The increase in cell volume and nuclear number of the koji-fungus Aspergillus oryzae contributes to its high enzyme productivity
- Microbiology Research Center for Sustainability (MiCS), Faculty of Life and Environmental Sciences, Tsukuba Institute for Advanced Research (TIAR), University of Tsukuba, Japan
- National Research Institute of Brewing, Japan
- Higuchi Matsunosuke Shoten Co., Ltd., Japan
- Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
- Department of Chemistry, School of Science, Kwansei Gakuin University, Japan
- Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Japan
- Department of Biotechnology, The University of Tokyo, Japan
- Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Japan
Figures
Figure 1
Increase in number of nuclei and cell volume in Aspergillus species.
(A) The nuclear distribution in the tip cells was categorized into classes I–III. The ratio of hyphae in each class was measured in Aspergillus species at 24, 48, and 72 hr of growth (n = 50). Data for A. oryzae RIB40 and A. nidulans are reproduced from a previous study (Yasui et al., 2020). (B) Colony morphology after 3 days of culture on the minimal medium. The nuclear distribution in the hyphae at the colony periphery stained with SYBR Green. Scale bars: hyphae, 20 μm; colonies, 1 cm. (C) 3D images of hyphae without increased nuclei (left) and with increased nuclei (right) in A. oryzae RIB40 from Video 1. Each nucleus is indicated with different colors by Imaris soft. Scale bar: 5 μm. (D) Comparison of nuclear size in class I hyphae (2.4 μm) and class III hyphae (1.6 μm), indicated by white lines. (E) Box plots of nucleus diameters in hyphae without increased nuclei and those with increased nuclei (n = 35, **p < 0.01, t-test). (F) Box plots of hyphal width at the colony periphery grown for 72 hr (n = 10–14, **p < 0.01, *p < 0.05, t-test). Strains with increased nuclei are marked in red, and those without are marked in blue. (G) Correlation between nuclear number and cell volume of hyphae at 100 μm from the tips in A. oryzae RIB40, RIB128, RIB915, and A. flavus.
Figure 2 with 1 supplement
Thick hyphae with increased nuclei emerge by branching.
(A) Time-lapse image sequence of A. oryzae RIB40 expressing H2B-GFP showing the emergence of thick hyphae with increased nuclei by branching from Video 2. Scale bar: 20 μm. Elapsed time is indicated in minutes. (B) Image sequence of successive nuclear division within the newly emerged branched hypha from Video 3. Scale bar: 10 μm. Elapsed time is indicated in minutes. (C) Time course of nuclear number per hypha. Nuclear numbers were counted with the start time of branching set as 0, every hour for 5–10 hr. Thick: hyphal width >7 μm, Thin: hyphal width <5 μm. (D) Branching from hyphae with increased nuclei generates both thick hyphae with increased nuclei (left) and thin hyphae without increased nuclei (right). Scale bars: 20 μm. (E) Image sequence of mycelial growth showing hyphae with increased nuclei (green) and without increased nuclei (white) from Video 4. Scale bars: 200 μm. Elapsed time is indicated in hours. (F) Line histogram of hyphal width at the colony periphery at 20, 40, and 60 hr, calculated from Video 4 (n = 40). (G) Correlation between hyphal width and maximum elongation rate calculated from Video 4 (n = 45). The maximum elongation rate is determined from elongation rates measured every hour over a 10-hr period. (H) Transmission electron microscopy (TEM) images of A. oryzae RIB40 hyphae. Hyphal diameter and cell wall thickness are indicated in white and black text, respectively. Scale bar: 1 μm. (I) Correlation plots of hyphal width and cell wall thickness based on TEM images. Thick hyphae (>7 μm) are marked in red, and thin hyphae (<7 μm) are marked in blue. (J) 3D surface images of A. oryzae RIB40 hyphal tips in the thick hypha (upper) and the thin hypha (lower) constructed using atomic force microscopy (AFM). (K) Surface roughness of cell walls in thick (red) and thin (blue) hyphae along the arrows in J.
Figure 2—figure supplement 1
Comparative characterization of thick and thin hyphae.
