PYRUVATE-CARBOXYLASE FROM CORYNEBACTERIUM-GLUTAMICUM - CHARACTERIZATION, EXPRESSION AND INACTIVATION OF THE PYC GENE

In addition to phosphoenolpyruvate carboxylase (PEPCx), pyruvate carbo xylase (PCx) has recently been found as an anaplerotic enzyme in the a mino-acid-producing bacterium Corynebacterium glutamicum, Using oligon ucleotides designed according to conserved regions of PCx amino acid s equences from other organisms, a 200 bp fragment central to the C. glu tamicum PCx gene (pyc) was amplified from genomic DNA by PCR, This fra gment was then used to identify and to subclone the entire C. glutamic um pyc gene, The cloned pyc gene was expressed in C. glutamicum, as ce lls harbouring the gene on plasmid showed four- to fivefold higher spe cific PCx activities when compared to the wild-type (WT). Moreover, in creased PCx protein Bevels in the pyc-plasmid-carrying strain were rea dily detected after SDS-PAGE of cell-free extracts. DNA sequence analy sis of the pyc gene, including its 5' and 3' flanking regions, and N-t erminal sequencing of the pyc gene product predicts a PCx polypeptide of 1140 amino acids with an M-r of 123 070. The amino acid sequence of this polypeptide shows between 62% and 45% identity when compared to PCx enzymes from other organisms. Transcriptional analyses revealed th at the pyc gene from C. glutamicum is monocistronic (3.5 kb mRNA) and that its transcription is initiated at an A residue 55 bp upstream of the translational start. Inactivation of the chromosomal pyc gene in C . glutamicum WT led to the absence of PCx activity and to negligible g rowth on lactate, indicating that PCx is essential for growth on this carbon source. Inactivation of both the PCx gene and the PEPCx gene in C. glutamicum led additionally to the inability to grow on glucose, i ndicating that no further anaplerotic enzymes for growth on carbohydra tes exist in this organism.