Aspergillus: A Powerful Protein Production Platform

Round 1

Reviewer 1 Report

The utilization of Aspergillus spp in protein production was described in this paper. The paper is well written and interesting for the broader scientific community. 

My minor remarks: 

Line 75: word Aspergillus is written with a mistake "Asper Gillus"

Line 240: Kainz et al. (2008) should be noted with reference number in square bracket

Author Response

Dear Reviewer,

Thank you for your positive comments.

Line 75 of the revised MS, the spelling has been corrected

Lines 246 and 254 of the revised MS, the reference for Kainz et al. (2008) was already present in square bracket ([74] line 248 of the former version) not at the end of the sentence but at the end of the paragraph since we developed the two steps described in this reference [82] in the revised MS.

Reviewer 2 Report

The review on Aspergillus being  powerful protein production platform by Fani etal. is very well written summarizing almost all the different research that has been carried out in various food and pharmaceutically relevant protein production in aspergillus ranging from heterologus expression of protein, genetic engineering in transcriptional reguation, glycosylation and secretion pathway. This reviewer liked the way the authors have summarized various work and making a table for individual approaches in recombinant protein production. The tables woud provide a great deal of references to the readers. 

Comment:

1) I would encourage the authors to make a new table summarizing if any CRISPR related modification used in aspergillus. It will be interesting for the readers.

2) I suggest authors to add in the future perpectives; what would scientist try to produce or use aspergillus for in the near future apart from the conventional recombinant protein production.

Author Response

Dear Reviewer,

Thank you for your very positive evaluation and your suggestions.

1) New references were added on the use of CRISPR and as our goal was not to focus on the molecular biology tools, we preferred to add them in a written rather than a tabular form (subsection 2.2, lines 103-108 of the revised MS).

Here is the added paragraph:

“Several molecular tools (e.g. synthetic promoters and terminators, selection markers, RNAi and CRISPR/Cas technologies), suitable for Aspergillus species, have also been developed, facilitating efficient and targeted manipulation of their genomes [29,30]. CRISPR/Cas, for example, a system developed to create site-specific double strand DNA breaks, has been successfully applied in editing the genome of A. niger [31–34], A. nidulans [34], A. oryzae [35], A. fumigatus [36] and other aspergilli [34].”

2) Concerning the comment about “some examples illustrating other applications than recombinant protein”, we introduced a sentence and a new reference recently published describing the current uses of Aspergillus and more generally ofr fungi (L 83-84 of the revised MS):

 “Several species have been used as prolific producers of other organic acids (e.g. itaconic), secondary metabolites and enzymes of biotechnological significance [11]”. Meyer, V.; Basenko, E.Y.; Benz, J.P.; Braus, G.H.; Caddick, M.X.; Csukai, M.; de Vries, R.P.; Endy, D.; Frisvad, J.C.; Gunde-Cimerman, N.; et al. Growing a circular economy with fungal biotechnology: a white paper. Fungal Biol. Biotechnol. 2020, 7, 5, doi:10.1186/s40694-020-00095-z

Two new sentences were added at the beginning of the conclusion (L493-496 of the revised MS):

“Filamentous fungi hold unlimited potential for industrial applications, from the development of meat-like products and biomaterials to bioremediation and biofuel production. One of their best qualities, largely exploited by the industry, is their innate capacity for secretion of enzymes, which facilitate downstream processing and product recovery.”

The future perspectives section was modified accordingly to the suggestion in a last paragraph (L501-516 of the revised MS):

“Advances in the molecular toolkit available for genetic manipulation of several Aspergillus species opened up the path for developing them into production systems for recombinant proteins. Nevertheless, due to a number of factors described in the review, aspergilli have not yet met the expected production levels. Many studies that focused on engineering different steps of protein synthesis and secretion, or generating protease-deficient strains, have resulted in a significant increase of protein yields. Additionally, optimization of the fungal fermentation process has further improved protein production. However, there are aspects of the fungal physiology that limit protein production and remain unclear. Continuous data input from “omics” studies sheds light on the complex fungal mechanisms related to protein quality control and secretion stress, as well as their impact on protein productivity. The knowledge generated from these studies combined with advances in the field of synthetic biology will soon place Aspergillus, and possibly other filamentous fungi, in the race for the most efficient recombinant protein production system. Its potential as a large-scale production platform not only for recombinant proteins, but also for organic acids, bioactive compounds, enzymes and peptides, as well as new perspectives related to the use of Aspergillus in waste treatment and bioremediation processes, prove that this fungus can provide sustainable solutions for multiple and diverse markets and industries.”