Assessment of effectiveness of Corynebacterium glutamicum promoters and their application for the enhancement of gene activity in lysine-producing bacteria

Resumo

A pNS-cat72 vector was constructed based on the replicative vector pNS2 and reporter gene cat of Tn9 transposone, which encodes chloramphenicol acetyltransferase, in order to clone various gene promoters and to assess their chloramphenicol resistance in Corynebacterium glutamicum strains. The strength of promoters of various genes in the Corynebacterium glutamicum strain GEN1-2 (lys^{CA279T, S317A}), which contains aspartokinase (which is resistant to lysine) and threonine feedback inhibition were studied using this vector. It was found that promoters of the genes eftu, sod, cspB and leuC provide higher level of chloramphenicol resistance than promoters of the genes lysC, pyc, tkt, fbp, which are involved in the control of lysine biosynthesis. It was shown that replacement of the natural promotor ddh by the promotor sod increases the level of transcription almost by ten times, whereas the diaminopimelate dehydrogenase activity is increases by three to four times, which results in a 9% enhancement in lysine production. The investigated set of promoters with different strengths is a necessary tool for the optimization of gene activities and the construction of metabolite-producing strains.