RESUMO
Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man(5)GlcNAc(2)-PP-Dol resulting in the formation of Man(6)GlcNAc(2)-PP-Dol, which is then further processed to the final precursor oligosaccharide Glc(3)Man(9)GlcNAc(2) for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the Δalg3 strain of S. cerevisiae. Inactivation of alg3 in S. commune resulted in the production of predominantly Man(3)GlcNAc(2) protein-linked N-glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms.
Assuntos
Agaricales , Glicoproteínas/biossíntese , Manosiltransferases , Proteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Schizophyllum , Agaricales/genética , Agaricales/crescimento & desenvolvimento , Agaricales/metabolismo , Sequência de Aminoácidos , Técnicas de Inativação de Genes , Glicosilação , Manosiltransferases/química , Manosiltransferases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizophyllum/genética , Schizophyllum/metabolismoRESUMO
Gene deletion in Schizophyllum commune is hampered by a low incidence of homologous integration. As a consequence, extensive screening is required to identify a transformant with the desired genotype. To alleviate this and to facilitate the assembly of deletion plasmids, vector pDelcas was constructed. This construct has a set of restriction sites, which allows for directional cloning of the flanking sequences at both sides of a nourseothricin resistance cassette. Moreover, it contains a phleomycin resistance cassette elsewhere in the plasmid, which is used to screen for transformants with an ectopic integration of the pDelcas derivative. The use of pDelcas derivatives in combination with an improved PCR screening protocol permitted the efficient identification of S. commune deletion strains. This procedure may also function in other basidiomycetes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11274-010-0356-0) contains supplementary material, which is available to authorized users.
RESUMO
The market for N-glycosylated therapeutic proteins represents multi-billion dollars in sales and is growing more than 10% each year. This requires cost-effective production platforms that display correct and homogeneous N-glycosylation. Based on recent results, we propose to use mushroom-forming basidiomycetes for the production of N-glycosylated therapeutic proteins.
Assuntos
Agaricales/metabolismo , Produtos Biológicos/biossíntese , Organismos Geneticamente Modificados/metabolismo , Proteínas Recombinantes/biossíntese , Agaricales/genética , Produtos Biológicos/genética , Biotecnologia/métodos , Glicosilação , Modelos Biológicos , Organismos Geneticamente Modificados/genética , Proteínas Recombinantes/genética , Tecnologia Farmacêutica/métodosRESUMO
N-linked glycans of Schizophyllum commune consist of Man(5-9)GlcNAc(2) structures. Lack of further glycan maturation is explained by the absence of genes encoding such functions in this and other homobasidiomycetes. N-linked glycans in vegetative mycelium and fruiting bodies of S. commune are mainly Man(7)GlcNAc(2) and Man(5)GlcNAc(2), respectively, suggesting more efficient mannose trimming in the mushroom.