(A) Microfluidic device for 2D observation of hyphae (upper) and overall image of A. oryzae RIB40 expressing H2B-GFP cultured within the device (lower). (B) Cross-sectional transmission electron microscopy (TEM) images. White dotted lines indicate nuclei (left). Enlarged view of the nuclear region (right). Scale bar: 2 μm. (C) Averaged Raman spectra (left) measured at different areas of thick and thin hyphae (right). (D) Mitochondrial staining using Rhodamine 123 in thick hyphae (upper) and thin hyphae (lower). (E) Young’s modulus of hyphal tips measured by atomic force microscopy (AFM). (F) Adhesion properties of hyphal tips measured by AFM.
Figure 3 with 1 supplement
Correlation between number of nuclei and enzyme secretion.
(A) Secreted protein per biomass in strains with increased nuclei (red) and without increased nuclei (blue) after 4 days of culture in minimal medium with 1% maltose (mean ± SE, n = 3, **p < 0.01, t-test). (B) Time course of α-amylase activity per biomass in A. oryzae RIB40 and RIB915 cultured in minimal medium with 1% maltose (mean ± SE, n = 3). (C) Measurement of α-amylase activity in a single hypha using a microfluidic device. A fluorescent substrate increases in fluorescence upon hydrolysis by α-amylase. ROIs for thick hypha are marked in red, and thin hypha in blue. Scale bar: 10 μm. (D) Temporal changes in fluorescence intensity measured in individual flow channels, as described in C. (E) Box plots of hyphal width in colonies grown in minimal medium with or without 1% yeast extract (n = 10–18, **p < 0.01, *p < 0.05, t-test). Conditions with increased nuclei are marked in red, and those without in blue. (F) Secreted protein per biomass in minimal medium with or without 1% yeast extract (mean ± SE, n = 3, **p < 0.01, t-test). (G) Ratio of class I–III hyphae in A. oryzae RIB915 colonies grown in minimal medium supplemented with 1% yeast extract or 0.1% individual amino acids (n = 50). (H) Correlation between the ratio of Class III hyphae and secreted protein per biomass in the 0.1% amino acid-supplemented medium. (I) Images of hyphae and nuclear distribution in A. oryzae RIB40 grown on minimal medium with or without 0.5 ng/ml rapamycin. Scale bars: 10 μm. (J) Ratio of class I–III hyphae in A. oryzae RIB40 cultured on minimal medium containing 0, 0.5, 5, or 100 ng/ml rapamycin (n = 50). (K) Box plots of hyphal width in the colonies cultured under the conditions in J (n = 11–18, **p < 0.01, t-test).
Figure 3—figure supplement 1
Single-hypha enzyme activity assay and the influence of YE, amino acids, and rapamycin on nuclear increase.
(A) Microfluidic device for isolating single hyphae and measuring enzymatic activity. The design was modified from a previous study (Maini Rekdal et al., 2024b). α-Amylase hydrolyzes the starch backbone, releasing a fluorescent signal. (B) Hyphal images stained with SYBR Green of A. oryzae RIB40, RIB915, and A. nidulans grown for 3 days on the minimal medium with or without 1% yeast extract. Scale bar: 20 μm. (C) Hyphal images stained with SYBR Green of A. oryzae RIB40 grown for 3 days on the minimal medium with 1% peptone, 1% casamino acid, 100 μM vitamin Bs or 10 mM nucleic acid. Scale bar: 10 μm. (D) Colonies and hyphae stained with SYBR Green of A. oryzae RIB915 grown for 3 days on the minimal medium with 0.1% amino acid, respectively. Scale bars: colony, 1 cm; hyphae, 10 μm. (E) Secreted protein per biomass in RIB915 cultured in the minimal medium with 0.1% amino acid (mean ± SE, n = 3, **p < 0.01, *p < 0.05, t-test). (F) Colony morphology of RIB40 cultured for 3 days on the minimal medium containing 0, 0.5, 5, or 100 ng/ml rapamycin. (G) Ratio of class I–III hyphae in A. oryzae RIB40 cultured on minimal medium with or without 100 ng/ml rapamycin and 1% yeast extract (n = 50).
Figure 4 with 1 supplement
Transcriptome analyses in hyphae with increased nuclei.
