Expression And Application Of The Genes Belonging To The CglI Restriction Modification System In Corynebacterium Glutamicum ATCC13032

Corynebacterium glutamicum is an important industrial microorganism and has been widely used for the industrial production of various amino acids.At present,metabolic engineering breeding has become the main way to obtain high-yield strains of C.glutamicum.However,the low DNA transformation efficiency on C.glutamicum is one of the major obstacles in metabolic engineering breeding.An important factor affecting the efficient transformation is the presence of restriction modification system in the C.glutamicum.Restriction and Modification System refers to a complex enzyme system consisting of a single subunit or a multi-subunit consisting of restriction enzyme and methyltransferase.These two enzymes usually appear in pairs with the same DNA recognition sites,but the opposite functions.Restriction endonucleases usually act on a DNA-specific site to cleave DNA,while methyltransferases protect the bacteria by methylating the DNA so that the restriction endonucleases no longer function at that site,thus protecting the cells from invasion by bacteriophage and foreign DNA.The cglI restriction modification consisting of the R.cglI,M.cglI and H.cglI genes in the model strain C.glutamicum ATCC13032 negatively affects transformation efficiency of exogenous DNA.In this study,in order to solve the low transformation rate of C.glutamicum,the construction of an E.coli intermediate host was carried out by chromosomal integration of the M.cglI gene.In order to express restriction endonuclease gene,a vector with tight regulation of gene expression was constructed and R.cglI was expressed.The substitutability of H.cglI gene was also explored in E.coli.The main research results are as follows:AhomologousrecombinationvectorpAU4-Larm-M.cglI-cat-Rarmforgene knockout/knockin was constructed.The three structural genes galE(epimerase),galT(galactosyltransferase),and galK(galactokinase)in the gal operon of E.coli were selected to form the sequence to be replaced.The left arm Lram is selected from the downstream region of the galT gene,covers 119 bp DNA sequence at the 3?-end of galT,and 0.79 kb DNA sequence after the stop codon of galT.The right arm Rarm is selected from the upstream region of the galT gene,covers 147 bp DNA sequence at the 5?-end of galT,and0.85 kb DNA sequence before the start codon of galT.The cat gene was used as selection marker for E.coli integrants.The ligament of Larm,the cat gene,the M.cglI gene and the Rarm was achieved by serial cloning operation in plasmid pAU4.Construction of The engineered host E.coli AU1 was constructed.The M.cglI gene encoding methyltransferase in the cglI restriction-modification system of C.glutamicum ATCC 13032 was integrated into the E.coli TG1 genome by homologous recombination for expression.The effect of M.cglI methylation on the transformation efficiency was verified by electroporation of plasmids from different sources into C.glutamicum ATCC13032.The transformation efficiency of C.glutamicum with pAU4 DNA extracted from E.coli TG1 was 1.80±0.21×10~2 cfu/?g plasmid DNA,while using pAU4 DNA extracted from E.coli AU1,the transformation efficiency reached up to 5.22±0.33×10~6cfu/?g plasmid DNA.The results showed that E.coli AU1 can confer plasmid specific M.cglI methylation patterns,making plasmids extracted from E.coli AU1 resistant to R.cglI restriction endonuclease and capable of efficient transformation into C.glutamicum ATCC 13032.A E.coli vector pAU10 with tight regulation of gene expression was constructed.This expression vector utilizes a combination of the highly repressible T7-LacO promoter/operator and the strong transcriptional terminator rrnBT2 upstream of the T7promoter to strictly control the transcription of the extremely toxic gene;In addition,the antisense RNA produced by the trp promoter/operator opposite to the T7 promoter blocks the translation of leaky mRNA.Without the supplementation of IPTG and L-tryptophan in the culture medium,transcription of the extremely toxic gene by the T7 promoter is highly repressed,and the trp promoter produces antisense RNA,which strictly suppressing the leakage expression of the extremely toxic protein in E.coli.After the addition of IPTG and L-tryptophan in the medium,the T7 promoter starts to efficiently transcribe the extremely virulence genes,and the presence of L-tryptophan shuts down the trp promoter without producing antisense RNA,which ensures that the vector is efficient expression of extremely protein in E.coli.The restriction enzyme BamHI was successfully expressed in E.coli,indicating that pAU10 is an excellent expression vector for expressing extremely toxic genes.The recombinant plasmid pAU10-R.cglI was constructed and transformed into E.coli JM109(DE3),resulting in E.coli JM109(DE3)/pAU10-R.cglI expression system.The size of the expressed protein size was approx.40 KDa in Western blot analysis,showing that the restriction endonuclease R.cglI was successfully expressed in E.coli.It was verified that the expression of the same family gene of H.cglI DEAD-family helicase-like can also assist the R.cglI restriction enzyme to exert its enzymatic cleavage function.The recombinant plasmid pAU4-R.cglI was transfected to E.coli Top10,and no transformants grew.The result showed that The homologous helicase of H.cglI helicase exists in E.coli and can also function as a helper of the R.cglI protein to cut heterologous DNA.In addition,the expression and purification of the H.cglI gene was successfully performed in E.coli.