Improvement in the
Production of L-Lysine by Over-expression of
Aspartokinase (ASK) in C. glutamicum ATCC 21799
Hilda Rastegari1,
Mohsen Chiani2, Azim Akbarzadeh2,
Sara Cheraghi3, Zahra Saffari2,
Mohammad Reza Mehrabi2, Ali Farhangi2
and Soheil Ghassemi2*
¹Department of Microbiology,
Azad University, Jahrom, ²Department of Pilot
Biotechnology, Pasteur Institute of Iran, ³Department of
Biochemistry, Payam_E_Noor University, Tehran,
Iran
*For correspondence: E-mail:
ghassemi_so@yahoo.com Tel: +98 21 6696
88 56; Fax: + 98 21 6646 51 32
Received: 30 April
2012
Revised accepted:
5 December 2012
Tropical
Journal of Pharmaceutical Research, February 2013;
12(1):
51-56
http://dx.doi.org/10.4314/tjpr.v12i1.9
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.
