Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p |
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Authors: | Uccelletti Daniela Staneva Dessislava Rufini Silvia Venkov Pencho Palleschi Claudio |
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Institution: | Department of Developmental and Cell Biology, University of Rome La Sapienza, Rome, Italy. |
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Abstract: | GDP-mannose is the mannosyl donor for the glycosylation reactions and is synthesized by GDP-mannose pyrophosphorylase from GTP and d-mannose-1-phosphate; in Saccharomyces cerevisiae this enzyme is encoded by the PSA1/VIG9/SRB1 gene. We isolated the Kluyveromyces lactis KlPSA1 gene by complementing the osmotic growth defects of S. cerevisiae srb1/psa1 mutants. KlPsa1p displayed a high degree of similarity with other GDP-mannose pyrophosphorylases and was demonstrated to be the functional homologue of S. cerevisiae Psa1p. Phenotypic analysis of a K. lactis strain overexpressing the KlPSA1 gene revealed changes in the cell wall assembly. Increasing the KlPSA1 copy number restored the defects in O-glycosylation, but not those in N-glycosylation, that occur in K. lactis cells depleted for the hexokinase Rag5p. Overexpression of GDP-mannose pyrophosphorylase also enhanced heterologous protein secretion in K. lactis as assayed by using the recombinant human serum albumin and the glucoamylase from Arxula adeninivorans. |
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Keywords: | KlPSA1 Glycosylation Secretion Kluyveromyces lactis GDP-mannose pyrophosphorylase |
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