The increase in cell volume and nuclear number of the koji-fungus Aspergillus oryzae contributes to its high enzyme productivity

In unicellular organisms, cell volume is tightly regulated in coordination with nuclear division timing (Amodeo and Skotheim, 2016; Nurse, 1975; Jorgensen et al., 2007). Similarly, in multinucleated filamentous fungi, a correlation exists between cell volume and nuclear number (Dynesen and Nielsen, 2003; Dundon et al., 2016). However, the number of nuclei varies across species (Roper et al., 2011), the factors governing nuclear number and cell volume remain largely unknown. Filamentous fungi are industrially significant microorganisms, and the strains selected through breeding provide ideal research models for studying these evolutionary processes.

Filamentous fungi secrete a variety of hydrolytic enzymes to support their growth, and humans have long harnessed this enzyme secretion ability for the production of a wide range of fermented foods and beverages (Hurst, 2023). Aspergillus oryzae has been used in Japan for over a thousand years to produce traditional fermented foods like sake, soy sauce, and miso (Kitamoto, 2015; Ichishima, 2016). A distinctive feature of the fermentation techniques is solid-state cultivation using substrates like rice, soybeans, and wheat bran. The inoculum of A. oryzae used in fermentation, known as ‘koji’, has been commercially produced for around 700 years (Yamashita, 2021). Koji is created by cultivating koji-fungus on grains, and koji starter manufacturers have selected and bred strains to improve the flavor and color of various fermented products. The A. oryzae strains currently in use are mainly preserved and managed by koji manufacturers and research institutions. A notable characteristic of A. oryzae is its high ability to secrete starch-degrading enzymes and proteases, which has led to extensive research into its genome and gene expression regulation (Machida et al., 2005; Machida et al., 2008; Gomi, 2019; Tanaka and Gomi, 2021; Wang et al., 2010; Ishida et al., 2000; Kitano et al., 2002).

Although A. oryzae shares 99.5% genomic similarity with Aspergillus flavus, which produces the carcinogenic aflatoxin (Rokas et al., 2007), there have been no reports of A. oryzae producing aflatoxins, and its safety has been confirmed at the molecular level (Kusumoto et al., 2000; Kobayashi et al., 2007). Phylogenomic analyses suggest that A. oryzae and A. flavus diverged approximately 50,000–189,000 years ago (Watarai et al., 2019; Chang and Ehrlich, 2010). Over the following millennia, human use of A. oryzae is thought to have driven significant genomic recombination, resulting in its evolution into a ‘cell factory’ specifically optimized for the breakdown of sugars and proteins (Gibbons et al., 2012).

Additionally, modern biotechnology utilizes filamentous fungi as cell factories to produce organic acids, enzymes, and pharmaceuticals (Meyer et al., 2016; Liu et al., 2023; Meyer et al., 2020). A. oryzae also shows high production capacity as a host for both homologous and heterologous protein production in modern biotechnology (Christensen et al., 1988; Saito et al., 2024). Recently, its application has expanded to include the production of pharmaceutical proteins and secondary metabolites (Huynh et al., 2020; Itoh et al., 2010). Furthermore, filamentous fungi involved in fermentation play a crucial role in creating a more sustainable food system, such as by upcycling agricultural by-products into food through fungal fermentation and using A. oryzae mycelium for alternative meats (Maini Rekdal et al., 2024b; Maini Rekdal et al., 2024a).

While basic and applied research on A. oryzae continues, fundamental questions remain unanswered, such as why A. oryzae exhibits high enzyme production capacity and why it excels as a host for heterologous expression. This study shows that the key lies in the increase in cell volume and nuclear number and analyzes the molecular mechanisms.

In A. oryzae some strains, hyphae grow about three times thicker and contain roughly ten times more nuclei than the initial hyphae (Figure 1). These thicker hyphae emerge from existing branches, where nuclear division is promoted, resulting in the formation of hyphae with an increased number of nuclei (Figure 2). A clear correlation was observed between hyphal cell volume and nuclear number (Figure 1G). This consistency aligns with findings in other organisms, from unicellular yeast to multicellular plants and animals, where the nuclear-to-cytoplasmic ratio is tightly regulated, allowing cell cycle progression only upon reaching a critical cell size (Amodeo and Skotheim, 2016; Nurse, 1975; Jorgensen et al., 2007). Both the G1/S and G2/M transitions are controlled by size checkpoints, with specific mechanisms differing between species (Futcher and Kellogg, 2024; Fantes and Nurse, 1977).

