Regulation Of Exogenous Protein Expression By Promoter Containing Cre Sequence

Carbon catabolite repression(CCR)effect is an important factor hindering the efficient expression of heterologous proteins.Through mutation of Catabolite responsive element(CRE)site on promoter,genetic modification of host strain,optimization of fermentation conditions and other methods,CCR effect can be accurately and effectively alleviated,and the transcription level of target gene in glucose as carbon source medium can be improved.It has important theoretical and application value.In this project,the promoter on the highly efficient expression vector p LY-3 was used as the research object,and the B.amyloliquefaciens were used as the host bacteria,and the Ccp A protein coding gene was first knocked out by homologous recombinant technology to study the effect of its deletion on the CCR effect,but it was found that the ccp A gene could not be knocked out,and the CCR effect of the bacterium could not be alleviated by constructing a gene deletion strain of ccp A.Then,by studying the effect of its mutation on the CCR effect by the mutation of the cre site on the promoter,the plasmids with single base mutations of the cre site conserved sequence and the plasmid with the complete deletion of the cre site conserved sequence were constructed,and the mutant plasmids were screened by green fluorescent protein,and the results showed that the G mutation of the 3rd base of the cre site conserved sequence to C could greatly increase the fluorescence intensity of GFP in the fermentation broth,compared with the plasmids at the cre site that were not mutated.The fluorescence intensity of GFP in the basic medium and fermentation medium with glucose as the carbon source increased by115.43% and 135%.In order to verify the effect of the conservative sequence mutation of the cre site on the transcription of the target gene,p LY-3 plasmid vectors carrying different exogenous protein genes were constructed,and their transcription levels were measured;the results showed that the 3rd base of the conservative sequence of the cre site was Compared with the control plasmid,the p Mcre-2G>C expression vector whose G mutation is C carries the transcription levels of alkaline protease,mesophilic amylase,aminopeptidase and keratinase genes,which are increased by 206%,117%,191% and 267%.Finally,the glucose concentration in the fermentation medium of the constructed recombinant strains 19030/p Mcre-2G>C-apr E and 19030/p Mcre-2G>C-ke was optimized,and the effect of glucose concentration on the transcription level of the target gene was analyzed.The results showed that When the glucose concentration in the medium is 5%,the promoter efficiency of the cre site mutation is the highest,which is most conducive to the transcription of the target gene,which provides a reference for further realizing the highefficiency expression of heterologous proteins.