Constitutive expression of a polygalacturonase from Aspergillus kawachii in Saccharomyces cerevisiae

ROJAS N.L.; ORTIZ, G. E.;; CHESINI, M.; GRAMISCI, B.R.; CAVALITTO, S; GHIRINGHELLI, P. D

San Miguel de Tucumán

Congreso; 45rd Annual Meeting Argentine Society for Biochemistry and Molecular Biology.; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. (SAIB).

Pectinases catalyze the hydrolysis of pectin and/or pectic acid. Among pectinases, endopolygalacturonases (PGase; E.C.3.2.1.15.) are the most important biocatalysts. A. kawachii expresses a PGase, namely PG1, active at acidic pH values. Low expression levels of wild type PG1 require cloning and over expression for its industrial applications. PG1 ORF was cloned into p416 and p426 S. cerevisiae expression vectors (after intron removal by PCR and partial digestion with restriction enzymes), generating the constitutive expression constructions p416GDP:PG1DI and p426GDP:PG1DI. E. coli Top10 cells were then transformed and clones were tested by restriction analysis and colony PCR using specific primers for Pg1. Positive clones showed no mutations and correct ORF. The p416GDP:PG1DI and p426GDP:PG1DI were used to transform S. cerevisiae INVSc1 and W303. Transformed yeast clones were analyzed by colony PCR. Positive clones were obtained and tested in terms of PG1 expression. Both strains expressed and exported an active PGase in YPD and SC URA- media. Expressed recombinant protein was recovered as a unique extracellular protein, has a MW of ~60 kDa and was identified as PG1 by hydrolysis of polygalacturonic acid at pH 2.5 but not at pH 5.