Genome characterization of two novel deep-sea sediment fungi, Penicillium pacificagyrus sp. nov. and Penicillium pacificasedimenti sp. nov., from South Pacific Gyre subseafloor sediments, highlights survivability.

BACKGROUND: Marine deep subsurface sediments were once thought to be devoid of eukaryotic life, but advances in molecular technology have unlocked the presence and activity of well-known closely related terrestrial and marine fungi. Commonly detected fungi in deep marine sediment environments includes Penicillium, Aspergillus, Cladosporium, Fusarium, and Schizophyllum, which could have important implications in carbon and nitrogen cycling in this isolated environment. In order to determine the diversity and unknown metabolic capabilities of fungi in deep-sea sediments, their genomes need to be fully analyzed. In this study, two Penicillium species were isolated from South Pacific Gyre sediment enrichments during Integrated Ocean Drilling Program Expedition 329. The inner gyre has very limited productivity, organic carbon, and nutrients. RESULTS: Here, we present high-quality genomes of two proposed novel Penicillium species using Illumina HiSeq and PacBio sequencing technologies. Single-copy homologues within the genomes were compared to other closely related genomes using OrthoMCL and maximum-likelihood estimation, which showed that these genomes were novel species within the genus Penicillium. We propose to name isolate SPG-F1 as Penicillium pacificasedimenti sp. nov. and SPG-F15 as Penicillium pacificagyrus sp. nov. The resulting genome sizes were 32.6 Mbp and 36.4 Mbp, respectively, and both genomes were greater than 98% complete as determined by the presence of complete single-copy orthologs. The transposable elements for each genome were 4.87% for P. pacificasedimenti and 10.68% for P. pacificagyrus. A total of 12,271 genes were predicted in the P. pacificasedimenti genome and 12,568 genes in P. pacificagyrus. Both isolates contained genes known to be involved in the degradation of recalcitrant carbon, amino acids, and lignin-derived carbon. CONCLUSIONS: Our results provide the first constructed genomes of novel Penicillium isolates from deep marine sediments, which will be useful for future studies of marine subsurface fungal diversity and function. Furthermore, these genomes shed light on the potential impact fungi in marine sediments and the subseafloor could have on global carbon and nitrogen biogeochemical cycles and how they may be persisting in the most energy-limited sedimentary biosphere.

Mnt1, an α-(1 → 2)-mannosyltransferase responsible for the elongation of N-glycans and O-glycans in Aspergillus fumigatus.

The fungal cell wall is necessary for survival as it serves a barrier for physical protection. Therefore, glycosyltransferases responsible for the synthesis of cell wall polysaccharides may be suitable targets for drug development. Mannose is a monosaccharide that is commonly found in sugar chains in the walls of fungi. Mannose residues are present in fungal-type galactomannan, O-glycans, N-glycans, glycosylphosphatidylinositol anchors, and glycosyl inositol phosphorylceramides in Aspergillus fumigatus. Three genes that are homologous to α-(1 â†’ 2)-mannosyltransferase genes and belong to the glycosyltransferase family 15 were found in the A. fumigatus strain, Af293/A1163, genome: cmsA/ktr4, cmsB/ktr7, and mnt1. It is reported that the mutant ∆mnt1 strain exhibited a wide range of properties that included high temperature and drug sensitivity, reduced conidia formation, leakage at the hyphal tips, and attenuation of virulence. However, it is unclear whether Mnt1 is a bona fide α-(1 â†’ 2)-mannosyltransferase and which mannose residues are synthesized by Mnt1 in vivo. In this study, we elucidated the structure of the Mnt1 reaction product, the structure of O-glycan in the Δmnt1 strain. In addition, the length of N-glycans attached to invertase was evaluated in the Δmnt1 strain. The results indicated that Mnt1 functioned as an α-(1 â†’ 2)-mannosyltransferase involved in the elongation of N-glycans and synthesis of the second mannose residue of O-glycans. The widespread abnormal phenotype caused by the disruption of the mnt1 gene is the combined result of the loss of mannose residues from O-glycans and N-glycans. We also clarified the enzymatic properties and substrate specificity of Mnt1 based on its predicted protein structure.

LaeA Controls Citric Acid Production through Regulation of the Citrate Exporter-Encoding cexA Gene in Aspergillus luchuensis mut. kawachii.