(A) Heatmap of gene expression in A. oryzae RIB40, RIB915, and A. nidulans grown in the minimal medium with or without 1% yeast extract. The top 500 genes with the largest variation in expression are shown. (B) Venn diagram of genes upregulated more than fourfold in the medium with yeast extract. (C) Gene ontology (GO) process analysis of genes uniquely upregulated in A. oryzae RIB915 grown in the medium with yeast extract. (D) Image sequence of cutting and collecting the targeted hypha by using laser microdissection. Scale bar: 100 μm. (E) Heatmap of gene expression in thick (n = 3) and thin (n = 2) hyphae dissected from A. oryzae RIB40. The top 300 genes with the largest expression differences are shown. (F) GO process analysis of genes upregulated more than fourfold in thick hyphae compared to thin hyphae. (G) Venn diagram of 449 genes upregulated in RIB915 with yeast extract and 558 genes upregulated in thick hyphae of RIB40. (H) GO annotations of 13 genes from the 21 shared genes in G. Genes related to cell wall processes are shown in green, cell membrane in blue, Ca²+ transport in yellow, and rRNA processing in orange. (I) Images of hyphae of RIB40 and ΔmsyA strains after 3 min of low osmotic stress. Scale bar: 200 μm. (J) Ratio of hyphal tip lysis under the hypoosmotic shock (mean ± SE, n = 50, **p < 0.01, t-test).
Figure 4—figure supplement 1
Expression changes of ribosomal genes and phenotypes of msyA and msyB deletion mutants.
(A) KEGG pathway visualization showing changes in ribosome-related gene expression in thick hyphae compared to thin hyphae. (B) Colonies and hyphae stained with SYBR Green of A. oryzae RIB915, RIB40, ΔmsyA, ΔmsyB, and ΔmsyAΔmsyB cultured in minimal medium for 3 days. Scale bar: 10 μm. (C) Images of hyphae of ΔmsyB and ΔmsyAΔmsyB strains after 3 min of low osmotic stress. Scale bar: 200 μm. (D) Images of hyphal tips stained with FM4-64 of RIB40 and ΔmsyA immediately after low osmotic stress (0 s) and 160 s later (upper). Scale bar: 5 μm. Enlarged images of the same hyphal tips (lower), showing outlines at 0 s in blue and at 160 s in green. (E) Time course of differences in hyphal width every 30 s for 150 s (mean ± SE, n = 3).
Figure 5 with 2 supplements
Comparison of industrial bred strains in A. oryzae, T. reesei, and P. chrysogenum.
(A) Phylogenetic clades A–F of A. oryzae TK strains. The strains with increased nuclei are indicated in red. (B) Colonies and hyphae stained with SYBR Green of strains in clades C, F, and G grown for 3 days on the minimal medium. Scale bar: 20 μm. (C) Sequence differences in the rseA gene among strains in clades C, F, and G. UTR regions are marked in pink, exons in purple, and nucleotide differences in white. (D) Colonies and hyphae stained with SYBR Green of RIB40 and the ΔrseA strain grown for 3 days on the minimal medium. Scale bar: 10 μm. (E) Colonies and hyphae stained with SYBR Green of RIB915 expressing rseA gene from RIB40 and TK-47 strains with increased nuclei, and TK-41 strain without increased nuclei. Scale bar: 20 μm. (F) Hyphal width of RIB915 and strains in E (mean ± S.E., n=14–23, ** p < 0.01, * p < 0.05, t-test). Strains with rseA from strains with increased nuclei are marked in red, and those without are marked in blue. (G) Hyphal images stained with SYBR Green of T. reesei (QM9414) and P. chrysogenum (IFO4688) grown for 3 days on the minimal medium with or without 1% yeast extract. Scale bar: 20 μm. (H) Ratio of class I–III hyphae in the T. reesei and P. chrysogenum control and bred strains cultured on the minimal medium with or without 1% yeast extract for 3 days (n = 50). (I) CMCase activity per biomass of T. reesei industrial and control strains under the conditions in G (mean ± SE, n = 3, **p < 0.01, t-test). (J) Protease activity per biomass of P. chrysogenum industrial and control strains under the conditions in G (mean ± SE, n = 3, **p < 0.01, t-test).
