Identification And Appplication Of Endogenous Expression Element 5'UTR In Corynebacterium Glutamicum

Corynebacterium glutamicum has unique advantages,such as no endotoxin,easy to achieve extracellular secretion of target protein,low extracellular hydrolase activity,and almost no secretion of endogenous protein,etc.,is an ideal host for heterologous protein expression.However,there are few high-efficiency expression vectors currently available in C.glutamicum,and the development of novel high-intensity expression vectors is of great significance for the industrial application of C.glutamicum.Aiming at these problems,this project has done the following research.Serveral high-intensity expression vectors were developed by identifying the 5'UTR and its downstream sequences of genes with high expression in C.glutamicum and constructing monocistronic expression module(MEM)and bicistronic expression module(BEM)respectively.First,based on previous study,12 genes were selected according to the abundance of its protein expression level in C.glutamicum,and successfully identified the 5'UTR and downstream sequences of these 12 genes through the 5'RACE method,9 of which have 5'UTR sequence,while other 3 genes lack the 5'UTR sequence.Different 5'UTR sequences were combined with the constitutive promoter PH36 to form a new MEM pH36-UTR.Using enhanced green fluorescent protein(EGFP)as a reporter protein,it was found that 5'UTR can significantly affect the transcription activity of the PH36 promoter and the translation eff-iciency of the reporter protein.Subsequently,the 5'UTR and its downstream 62 bp sequence were combined with the promoter PH36 to form a MEM pH36-BUTR.Compared with BEM,the fluorescence intensity increased by 1.2 to 27.8 times respectively.Then,the promoter PH36 was replaced with another high-intensity inducible promoter Ptac to form a new BEM pTac-BUTR,three high-intensity expression vectors were developed and named as pTac-B2826/B2473/B2328-egfp.Among them,the fluorescence intensity of pTac-B2826-egfp expressing EGFP is about 3.6 times that of pTac-positive-egfp.EGFP protein was replaced with VHH protein(variable domain of heavy chain of heavy-chain antibody),which was successfully and efficiently secreted in C.glutamicum.All those results confirmed the stability of the developed expression vectors in C.glutamicum.The expression mechanism of genes without 5'UTR sequence was explored.The 62 bp downstream of the start codon of 12 genes were directly combined with promoter Ptac to form BEM pTac-LUTR.It was found that the downstream 62 bp sequence of those genes lacking the 5'UTR sequence had a significantly higher impact on the expression module than the downstream 62 bp from those genes without the 5'UTR sequence.At the same time,the start codons of all 62 bp sequences were replaced with AUG,and it was found that compared with the 62 bp sequence with the start codon GUG,its expression ability was further improved.Which shows that the start codon AUG plays an important role in the gene expression without 5'UTR sequence.The C-terminal domain of protein ApxIII(A.pleuropneumoniae toxin III,ApxIII-C)was expressed and secreted in C.glutamicum using the high expression vector selected above.The full-length protein ApxIII cannot be efficiently expressed in C.glutamicum,and it has a strong inhibitory on bacterial growth.The ApxIII protein is divided into five domains,one of them C-terminal domain(ApxIII-C)was reported have the same immune activity with ApxIII protein.And ApxIII-C protein was expressed successfully using the highest intensity expression vector pTac-B2826 in C.glutamicum.Subsequently,the signal peptide and the fermentation conditions were further optimized trying to improve the expression level of ApxIII-C protein.It was found that the CSPB2 signal peptide,the Sec secretory pathway,can achieve the best secretory expression of the ApxIII-C protein C.glutamicum.Under the best fermentation condition,the expression of ApxIII-C protein produced by C.glutamicum reached 87.56 mg·L-1 in shake flask.