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

Reviewer #1 (Public review):

Filamentous fungi are established work horses in biotechnology with Aspergillus oryzae as a prominent example with a thousand-year of history. Still the cell biology and biochemical properties of the production strains is not well understood. The paper of the Takeshita group describes the change in nuclear numbers and correlate it to different production capacities. They used microfluidic devices to really correlate the production with nuclear numbers. In addition, they used microdissection to understand expression profile changes and found an increase of ribosomes. The analysis of two genes involved in cell volume control in S. pombe did not reveal conclusive answers to explain the phenomenon. It appears that it is a multi-trait phenotype. Finally, they identified SNPs in many industrial strains and tried to correlate them to the capability of increasing their nuclear numbers.

The methods used in the paper range from high quality cell biology, Raman spectroscopy to atomic force and electron microscopy and from laser microdissection to the use of microfluidic devices to study individual hyphae.

This is a very interesting, biotechnologically relevant paper with the application of excellent cell biology.

Comments on revised version:

The authors addressed all suggestions satisfactorily.

Reviewer #2 (Public review):

Summary:

In the study presented by Itani and colleagues it is shown that some strains of Aspergillus oryzae - especially those used industrially for the production of sake and soy sauce - develop hyphae with a significantly increased number of nuclei and cell volume over time. These thick hyphae are formed by branching from normal hyphae and grow faster and therefore dominate the colonies. The number of nuclei positively correlates with the thicker hyphae and also the amount of secreted enzymes. The addition of nutrients such as yeast extract or certain amino acids enhanced this effect. Genome and transcriptome analyses identified genes, including rseA, that are associated with the increased number of nuclei and enzyme production. The authors conclude from their data involvement of glycosyltransferases, calcium channels and the tor regulatory cascade in regulation of cell volume and number of nuclei. Thicker hyphae and an increased number of nuclei was also observed in high-production strains of other industrially used fungi such as Trichoderma reesei and Penicillium chrysogenum, leading to the hypothesis that the mentioned phenotypes are characteristic of production strains which is of significant interest for fungal biotechnology.

Strengths:

The study is very comprehensive and involves application of divers state-of-the-art cell biological, biochemical and genetic methods. Overall, the data are properly controlled and analyzed, and the figures and movies are of excellent quality.The results are particularly interesting with regard to the elucidation of molecular mechanisms that regulate the size of fungal hyphae and the number of nuclei. For this, the authors have discovered a very good model: (regular) strains with a low number of nuclei and strains with high number of nuclei. Also, the results can be expected to be of interest for the further optimization of industrially relevant filamentous fungi.

In the revision the authors addressed all my comments and as a result produced an even stronger study.

Reviewer #3 (Public review):

Summary:

The authors seek to determine the underlying traits that support the exceptional capacity of Aspergillus oryzae to secrete enzymes and heterologous proteins. To do so, they leverage the availability of multiple domesticated isolates of A. oryzae along with other Aspergillus species to perform comparative imaging and genomic analysis.

Strengths:

The strength of this study lies in the use of multifaceted approaches to identify significant differences in hyphal morphology that correlate with enzyme secretion, which is then followed by the use of genomics to identify candidate functions that underlie these differences.

Weaknesses:

The authors addressed all suggestions satisfactorily.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Recommendations for the authors):

The authors addressed all suggestions satisfactorily.

Reviewer #2 (Recommendations for the authors):

The authors have adequately dealt with the comments.

Reviewer #3 (Recommendations for the authors):

(1) Line 157. Although the authors have added a statement acknowledging that addition of YE increased hyphal width and secretion in A. nidulans without increasing nuclear number, they have not indicated how this result might impact their model. It might just boil down to variation between the different Aspergilli, but it merits attention.

(2) Line 341. To extend the argument, you might consider adding this citation (https://elifesciences.org/articles/76075), which provides evidence that nuclear size might scale with osmotic pressure based on the density of macromolecules in the nucleus vs. cytoplasm.

Thanks for the suggestion.

L341 This is likely related to the phenomenon in which a decrease in cell size is accompanied by a reduction in nuclear size (66).

(3) Line 343. Neurospora crass hyphal cells can exceed 100 nuclei...

Changed.