Figure 5—figure supplement 1
A. oryzae TK strains phenotypes, rseA gene variants, and ectopic expression.
(A) Colonies and hyphae stained with SYBR Green of strains from clades A, B, and E cultured on the minimal medium (left) with yeast extract (right) for 3 days. Scale bar: 20 μm. (B) Alignment of rseA orthologs in A. oryzae RIB40, TK-41, TK-42, A. sojae, and A. flavus. Amino acid substitution T66K in A. oryzae is highlighted in black. (C) Predicted structure of A. oryzae RseA by AlphaFold2. Orange indicates the Glycosyltransferase-like family 2 domain, and yellow indicates a membrane-bound protein region predicted by InterPro. T66K and L266 are highlighted in red. (D) Hyphae at the colony periphery of RIB40 and ΔrseA cultured on the minimal medium for 3 days. Scale bar: 300 μm. (E) Ratio of class I–III hyphae in RIB915 and the strain expressing rseA of RIB40 (with increased nuclei), TK-47 (with increased nuclei), or TK-41 (without increased nuclei) cultured on the minimal medium for 3 days.
Figure 5—figure supplement 2
Phenotype of industrial control strains.
(A, B) Colonies and hyphae stained with SYBR Green of the T. reesei control strain (QM6a) (A) and the P. chrysogenum control strain (JCM14249) cultured on the minimal medium with or without 1% yeast extract for 3 days. Scale bar: 20 μm. (C) Box plots of hyphal width of industrial strains and their controls under the same conditions in G (n = 11–15, **p < 0.01, t-test). (D) Box plots of signal intensity of GFP-histon in A. oryzae RIB40 (n = 10). (E) Box plots of spore size in A. oryzae RIB40 and RIB915 (n = 100).
Figure 6
Working model of hyphal cell volume, nuclear number increase, and enhanced enzyme productivity.
(A) Molecular mechanism by which cell volume and nuclear number regulate each other and each increases simultaneously. (B) An increase in the number of nuclei per hyphal cell enhances transcription, translation, and enzyme secretion per cell.
Videos
Video 1
3D images of hyphae without increased nuclei (left) and with increased nuclei (right) in A. oryzae RIB40.
Each nucleus is indicated with different colors by Imaris soft. Scale bar: 5 μm.
Video 2
Emergence of thick hyphae with increased nuclei by branching in A. oryzae RIB40 expressing H2B-GFP.
Scale bar: 20 μm. Elapsed time is indicated in minutes.
Video 3
Successive nuclear division within the newly emerged thick branched hypha in A. oryzae RIB40 expressing H2B-GFP.
Scale bar: 10 μm. Elapsed time is indicated in minutes.
Video 4
Mycelial growth of hyphae with increased nuclei (green) and without increased nuclei (white).
Scale bars: 200 μm. Elapsed time is indicated in hours.
Video 5
Amylase activity was monitored with a fluorescent substrate under conditions where a single thick or thin hypha grew in a microfluidic channel.
Scale bar: 10 μm. Elapsed time is indicated in minutes.
Video 6
Each hypha was dissected using laser microdissection and collected separately.
Additional files
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Supplementary file 1
Annotated data of RNA-seq in A. oryzae RIB40 and RIB915, and A. nidulans grown in the minimal medium with or without yeast extract.
- https://cdn.elifesciences.org/articles/107043/elife-107043-supp1-v1.xlsx
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Supplementary file 2
Annotated data of RNA-seq in A. oryzae RIB40 thick or thin hyphae.
- https://cdn.elifesciences.org/articles/107043/elife-107043-supp2-v1.xlsx
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Supplementary file 3
Annotated data of RNA-seq common in upregulated gene in A. oryzae RIB915 grown with yeast extract and in A. oryzae RIB40 thick hyphae.
- https://cdn.elifesciences.org/articles/107043/elife-107043-supp3-v1.xlsx
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Supplementary file 4
SNP analysis of ORFs in clade F between TK-32 and TK-38.
- https://cdn.elifesciences.org/articles/107043/elife-107043-supp4-v1.xlsx
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Supplementary file 5
SNP analysis of ORFs in clade G between TK-41 and TK-47.
- https://cdn.elifesciences.org/articles/107043/elife-107043-supp5-v1.xlsx
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Supplementary file 6
Strains used in this study.
- https://cdn.elifesciences.org/articles/107043/elife-107043-supp6-v1.xlsx
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Supplementary file 7
Composition of minimal medium.
- https://cdn.elifesciences.org/articles/107043/elife-107043-supp7-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107043/elife-107043-mdarchecklist1-v1.docx
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The increase in cell volume and nuclear number of the koji-fungus Aspergillus oryzae contributes to its high enzyme productivity
eLife 14:RP107043.
https://doi.org/10.7554/eLife.107043.4
