RESUMEN
The addition of mannose residues to glycoproteins and glycolipids in the Golgi is carried out by mannosyltransferases. Their activity depends on the presence of GDP-mannose in the lumen of the Golgi. The transport of GDP-mannose (mannosyl donor) into the Golgi requires a specific nucleotide sugar transport present in the Golgi membrane. Here, we report the identification and functional characterization of the putative GDP-mannose transporter in Aspergillus niger, encoded by the gmtA gene (An17g02140). The single GDP-mannose transporter was identified in the A. niger genome and deletion analysis showed that gmtA is an essential gene. The lethal phenotype of the gmtA could be fully complemented by expressing an YFP-GmtA fusion protein from the endogenous gmtA promoter. Fluorescence studies revealed that, as in other fungal species, GmtA localized as punctate dots throughout the hyphal cytoplasm, representing Golgi bodies or Golgi equivalents. SrgC encodes a member of the Rab6/Ypt6 subfamily of secretion-related GTPases and is predicted to be required for the Golgi to vacuole transport. Loss of function of the srgC gene in A. niger resulted in strongly reduced growth and the inability to form conidiospores at 37°C and higher. Furthermore, the srgC disruption in the A. niger strain expressing the functional YFP-GmtA fusion protein led to an apparent 'disappearance' of the Golgi-like structures. The analysis suggests that SrgC has an important role in maintaining the integrity of Golgi-like structures in A. niger.
Asunto(s)
Aspergillus niger/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Aparato de Golgi/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Aspergillus niger/genética , Aspergillus niger/crecimiento & desarrollo , Aspergillus niger/ultraestructura , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Esenciales , Genes Fúngicos , Guanosina Difosfato Manosa/metabolismo , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/genética , Proteínas Recombinantes de Fusión/genética , Alineación de SecuenciaRESUMEN
Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512-Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch. Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous protein that is secreted as a fusion with the glucoamylase protein.
