Cloning of a protopectinase gene of Trichosporon penicillatum and its expression in Saccharomyces cerevisiae

Abstract

A protopectinase (PPase)-encoding gene, PSE3, from Trichosporon penicillatum was cloned by colony hybridization using two oligonucleotide probes synthesized from the N-terminal amino acid sequences of native PPase SE1 and one peptide from a lysyl endopeptidase digest. Nucleotide sequencing revealed that PSE3 contains an ORF encoding a 367 amino acid protein. Mature PPase SE3 is composed of 340 amino acids and the N-terminus of the ORF appeared to correspond to a signal peptide and a propeptide processed by a KEX2-like proteinase. The deduced amino acid sequence of PSE3 was 65.4, 56.7, 58.1, 61.8 and 48.9% homologous to the polygalacturonases of Aspergillus oryzae, Aspergillus niger, Aspergillus tubigensis, Cochliobolus carbonum and Fusarium moniliforme, respectively. One domain, which might interact with polygalacturonic acid, is highly conserved not only in fungal polygalacturonases but also in bacterial and plant polygalacturonases. PSE3 was expressed in Saccharomyces cerevisiae, but three forms (the mature form, a glycosylated form and an uncharacterized processed form) of PPase SE3 were present among the PSE3 products.