Microbial Steroids: Novel Frameworks and Bioactivity Profiles
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript of Valery et al. presents a review of steroidal natural products derived from microorganisms, with a strong emphasis on endophytic and marine-derived fungi, particularly species of Aspergillus and Penicillium. The authors compile an impressive number of structurally diverse steroids, including highly rearranged, secosteroidal, and hybrid architectures, and summarize their reported biological activities across a broad spectrum of assays. The breadth of chemical space covered—ranging from classical ergostane derivatives to Diels–Alder adducts and heterocycle-fused frameworks—underscores the remarkable biosynthetic capacity of fungi and highlights their value as sources of bioactive metabolites. The manuscript is information-rich and well referenced, and the figures comprehensively illustrate the structural diversity of the compounds discussed. I only have a few comments. I suggest that authors add a part to give more details about on future directions (e.g., genome mining, biosynthetic gene clusters, coculture strategies, or synthetic biology). For example, an increasing number of natural products have been found based on genomic mining and subsequent experimental verification. With the continuous development of sequencing technology, there are increasingly more genomic resources stored in public data sets. The hidden potential for synthesizing natural products within these resources remains to be further explored. I think that more discussion about future directions about discovering novel microbial steroids would improve the quality of this review.
Author Response
Reviewer 1
Response:
We sincerely thank the reviewer for the positive and encouraging evaluation of our manuscript and for highlighting the breadth of chemical diversity and biosynthetic capacity of microbial steroids presented in this review. We particularly appreciate the constructive suggestion to expand the discussion on future directions for the discovery of novel microbial steroidal natural products.
In response to this comment, we have added a new dedicated subsection on future perspectives and emerging strategies in microbial steroid discovery (Section 5. Future Directions in Microbial Steroid Research). This section now discusses:
The role of genome mining and bioinformatic analysis in identifying cryptic and silent biosynthetic gene clusters related to steroid biosynthesis;
Advances in high-throughput sequencing technologies and the growing availability of fungal and bacterial genome datasets in public repositories;
The use of co-culture and OSMAC (One Strain–Many Compounds) strategies to activate otherwise silent metabolic pathways;
Emerging approaches in synthetic biology and pathway engineering, including heterologous expression and refactoring of biosynthetic gene clusters, to access new steroidal scaffolds and improve yields.
We believe that the inclusion of this forward-looking discussion strengthens the manuscript by placing the compiled chemical and biological data into a broader context and by highlighting promising avenues for future discovery and development of microbial steroids. We are grateful to the reviewer for this valuable suggestion, which has significantly enhanced the scope and impact of the review.
With kind regards
Valery M Dembitsky
P.S.
All corrections and additions, in accordance with the requirements of all three reviewers, have been made directly into the text and highlighted in blue.
Reviewer 2 Report
Comments and Suggestions for Authors
The manuscript provides an extensive and well-documented overview of steroidal metabolites produced by fungal and microbial sources, with a strong emphasis on structural novelty and reported bioactivities. The topic is relevant, timely, and suitable for a high-impact journal. However, in its current form, the article is largely descriptive, excessively long, and lacks sufficient critical synthesis, conceptual framing, and comparative analysis, which limits its scientific impact. My observations are listed below:
The abstracts lacks a critical message (what is learned, what is missing in the field, why this review matters now).
Section 2. The Genus Aspergillus - The section reads as a catalogue of compounds, not a critical review
There are no comparison between species, compound classes, or bioactivities, also there are limited discussion of biosynthetic logic or structure–activity trends. Which scaffolds recur?
which bioactivities appear most promising?, which findings are isolated vs. generalizable?
Section 3. Genus Penicillium - Structural novelty is emphasized, but biological relevance is unevenly discussed. Maybe the authors may group compounds by structural motif or biological function and explicitly discuss which compounds stand out and why
Section 4. Steroids from Miscellaneous Microorganisms - Please clarify why these organisms are grouped together. Also the authors should add a comparative paragraph linking these findings to Aspergillus and Penicillium.
Author Response
Reviewer 2
We sincerely thank the reviewer for the thorough and constructive evaluation of our manuscript and for recognizing the relevance, timeliness, and breadth of the review.
We fully agree that, in its original form, the manuscript placed strong emphasis on descriptive coverage and structural diversity, and that additional critical synthesis and conceptual framing would substantially enhance its scientific impact. In response to the reviewer’s comments, we have revised the manuscript extensively to strengthen comparative analysis, highlight recurring themes, and improve clarity and focus. Our detailed responses are provided below.
Comment 1: Abstract lacks a critical message (what is learned, what is missing, and why the review matters now).
Response:
We agree with this assessment. The abstract has been fully rewritten to emphasize:
- Key insights gained from comparing microbial steroid scaffolds across taxa,
- Current limitations in understanding biosynthetic logic and biological relevance,
- Why this review is timely in the context of genome mining, synthetic biology, and renewed interest in microbial steroids as drug leads.
