Expression of the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene together with the Erwinia pectate lyase and polygalacturonase genes in Saccharomyces cerevisiae.
Recombinant Saccharomyces cerevisiae strains capable of simultaneous secretion of bacterial glucanase and pectinase enzymes have been developed. The Butyrivibrio fibrrisolvens endo-beta-1,4-glucanase gene (end1), the Erwinia chrysanthemi pectate lyase gene (pelE) and E. carotovora polygalacturonase gene (peh1) were each inserted between a yeast expression-secretion cassette and yeast gene terminator, and cloned into yeast-centromeric shuttle vectors. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of glucanase and pectinases was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). These YCplac111-based constructs, designated END1, PEL5, AND PEH1, respectively, were transformed into S. cerevisiae. The END1, PEL5 and PEH1 constructs were co-expressed in laboratory strains of S. cerevisiae as well as in wine and distillers' yeasts. DNA-RNA hybridization analysis showed the presence of END1, PEL5 and PEH1 transcripts. Carboxymethylcellulose and polypectate agarose assays revealed the production of biologically active endo-beta-1,4-glucanase, pectate lyase and polygalacturonase by the S. cerevisiae transformants. Interestingly, although the same expression-secretion cassette was used in all three constructs, time-course assays indicated that the pectinases were secreted before the glucanase. It is tempting to speculate that the bulkiness of the END1-encoded protein and the five alternating repeats of Pro-Asp-Pro-Thr(Gln)-Pro-Val-Asp within the glucanase moiety could be involved in the delayed secretion of the glucanase.
van Rensburg
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Department of Microbiology, University of Stellenbosch, South Africa.
van Zyl
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Pretorius
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001438:Bacteroidaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism; D011096:Polygalacturonase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D010368:Pectins; D002482:Cellulose; D011096:Polygalacturonase; D002480:Cellulase; D011133:Polysaccharide-Lyases; C023911:pectate lyase