Open AccessCloning and expression of thermophilic catechol 1,2‐dioxygenase gene ( catA ) from Streptomyces setonii
Author(s) -
An HaeReun,
Park HyunJoo,
Kim EungSoo
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb10491.x
Subject(s) - biology , escherichia coli , microbiology and biotechnology , dna , molecular cloning , open reading frame , gene , heterologous expression , biochemistry , genetics , recombinant dna , peptide sequence
Streptomyces setonii (ATCC 39116) degrades various single aromatic compounds such as phenol or benzoate via an ortho ‐cleavage pathway using catechol 1,2‐dioxygenase (C12O). A PCR using degenerate primers based on the conserved regions of known C12O‐encoding genes amplified a 0.45‐kbp DNA fragment from S. setonii total DNA. A Southern hybridization analysis and size‐selected DNA library screening using the 0.45‐kbp PCR product as a probe led to the isolation of a 6.4‐kbp S. setonii DNA fragment, from which the C12O‐encoding genetic locus was found to be located within a 1.4‐kbp DNA fragment. A complete nucleotide sequencing analysis of the 1.4‐kbp DNA fragment revealed a 0.84‐kbp open reading frame, which showed a strong overall amino acid similarity to the known high‐G+C Gram‐positive (but significantly less to the Gram‐negative) bacterial mesophilic C12Os. The heterologous expression of the cloned 1.4‐kbp DNA fragment in Escherichia coli demonstrated that this C12O possessed a thermophilic activity within a broad temperature range (up to 65°C) and showed a higher activity against 3‐methylcatechol than catechol or 4‐methylcatechol, but no activity against protocatechuate.