current industrial B.subtilis, to lower its spend became one of the key strategies for developing cheap B.subtilis preparation with high spores concentration.The first aim of this work was to increase industrial B. subtilis WHK-Z12spores production to lower its cost by optimizing the nutritional conditions through a statistical approach. Our preliminary experiments showed that corn starch, corn flour and wheat bran were the best carbon sources, while corn steep liquor, soybean flour and yeast extract were the suitable nitrogen sources. Using Plackett-Burman design, corn steep liquor, soybean flour and yeast extract were found to be the key medium ingredients for enhancing spore production, and were studied for further optimization using central composite design. The key medium components in our optimization medium for the highest predictable spore yield were16.18g/1of corn steep liquor,17.53g/1of soybean flour and8.14g/1of yeast extract. In our verification experiment, the improved medium produced spores as high as1.52±0.06×1010spores/ml under flask cultivation conditions, and1.56±0.07×1010spores/ml could be achieved in a30-1fermenter after40h of cultivation. To the best of our knowledge, these results compared favorably by an increase of100%to the documented spore yields produced by B. subtilis strains.The second intention of this study was to develop an optimal strategy for preventing B. subtilis168cell lysis during its high temperature submerged culture through knocking-out its several lytic enzyme related genes by multiple genes inactivation method. For convenience research, B.subtilis168that is easy for genetic manipulation was chosen as the host. The following lytic enzyme related genes were studied. xpf(also named pcf) coded a PBSX RNA polymerase sigma factor-like (Pcf) in prophage PBSX. Pcf was necessary for transcription of PBSX lytic enzymes (Xh1A, Xh1B, XlyA and XepA). skfA coded a sporulating killing factor (SkfA) in prophage1, and SkfA could kill sister cells under nutrition limitation. lytC coded a major vegetative autolysin (LytC) in B. subtilis. sdpC coded a sporulating delay protein (SdpC), which could strengthen the activity of SkfA. In prophage skin element cw1A coded an autolysin (Cw1A), yqxH coded a protein similar to holing (YqxH), yqxG coded a protein similar to phage-related lytic exoenzyme (YqxG). The DNA fragment containing cwlA, yqxH, yqxG was named as yGlA in this work. Then, a serial of mutants of B.subtilis168were constructed by using five knocking-out vectors (pNNB194-△xpf, pNNB194-△skfA, pNNB194-△lytC, pNNB194-AsdpC and pNNB194-△yGlA), and we evaluated the effects of these mutants on preventing cell lysis in B.subtilis168by monitoring their growth speed, biomass, viable cell concentration cultured at37℃culture. We found some interesting results:a.△skfA, AlytC,△sdpC or Axpf could resist to cell lysis, while AskfA could effectively inhibit cell lysis among them. However,△yGIA had little effect on lysis. b.△skfA△sdpC became the optimal strategy for preventing B.subtils cell lysis under high temperature culture with its rapid growth speed of colony, the highest biomass and viable cell counts. c.△skfA,△lytC and AsdpC did some damage to spore production,△yGIA had no effect on spore production, while Axpf could be helpful for increasing spore production. These findings will provide some theorial and technical bases for controlling B.subtlis cell lysis during industrial production through strains improvement, especially under high temperature culture. |
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