| Corynebaterium glutamicum as a potential host in the production of recombinant protein has aroused people's concern,which possess some particular advantages such as no endotoxins,more strong ability in secreting protein into culture and less protease in the culture supernatant compared with Escherichia coli.But it lacks expression vectors to express target genes,and those existed have low efficiency on expression level.The shortage also constrained the further application of metabolic engineering and synthetic biology on this strain.The advance of omics technologies and further understanding of genetic elements provide us with more ideas to construct expression vectors.In this study,with the help of omics information and the understanding of genetic expression cassette,strong expression parts which initiate the expression of genes could be constructed by using genomic sequence material and a creative recombinant expression cassette,achieving a series of efficient expression vectors.Major results above:?1?By analyzing the C.glutamicum transcriptomic data,six genes steadily transcribed varying different dissolved oxygen concentrations were identified from genome,and with the help of bioinformatics prediction of promoter regions,the conventional expression parts,promoter-5'UTR,were constructed.Then expression parts were assembled with reporter gene,enhanced green fluorescent protein?egfp?,in a probe vector to be evaluated for their activities.Results showed that all 6 expression parts could to some extent initiate the expression of egfp.The expression part derived from the tuf gene of C.glutamicum led to the highest expression level of egfp which was similar to that of a reported strong part Pgro.?2?In order to further improve the efficiency of expression part,according to the C.glutamicum proteomic data,12 genes with high protein abundance were identified from genome.The architecture of expression part was also changed to “promoter-5'UTR-5'coding sequenceSD2”,forming a bacterial bicistronic expression cassette.This novel expression part preserves 38-bp 5'coding sequence of source gene,recovering the original ribosome translation initiation region which plays an important roles in expression of target gene.Meanwhile the 12 sequences corresponding to the high-protein-abundance genes were also used to construct expression parts in conventional architecture and a modified architecture,respectively.By comparison,bicistronic expression parts showed obviously higher activities than conventional expression parts,leading to 1.4-790 fold EGFP expression level of the convention,which was due to the improvement of both transcriptional level and translation efficiency.The improvement of translation efficiency contribute more to final change of expression level,which may be attributed to the preservation of translation initiation region in bicistronic expression parts.Besides,we proved that the improvement comes from the whole design of bicistronic expression part but not a specific sequence.The strongest bicistronic expression parts was obtained and it is 1.35 times the EGFP expression level of the constitutive-expressed pXMJ19 which is a classic frequently-used expression vectors in C.glutamicum.?3?Expression parts derived from C.glutamicum genome were used in Escherichia coli to express egfp,testing the efficiency of parts working in heterologous environment.Those expression parts were also available in another bacterium,providing the possibility of interchangeably using expression parts among various bacteria.In E.coli,the bicistronic expression parts still showed superiority over respective conventional expression parts.Another gene,single-chain fragment variable?scFv?was used as target gene in the test of expression parts.Plasmid pXMJ19 constitutive derivate fail to work,but the strongest bicistronic expression parts performed the high-level expression of scFv.More than 100 mg·L-1 of scFv was produced in shake flask level cultivation,higher than the previously reported level of 18.12 mg·L-1. |
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