De Jesus, Jordan Paul V.

March 27, 2015

CAS 02 601 P

Sir. Bigol

Strain improvement Finals: Strain improvement of C. Glutamicum

Introduction
Corynebacterium glutamicum is a soil-dwelling Gram positive bacterium that
belongs to a group of bacteria that ahas a high G.C content which is the
Actinobacteria. C. glutamicum has the ability to secrete amino acids such as Lglutamate and L-lysine. Due to the high industrial importance of these amino acids as
food flavor enhancer and food additives C. glutamicum is an organism most studied by
researchers. Likewise, C. glutamicum is then a model organism for other members of
the suborder Corynebacterianeae. This suborder includes important pathogens such
as Mycobacterium tuberculosis,Mycobacterium leprae, and C. diphtheriae.
The Corynebacterianeae has a cell wall components that give unique
characteristics to the cell wall. Though Corynebacterianeae has a thick peptidoglycan
layer like other Gram positive bacteria, they have a second permeability barrier formed
by a bilayer of mycolic acids on the cell surface. Mycoloyl residues are covalently liked
to arabinogalactan, another cell wall component of Corynebacterianeae. The cell
morphology ofCorynebacterianeae is diverse. The name giving coryne-form (clubshaped) is often observed, however, cells can form as classical rods or cocci,
depending on growth conditions.
The members of the coryneform group are found to be the lack of pathogenecity,
lack of spore forming ability, high growth rates, relatively limited growth requirements,
absence of native extracellular protease secretion and relative stability of the
corynebacterium genome itself. Due to the up to date researches in this bacteria coms
the robust industrial processes that makes it more competitive with those of E. Coli, B.
Subtilis, or yeast based processes.
Bacteria belonging to the genus Corynebacterium are due to their ability to
produce and secrete a number of industrially important amino acids and nucleotides
used in industrial production processes, in particular for large scale production of the
amino acids glutamate and lysine. Lysine is produced in an aerobic fermentation
process using the bacterium Corynebacterium glutamicum or Escherichia coli. Lysine is
an essential amino acid for animals, and since the content of lysine often is suboptimal
in corn, barley and wheat, the feedstuffs traditionally used as the major ingredients for

animal feed, this amino acid often becomes limiting for feed efficiency. Supplementing
lysine in concentrations between 0.5% and 1% to the feed leads to an optimized protein
utilization of the feed which improves the growth of especially pigs and poultry with up to
20%. In addition to the economical benefit from the increased productivity, less nitrogen
is released to the environment, which is an issue that has received a lot of attention in
recent years.
Due to the presesnce of a thick wall, C. Glutamicum was described to be
subjected in some techniques such as chemically induced DNA uptake system,
protoplast transformation or electro transformation, in addition to transduction and
conjugation techniques.
Strain improvement of C. Glutamicum
The over expression of each gene lysC, dap A, dap B and asd revealed that sole
over expression of wild type dihydropicolinate synthase resulted in lysine formation but
in a lower amount (11mM) and the other enzymes had no effect on lysine secretion in
C. Glutamicum. (L. Eggeling et al. 1991). In this particular research, researchers will be
using the the classical mutagenesis technique which will allow for the over expression of
the dap A synthase which is a gene of C. Glutamicum.
The goal of this research is to improve the L-lysine yield of C. Glutamicum
through the over expression of the dap A synthase.
The researchers will be using the C. Glutamicum strain ATCC 13032 which will
be isolated in auxotrophic mutants through the penicillin selection technique. The strains
will be cultured in the medium and will be fermented in the bioreactor. After the
fermentation process, the glucose content wil be determined through the anthrone
method and the residual sugars will be determined in the supernatant fluid through the
colorometric DNS method of Miller. The bacterial cells will then be subjected to
centrifugation in preparation for the PCR and Agarose gel electrophoresis for the DNA
separation technique. After which, the separated DNA fragments will be subjected for
the restriction digestion of the selected gene and ligation.

References:
Cited from: Faculty of Biology, Department I Microbiology Ludwig Maximillians
University; Bacterial Cell biology
Retrieved at: http://www.bacteriology.bio.lmu.de/research/coryne/index.html
Meenu S., Muralidhara Rao et al. (2011) Approaches improving the production of Llysine in C, glutamicum. Int. J. Res. Pharm. Sci. : 2(4)
Ramesh Malothu, Meenu Singh et al. (2012) OVER EXPRESSION OF
ASPARTOKINASE GENE IN CORYNEBACTERIUM GLUTAMICUM FOR HIGHER
PRODUCTION
LEVELS OF L-LYSINE; International Journal of Biological &
Pharmaceutical Research.; 3(6): 758-761.
Mateos L, Ordonez E. Et al. (2006) C. Glutamicum as a model bacterium for the
bioremediation of arsenic. International Microbiology; 9: 207-2015