The Effect Of CcpA Deficiency On Carbon Metabolism And Aminopeptidase In Bacillus Cereus CZ

Bacillus cereus CZ,a self screening bacterium in our experiment,can produce highly active extracellular L-leucine aminopeptidase.It was found that carbon source had a significant effect on the production of aminopeptidase in previous fermentation optimization,while the other factors had little effect.Since the ccp A gene is the global regulatory factor responsible for the carbon metabolism of the central carbon metabolism in Gram-positive bacteria with low GC content,our laboratory intends to explore whether there is some connection between Ccp A and aminopeptidase,and attempts to increase the production of aminopeptidase.The temperature sensitive plasmid p KSV7 was used to obtain the ccp A defective mutant—CZ?ccp A and construct ccp A gene complementation strain CZ1 for phenotypic validation of knockout strains.There was no difference in the growth and metabolism of the three strains in the LB medium,indicating that the deletion of ccp A did not affect the activity of the strain.Shaking flask fermentation experiments showed that the deficiency of ccp A could partly relieve the inhibition of biomass of the strain by high concentration of nitrogen,on the other hand,it could improve the production of aminopeptidase.When fermenting with non-PTS carbon source such as D-xylose and L-arabinose,the activity of aminopeptidase of CZ?ccp A was higher than that of CZ and CZ1,especially when the D-xylose was used as a single carbon source and 1.5 percent urea as nitrogen source for 84 h,the enzyme activity of the deleted mutant strain reached 337.24 U·m L-1,which was 48.25 percent higher than that of wild strain CZ.During the fermentation of sodium citrate,the pH of CZ?ccpA was significantly different from CZ and CZ1.When Mannan oligosaccharides were used as carbon source,the CZ and CZ1 died rapidly while CZ?ccp A grew normally,etc.In view of these results,we carried out further experimental research.By using Real-Time PCR analysis,it was found that the ccp A gene deletion strain of Bacillus cereus CZ can reduce glycolysis and improve the expression flux of TCA cycle,which would weaken the carbon overflow and make carbon source more effectively utilized.In addition,the enzyme production of CZ?ccp A was lowerthan that of CZ and CZ1 when PTS carbon source was used,but the result was just opposite when non-PTS carbon source was used,which may be related to the regulation of glutamate synthase by glt AB operon,and the mechanism of aminopeptidase production is reasonably speculated in this paper,but it can not be verified exactly because of the time relationship.