The putative methyltransferase LaeA is a global regulator of metabolic and development processes in filamentous fungi. We characterized the homologous laeA genes of the white koji fungus Aspergillus luchuensis mut. kawachii (A. kawachii) to determine their role in citric acid hyperproduction. The ΔlaeA strain exhibited a significant reduction in citric acid production. Cap analysis gene expression (CAGE) revealed that laeA is required for the expression of a putative citrate exporter-encoding cexA gene, which is critical for citric acid production. Deficient citric acid production by a ΔlaeA strain was rescued by the overexpression of cexA to a level comparable with that of a cexA-overexpressing ΔcexA strain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates the expression of cexA via methylation levels of the histones H3K4 and H3K9. These results indicate that LaeA is involved in citric acid production through epigenetic regulation of cexA in A. kawachiiIMPORTANCEA. kawachii has been traditionally used for production of the distilled spirit shochu in Japan. Citric acid produced by A. kawachii plays an important role in preventing microbial contamination during the shochu fermentation process. This study characterized homologous laeA genes; using CAGE, complementation tests, and ChIP-qPCR, it was found that laeA is required for citric acid production through the regulation of cexA in A. kawachii The epigenetic regulation of citric acid production elucidated in this study will be useful for controlling the fermentation processes of shochu.

A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii.

We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1-based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii. The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.

Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii.

Aspergillus luchuensis mut. kawachii (A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpA ΔyhmA strain, we constructed an S-tagged ctpA (ctpA-S) conditional expression strain in the ΔyhmA background using the Tet-On promoter system. Knockdown of ctpA-S in ΔyhmA resulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis in A. kawachiiIMPORTANCE Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from Aspergillus luchuensis mut. kawachii Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

Multi-gene phylogenetic analysis reveals that shochu-fermenting Saccharomyces cerevisiae strains form a distinct sub-clade of the Japanese sake cluster.

Shochu is a traditional Japanese distilled spirit. The formation of the distinguishing flavour of shochu produced in individual distilleries is attributed to putative indigenous yeast strains. In this study, we performed the first (to our knowledge) phylogenetic classification of shochu strains based on nucleotide gene sequences. We performed phylogenetic classification of 21 putative indigenous shochu yeast strains isolated from 11 distilleries. All of these strains were shown or confirmed to be Saccharomyces cerevisiae, sharing species identification with 34 known S. cerevisiae strains (including commonly used shochu, sake, ale, whisky, bakery, bioethanol and laboratory yeast strains and clinical isolate) that were tested in parallel. Our analysis used five genes that reflect genome-level phylogeny for the strain-level classification. In a first step, we demonstrated that partial regions of the ZAP1, THI7, PXL1, YRR1 and GLG1 genes were sufficient to reproduce previous sub-species classifications. In a second step, these five analysed regions from each of 25 strains (four commonly used shochu strains and the 21 putative indigenous shochu strains) were concatenated and used to generate a phylogenetic tree. Further analysis revealed that the putative indigenous shochu yeast strains form a monophyletic group that includes both the shochu yeasts and a subset of the sake group strains; this cluster is a sister group to other sake yeast strains, together comprising a sake-shochu group. Differences among shochu strains were small, suggesting that it may be possible to correlate subtle phenotypic differences among shochu flavours with specific differences in genome sequences. Copyright © 2017 John Wiley & Sons, Ltd.

Construction of a Cosmid-Based Ultraefficient Genomic Library System for Filamentous Fungi of the Genus Aspergillus.

Filamentous fungi of the genus Aspergillus include producers of industrially important organic acids, enzymes, and secondary metabolites, as well as pathogens of many plants and animals. Novel genes in the Aspergillus genome are potentially crucial for the fermentation and drug industries (e.g., agrochemicals and antifungal drugs). A research approach based on classical genetics is effective for identifying functionally unknown genes. During analyses based on classical genetics, mutations must be identified easily and quickly. Herein, we report the development of a cosmid-based plasmid pTOCK1 and the use of a genomic library of Aspergillus nidulans constructed using pTOCK1. The cosmid-based genomic library was used for convenient auxotrophic mutants (pyroA and pabaB), as well as mutants with abnormal colony morphology (gfsA) and yellow conidia (yA), to obtain library clones complementary to these phenotypes. The complementary strain could be obtained through a single transformation, and the cosmid could be rescued. Thus, our cosmid library system can be used to identify the causative gene in a mutant strain.

