Cloning and expression of the α-L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

Abstract

First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abfB) was amplified with the polymerase chain reaction technique. The abfB DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1(P)) and terminator (PGK1(T)) sequences on a multicopy episomal plasmid. The resulting construct PGK1(P)abfB-PGK1(T) was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf2) that is 9.4% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.