Co-expression of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in Saccharomyces cerevisiae.
A cDNA fragment encoding the Phanerochaete chrysosporium cellobiohydrolase (cbh1-4) was amplified and cloned with the aid of the polymerase chain reaction (PCR) technique. The cbh1-4 gene and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase (end1) gene were successfully expressed in Saccharomyces cerevisiae under the control of the phosphoglycerate kinase-I (PGK1) and alcohol dehydrogenase-II (ADH2) gene promoters and terminators, respectively. The native P. chrysosporium signal sequence mediated secretion of cellobiohydrolase in S. cerevisiae, whereas secretion of the endo-beta-1,4-glucanase was directed by the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1S). These constructs, designated CBH1 and END1, respectively, were expressed separately and jointly in S. cerevisiae. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of CBH1 and END1 synergistically enhanced cellulose degradation by S. cerevisiae.
van Rensburg
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Department of Microbiology and Institute for Wine Biotechnology, University of Stellenbosch, Stellenbosch 7600, South Africa.
van Zyl
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Pretorius
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D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D017931:DNA Primers; D015870:Gene Expression; D005798:Genes, Bacterial; D005800:Genes, Fungal; D005838:Genotype; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D002480:Cellulase; C028020:licheninase; D043366:Cellulose 1,4-beta-Cellobiosidase