In A. nidulans, it is known that the duration of the G1 and G2 phases is regulated by temperature (Bergen and Morris, 1983). While the mechanisms governing the G1/S phase transition remain unclear (Dörter and Momany, 2016), the G2/M phase transition has been shown to involve the following: During the interphase of A. nidulans, NimX (CDK1) binds to NimE (cyclin B), and its activity is regulated through phosphorylation by AnkA (Wee1 kinase) and NimT (Cdc25 phosphatase) (Gould and Nurse, 1989; Wu et al., 1998; Ye et al., 1997). The activated NimX–NimE complex subsequently dephosphorylates by NimT and activates NimA (Never-in-Mitosis A) protein kinase (Shen and Osmani, 2013; Osmani et al., 1987). NimA is essential for the transition from G2 to mitosis, and its expression is tightly regulated during the cell cycle (Osmani et al., 1987). The mRNA level of nimA and nimT begins to increase in G2, reaches a plateau during late G2 and M phases, and is sharply degraded at the end of mitosis upon re-entry into interphase (Osmani et al., 1987; Osmani et al., 1991), meaning high expression levels of nimA and nimT result in a longer G2 phase. In thick hyphae, nimA and nimT mRNA was hardly detected, suggesting that thick hyphae sense cell volume in some way and subsequently shorten the G2 phase (Figure 6A). The expression level of nimE was low in both thick and thin hyphae, with no significant difference observed. As known in other organisms, its function is likely regulated through phosphorylation and protein degradation.

Figure 6

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Branching is thought to occur through the degradation and reconstruction of the cell wall at the branching site (Harris, 2008). SNP analysis between industrial strains with different phenotypes identified RseA, a predicted glycosyltransferase (Figure 5). Deletion of rseA caused defects in cell wall synthesis in A. nidulans, leading to increased enzyme secretion (Ogawa et al., 2021; Feng et al., 2017). Although the targets of RseA are unknown, RseA could be involved in the cell wall integrity by modifying glycosyl chains on enzymes responsible for cell wall synthesis and degradation. Mutations in rseA may impair the regulation of cell wall loosening at the branching site, resulting in the formation of thicker hyphae. In fact, in the thick hyphae of RIB40, localized imbalances in cell wall components have been demonstrated by transmission electron microscopy and AFM.

Thicker hyphae can only form if high turgor pressure is maintained within the hyphae, in addition to changes in cell wall remodeling during branching. msyA and msyB, whose orthologues are calcium channels involved in the regulation of cell volume, were identified among the genes that show a significant increase in expression in thicker hyphae (Figure 4; Nakayama et al., 2012; Nakayama et al., 2014). Analysis of the knockout strains demonstrated the function of cell volume regulation adapted to increased turgor pressure in hypoosmotic conditions. The function of these calcium channels is likely crucial for maintaining the balance of turgor pressure and cell volume.

The TOR pathway is a central regulator of cellular growth, responding to a variety of nutrients, such as amino acids, and carbon sources, as well as environmental factors like oxygen levels, osmotic pressure, pH, and temperature stress (Urban et al., 2007; De Virgilio and Loewith, 2006). Upon sensing these inputs, the TOR pathway influences translation activity, metabolic flow, and cell size (De Virgilio and Loewith, 2006; Kim and Guan, 2019). In both yeast and mammalian models, amino acids like branched-chain amino acids, glutamine, and alanine are crucial for nutrient sensing, forming the basis for TOR signaling activation (Kim and Guan, 2019; Saxton and Sabatini, 2017; Meng et al., 2020). These amino acids are like those contributing to nuclear increase in the RIB915 strain (Figure 3G). Furthermore, TOR inhibition by rapamycin in the RIB40 strain suppressed the formation of hyphae with increased nuclei (Figure 3I–K). Consistent with the activation of translation by the TOR pathway, the expression of ribosome-related genes was elevated in thicker hyphae . The TOR pathway directly regulates mitochondrial activity (Düvel et al., 2010). Thick hyphae exhibited heightened mitochondrial density and activity , suggesting that the TOR pathway supports the high ATP demand associated with rapid biosynthesis.

