Construction Of Gene Deletion Systems In Corynebacterium Glutamicum

Corynebacterium glutamicum belongs to actinomycetales, is a high G+C content, gram-positive bacterium. It has been widely used for production of different amino acids. In recent years, metabolic engineering has been used to construct amino acid producer. Therefore, it is urgent to develop expression vectors and gene-deletion vector suitable for C. glutamicum. This study was focused on the construction of novel gene-deletion vectors of C. glutamicum. The main results are listed below:(1) The expression of sacB gene coding levansucrase under different promoter was measured in C. glutamicum. It showed the native promoter of sacB failed to efficiently promote its expression in C. glutamicum; PlacM effectively promote its expression in C. glutamicum. The activity of levansucrase expressed in C.glutamicum showed PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum.(2) Using pDXW-3 as gene-deletion vectors, aceE gene of C. glutamicum ATCC 13032 was deleted successfully. Compared with the currently available pK18mobsacB/pK19mobsacB vectors, pDXW-3 is more suitable for gene deletion and allelic exchange by homologous recombination in C. glutamicum ATCC 13032.(3) Based on the recombination/site-specific recombination system, a second novel gene deletion system in C. glutamicum was constructed. This system contains several vectors, such as pDTW109, pDTW201 and pDTW202. The vector pDTW109 harbors oriE for replication in Escherichia coli, rep (TS) for temperature-sensitive replication in C. glutamicum, cat under tacM promoter for chloromycetin resistance, cre for expressing recombinase. pDTW109 in C. glutamicum could be lost by culturing at high temperature. pDTW201 harbors a kan cassette flanking with two loxP sites, while pDTW202 harbors a kan cassette flanking with loxPLE/loxPRE.(4) Using the recombination/site-specific recombination system, the aceE gene in C. glutamicum ATCC 14067 was deleted, resulting YTW-101. YTW-101 could accumulate pyruvate, and therefore be used as a candidate for constructing amino acid producer. The ilvA gene in YTW-101 was further deleted, resulting YTW-102. YTW-102 could be a good candidate for constructing L-valine and L-leucine producer.