Characterization of a novel 2,4,6-trichlorophenol-inducible gene encoding chlorophenol O-methyltransferase from Trichoderma longibrachiatum responsible for the formation of chloroanisoles and detoxification of chlorophenols

Abstract

De novo sequencing of eight internal peptides of purified chlorophenol O-methyltransferase, or CMT1 (before named as CPOMT), from Trichoderma longibrachiatum was performed by MALDI-TOF/TOF and ESI-IT. A novel gene (cmt1) encoding CMT1 was cloned by using a PCR approach based on the amino acid sequence of two internal peptides. The gene (1637 bp) encoded a protein of 468 amino acids with a deduced molecular mass of 52.4 kDa, and a theoretical isoelectric point of 5.93. This gene contains four introns, whose location was confirmed by comparison of cDNA and chromosomal sequences. The expression of cmt1 gene was induced at transcriptional level by exposure of fungal mycelia to 2,4,6-trichlorophenol (2,4,6-TCP). Putative homologous genes were detected in many different fungal strains, including other Trichoderma species. Partial silencing of cmt1 gene resulted in a 48.9% (+/-5.2) decrease of CMT1 activity levels in a T. longibrachiatum At37 transformant strain by comparison with the wild type, whereas a decrease of up to 53.0% was observed in the levels of 2,4,6-TCA produced in liquid cultures. Efficient expression of cmt1 gene in Escherichia coli unequivocally confirmed that it encodes a CMT1 enzyme.