Cloning and characterization of a chitosanase gene from the plant pathogenic fungus Fusarium solani

The plant pathogenic fungus Fusarium solani f. sp. phaseoli SUF386 secretes a chitosanase in the absence of exogenous chitosan. Based on partial amino acid sequences of the purified chitosanase, two degenerate oligonucleotides were synthesized and used as reverse transcription-mediated PCR (RT-PCR) primers to amplify a 500-bp fragment of corresponding cDNA. The PCR product was used as a probe to isolate the genomic copy of the gene (csn). F. solani csn has an open reading frame encoding a polypeptide of 304 amino acid residues with a calculated molecular mass of 31,876 Da and containing a putative 19-amino acid residue signal sequence. Comparison between the genomic and cDNA sequences revealed that three introns are present in the coding region. Southern blot analysis results indicated that csn is present as a single copy in the genome of F. solani SUF386, The cDNA fragment corresponding to the mature enzyme was introduced into E. coli using an expression vector driven by the T7 promoter. The resulting E. coli transformant overproduced proteins with chitosanolytic activity.