Author/Authors :
Rastegari, Hilda islamic azad university - Department of Microbiology, ايران , Chiani, Mohsen Pasteur Institute of Iran - Department of Pilot Biotechnology, ايران , Akbarzadeh, Azim Pasteur Institute of Iran - Department of Pilot Biotechnology, ايران , Cheraghi, Sara payame noor university - Department of Biochemistry, تهران, ايران , Saffari, Zahra Pasteur Institute of Iran - Department of Pilot Biotechnology, ايران , Mehrabi, Mohammad Reza Pasteur Institute of Iran - Department of Pilot Biotechnology, ايران , Farhangi, Ali Pasteur Institute of Iran - Department of Pilot Biotechnology, ايران , Ghassemi, Soheil Pasteur Institute of Iran - Department of Pilot Biotechnology, ايران
Abstract :
Purpose: To clone Corynebacterium glutamicum ATCC21799 aspartokinase gene (EC 2.7.2.4) using shuttle expression vector pEKEx2 in order to increase lysine production. Methods: C. glutamicum DNA was extracted and used for amplification of aspartokinase gene (ask) by cloning into an E. coli/C. glutamicum shuttle expression vector, pEKEx2. Initially, the recombinant vector transformed into E. coli DH5α and then into C. glutamicum. Results: Electrophoresis of recombinant protein by SDS-PAGE showed that the molecular weight of the recombinant protein was 42 KD. The induction of recombinant vector by IPTG had an inhibitory effect on cell growth due to over-expression of the cloned gene. The results of lysine assay by Chinard method showed that lysine production increased about two-fold, compared with the parent strain, as a result of increased copy numbers of lysC gene in recombinant strain. Conclusion: A two-fold increase in lysine production was observed by cloning of the ASK gene in C. glutamicum rather than in E. coli, due to the presence of lysine exporter channel which facilitates lysine extraction.
Keywords :
LysC gene , Corynebacterium glutamicum , L , lysine , Cloning , Aspartokinase , E. coli