Identification of a putative α-galactoside ß-(1 → 3)-galactosyltransferase involved in the biosynthesis of galactomannan side chain of glucuronoxylomannogalactan in Cryptococcus neoformans.

The cell surface of Cryptococcus neoformans is covered by a thick capsular polysaccharide. The capsule is the most important virulence factor of C. neoformans; however, the complete mechanism of its biosynthesis is unknown. The capsule is composed of glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). As GXM is the most abundant component of the capsule, many studies have focused on GXM biosynthesis. However, although GXMGal has an important role in virulence, studies on its biosynthesis are scarce. Herein, we have identified a GT31 family ß-(1 → 3)-galactosyltransferase Ggt2, which is involved in the biosynthesis of the galactomannan side chain of GXMGal. Comparative analysis of GXMGal produced by a ggt2 disruption strain revealed that Ggt2 is a glycosyltransferase that catalyzes the initial reaction in the synthesis of the galactomannan side chain of GXMGal. The ggt2 disruption strain showed a temperature-sensitive phenotype at 37°C, indicating that the galactomannan side chain of GXMGal is important for high-temperature stress tolerance in C. neoformans. Our findings provide insights into complex capsule biosynthesis in C. neoformans.

Discovery of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in Aspergillus fumigatus mycelium.

The cellular surface of the pathogenic filamentous fungus Aspergillus fumigatus is enveloped in a mannose layer, featuring well-established fungal-type galactomannan and O-mannose-type galactomannan. This study reports the discovery of cell wall component in A. fumigatus mycelium, which resembles N-glycan outer chains found in yeast. The glycosyltransferases involved in its biosynthesis in A. fumigatus were identified, with a focus on two key α-(1→2)-mannosyltransferases, Mnn2 and Mnn5, and two α-(1→6)-mannosyltransferases, Mnn9 and Van1. In vitro examination revealed the roles of recombinant Mnn2 and Mnn5 in transferring α-(1→2)-mannosyl residues. Proton nuclear magnetic resonance (1H-NMR) analysis of cell wall extracts from the ∆mnn2∆mnn5 strain indicated the existence of an α-(1→6)-linked mannan backbone in the A. fumigatus mycelium, with Mnn2 and Mnn5 adding α-(1→2)-mannosyl residues to this backbone. The α-(1→6)-linked mannan backbone was absent in strains where mnn9 or van1 was disrupted in the parental ∆mnn2∆mnn5 strain in A. fumigatus. Mnn9 and Van1 functioned as α-(1→6)-linked mannan polymerases in heterodimers when co-expressed in Escherichia coli, indicating their crucial role in biosynthesizing the α-(1→6)-linked mannan backbone. Disruptions of these mannosyltransferases did not affect fungal-type galactomannan biosynthesis. This study provides insights into the complexity of fungal cell wall architecture and a better understanding of mannan biosynthesis in A. fumigatus. IMPORTANCE: This study unravels the complexities of mannan biosynthesis in A. fumigatus, a key area for antifungal drug discovery. It reveals the presence of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in A. fumigatus mycelium, offering fresh insights into the fungal cell wall's design. Key enzymes, Mnn2, Mnn5, Mnn9, and Van1, are instrumental in this process, with Mnn2 and Mnn5 adding specific mannose residues and Mnn9 and Van1 assembling the α-(1→6)-linked mannan structures. Although fungal-type galactomannan's presence in the cell wall is known, the existence of an α-(1→6)-linked mannan adds a new dimension to our understanding. This intricate web of mannan biosynthesis opens avenues for further exploration and enhances our understanding of fungal cell wall dynamics, paving the way for targeted drug development.

The Metabolic Regulation of Amino Acid Synthesis Counteracts Reactive Nitrogen Stress via Aspergillus nidulans Cross-Pathway Control.

