title: Key enzymes of the protocatechuate branch of the beta-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum.
abstract: Although the protocatechuate branch of the beta-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 locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. 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 of C. 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 by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the beta-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Xihui
authors.initials: X
authors.lastname: Shen
authors.forename: Shuangjiang
authors.initials: S
authors.lastname: Liu
mesh_terms: D000272:Adipates / Q000378:metabolism*; D019295:Computational Biology / Q000379:methods; D048230:Corynebacterium glutamicum / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D006841:Hydrocarbons, Aromatic / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism*; D009154:Mutation; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011520:Protocatechuate-3,4-Dioxygenase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000272:Adipates; D006841:Hydrocarbons, Aromatic; D062385:Hydroxybenzoates; D011994:Recombinant Proteins; C027316:3-oxoadipic acid; C009091:protocatechuic acid; D011520:Protocatechuate-3,4-Dioxygenase