Industrial Production of Antibiotics in Fungi: Current State, Deciphering the Molecular Basis of Classical Strain Improvement and Increasing the Production of High-Yielding Strains by the Addition of Low-Molecular Weight Inducers
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsFermentation of Antibiotics in Fungi: Current State, Deciphering the Molecular Basis of Classical Strain Improvement and Increasing the Production of High-Yielding Strains by the Addition of Low-Molecular Weight Inducers
This article mainly describes the current situation of using fungi to produce antibiotics. It introduces the types of antibiotics according to the production methods, the improvement of industrial antibiotic production strains, the industrial production of antibiotics, and the role of low molecular weight inducers in the production of antibiotics from high-yield strains. Key strategies for high production of fungal secondary metabolites were also mentioned. The use of genetic engineering methods to carry out targeted modifications when creating new antibiotic-producing strains and to increase the production of classical improved strains provides the possibility and basis. However, there are still the following issues that need clarification from the author:
1.Abstract section: Although the abstract notes that "key changes" have occurred in the strain improvement program, it lacks specific elaboration. The authors would be wise to provide a succinct overview of these changes or highlight specific multi-omics applications that could help improve antibiotic production.
2. Authors should clarify technical terms and concepts to make it easier for readers to understand the article. For example, it would be helpful to provide a more comprehensive explanation of the importance of Paul Ehrlich's "magic bullet" in relation to the evolution of antibiotics.
3.Lines 93-95, The author should furnish examples that illustrate how antibiotics can be altered or adapted to effectively counter resistance mechanisms.
4. Lines 217-220，provide a concise explanation of how bioinformatics analysis aids in predicting undiscovered sites containing Secondary Metabolite (SM) Biosynthetic Gene Clusters (BGCs) and its contribution to uncovering latent antibiotic products.
5. Part 4, the importance of achieving increased yield levels through sequential mutagenesis is emphasized, while the specific magnitude of these increases in high yield levels needs to be detailed. Additionally, it is necessary to explore the approximate scope or technical limits of the method, such as the possible number of iterations。
6.Lines 560-562 ，the authors would do well to carefully describe the potential for developing new antibiotics based on fungal short messages. Assess the distinctive value or unique characteristics that these new antibiotics might possess
7. In Part 6, lines 617-623, the authors should consider elucidating and broadening the discussion concerning co-culture technology. This expansion could offer further insights into the advancements and potential applications of this technology in activating dormant fungal Biosynthetic Gene Clusters (BGCs). Additionally, highlighting the significant strides made in this area would enrich the discussion.
8. Conclusion: Line 981. Are “fey changes” a spelling mistake?
9. Considering the overall structure of the article, the fifth section and those following it appear somewhat disorganized, leading to excessive length. It's advisable to consolidate paragraphs to streamline the content.
Comments on the Quality of English LanguageModerate editing of English language required
Author Response
Responses to Reviewer #1
Question: This article mainly describes the current situation of using fungi to produce antibiotics. It introduces the types of antibiotics according to the production methods, the improvement of industrial antibiotic production strains, the industrial production of antibiotics, and the role of low molecular weight inducers in the production of antibiotics from high-yield strains. Key strategies for high production of fungal secondary metabolites were also mentioned. The use of genetic engineering methods to carry out targeted modifications when creating new antibiotic-producing strains and to increase the production of classical improved strains provides the possibility and basis. However, there are still the following issues that need clarification from the author:
Response: I am extremely grateful to the reviewer for the detailed and critical analysis of the manuscript, valuable comments and advices.
Q: 1.Abstract section: Although the abstract notes that "key changes" have occurred in the strain improvement program, it lacks specific elaboration. The authors would be wise to provide a succinct overview of these changes or highlight specific multi-omics applications that could help improve antibiotic production.
Re: Thank you for your important comment. I expanded the abstract, provided a succinct overview of "key changes" have occurred in the strain improvement program
Q: 2. Authors should clarify technical terms and concepts to make it easier for readers to understand the article. For example, it would be helpful to provide a more comprehensive explanation of the importance of Paul Ehrlich's "magic bullet" in relation to the evolution of antibiotics.
Re: Thanks for the important note. I expanded the Introduction, where I pointed out the importance of Paul Ehrlich's "magic bullet" in relation to the evolution of antibiotics.
Q: 3.Lines 93-95, The author should furnish examples that illustrate how antibiotics can be altered or adapted to effectively counter resistance mechanisms.
