THE PECTIN LYASE-ENCODING GENE (PNL) FAMILY FROM GLOMERELLA-CINGULATA- CHARACTERIZATION OF PNLA AND ITS EXPRESSION IN YEAST

Oligodeoxyribonucleotide primers were designed from conserved amino ac id (aa) sequences between pectin lyase D (PNLD) from Aspergillus niger and pectate lyases A and E (PELA/E) from Erwinia chrysanthemi. The po lymerase chain reaction (PCR) was used with these primers to amplify g enomic DNA from the plant pathogenic fungus Glomerella cingulata. Thre e different 220-bp fragments with homology to PNL-encoding genes from A. niger, and a 320-bp fragment with homology to PEL-encoding genes fr om Nicotiana tabacum and E. carotovora were cloned. One of the 220-bp PCR products (designated pnlA) was used as a probe to isolate a PNL-en coding gene from a lambda genomic DNA library prepared from G. cingula ta. Nucleotide (nt) sequence data revealed that this gene has seven ex ons and codes for a putative 380-aa protein. The nt sequence of a cDNA clone, prepared using PCR, confirmed the presence of the six introns. The positions of the introns were different from the sites of the fiv e introns present in the three PNL-encoding genes previously sequenced from A. niger. PNLA was synthesised in yeast by cloning the cDNA into the expression vector, pEMBLYex-4, and enzymatically active protein w as secreted into the culture medium. Significantly higher expression w as achieved when the context of the start codon, CACCATG, was mutated to CAAAATG, a consensus sequence commonly found in highly expressed ye ast genes. The produced protein had an isoelectric point (pI) of 9.4, the same as that for the G. cingulata pnlA product. The putative prote in product was 55-62% identical at the aa level to the PNL from A. nig er,but only 21-26% identical to PEL from Erwinia spp. and N. tabacum.