Shunsuke Masuo

Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions

The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414–12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2‐DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans.

Mechanism of de novo branched-chain amino acid synthesis as an alternative electron sink in hypoxic Aspergillus nidulans cells.

ABSTRACT Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD+ and NADPH/NADP+ under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD+ and NADP+ regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

Global gene expression analysis of Aspergillus nidulans reveals metabolic shift and transcription suppression under hypoxia

Hypoxia imposes a challenge upon most filamentous fungi that require oxygen for proliferation. Here, we used whole genome DNA microarrays to investigate global transcriptional changes in Aspergillus nidulans gene expression after exposure to hypoxia followed by normoxia. Aeration affected the expression of 2,864 genes (27% of the total number of genes in the fungus), of which 50% were either induced or repressed under hypoxic conditions. Up-regulated genes included those for glycolysis, ethanol production, the tricarboxylic acid (TCA) cycle, and for the γ-aminobutyrate (GABA) shunt that bypasses two steps of the TCA cycle. Ethanol and lactate production under hypoxic conditions indicated that glucose was fermented to these compounds via the glycolytic pathway. Since the GABA shunt bypasses the NADH-generating reaction of the TCA cycle catalyzed by oxoglutarate dehydrogenase, hypoxic A. nidulans cells eliminated excess NADH. Hypoxia down-regulated some genes involved in transcription initiation by RNA polymerase II, and lowered the cellular mRNA content. These functions were resumed by re-oxygenation, indicating that A. nidulans controls global transcription to adapt to a hypoxic environment. This study is the first to show that hypoxia elicits systematic transcriptional responses in A. nidulans.

Hydrolase Controls Cellular NAD, Sirtuin, and Secondary Metabolites

ABSTRACT Cellular levels of NAD+ and NADH are thought to be controlled by de novo and salvage mechanisms, although evidence has not yet indicated that they are regulated by NAD+ degradation. Here we show that the conserved nudix hydrolase isozyme NdxA hydrolyzes and decreases cellular NAD+ and NADH in Aspergillus nidulans. The NdxA-deficient fungus accumulated more NAD+ during the stationary growth phase, indicating that NdxA maintains cellular NAD+/NADH homeostasis. The deficient strain also generated less of the secondary metabolites sterigmatocystin and penicillin G and of their gene transcripts than did the wild type. These defects were associated with a reduction in acetylated histone H4 on the gene promoters of aflR and ipnA that are involved in synthesizing secondary metabolites. Thus, NdxA increases acetylation levels of histone H4. We discovered that the novel fungal sirtuin isozyme SirA uses NAD+ as a cosubstrate to deacetylate the lysine 16 residue of histone H4 on the gene promoter and represses gene expression. The impaired acetylation of histone and secondary metabolite synthesis in the NdxA-deficient strain were restored by eliminating functional SirA, indicating that SirA mediates NdxA-dependent regulation. These results indicated that NdxA controls total levels of NAD+/NADH and negatively regulates sirtuin function and chromatin structure.

Conserved and specific responses to hypoxia in Aspergillus oryzae and Aspergillus nidulans determined by comparative transcriptomics.

Hypoxia imposes stress on filamentous fungi that require oxygen to proliferate. Global transcription analysis of Aspergillus oryzae grown under hypoxic conditions found that the expression of about 50% of 4,244 affected genes was either induced or repressed more than 2-fold. A comparison of these genes with the hypoxically regulated genes of Aspergillus nidulans based on their predicted amino acid sequences classified them as bi-directional best hit (BBH), one-way best hit (extra homolog, EH), and no-hit (non-syntenic genes, NSG) genes. Clustering analysis of the BBH genes indicated that A. oryzae and A. nidulans down-regulated global translation and transcription under hypoxic conditions, respectively. Under hypoxic conditions, both fungi up-regulated genes for alcohol fermentation and the γ-aminobutyrate shunt of the tricarboxylate cycle, whereas A. oryzae up-regulated the glyoxylate pathway, indicating that both fungi eliminate NADH accumulation under hypoxic conditions. The A. oryzae NS genes included specific genes for secondary and nitric oxide metabolism under hypoxic conditions. This comparative transcriptomic analysis discovered common and strain-specific responses to hypoxia in hypoxic Aspergillus species.

Distinct Septin Heteropolymers Co-Exist during Multicellular Development in the Filamentous Fungus Aspergillus nidulans

Septins are important components of the cytoskeleton that are highly conserved in eukaryotes and play major roles in cytokinesis, patterning, and many developmental processes. Septins form heteropolymers which assemble into higher-order structures including rings, filaments, and gauzes. In contrast to actin filaments and microtubules, the molecular mechanism by which septins assemble is not well-understood. Here, we report that in the filamentous fungus Aspergillus nidulans, four core septins form heteropolymeric complexes. AspE, a fifth septin lacking in unicellular yeasts, interacts with only one of the core septins, and only during multicellular growth. AspE is required for proper localization of three of the core septins, and requires this same subset of core septins for its own unique cortical localization. The ΔaspE mutant lacks developmentally-specific septin higher-order structures and shows reduced spore production and slow growth with low temperatures and osmotic stress. Our results show that at least two distinct septin heteropolymer populations co-exist in A. nidulans, and that while AspE is not a subunit of either heteropolymer, it is required for assembly of septin higher-order structures found in multicellular development.

