Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species

Background: The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g.against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers' yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified.Results: Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them,Delta tpsA,Delta tppA and Delta tppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was Delta tppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides Delta tppA, the most studied deletion mutant in this work was Delta tppB. This gene encodes a protein conserved in filamentous Ascomycota. The Delta tppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress.Conclusion: A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase.