Study On The Sporulation Mechanism And Enhancement Of Sporulation Ability Of Clostridium Tyrobutyricum

Clostridium tyrobutyricum ATCC 25755(Ct)is a strictly anaerobic,spore-producing and non-pathogenic microorganism,which has been widely used in the cheap biomass fermentation because of its high production and selectivity of butyrate and butanol.C.tyrobutyricum also can be used for animal breeding with the advantages of enhancing animal intestinal health,increasing animal weight,reducing the feed conversion ratio and reducing antibiotic pollution.Under some conditions,C.tyrobutyricum forms highly stress-resistant spores to resist adverse environments,increase the survival level of the strain.However,the sporulation rate of C.tyrobutyricum is low and the sporulation mechanism is unclear.The sporulation mechanism was described first by studying the function and relationship of spo0A and sigma factors.Later the sporulation level was improved by optimizing the medium component and culture condition.Finally,the gene that could improve the sporulation ability of the strain was found and further improve the sporulation level through genetic engineering manipulation.The master gene spo0A and four sigma factors in sporulation program of C.tyrobutyricum were explored firstly.It was found that the strain lost the ability of sporulation formation and the expression level of genes involved in spore formation decreased significantly after spo0A knockout.However,the sporulation level of spo0A overexpression strain decreased to 2%of C.tyrobutyricum with the cell morphology changed significantly,and the expression level of sigma factor also decreased.Afterσ~F,σ~E,σ~G andσ~K were knocked out,no spore was produced.When sigK was knocked out,the expression levels of spo0A decreased significantly,while the transcription levels of sigF,sigE and sigG were down regulated,indicating that sigK played a role in early sporulation stage by affecting the transcription of spo0A in C.tyrobutyricum.The sporulation level,spores heat resistance and germination performance of C.tyrobutyricum were first evaluated,and the Reinforced Clostridial Medium(RCM)was selected with highest sporulation level on the third day reaching 9.3×10~4 CFU/mL.Next,the medium composition and culture conditions was optimized using single factor test and found that the spores could reach 7.61×10~7 CFU/mL when the medium composition were supplement with 10g/L xylose as carbon source,10 g/L fish meal as nitrogen source,fermentation temperature35℃,pH 5.0,1.5%inoculation size and 150 r/min rotate speed.After amplification culture in the 5 L fermentation tank three days,with 40 g/L xylose as the carbon source,the spore reached3.58×10~8 CFU/mL and the spore yield was as high as 95.6%.Nxet,the genome sequence of C.tyrobutyricum was analyzed and one global factor abrB(antibiotic resistance protein B)gene involved in sporulation was identified.The regulation ability of abrB in sporulation was found to through regulating spo0A and sigma factor.In addition,abrB also indirectly affected sporulation by affecting the fab operon participated in fatty acid synthesis pathway.abrB gene knockout significantly reduced the biofilm formation level and affected the strain resistance to low pH,high osmotic pressure and product concentration.Compared with the C.tyrobutyricum,Ct/ΔabrB significantly reduced the utilization capacity of xylose and mannose by 22.33%and 29.04%respectively,but had no effect on the fermentation capacity of glucose and fructose.In glucose/xylose mixed fermentation,abrB deficiency alleviated the inhibition of carbon metabolism,strengthened the xylose consumption ability and greatly increased the yield of butyrate(13.11 g/L vs.7.00 g/L).In glucose/mannose mixed fermentation,Ct/ΔabrB enhanced the acetate reassimilation ability,and the yield of butyrate increased by 12.61%compared to C.tyrobutyricum.In summary,this study explored the effects of the key sporulation gene spo0A and sigma factor of C.tyrobutyricum ATCC 25755 on spore formation.The role of the sporulation regulator Abr B on the sporulation was also identified and explored,enriching the sporulation of C.tyrobutyricum and its regulatory mechanism.At the same time,through the optimization of medium and culture conditions,the sporulation level was increased by 3.8×10~4 times.The cost of spore production was significantly reduced,which provided technical support for the large-scale preparation of probiotic preparations by C.tyrobutyricum...