Cloning and expression of an Aspergillus kawachii endo-1,4-beta-xylanase gene in Saccharomyces cerevisiae.
First-strand cDNA was prepared from mRNA isolated from Aspergillus kawachii IFO4308 and the beta-xylanase gene (xynC) amplified by using the polymerase chain reaction (PCR) technique. This gene was inserted between the yeast phosphoglycerate kinase (PGK1) gene promoter (PGK1p) and terminator (PGK1T) sequences. The PGK1p-xynC-PGK1T construct (designated XYN3) was cloned into a multicopy episomal plasmid and the XYN3 gene was expressed in Saccharomyces cerevisiae. Functional beta-xylanase (Xyn3) was produced and secreted by the recombinant yeast. Xyn3 was stable between 30 and 50 degrees C, and the optimum temperature and pH were shown to be at 60 degrees C and lower than pH 3, respectively. An autoselective furl::LEU2 XYN3 recombinant strain was developed that allowed beta-xylanase production at a level of 300 nkat/ml in a non-selective complex medium.
Crous
J M
JM
Department of Microbiology, University of Stellenbosch, South Africa.
Pretorius
I S
IS
van Zyl
W H
WH
D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D043364:Endo-1,4-beta Xylanases; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014995:Xylosidases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases