Cloning and expression of thermophilic catechol 1,2-dioxygenase gene (catA) fromStreptomyces setonii

• DOI: 10.1111/j.1574-6968.2001.tb10491.x
• Publication Date: 2006-01-09T14:48:12Z
• Authors: Hae-Reun An; Hyun-Joo Park; Eung-Soo Kim
• Keywords: 0106 biological sciences; Base Sequence; Molecular Sequence Data; Temperature; Sequence Analysis, DNA; 01 natural sciences; Catechol 1,2-Dioxygenase; Streptomyces; Dioxygenases; 3. Good health; Escherichia coli; Oxygenases; Amino Acid Sequence; Cloning, Molecular; Plasmids

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 degrees C) and showed a higher activity against 3-methylcatechol than catechol or 4-methylcatechol, but no activity against protocatechuate.