| Abstract: | Although the protocatechuate branch of the β-ketoadipate pathway in Gram− bacteria has been well studied, this branch is less understood in Gram+ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate,p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing
cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this
bacterium revealed that the genetic locusncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 andncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). RecombinantEscherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted
inC. glutamicum, the ability to degrade and assimilate protocatechuate,p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability
to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity ofC. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate
3,4-dioxygenase, was encoded byncg12314 andncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining
the genome data publically available in the Gen Bank. The functional identification of genes and their unique organization
inC. glutamicum provided new insight into the genetic diversity of aromatic compound degradation. |
