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.