Cell volume and nuclear number mutually regulate each other, meaning that thicker hyphae always possess more nuclei. For the formation of thicker hyphae with numerous nuclei, an increase in cell volume and a corresponding increase in nuclei must occur simultaneously (Figure 6A). In our model, cell wall loosening at a branching site and regulation of cell volume by turgor pressure constitute necessary conditions for increasing cell volume and maintaining thick hyphae. RseA and MsyA may be involved in these processes. At the same time, enhanced translational capacity, possibly due to increased expression of ribosomal genes associated with TOR activation, and mechanisms that accelerate the cell cycle represent another essential condition that enables an increase in nuclear number. Both genetic potential and nutritional environmental signals are likely required for the formation of thick hyphae with a high number of nuclei. In the A. oryzae RIB915 strain, the number of nuclei and cell volume increased in response to specific amino acids, whereas in the A. oryzae RIB40 and other strains, both increased even without the addition of such amino acids, suggesting that they possess the genetic potential to respond independently of nutritional signals. When thick hyphae were cultured on fresh medium, thin hyphae initially emerged, suggesting the necessity of sustained high metabolic activity. Weakening the cell wall by treatment with a low concentration of calcofluor white did not lead to hyphal thickening or an increase in nuclear number. On the contrary, thick hyphae have thicker cell walls (Figure 2H–K), which are necessary to maintain the increased cell volume.

A. nidulans and A. flavus did not exhibit an increase in nuclear number. A. sojae displayed increased nuclear numbers, whereas A. kawachii and A. luchuensis did not (Figure 1). The 20 strains of A. oryzae selected from different clades showed an increase in nuclear number in the minimal medium supplemented with yeast extract. In the medium without yeast extract, there were strains that showed an increase in nuclear number and others that did not. These differences might be due to variations in their applications or the selection of strains with diverse characteristics, such as not only enzyme activity but also their impact on the flavor, aroma, and color of the final products. Additionally, there might be dominant mutations that activate the TOR pathway even in the minimal medium. Multiple gene mutations were identified as contributing to the phenotype of increased hyphal volume and nuclear number, suggesting that the phenotype was not the result of a single mutation but rather the gradual accumulation of mutations through breeding. The koji-fungus strains used to produce high-quality sake and soy sauce have been selectively bred over many years, long before the advent of modern biotechnology, to enhance their saccharolytic and proteolytic activities. Modern biotechnological bred strains such as T. reesei QM9414 and P. chrysogenum IFO4688 also exhibit a phenotype of thicker hyphae with increased nuclear numbers under nutrient-rich conditions (Figure 5). This consistency suggests that by breeding naturally multinucleate filamentous fungi under nutrient-rich conditions, it is possible to obtain strains with increased nuclear numbers and enhanced enzyme or compound production.

A previous study reported an increase in the number of nuclei in A. nidulans (Clutterbuck, 1970; Vinck et al., 2005). Here, we examined the nuclear distribution of A. nidulans grown on the culture media; however, we did not find class III hyphae as observed in A. oryzae. Even in A. nidulans, conidiophore stalks contain a high number of nuclei. It has been shown that A. oryzae has a taller conidiophore stalk (Wada et al., 2012). In the thick hyphae of A. oryzae, the expression level of flbA, an early regulator of conidiophore development (Lee and Adams, 1994), was elevated. This suggests that differentiation to aerial hyphae may be involved in the increase of hyphal volume and nuclear number.

We investigated whether the reduction in nuclear size observed in thick hyphae was due to a change from diploid to haploid status. However, no difference in GFP-histone fluorescence intensity was detected between thick and thin hyphae (Figure 5—figure supplement 2D). In both RIB40 and RIB915 strains, no significant difference in conidial size was observed despite the large difference in the number of nuclei within the hyphae (Figure 5—figure supplement 2E). These results suggest that both thick and thin hyphae remain haploid, and that the smaller nuclear size observed in thick hyphae is likely due to a higher nuclear density. This is likely related to the phenomenon in which a decrease in cell size is accompanied by a reduction in nuclear size (Lemière et al., 2022).