Nitric oxide (NO) is a natural reactive nitrogen species (RNS) that alters proteins, DNA, and lipids and damages biological activities. Although microorganisms respond to and detoxify NO, the regulation of the cellular metabolic mechanisms that cause cells to tolerate RNS toxicity is not completely understood. We found that the proline and arginine auxotrophic proA5 and argB2 mutants of the fungus Aspergillus nidulans require more arginine and proline for normal growth under RNS stress that starves cells by accumulating fewer amino acids. Fungal transcriptomes indicated that RNS stress upregulates the expression of the biosynthetic genes required for global amino acids, including proline and arginine. A mutant of the gene disruptant, cpcA, which encodes the transcriptional regulation of the cross-pathway control of general amino acid synthesis, did not induce these genes, and cells accumulated fewer amino acids under RNS stress. These results indicated a novel function of CpcA in the cellular response to RNS stress, which is mediated through amino acid starvation and induces the transcription of genes for general amino acid synthesis. Since CpcA also controls organic acid biosynthesis, impaired intermediates of such biosynthesis might starve cells of amino acids. These findings revealed the importance of the mechanism regulating amino acid homeostasis for fungal responses to and survival under RNS stress.

Overexpression of the DHA1 family, ChlH and ChlK, leads to enhanced dicarboxylic acids production in koji fungi, Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A. luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A. luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25 mM and 50 mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus.

Complete genome sequence of Bacillus subtilis subsp. natto NARUSE using PacBio sequencing.

We report the complete genome sequence of Bacillus subtilis subsp. natto NARUSE, which has been traditionally employed for fermenting soybeans in Japan. The genome was sequenced using the PacBio system, yielding a sequence, yielding a sequence length of 4,148,793 nucleotides for the circular chromosome and 62,770 nucleotides for the plasmid.

Identification of galactofuranose antigens such as galactomannoproteins and fungal-type galactomannan from the yellow koji fungus (Aspergillus oryzae).

Filamentous fungi belonging to the genus Aspergillus are known to possess galactomannan in their cell walls. Galactomannan is highly antigenic to humans and has been reported to be involved in the pathogenicity of pathogenic filamentous fungi, such as A. fumigatus, and in immune responses. In this study, we aimed to confirm the presence of D-galactofuranose-containing glycans and to clarify the biosynthesis of D-galactofuranose-containing glycans in Aspergillus oryzae, a yellow koji fungus. We found that the galactofuranose antigen is also present in A. oryzae. Deletion of ugmA, which encodes UDP-galactopyranose mutase in A. oryzae, suppressed mycelial elongation, suggesting that D-galactofuranose-containing glycans play an important role in cell wall integrity in A. oryzae. Proton nuclear magnetic resonance spectrometry revealed that the galactofuranose-containing sugar chain was deficient and that core mannan backbone structures were present in ΔugmA A. oryzae, indicating the presence of fungal-type galactomannan in the cell wall fraction of A. oryzae. The findings of this study provide new insights into the cell wall structure of A. oryzae, which is essential for the production of fermented foods in Japan.

Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii.

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A. luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

Identification of an α-(1→6)-Mannosyltransferase Contributing To Biosynthesis of the Fungal-Type Galactomannan α-Core-Mannan Structure in Aspergillus fumigatus.

Fungal-type galactomannan, a cell wall component of Aspergillus fumigatus, is composed of α-(1→2)-/α-(1→6)-linked mannan and ß-(1→5)-/ß-(1→6)-linked galactofuran side chains. Recently, CmsA and CmsB were identified as the α-(1→2)-mannosyltransferases involved in the biosynthesis of the α-core-mannan. However, the α-(1→6)-mannosyltransferase involved in the biosynthesis of the α-core-mannan has not been identified yet. In this study, we analyzed 9 putative α-(1→6)-mannosyltransferase gene disruption strains of A. fumigatus. The ΔanpA strain resulted in decreased mycelial elongation and reduced conidia formation. Proton nuclear magnetic resonance analysis revealed that the ΔanpA strain failed to produce the α-core-mannan of fungal-type galactomannan. We also found that recombinant AnpA exhibited much stronger α-(1→6)-mannosyltransferase activity toward α-(1→2)-mannobiose than α-(1→6)-mannobiose in vitro. Molecular simulations corroborated the fact that AnpA has a structure that can recognize the donor and acceptor substrates suitable for α-(1→6)-mannoside bond formation and that its catalytic activity would be specific for the elongation of the α-core-mannan structure in vivo. The identified AnpA is similar to Anp1p, which is involved in the elongation of the N-glycan outer chain in budding yeast, but the building sugar chain structure is different. The difference was attributed to the difference in substrate recognition of AnpA, which was clarified by simulations based on protein conformation. Thus, even proteins that seem to be functionally identical due to amino acid sequence similarity may be glycosyltransferase enzymes that make different glycans upon detailed analysis. This study describes an example of such a case. IMPORTANCE Fungal-type galactomannan is a polysaccharide incorporated into the cell wall of filamentous fungi belonging to the subphylum Pezizomycotina. Biosynthetic enzymes of fungal-type galactomannan are potential targets for antifungal drugs and agrochemicals. In this study, we identified an α-(1→6)-mannosyltransferase responsible for the biosynthesis of the α-core-mannan of fungal-type galactomannan, which has not been known for a long time. The findings of this study shed light on processes that shape this cellular structure while identifying a key enzyme essential for the biosynthesis of fungal-type galactomannan.

