Verlag des Forschungszentrums Jülich
JUEL-4054
In order to clone the exporter gene from C. glutamicum a transposon mutant was
isolated which was unable to excrete L-isoleucine. Cloning and sequencing of the
insertion site of the transposon revealed a gene cluster consisting of three genes.
Two of these genes code for highly hydrophobic proteins with a molecular weight of
27.3 KDa and 11.5 KDa, respectively. The presence of seven putative trans-
membrane helices in the larger and four in the smaller protein suggests that both are
integral membrane proteins. Adjacent, but transcribed divergently to the two genes, a
possible regulatory gene (Irp) is located. Functional analysis with defined mutants
was performed determining the active export rates. The export rate for L-isoleucine in
a strain deleted of both genes was not detectable. Overexpression of both genes
resulted in an increased export rate of 7.8 nmol min-1 mg-1 dw compared to the wild
type with 4.2 nmol min-1 mg-1 dw. Both genes are required to mediate isoleucine
export. The gene of the possible regulator Lrp is essential for the export suggesting
an activating role for the expression of the carrier genes. The branched-chain amino
acids L-Ieucine and L-valine are also excreted by this system. According to the
tunction of the proteins as export carriers for branched-chain amino acids, they were
named BrnFE. Database analyses resulted in the identitication of more than 25
sequences with identities to BrnF and BrnE. In all cases the genomic organisation
was identical to brnF located upstream of brn E. The homologous proteins are now
summarized as the LIV-E family of transporters. Interestingly, C. glutamicum
possesses a second paralogous BrnFE system of presently unknown function.
Kennerknecht, Nicole
Untersuchungen zum Export verzweigtkettiger Aminosäuren in Corynebacterium glutamicum
IV, 133 S., 2003
The essential amino acid L-isoleucine plays an important role in the physiology of
humans and animals. In the few last years there have been many attempts to
overproduce L-isoleucine with the Gram-positive bacterium Corynebacterium
glutamicum. Physiological experiments revealed the existence of an active export
carrier for this amino acid. Therefore, the identitication and isolation of the coding
gene and subsequent functional analysis was the aim of this work.
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