Re: Thank you for your question. As an example of overcoming the emerging resistance of microorganisms to used antibiotics, I provided the antibiotic ceftaroline (Teflaro®, Teva Pharmaceuticals USA, Inc. NJ in the US; and Zinforo®, AstraZeneca, Cambridge, UK in Europe), the 5th generation cephalosporin, which has become active against methicillin-resistant Staphylococcus aureus (MRSA), previously resistant to beta-lactam antibiotics, including penicillins, cephalosporins, and carbopenems. This was due to the affinity of ceftaroline to penicillin-binding protein 2a (PBP2a), which did not bind other beta-lactams [1]
- Fernandez, R.; Paz, L.I.; Rosato, R.R.; Rosato, A.E. Ceftaroline Is Active against Heteroresistant Methicillin-Resistant Staphylococcus aureus Clinical Strains despite Associated Mutational Mechanisms and Intermediate Levels of Resistance. Antimicrob. Agents Chemother. 2014, 58, 5736, doi:10.1128/AAC.03019-14.
Q: 4. Lines 217-220，provide a concise explanation of how bioinformatics analysis aids in predicting undiscovered sites containing Secondary Metabolite (SM) Biosynthetic Gene Clusters (BGCs) and its contribution to uncovering latent antibiotic products.
Re: Thanks for your question. In the indicated place in the article, I wrote how bioinformatics analysis aids in predicting novel BGCs and its contribution to uncovering latent antibiotic products.
Q: 5. Part 4, the importance of achieving increased yield levels through sequential mutagenesis is emphasized, while the specific magnitude of these increases in high yield levels needs to be detailed. Additionally, it is necessary to explore the approximate scope or technical limits of the method, such as the possible number of iterations。
Re: Thanks for your comment. I have added in Part 4 the specific magnitude in the increase of secondary metabolite production in improved high-yielding strains. 100-1000 or more times. More detailed information specifically on individual fungal producers is given in section 5 (Table 1), in the place where these producers are described. In addition, earlier in the introduction, I already gave the same data:
“Therefore, on the basis of wild-type (WT) strains, improved high-yielding (HY) producers were obtained, in which the yield of the target SM was increased by 100–1000 or more times” (c)
Technical limits of the method in terms of number of iterations are given from relevant literature sources in Part 4:
“In many programs, it was possible to implement 10–50 or more rounds of mutagenesis until the technological limit of the method is reached” (с)
Q: 6.Lines 560-562 ，the authors would do well to carefully describe the potential for developing new antibiotics based on fungal short messages. Assess the distinctive value or unique characteristics that these new antibiotics might possess
Re: Thanks for your comment. I studied the available literature in detail, described medicinal compounds from fungi that are in clinical trials, described the targets and mechanism of action.
Q: 7. In Part 6, lines 617-623, the authors should consider elucidating and broadening the discussion concerning co-culture technology. This expansion could offer further insights into the advancements and potential applications of this technology in activating dormant fungal Biosynthetic Gene Clusters (BGCs). Additionally, highlighting the significant strides made in this area would enrich the discussion.
Re: Thank you for this question. The answer allowed me to expand the information on co-cultivation for the production of new secondary metabolites. In particular, I provide information on what media and how many new compounds were obtained during co-cultivations. In addition, I added information about the known molecular basis of this phenomenon. In particular, information from last year’s Science Advances article DOI: 10.1126/sciadv.abo6094, which showed the role of the global regulation system of secondary metabolism in changing the profile of SMs during co-cultivation.
Q: 8. Conclusion: Line 981. Are “fey changes” a spelling mistake?
Re: 8. Corrected to “key changes”. Sorry for the missed typo.
Q: 9. Considering the overall structure of the article, the fifth section and those following it appear somewhat disorganized, leading to excessive length. It's advisable to consolidate paragraphs to streamline the content.
Re: Corrected. I have combined most of the paragraphs from section 5 to the end of the review
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author made a review on the production of fungal antibiotics and took beta-lactams and their producers as main examples. Although the work provides useful information, its quality should be improved before consideration for acceptance. Below are some suggestions.
1) The title highlights the term “Fermentation of Antibiotics in Fungi” rather than production of fungal antibiotics. Therefore, fermentation parameters, such as cultivation media, conditions, types of bioreactors, kinetics, and modes, should be discussed. However, the author neglected these aspects.
2) The author emphasized the importance of mutagenesis as a “Classical Strain Improvement”, whereas conditions of mutagenesis that had been successfully applied did not show in this work. In addition, like other bioproducts produced by fungi, antibiotics are now obtained from strains that are genetically modified by metabolic engineering and biosynthetic biology methods. New methods should be included as well.
3) Another problem is the description of “Low-Molecular Weight Inducers”. The author used large content to introduce the role of low-molecular weight compounds in the interaction between fungi and other species. Effect of polyamines on secondary metabolism in fungi was also included. However, the term “High-Yielding” is not defined in the work and we do not know which strain is “High-Yielding”. Therefore, the section “6 Role of Low Molecular Weight Inductors in the Production of Antibiotics in HY strains” seems unclear.