Chromosome-level genome sequence data and analysis of the white koji fungus, Aspergillus luchuensis mut. kawachii IFO 4308.

Aspergillus luchuensis mut. kawachii is used primarily in the production of shochu, a traditional Japanese distilled alcoholic beverage. Here, we report the chromosome-level genome sequence of A. luchuensis mut. kawachii IFO 4308 (NBRC 4308) and a comparison of the sequence with that of A. luchuensis RIB2601. The genome of strain IFO 4308 was assembled into nine contigs consisting of eight chromosomes and one mitochondrial DNA segment. The nearly complete genome of strain IFO 4308 comprises 37,287,730 bp with a GC content of 48.85% and 12,664 predicted coding sequences and 267 tRNAs. Comparison of the IFO 4308 and RIB2601 genomes revealed a highly conserved structure; however, the IFO 4308 genome is larger than that of RIB2601, which is primarily attributed to chromosome 5. The genome sequence of IFO 4308 was deposited in DDBJ/ENA/GenBank under accession numbers AP024425-AP024433.

Chromosome-Level Genome Sequence of Aspergillus puulaauensis MK2, a Fungus Isolated from a Dead Hard Tick.

Aspergillus puulaauensis strain MK2 was isolated from a dead hard tick (Haemaphysalis longicornis). Here, we determined the chromosome-level genome sequence of A. puulaauensis MK2.

Chromosome-Level Genome Sequence of the Black Koji Fungus Aspergillus luchuensis RIB2601.

Aspergillus luchuensis is used for the production of awamori and shochu, which are traditional Japanese distilled alcoholic beverages. Here, we determined the chromosome-level genome sequence of A. luchuensis RIB2601.

Chromosome-Level Genome Sequence of Aspergillus chevalieri M1, Isolated from Katsuobushi.

In this study, we report the chromosome-level genome sequence of the osmophilic filamentous fungus Aspergillus chevalieri M1, which was isolated from a dried bonito, katsuobushi. This fungus plays a significant role in the fermentation and ripening process. Thus, elucidating the sequence data for this fungus will aid in subsequent genomic research on the fungi involved in katsuobushi production.

Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

Citrate exporter CexA plays a key role in the production of citric acid in fungi; however, its role in intracellular metabolism has remained unclear. In this study, we comparably characterized homologous cexA genes in the white koji fungus Aspergillus luchuensis mut. kawachii and the yellow koji fungus Aspergillus oryzae, which exhibit high and low abilities, respectively, to produce citric acid. Disruption of cexA caused a significant decline of both extracellular and intracellular citric acid accumulation in Aspergillus kawachii, while overexpression of the A. kawachii cexA gene (AkcexA) into A. oryzae significantly enhanced both extracellular and intracellular citric acid accumulation in A. oryzae to a level comparable to that of A. kawachii. In addition, overexpression of two intrinsic cexA homologs (AocexA and AocexB) in A. oryzae also enhanced its extracellular and intracellular citric acid accumulation. Comprehensive analysis of intracellular metabolites from an AkcexA-overexpressing strain of A. oryzae compared with its control strain identified metabolic changes associated with intracellular citric acid accumulation via the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle. Our results indicate that citric acid export enhances not only extracellular citric acid accumulation but also intracellular metabolic fluxes to generate citric acid.