Metagenomic analyses reveal phylogenetic diversity of carboxypeptidase gene sequences in activated sludge of a wastewater treatment plant in Shanghai, China

Activated sludge of wastewater treatment plants carries a diverse microflora. However, up to 80-90 % of microorganisms in activated sludge cannot be cultured by current laboratory techniques, leaving an enzyme reservoir largely unexplored. In this study, we investigated carboxypeptidase diversity in activated sludge of a wastewater treatment plant in Shanghai, China, by a culture-independent metagenomic approach. Three sets of consensus degenerate hybrid oligonucleotide primers (CODEHOPs) targeting conserved domains of public carboxypeptidases have been designed to amplify carboxypeptidase gene sequences in the metagenomic DNA of activated sludge by PCR. The desired amplicons were evaluated by carboxypeptidase sequence clone libraries and phylogenetic analyses. We uncovered a significant diversity of carboxypeptidases present in the activated sludge. Deduced carboxypeptidase amino acid sequences (127-208 amino acids) were classified into three distinct clusters, alpha, beta, and gamma. Sequences belonging to clusters alpha and beta shared 58-97 % identity to known carboxypeptidase sequences from diverse species, whereas sequences in the cluster gamma were remarkably less related to public carboxypeptidase homologous in the GenBank database, strongly suggesting that novel carboxypeptidase families or microbial niches exist in the activated sludge. We also observed numerous carboxypeptidase sequences that were much closer to those from representative strains present in industrial and sewage treatment and bioremediation. Thermostable and halotolerant carboxypeptidase sequences were also detected in clusters alpha and beta. Coexistence of various carboxypeptidases is evidence of a diverse microflora in the activated sludge, a feature suggesting a valuable gene resource to be further explored for biotechnology application.