Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p.
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.
Uccelletti
Daniela
D
Department of Developmental and Cell Biology, University of Rome La Sapienza, Rome, Italy.
Staneva
Dessislava
D
Rufini
Silvia
S
Venkov
Pencho
P
Palleschi
Claudio
C
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000235:genetics; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D006031:Glycosylation; D006593:Hexokinase / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D006593:Hexokinase; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase