Cloning of the corynebacterium glutamicum ddh gene in order to increasing the lysine production

ABSTRACT

Due to wide range of application of L-Lysine, as an essential amino acid, in different industries, the demand for this amino acid has been increased and this has been led to the development of research on industrial production of L-Lysine. In this study, we tried to increase the yield of production of L-Lysine by genetic optimization. After inspection of different effective enzymes in Lysine biosynthesis pathway, Diaminopimelate dehydrogenase [EC 1.4.1.16] enzyme was selected. This enzyme would be coded by ddh gene. After chromosomal DNA extraction of C.glutamicum ATCC 21799 and designing of primers, the gene fragment was separated from genome by PCR withpfu DNA polymerase enzyme and was cloned in TAcloning vector for facilitation of cloning and determination of nucleotide sequence. Then, enzymatic digestion of TAcloning vector and pET28a vector were performed by EcoRI and Sail restriction enzymes which their products were ddh gene and linear vector. Ligation reaction was accomplished and for checking the accuracy of ligation, it was transformed into E.coliDH5a and finally in expression host, E.coli BL21 [DE3]. The 1150bp band in PCR products and enzymatic digestion of extracted vectors with EcoRI and Sail, sequencing and SDS-PAGE confirmed the accuracy of cloning. Recombinant bacterial colonies were investigated and confirmed by two methods [PCR and enzymatic digestion]. This study showed significant increased expression rate of DAP dehydrogenase enzyme in this expression vector for the first time