Identification Of Strong Promoters In Corynebacterium Glutamicum Using Two-dimensional Electrophoresis And QPCR Technology

Corynebacterium glutamicum,a species of the class Actinobateria,is used for the industrial production of various amino acids.Due to its convenient biotechnological properties,it also has great potential for the large-scale production of other desirable bacterial metabolites.Corynebacterium glutamicum,as a host of the production of industrial recombinant protein,has been gone for a deeper study,for its fast growing as well as be recognized as safe.In order to produce more recombination protein,there is a need to develop an excellent performance of "gene expression box" of Corynebacterium glutamicum.The "gene expression box" include:host,plasmid,promoter;secretion pathway and other gene elements related to gene expression.The promoter is a start signal at the beginning of a gene cluster that directs the RNA polymerase to initiate RNA synthesis.As an important element of transcription,the activity of the promoter have a great influence in the start of transcription,action timing as well as the expression of exogenous gene.Therefore,it can further influence the level of transcription of exogenous gene in the host,and finally affect the expression of the target production.To some degree,use of the strong promoter can increase expression of exogenous genes.However,there is few report on identifying endogenous strong promoters in Corynebacterium glutamicum.In this study,we screen and identify the endogenous strong promoter in Corynebacterium glutamicum-in order to provide a powerful element for the construct a cell factory of Corynebacterium glutamicum.In the preliminary work,we performed two-dimensional electrophoresis of the whole cell proteins of the strain Corynebacterium glutamicum ATCC13032,analyzed some high expression protein spot,and predicted the promoters using the website.In this study,we chose 15 promoters and performed identification of the endogenous strong promoter by the comparing the predicted promoters and the strong promoter Ptac-Min Corynebacterium glutamicum.In this study,the main results are follows:(1)The recombinant promoter-probe vectors were successfully constructed by connecting these predicted promoters with the promoter-probe vector pDXW-11.(2)The recombination vector were transform into E.coli BL21(DE3),and the transformer were further cultiviated using LB plate containing chloramphenicol.The result showed that all the transformants can grow well in LB medium containing chloramphenicol,indicating(3)that all the promoter have activity in E.coli.(4)The successfully verified recombinant plasmid was transformed into Corynebacterium glutamicum,and chloramphenicol was also used as the selection condition.After screening,all promoters can grow normally in the LBHI medium which contained chloramphenicol,indicating that they all have activity in Corynebacterium glutamicum.(5)Using the real-time quantitative PCR and the chloramphenicol gradient test as well as enzyme activity detection to further test the activity of the 15 promoters.We found that the chloramphenicol tolerance of the promoter P1937 was 40 ?g/mL,and its chloramphenicol acyltransferase activity was 11.2 times higher than that of the strong promoter Ptac-M,also the qPCR result showed that he transcription level of the cat gene under the control of P1937 was 2.07times higher than that of the strong promoter Ptac-M.