ISSN 0003-6838, Applied Biochemistry and Microbiology, 2017, Vol. 53, No. 8, pp. 781–785. © Pleiades Publishing, Inc., 2017.
Original Russian Text © I.P. Tokmakova, L.E. Ryabchenko, T.V. Gerasimova, S.V. Kameneva, A.S. Yanenko, 2016, published in Biotekhnologiya, 2016, Vol. 32, No. 6, pp. 53–59.
Mutations in the fusA Gene Encoding Elongation Factor G
in the Coryneform Bacterium Lead to Increased Lysine Production
I. P. Tokmakova*, L. E. Ryabchenko, T. V. Gerasimova, S. V. Kameneva, and A. S. Yanenko**
The State Research Institute for Genetics and Selection of Industrial Microorganisms (GosNIIgenetika), Moscow, 117545 Russia
Received November 30, 2016
Abstract⎯Resistance to fusidic acid in Corynebacterium glutamicum and Brevibacterium flavum is associated
with mutations in the fusA gene, which encodes the elongation factor G (EF-G). Two to ten percent of fusidic
acid-resistant clones were shown to produce more lysine than parent strains. Sequencing of the fusA gene in
clones with a high level of lysine production made it possible to find two mutations in the gene at position
1383—С1383G and С1383А. These mutations cause amino acid replacement at position 461 in the protein
EF-G, namely, histidine is substituted by glutamine (H461Q). The mutation С1383G was introduced in the
chromosomal copy of the fusA gene in C. glutamicum and B. flavum strains by homologous recombination.
All clones containing the mutant variant of the fusA gene produced 10% more lysine than the parent strains.
Keywords: Brevibacterium flavum, Corynebacterium glutamicum, elongation factor G, fusA gene, L-lysine syn-
thesis, mutations in fusA, resistance to fusidic acid
L-lysine is an essential amino acid, and it is the
most popular feed additive for animal husbandry and
poultry. The main method of producing L-lysine is
sugar fermentation by bacteria such as Corynebacte-
rium glutamicum and Brevibacterium sp.  and Esch-
erichia coli . Genetically modified bacterial strains
with increased productivity, which is achieved either
by genetic engineering or traditional breeding meth-
ods, are usually used to increase L-lysine production.
One approach to increase strain productivity is to
obtain mutants resistant to antibiotics that influence
the ribosomal complex . For example, resistance to
kanamycin of the strain C. glutamicum KFCC10881-
СЈР5103 was accompanied by an 8% increase in lysine
productivity in comparison with the parent strain .
Fusidic acid belongs to a group of antibiotics that
inhibit protein synthesis on ribosomes. It is known
that the resistance to fusidic acid of Staphylococcus
aureus is due to mutations in the fusA gene, which
encodes elongation factor G (EF-G) of the ribosomal
complex . However, there are no published data on
the mutant lysine-producing bacteria (Brevibacterium
or Corynebacterium), which are resistant to fusidic
acid. Therefore, for the purposes of breeding of the
strains, it was considerably interesting to study the
relationship between fusidic acid resistance and lysine
The goal of the present work was to study lysine
synthesis by coryneform bacterial strains (C. glutam-
icum and B. flavum) resistant to fusidic acid.
MATERIALS AND METHODS
Bacterial Strains, Plasmids, And Culture Condition
The strain C. glutamicum GEN1-6, a C. glutamicum
АТСС13032-derived strain, was obtained by replacing
the native allele of the lysC gene by the mutant lysC
(resistance to feedback inhibition) via homologous
For plasmid design the standard strain of E. coli
XLI Blue was used as an intermediate host. The E. coli
strains were grown at 37°C in LB medium of the fol-
lowing composition, g/L: tripton, 10; yeast extract,5;
and NaCl, 5; the solid medium additionally contained
bacto agar (15 g/L). All reagents and substrates were pur-
chased from Sigma-Aldrich Co. (Merck). If necessary,
antibiotics were added to the medium: kanamycin (Sin-
tez, Russia) (up to a concentration of 50 μg/mL) and
ampicillin (Sintez, Russia) (up to 100 μg/mL).
Strains of C. glutamicum and B. flavum were grown
on medium 2 × LB with 1% maltose at 30°C. If nec-
essary, antibiotics were added to the medium: kana-
mycin (17 mg/mL) and fusidic acid (5 mg/mL)
The bacterial strains and plasmids used in this work
are given in Table 1.
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