The revised abstract now clearly articulates what the field has learned, what gaps remain, and how this review advances the field at this time.
Comment 2: Section 2 (Genus Aspergillus) reads as a catalogue rather than a critical review; lacks comparisons, biosynthetic logic, and structure–activity trends.
Response:
We appreciate this important point and have substantially revised Section 2 to move beyond a purely descriptive format. Specifically:
- Compounds are now grouped by recurring structural scaffolds (e.g., ergostanes, secosteroids, rearranged and hybrid frameworks).
- We added comparative analysis across Aspergillus species, highlighting which steroidal motifs recur and which appear species- or strain-specific.
- A new synthesis paragraph discusses:
- Recurrent biosynthetic features (e.g., oxidative ring cleavage, skeletal rearrangements),
- Emerging structure–activity trends, particularly in cytotoxic, anti-inflammatory, and enzyme-inhibitory assays,
- Which bioactivities appear robust across multiple scaffolds versus isolated observations.
This revision transforms Section 2 into a conceptual overview of steroid biosynthesis and bioactivity within Aspergillus rather than a compound list.
Comment 3: Section 3 (Genus Penicillium) emphasizes structural novelty but unevenly discusses biological relevance; compounds should be grouped and critically evaluated.
Response:
We fully agree and have reorganized Section 3 accordingly:
- Steroids are now classified by structural motifs and functional features, such as polyoxygenated steroids, rearranged skeletons, and hybrid metabolites.
- Biological activities are critically evaluated, with explicit discussion of:
- Which compounds exhibit potent or reproducible bioactivities,
- Which activities are weak, isolated, or insufficiently validated.
- We added a highlight section identifying Penicillium-derived steroids that stand out as promising leads, along with reasons for their relevance (potency, novelty, or mechanism).
This revision provides a clearer link between structural novelty and biological significance.
Comment 4: Section 4 (Miscellaneous Microorganisms) – unclear rationale for grouping; lacks comparison with Aspergillus and Penicillium.
Response:
We thank the reviewer for pointing this out. In the revised manuscript:
- We now explicitly clarify the rationale for grouping these organisms, emphasizing their shared contribution to expanding steroid chemical space beyond the dominant genera.
- A new comparative synthesis paragraph has been added at the end of Section 4, directly contrasting:
- Structural trends observed in miscellaneous microbes versus Aspergillus and Penicillium,
- Differences in biosynthetic complexity, frequency of rearranged skeletons, and reported bioactivities.
- This comparison highlights how non-canonical producers complement major fungal genera by contributing rare frameworks and unique modifications.
Overall Revisions and Conceptual Improvements
In response to Reviewer 2’s overarching critique, we have implemented the following global improvements:
- Reduced redundancy and improved narrative flow to address manuscript length.
- Added comparative tables and synthesis paragraphs where appropriate.
- Strengthened conceptual framing, especially regarding biosynthetic logic, scaffold recurrence, and translational relevance.
- Introduced a dedicated “Future Directions” section outlining genome mining, BGC analysis, co-culture strategies, and synthetic biology approaches.
We believe these revisions significantly enhance the critical depth, coherence, and scientific impact of the manuscript. We are grateful to the reviewer for these insightful suggestions, which have greatly improved the quality of the review.
With kind regards
Valery M. Dembitsky
P.S.
All corrections and additions, in accordance with the requirements of all three reviewers, have been made directly into the text and highlighted in blue.
Reviewer 3 Report
Comments and Suggestions for Authors
This review summarizes recent discoveries and structural classes of fungal-derived steroids, emphasizing their chemical diversity and bioactivity profiles, providing a valuable reference for the future research on fungal-derived steroids. However, some modifications should be considered before publication.
- Please check whether "bacteria" is appropriate in the keywords, and whether "microbial sources" or "fungal sources" is more accurate in Line 53.
- Please supplement the time range of the reviewed literatures in the Abstract and Introduction sections.
- Are the images in the manuscript copyrighted by the author? If not, please add references or online sources.
- The Abstract mentions "Endophytic and marine-derived fungi." Please expand relevant content on marine fungi around Line 35.
- In the Introduction, please add a summary of reported reviews on "Microbial Steroids" (if none, please state) and explain the novelty that distinguish the manuscript from others.
- In the Conclusion section, please provide statistical data and diagrams to analyze the compounds reviewed in the manuscript. For example, literature/compound counts listed, the proportion of compounds isolated from different genera, the proportion of compounds with various bioactivities, structure-activity relationship, and so on. Overall, analyze the data of the manuscript to provide references for other researchers, rather than merely listing information.
- Please explain why Section 2.1 is separately subdivided. Additionally, are Lines 75–93 describing a single fungal strain? If so, please merge them into one paragraph. If not, please clarify the sources and names of the strains.
