Noncatalytic docking domains of cellulosomes of anaerobic fungi

A method is presented for the specific isolation of genes encoding cellulos ome components from anaerobic fungi. The catalytic components of the cellul osome of anaerobic fungi typically contain, besides the catalytic domain, m ostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking dom ain (NCDD) interspaced by short linkers. Degenerate primers were designed t o anneal to the highly conserved region within the NCDDs of the monocentric fungus Piromyces sp. strain E2 and the polycentric fungus Orpinomyces sp. strain PC-2. Through PCR using cDNA from Orpinomyces sp. and genomic DNA fr om Piromyces sp. as templates, respectively, 9 and 19 PCR products were iso lated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indic ated the presence of three structural subfamilies within fungal NCDDs. Furt hermore, evidence is presented that the NCDDs in CeIC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.