The number of nuclei in filamentous fungi varies greatly among species (Roper et al., 2011). For example, Neurospora crassa hyphal cells can exceed 100 nuclei, which correlates with its exceptionally rapid growth rate and high protein secretion capacity (Havlik et al., 2017). An increase in the number of nuclei per cell is expected to enhance transcription and translation per cell, thereby improving enzyme production and secretion capacity (Figure 6B). Whereas maintaining a high nuclear number requires a substantial amount of energy, and in conditions where external nutrient supply is insufficient, cells rapidly die. Thus, the tendency to maintain a high nuclear number could be a selective pressure unlikely to occur in nature. The regulation of nuclear number and its ecological strategy are intriguing in other fungi such as N. crassa, which rapidly spreads after wildfires (Jacobson et al., 2004), and arbuscular mycorrhiza fungi that form symbiotic relationships with plants and contain thousands of nuclei within hyphae lacking septa (Kokkoris et al., 2020). In any case, koji-fungus strains were bred specifically for high enzyme production in nutrient-rich environments, and we propose that some of them acquired the specialized trait of increased cell volume and nuclear number through selection. This study elucidates the evolutionary processes driven by breeding selection in A. oryzae and the underlying mechanisms contributing to high enzyme productivity. These findings not only offer new approaches for optimizing filamentous fungi for bioindustry applications but also lead to the elucidation of the molecular mechanisms regulating cell volume and nuclear number in multinucleated organisms.

The RNA-seq data have been deposited in DDBJ as BioProject PRJDB19992.

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Author details

1. Ayaka Itani

   Microbiology Research Center for Sustainability (MiCS), Faculty of Life and Environmental Sciences, Tsukuba Institute for Advanced Research (TIAR), University of Tsukuba, Tsukuba, Japan

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

2. Haruto Motomura

   Microbiology Research Center for Sustainability (MiCS), Faculty of Life and Environmental Sciences, Tsukuba Institute for Advanced Research (TIAR), University of Tsukuba, Tsukuba, Japan

   Contribution

   Formal analysis, Validation, Investigation

   Competing interests

   No competing interests declared

3. Ken Oda

   National Research Institute of Brewing, Higashi-hiroshima, Japan

   Contribution

   Resources

   Competing interests

   No competing interests declared

4. Hideyuki Yamashita

   Higuchi Matsunosuke Shoten Co., Ltd., Osaka, Japan

   Contribution

   Resources

   Competing interests

   employee of Higuchi Matsunosuke Shoten Co., Ltd

5. Kanae Sakai

   Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

6. Ken-ichi Kusumoto

   Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Shinsuke Shigeto

   Department of Chemistry, School of Science, Kwansei Gakuin University, Hyogo, Japan

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2035-2068

8. Takehiko Ichikawa

   Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan

   Contribution

   Methodology

   Competing interests

   No competing interests declared

9. Hosain Mohammad Mubarak

   Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan

   Contribution

   Methodology

   Competing interests

   No competing interests declared

10. Takeshi Fukuma

    Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan

    Contribution

    Methodology

    Competing interests

    No competing interests declared

11. Takuya Katayama

    1. Department of Biotechnology, The University of Tokyo, Tokyo, Japan
    2. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan

    Contribution

    Resources, Methodology

    Competing interests

    No competing interests declared

12. Jun-ichi Maruyama

    1. Department of Biotechnology, The University of Tokyo, Tokyo, Japan
    2. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan

    Contribution

    Resources, Supervision, Methodology

    Competing interests

    No competing interests declared

13. Shunsuke Masuo

    Microbiology Research Center for Sustainability (MiCS), Faculty of Life and Environmental Sciences, Tsukuba Institute for Advanced Research (TIAR), University of Tsukuba, Tsukuba, Japan

    Contribution

    Resources, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-0454-3315

14. Naoki Takaya

    Microbiology Research Center for Sustainability (MiCS), Faculty of Life and Environmental Sciences, Tsukuba Institute for Advanced Research (TIAR), University of Tsukuba, Tsukuba, Japan

    Contribution

    Conceptualization, Resources, Project administration

    Competing interests

    No competing interests declared

15. Norio Takeshita

    Microbiology Research Center for Sustainability (MiCS), Faculty of Life and Environmental Sciences, Tsukuba Institute for Advanced Research (TIAR), University of Tsukuba, Tsukuba, Japan

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    takeshita.norio.gf@u.tsukuba.ac.jp

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2666-4991

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