| Corynebacterium glutamicum is widely used in industrial production of various amino acids.Because C.glutamicum has protein secreting ability but does not produce extracellular proteases,it is considered to be an ideal recombinant protein-producing host bacterium.It is particularly important for C.glutamicum expression system to efficiently express recombinant protein in C.glutamicum.Promoter is an important component of gene expression vector and an important cis element of gene expression regulation.The strong promoter is a key gene regulatory element in the construction of recombinant protein overexpression by genetic engineering and is also an important gene regulatory element in the metabolic engineering of host bacteria.The study on promoters is of great significance for the construction of genetic engineering vectors,metabolic engineering strains and expression of target proteins.Identifying strong promoters can provide efficient gene expression regulatory elements for C.glutamicum metabolic engineering breeding and recombinant protein production,but the existing promoters have low transcriptional activity,which can not meet the needs of C.glutamicum expression system modification.The identification of endogenous strong promoter of C.glutamicum is rarely reported.In this study,proteins with high abundance expression were selected based on 2-d electropHoresis of C.glutamicum strain.After the 5’-end upstream sequence of the target protein gene was determined,the promoter sequences were firstly predicted by bioinformatics method,the promoter elements were analyzed and predicted,and the transcriptional activity of the promoter sequence was verified by experimental methods.By this method,the endogenous gene promoter of C.glutamicum ATCC13032 strain was screened and predicted.Firstly,two-dimensional electropHoresis of the whole protein was carried out on the cells of the C.glutamicum ATCC13032 strain and the high-expression protein spots were analyzed by mass spectrometry.16 high-abundance expressed protein spots were selected,and their corresponding proteins were obtained by using the protein analysis software based on the genomics data.A total of 55 high-abundance expressed proteins were screened out.Secondly,the promoter prediction software was used to predict the sequences of 55 protein-coding genes.The promoter prediction results showed that the promoter transcriptional activity of C.glutamicum Cgl0229,Cgl0841,Cgl0864,Cgl1249,Cgl1607,Cgl1615,Cgl1805,Cgl2060,Cgl2267,Cgl2988,Cgleno,Cglgap and Cgl1dh genes was strong.The Ptac-M promoter is a known exogenous strong promoter mutated from the E.coli Ptac strong promoter with high transcriptional activity in C.glutamicum strain.We took the Ptac-M strong promoter as a reference object and compared the predicted screened promoter with the Ptac-M strong promoter to determine the transcriptional activity of the promoter.Then,the predicted promoters P0229、P0841、P0864、P1249、P1607、P1615、P1805、P2060、P2267、P2988、Peno、Pgap、Pldh and Ptac-M were inserted into the promoter-probe vector pDXW-11 with cat as the reporter gene,and the new recombinant promoter-probe vector was successfully constructed.The competent cells of ATCC13032 strain were transformed to obtain the engineered strain C.glutamicum ATCC13032,Cglutamicum/pDXW-11-P0062.C.glutamicum/pDXW-11-P0215,C.glutamicum/pDXW-11-P0229,C.glutamicum/pDXW-11-P0841,C.glutamicum/pDXW-11-P0864,C.glutamicum/pDXW-11-P1249,C.glutamicum/pDXW-11-P1607,C.glutamicum/pDXW-11-P1615,C.glutamicum/pDXW-11-P1805,C.glutamicum/pDXW-11-P2060,C.glutamicum/pDXW-11-P2267,C.glutamicum/pDXW-11-P2988,C.glutamicum/pDXW-11-Peno,C.glutamicum/pDXW-11-Pgap,C.glutamicum/pDXW-11-P/dh and C.glutamicum/pDXW-11-Ptac-M.Finally,we verified the transcriptional activity of 16 promoter sequences by detecting the chlorampHenicol tolerance of each engineered strain,the chlorampHenicol acyltransferase activity of the reporter protein CAT,and the transcriptional level of chlorampHenicol acetyltransferase gene CAT by fluorescence quantitative PCR.The final transcriptional activity of the promoter fragment was from high to low:P0864>Ptac-M>P 1607>P0841>P 1249>P 1615>Pgap>P0215>P0229>P2060>P0062>P2267>P2988>Peno>Pldh>P1805. |
