Expression of a Trichoderma reesei beta-xylanase gene (XYN2) in Saccharomyces cerevisiae.
The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-beta-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide. The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively. The 33-amino-acid leader peptide of the Xyn2 beta-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous beta-xylanase. The molecular mass of the recombinant beta-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium. These autoselective S. cerevisiae strains produced 1,200 and 160 nkat of beta-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively. The recombinant enzyme showed highest activity at pH 6 and 60 degrees C and retained more than 90% of its activity after 60 min at 50 degrees C.
la Grange
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DC
Department of Microbiology, University of Stellenbosch, South Africa.
Pretorius
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van Zyl
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WH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*; D014995:Xylosidases / Q000235:genetics*
D011994:Recombinant Proteins; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases