Procedure form converting a strain of yeast that does not produce endopolygalacturonase into one that does uses targeted modification of its genome

摘要

The yeast conversion procedure, especially for use with Saccharomyces cerevisiae, consists of introducing a carrier plasmide of a sequence representing the coding ORF (Open Reading Frame) for the endopolygalacturonase enzyme as well as a promoter devoid of silencing sequences. The plasmide used for the procedure is preferably pRPGL1-2. An independent claim concerns the procedure for obtaining a plasmide carrying the necessary genetic material for the expression and amplification of endopolygalacturonase by PCR amplification with U1 and A1 primers of a DNA genome fragment of about 1500pb taken from the X2180 yeast strain (Saccharomyces cerevisiae), subjecting the ends of the amplified portion to phosphorylation, opening the pFL45 plasmide by the BamHl and Xbal restriction enzymes, and joining the amplified fragments to the pFL45 plasmide after linearisation.