Expression of the Corynebacterium glutamicum panD gene encoding L-aspartate-alpha-decarboxylase leads to pantothenate overproduction in Escherichia coli.
The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panD mutant strain. Sequence analysis revealed that the coding region of panD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A defined C. glutamicum panD mutant completely lacked L-aspartate-alpha-decarboxylase activity and exhibited beta-alanine auxotrophy. The C. glutamicum panD (panDC. g.) as well as the E. coli panD (panDE.c.) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of L-aspartate-alpha-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed in E. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of the panDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of the panDC.g. gene was sufficient beta-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external beta-alanine supplementation. The enhanced expression of panDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)-1 h-1.
Dusch
N
N
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, D-33501 Bielefeld, Germany.
Pühler
A
A
Kalinowski
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010205:Pantothenic Acid / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011995:Recombination, Genetic; D016415:Sequence Alignment
D010205:Pantothenic Acid; C019104:aspartate-alpha-decarboxylase; D005968:Glutamate Decarboxylase