Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1)

The yeast Kluyveromyces marxianus strain BKM Y-719 produces an efficient pe ctin-degrading endopolygalacturonase (EPG) that cleaves the internal alpha- 1,4-D-glycosidic linkages to yield oligomers of varying sizes. The EPG1 gen e encoding this industrially important EPG was cloned by using the polymera se chain reaction (PCR) technique and degenerate primers to generate a 135 bp DNA fragment with which a genomic library was screened. The cloned fragm ent contained an open reading frame (ORF) of 1083 bp, encoding a 361 amino acid polypeptide. The predicted amino acid (aa) sequence of EPG showed simi larity with polygalacturonases (PGs) of fungi. Analysis of the aa sequence indicated that the first 25 aa constitute a signal sequence and a motif (C( 218)XGGHGXSIGSVG(230)) that is usually associated with a PG active site. Pu lsed-field gel electrophoresis resolved chromosomal bands for K. marxianus BKM Y-719 and using chromoblotting it seems that EPG1 is present as only a single copy in the genome. The nucleotide sequence of K. marxianus BKM Y-71 9 EPG1 was submitted to the EMBL under Accession No. AJ000076. Copyright (C ) 1999 John Wiley & Sons, Ltd.