Ashbya gossypii as a model system to study septin organization by single-molecule localization microscopy.

Heteromeric complexes of GTP-binding proteins from the septin family assemble into higher order structures that are essential for cell division in many organisms. The correct organization of the subunits into filaments, gauzes, and rings is the basis of septin function in this process. Electron microscopy and polarization fluorescence microscopy contributed greatly to the understanding of the dynamics and organization of such structures. However, both methods show technical limitations in resolution and specificity that do not allow the identification of individual septin complexes in assemblies in intact cells. Single-molecule localization-based fluorescence superresolution microscopy methods combine the resolution of cellular structures at the nanometer level with highest molecular specificity and excellent contrast. Here, we provide a protocol that enables the investigation of the organization of septin complexes in higher order structures in cells by combining advantageous features of the model organism Ashbya gossypii with single-molecule localization microscopy. Our assay is designed to investigate the general assembly mechanism of septin complexes in cells and is applicable to many cell types.

Ashbya gossypii beyond industrial riboflavin production: A historical perspective and emerging biotechnological applications.

The filamentous fungus Ashbya gossypii has been safely and successfully used for more than two decades in the commercial production of riboflavin (vitamin B2). Its industrial relevance combined with its high genetic similarity with Saccharomyces cerevisiae together promoted the accumulation of fundamental knowledge that has been efficiently converted into a significant molecular and in silico toolbox for its genetic engineering. This synergy has enabled a directed and sustained exploitation of A. gossypii as an industrial riboflavin producer. Although there is still room for optimizing riboflavin production, the recent years have seen an abundant advance in the exploration of A. gossypii for other biotechnological applications, such as the production of recombinant proteins, single cell oil and flavour compounds. Here, we will address the biotechnological potential of A. gossypii beyond riboflavin production by presenting (a) a physiological and metabolic perspective over this fungus; (b) the molecular toolbox available for its manipulation; and (c) commercial and emerging biotechnological applications for this industrially important fungus, together with the approaches adopted for its engineering.

Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway.

The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4-18GlcNAc2, mostly containing neutral core-type N-glycans with 8-10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4-7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.

Yap1-dependent oxidative stress response provides a link to riboflavin production in Ashbya gossypii.

Ashbya gossypii is a natural overproducer of riboflavin. Overproduction of riboflavin can be induced by environmental stress, e.g. nutritional or oxidative stress. The Yap-protein family has a well-documented role in stress response. Particularly, Yap1 has a major role in directing the oxidative stress responses. The A. gossypii YAP-family consists of only three genes in contrast to its closest relative Eremothecium cymbalariae, which has four YAP-homologs. Gene order at Eremothecium YAP-loci is conserved with the reconstructed yeast ancestor. AgYap1p is unique amongst Yap-homologs as it lacks the cysteine-rich domains (CRDs). AgYAP1 expression is inducible and GFP-AgYap1 localizes to the nucleus. Agyap1 mutants displayed higher sensitivity against oxidative stress - H(2)O(2) and menadione - and are strongly reduced in riboflavin production. High levels of cAMP, which also reduce riboflavin production, show a synergistic effect on this sensitivity. AgYAP1 and a chimera of AgYAP1 (with the DNA-binding domain) and ScYAP1 (with the CRDs) can both complement the Scyap1 oxidative stress sensitivity. This suggests that the DNA-binding sites of ScYap1 are conserved in A. gossypii. Expression of AgRIB4, which contains three putative Yap1-binding sites, assayed via a lacZ-reporter gene was strongly reduced in an Agyap1 mutant suggesting a direct involvement of AgYap1 in riboflavin production. Furthermore, our data show that application of H(2)O(2) stress leads to an increase in riboflavin production in a Yap1-dependent manner.

Molecular and structural characterization of GTP-cyclohydrolase II in Eremothecium ashbyi NRRL Y-1363: cDNA cloning, comparative sequence analysis and molecular modeling.

GTP-cyclohydrolase II (GCH II) encoded by RIB1 gene catalyzes the first committed step in the riboflavin biosynthetic pathway. We report here the cloning and characterization of the entire RIB1 ORF (EaRIB1) of 942bp by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE-PCR) in Eremothecium ashbyi where it was found to be present as a single-copy gene. EaRIB1 sequence is available at the GenBank Accession Number EF565374. The putative peptide of 313-aa has a high similarity of 60-70% with GCH II sequences from other ascomycete fungi. Gene expression and alignment studies confirmed the functional annotation of this gene. Homology model was developed with Escherichia coli (PDB 2BZ1) as template to identify the catalytic domains and to explore its functional architecture. We report here the first three-dimensional model of any fungal GCH II which due to its absence in humans assumes significance for anti-fungal drug targeting.