Author Response
Reviewer 3
We sincerely thank the reviewer for their careful evaluation of our manuscript and for the constructive comments, which have helped us improve the clarity, balance, and analytical depth of the review. All suggestions have been carefully considered and addressed as detailed below.
General Comment on Keywords and Terminology
Reviewer comment:
Please check whether “bacteria” is appropriate in the keywords, and whether “microbial sources” or “fungal sources” is more accurate in Line 53.
Response:
We agree with the reviewer. Although a small number of bacterial-derived steroids are discussed, the manuscript predominantly focuses on fungal metabolites. Accordingly, the keyword list has been revised by removing “bacteria” and replacing it with “fungal steroids” and “microbial steroids”, as appropriate. In Line 53, the wording has been corrected to “fungal sources” to accurately reflect the scope of the review.
1. Time Range of the Reviewed Literature
Reviewer comment:
Please supplement the time range of the reviewed literature in the Abstract and Introduction sections.
Response:
We appreciate this suggestion. The time span of the literature surveyed (approximately 2000–2024) has now been explicitly stated in both the Abstract and the Introduction, clarifying the temporal scope of the review and emphasizing its focus on recent and emerging discoveries.
2. Copyright of Figures
Reviewer comment:
Are the images in the manuscript copyrighted by the author? If not, please add references or online sources.
Response:
All figures in the manuscript were redrawn by the authors based on the original literature sources and are therefore original schematic representations, not reproductions. Appropriate literature citations have been added to each figure caption to acknowledge the original sources of the compounds and structures, in accordance with MDPI guidelines.
3. Expansion of Marine Fungi Content
Reviewer comment:
The Abstract mentions “endophytic and marine-derived fungi.” Please expand relevant content on marine fungi around Line 35.
Response:
We agree and have expanded the discussion on marine-derived fungi in the Introduction (around Line 35). The revised text now highlights:
- The ecological uniqueness of marine fungal habitats,
- The frequent occurrence of structurally rearranged and halogenated steroids,
- The contribution of marine fungi to chemically distinct steroidal frameworks compared to terrestrial counterparts.
This expansion aligns the Introduction more closely with the emphasis stated in the Abstract.
4. Contextualization with Previous Reviews and Novelty
Reviewer comment:
Please add a summary of reported reviews on “Microbial Steroids” (if none, please state) and explain the novelty that distinguishes the manuscript from others.
Response:
A new paragraph has been added to the Introduction summarizing existing reviews related to microbial and fungal steroids, noting that most prior works either:
- Focus on specific genera,
- Emphasize biosynthesis or ecology,
- Or treat steroids as a minor subset of broader fungal metabolite reviews.
We explicitly state that no comprehensive review dedicated to structurally diverse microbial steroids with integrated bioactivity analysis currently exists. The novelty of our manuscript lies in:
- Its broad comparative coverage across multiple fungal genera,
- Its emphasis on unusual steroid frameworks (secosteroids, rearranged steroids, hybrids),
- And the integration of chemical diversity with bioactivity trends and future discovery strategies.
5. Data Analysis and Visualization in the Conclusion
Reviewer comment:
Please provide statistical data and diagrams in the Conclusion to analyze the compounds reviewed.
Response:
We fully agree and have substantially revised the Conclusion section. The revised version now includes:
- Quantitative summaries of the number of compounds reviewed,
- Distribution of compounds across major genera (e.g., Aspergillus, Penicillium, others),
- Classification of compounds by major bioactivity types (cytotoxic, antimicrobial, anti-inflammatory, etc.),
- A brief discussion of emerging structure–activity trends.
In addition, summary tables and schematic diagrams have been added to visually represent these distributions, providing a clearer analytical framework and facilitating comparison for future researchers.
6. Clarification of Section 2.1 Subdivision
Reviewer comment:
Please explain why Section 2.1 is separately subdivided. Are Lines 75–93 describing a single fungal strain?
Response:
Thank you for pointing this out. Section 2.1 was subdivided to emphasize a distinct subgroup of steroidal metabolites derived from closely related Aspergillus strains sharing biosynthetic features. However, we agree that the original presentation lacked clarity.
The text in Lines 75–93 has now been revised and consolidated, with explicit clarification of:
- The number of strains involved,
- Their taxonomic identities,
- And the rationale for grouping them together.
Where appropriate, compounds derived from a single strain are now discussed within a single unified paragraph, improving readability and coherence.
Once again, we thank the reviewer for these insightful comments, which have significantly strengthened the manuscript in terms of clarity, rigor, and analytical depth.
P.S.
All corrections and additions, in accordance with the requirements of all three reviewers, have been made directly into the text and highlighted in blue.
With kind regards
Valery M. Dembitsky
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
Agree