4) Many sentences are redundant. The author should express his idea more concisely. For example, the story “magic bullet” repeated many times, which, in my opinion, is not the key idea of this work. Besides, the “seventh bullet” is not a good metaphor for new techniques for improving the yield of antibiotics. There are other repeated sentences existing in the article. Please revise the manuscript.
5) Many figures give little information, such as Fig 2, 3, 5, 7, 8, 9. Enrich them, or just delete them.
6) Section 8, it is better to provide “Prospects/ perspectives” than “Conclusions”.
Comments on the Quality of English LanguageSome grammar errors exist. Please revise the manuscript carefully.
Author Response
Responses to Reviewer #2
Question: The author made a review on the production of fungal antibiotics and took beta-lactams and their producers as main examples. Although the work provides useful information, its quality should be improved before consideration for acceptance. Below are some suggestions.
Response: I am extremely grateful to the reviewer for taking the time to read my review, constructive fruitful critical analysis, important comments and suggestions.
Q: 1) The title highlights the term “Fermentation of Antibiotics in Fungi” rather than production of fungal antibiotics. Therefore, fermentation parameters, such as cultivation media, conditions, types of bioreactors, kinetics, and modes, should be discussed. However, the author neglected these aspects.
Re: Thank you for your valuable comment. Indeed, the title does not correspond to the content, since fermentation parameters are not considered in the review. Considering that the listed parameters require writing another review to fully cover these parameters, I changed the title. Corrected “Fermentation” to “Industrial Production”.
Q: 2) The author emphasized the importance of mutagenesis as a “Classical Strain Improvement”, whereas conditions of mutagenesis that had been successfully applied did not show in this work.
In addition, like other bioproducts produced by fungi, antibiotics are now obtained from strains that are genetically modified by metabolic engineering and biosynthetic biology methods. New methods should be included as well.
Re: As you correctly noted in the previous question, the title did not quite correspond to the material presented in the review. In its current form the name is “Induscrial Production of Antibiotics in Fungi: Current State, Deciphering the Molecular Basis of Classical Strain Improvement and Increasing the Production of High-Yielding Strains by the Addition of Low-Molecular Weight Inducers”.
And speaking about obtaining industrial fungal producers of antibiotics, section 4 says:
“The powerful genetic engineering tool has great potential in terms of targeting and the diversity of its capabilities [1]. However, none of them led to the creation of an industrial antibiotic-producing fungal strain (as distinct from the production of primary metabolites or enzymes) [2–5].” (c)
In this regard, the only current method for obtaining industrial high-yielding strain from a wild-type isolate still remains classical methods. Strains that are genetically modified by metabolic engineering and biosynthetic biology methods produce several orders of magnitude less of the target secondary metabolite.
It must be said that currently numerous genetic engineering technologies are used to obtain producers of secondary metabolites based on fungal strains already improved by classical methods. But this is a topic that lies beyond the scope of this review and has independent review value. For example, a recent review was written on this topic https://doi.org/10.3390/jof9080834, which has about 300 literary sources. The current review already includes over 350 literary sources, which is enough to combine information on a given topic. As a result, I limited myself to citation reviews written on genetically engineered improvement of fungal strains, while I focused on classical improvement methods. For the same reason, this review does not include conditions of mutagenesis that had been successfully applied to obtain industrial fungal strains.
- Mózsik, L.; Iacovelli, R.; Bovenberg, R.A.L.; Driessen, A.J.M. Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi. Front. Bioeng. Biotechnol. 2022, 10, doi:10.3389/FBIOE.2022.901037.
- Wang, Q.; Zhong, C.; Xiao, H. Genetic Engineering of Filamentous Fungi for Efficient Protein Expression and Secretion. Front. Bioeng. Biotechnol. 2020, 8, 526947, doi:10.3389/FBIOE.2020.00293/BIBTEX.
- Jin, F.J.; Hu, S.; Wang, B.T.; Jin, L. Advances in Genetic Engineering Technology and Its Application in the Industrial Fungus Aspergillus oryzae. Front. Microbiol. 2021, 12, 644404, doi:10.3389/FMICB.2021.644404/BIBTEX.
- Wösten, H.A.B. Filamentous fungi for the production of enzymes, chemicals and materials. Curr. Opin. Biotechnol. 2019, 59, 65–70, doi:10.1016/J.COPBIO.2019.02.010.
- Salazar-Cerezo, S.; de Vries, R.P.; Garrigues, S. Strategies for the Development of Industrial Fungal Producing Strains. J. Fungi 2023, Vol. 9, Page 834 2023, 9, 834, doi:10.3390/JOF9080834.