Bacterial fermentation platform for producing artificial aromatic amines

Aromatic amines containing an aminobenzene or an aniline moiety comprise versatile natural and artificial compounds including bioactive molecules and resources for advanced materials. However, a bio-production platform has not been implemented. Here we constructed a bacterial platform for para-substituted aminobenzene relatives of aromatic amines via enzymes in an alternate shikimate pathway predicted in a Pseudomonad bacterium. Optimization of the metabolic pathway in Escherichia coli cells converted biomass glucose to 4-aminophenylalanine with high efficiency (4.4 g L−1 in fed-batch cultivation). We designed and produced artificial pathways that mimicked the fungal Ehrlich pathway in E. coli and converted 4-aminophenylalanine into 4-aminophenylethanol and 4-aminophenylacetate at 90% molar yields. Combining these conversion systems or fungal phenylalanine decarboxylases, the 4-aminophenylalanine-producing platform fermented glucose to 4-aminophenylethanol, 4-aminophenylacetate, and 4-phenylethylamine. This original bacterial platform for producing artificial aromatic amines highlights their potential as heteroatoms containing bio-based materials that can replace those derived from petroleum.

NO-inducible nitrosothionein mediates NO removal in tandem with thioredoxin.

Nitric oxide (NO) is a toxic reactive nitrogen species that induces microbial adaption mechanisms. Screening a genomic DNA library identified a new gene, ntpA, that conferred growth tolerance upon Aspergillus nidulans against exogenous NO. The gene encoded a cysteine-rich 23-amino-acid peptide that reacted with NO and S-nitrosoglutathione to generate an S-nitrosated peptide. Disrupting ntpA increased amounts of cellular S-nitrosothiol and NO susceptibility. Thioredoxin and its reductase denitrosated the S-nitrosated peptide, decreased cellular S-nitrosothiol and conferred tolerance against NO, indicating peptide-mediated catalytic NO removal. The peptide binds copper(I) in vitro but is dispensable for metal tolerance in vivo. NO but not metal ions induced production of the peptide and ntpA transcripts. We discovered that the thionein family of peptides has NO-related functions and propose that the new peptide be named NO-inducible nitrosothionein (iNT). The ubiquitous distribution of iNT-like polypeptides constitutes a potent NO-detoxifying mechanism that is conserved among various organisms.

Aspergillus oryzae pathways that convert phenylalanine into the flavor volatile 2-phenylethanol

The filamentous fungus Aspergillus oryzae RIB40 produced 2-phenylethanol (PE) when cultured in minimum medium containing l-phenylalanine as a sole source of nitrogen. The fungus accumulated less PE in the absence of l-phenylalanine, indicating that it converted l-phenylalanine to PE. The PE production associated with fungal glucose consumption was repressed by exogenous ammonium, indicating that nitrogen-metabolite repression controls the pathway that produces PE. We identified the A. oryzae ppdA gene that is expressed at high levels in the presence of exogenous l-phenylalanine and its encoded protein was an active phenylpyruvate decarboxylase. The fungal genome encodes predicted aminotransferases of phenylalanine and PE dehydrogenases, which, together with PpdA, are likely to constitute an Erlich pathway similar to that in Saccharomyces cerevisiae that produces PE. We also identified an A. oryzae aromatic amino acid decarboxylase (AadA) that converted l-phenylalanine to phenylethylamine (PEA), and phenylalanine-inducible PEA oxidase activity in fungal cell extracts, and found that both constitute an alternative pathway through which PEA generates PE. Incubating fungal cultures with l-[(2)H8] phenylalanine to distinguish PE produced by these pathways, indicated that the fungus produced PE by both pathways, but to a greater extent by the Erlich pathway. Gene disruption of ppdA and aadA showed that both pathways participate in the fungal conversion of l-phenylalanine to PE.

Thiamine synthesis regulates the fermentation mechanisms in the fungus Aspergillus nidulans

Thiamine pyrophosphate (TPP) is a critical cofactor and its biosynthesis is under the control of TPP availability. Here we disrupted a predicted thiA gene of the fungus Aspergillus nidulans and demonstrated that it is essential for synthesizing cellular thiamine. The thiamine riboswitch is a post-transcriptional mechanism for TPP to repress gene expression and it is located on A. nidulans thiA pre-messenger RNA. The thiA riboswitch was not fully derepressed under thiamine-limited conditions, and fully derepressed under environmental stressors. Upon exposure to hypoxic stress, the fungus accumulated more ThiA and NmtA proteins, and more thiamine than under aerobic conditions. The thiA gene was required for the fungus to upregulate hypoxic branched-chain amino acids and ethanol fermentation that involve enzymes containing TPP. These findings indicate that hypoxia modulates thiA expression through the thiamine riboswitch, and alters cellular fermentation mechanisms by regulating the activity of the TPP enzymes. Graphical abstract Upon exposure to hypoxic stress (B; –O2), the fungus accumulated more ThiA and NmtA proteins that involve in thiamine biosynthesis (A), and more thiamine than under aerobic conditions (B;+O2).