Cloning and expression of the alpha-L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae.
First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the alpha-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 (PGK1P) and terminator (PGK1T) sequences on a multicopy episomal plasmid. The resulting construct PGK1P-abfB-PGK1T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional alpha-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 94% identical to the alpha-L-arabinofuranosidase B of A. niger N400. Maximum alpha-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.
Crous
J M
JM
Department of Microbiology, University of Stellenbosch, South Africa.
Pretorius
I S
IS
van Zyl
W H
WH
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004271:DNA, Fungal; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase