Verlag des Forschungszentrums Jülich
JUEL-3946
Tilg, Yvonne
Lipidsynthese in Corynebacterium glutamicum
V, 133 S., 2002
The Gram-positive soil bacterium C. glutamicum is used for the industrial production of
amino acids as L-glutamate and L-Iysine. The cell wall of this bacterium and related
Corynebacterianeae is in addition to the cytoplasmic membrane built up of a second distinct
lipid layer, e.g. the mycolic acid layer. It was shown that these two lipid layers are decisive for
the transport of solutes and are likely to influence the efflux of the amino acids. Since the
lipid synthesis of the bacterium is not entirely known, the present work describes the isolation
of acyl-CoA carboxylases which catalyze the initial reaction of the lipid biosynthesis as well
as the characterization of the enzyme activity.
In addition to the gene accBC coding for a putative α-subunit of an acyl-CoA carboxylase the
four further genes accDA, dtsR1, dtsR2 and dtsR3 were isolated and sequenced. The
deduced amino acid sequences of the genes exhibit high identities to β-subunits of acyl-CoA
carboxylases. Enzymatic investigations revealed that AccBC and AccDA built up a functio-
nally enzyme complex. The coexpression of accBC and accDA causes a threefold higher
carboxylation of acetyl-CoA to malonyl-CoA, the precursor of the fatty acid biosynthesis, with
a specific activity of 161 mU / mg protein. The enzyme carboxylates propionyl-CoA as weil.
The enzyme is also proved to be essential for C. glutamicum since it is not possible to
inactivate accBC or accDA. The inactivation of dtsR1, dtsR2 or dtsR3 led to evident growth
limitations, respectively. The growth of each mutant strain is partly restored by
supplementation with different fatty acids. This points to a function of the gene products in
the lipid biosynthesis. The isolation of the biotinylated AccBC protein by avidin affinity
chromatography resulted in the coisolation of proteins identified as DtsR1 , DtsR2 and DtsR3.
Thereby it is shown that one α-subunit can interact with several ß-subunits. In conclusion
C. glutamicum could have different acyl-CoA carboxylases whose common a-subunit
catalizes the CO2-fixation and whose β-subunits are responsible for different yet unknown
substrate specifities.
The L-glutamate and L-Iysine export is influenced by overexpression of the acyl-CoA
carboxylase genes. After mutual overexpression of accBC and dtsR1 only 30% L-glutamate
has accumulated. The mutual overexpression of accBC and accDA decreased the L-Iysine
accumulation by 11 %. Thus the amino acid efflux seems to be correlated with the lipid
composition of the membrane.
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