Q: 3) Another problem is the description of “Low-Molecular Weight Inducers”. The author used large content to introduce the role of low-molecular weight compounds in the interaction between fungi and other species. Effect of polyamines on secondary metabolism in fungi was also included. However, the term “High-Yielding” is not defined in the work and we do not know which strain is “High-Yielding”. Therefore, the section “6 Role of Low Molecular Weight Inductors in the Production of Antibiotics in HY strains” seems unclear.
Re: Thank you very much for your comment. The term “High-Yielding” is one of the key ones in this review, so it is described in detail in the abstract: “The majority of the antibiotic market comes from the fermentation of high-yielding (HY) fungal strains. These strains have been obtained since the 1950s from wild-type (WT) isolates as a result of classical strain improvement (CSI) programs primarily involving multi-round random mutagenesis and screening.” (c)
In addition, this term is discussed extensively in the Introduction, and is present in Figure 1, Table 1. In order not to duplicate information, I did not describe it in section 6.
Q: 4) Many sentences are redundant. The author should express his idea more concisely. For example, the story “magic bullet” repeated many times, which, in my opinion, is not the key idea of this work. Besides, the “seventh bullet” is not a good metaphor for new techniques for improving the yield of antibiotics. There are other repeated sentences existing in the article. Please revise the manuscript.
Re: The story of the magic bullet is given in the Introduction, from the point of view of describing the emergence of the terminology introduced by Ehrlich. In addition, in the Introduction I gave a brief history of antibiotics from the point of view of the magic bullet.
In addition, I return to it in Chapter 7 in order to introduce an analogy for events arising during random mutagenesis. However, this is not a repetition, but an expansion of terminology, which is a new vision that I took the liberty of presenting in this review. To illustrate this analogy, the term magic bullet is also present in Figures 8 and 9.
Q: 5) Many figures give little information, such as Fig 2, 3, 5, 7, 8, 9. Enrich them, or just delete them.
Re: Thank you very much for your comment. All Figures in this review are original and not borrowed from other sources. In my review, I show both complex Figures and more unloaded ones. I did not overload the drawings with additional information if it was not needed there. Figures accompany the text and make it easier to perceive information. I came up with them in parallel with the text, so there is a relationship. Please allow me not to change the Figures, since they form a single whole with the text. By changing the Figures, you will have to change the text. I will certainly change the Figures, or add information to them if you suggest or point out constructive additions or errors. However, simply adding for the sake of adding does not produce useful information. On the contrary, it can make perception difficult. The simplicity of Figures, like a mathematical formula, does not mean that it needs to be improved.
Q: 6) Section 8, it is better to provide “Prospects/ perspectives” than “Conclusions”.
Re: Thank you for your offer. Corrected to Perspectives.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsSummary: The review article by Alexander A. Zhgun is a well-constructed article that provides concise details about antibiotics. This research article makes a substantial contribution to the field of antibiotic production through fermentation in fungi.
Review: Overall, the review is well-written, offering a thorough examination of existing knowledge related to antibiotic fermentation in fungi. The author has successfully contextualized their research within the broader scientific landscape, highlighting gaps in understanding and laying the groundwork for their specific contributions. The authors meticulously detail the outcomes of classical strain improvement (CSI) efforts and the impact of low-molecular-weight compounds (LMWC) on antibiotic production. This review article is beneficial and would interest several researchers and pharmaceutical companies working in the field of antibiotics.
Author Response
Responses to Reviewer #3
Question: Summary: The review article by Alexander A. Zhgun is a well-constructed article that provides concise details about antibiotics. This research article makes a substantial contribution to the field of antibiotic production through fermentation in fungi.
Response: I am extremely grateful to the reviewer for taking the time to read my review in detail and for the high rating, which is extremely important to me
Q: Review: Overall, the review is well-written, offering a thorough examination of existing knowledge related to antibiotic fermentation in fungi. The author has successfully contextualized their research within the broader scientific landscape, highlighting gaps in understanding and laying the groundwork for their specific contributions. The authors meticulously detail the outcomes of classical strain improvement (CSI) efforts and the impact of low-molecular-weight compounds (LMWC) on antibiotic production. This review article is beneficial and would interest several researchers and pharmaceutical companies working in the field of antibiotics.
Re: Thank you very much for your deep understanding of the processes occurring during classical strain improvement, and those research tasks that my scientific group was concerned with studying the impact of low-molecular-weight compounds (LMWC) on antibiotic production.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI have no comments and suggestions for authors.
Comments on the Quality of English LanguageI have no comments on the quality of English language.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author responded to the comments and revised the manuscript accordingly. I think the article is suitable for consideration for pulication.
