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Nice work. The authors have addressed all of the reviewers' comments. This manuscript is ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Fanglin Guan, a PeerJ Section Editor covering this Section #]
no comment
no comment
no comment
The current version of the manuscript is significantly improved compared to the original. The authors responded to my questions and made the requested changes to the manuscript. Considering the above, I recommend the current version of MS for publication. Congratulations!
I reviewed the revision of the manuscript and the rebuttal letter. The authors addressed most of my comments adequately and in the cases they have not agreed with my criticism, they explained it convincingly, and they added additional arguments to the Discussion to prevent misunderstanding. I believe that the revised manuscript is suitable for publication in PeerJ.
see Basic Reporting
see Basic Reporting
No comments on the revised manuscript.
No more comment.
No comment.
Thanks for the detailed answer to the reviewers.
I see that all my remarks were considered. I have no more comments.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The topic and scope of the research presented in this work are interesting from both a scientific and an application perspective. I agree with the authors' statement that " …The high diversity of macrolactone congeners seems to result from an ancient but ongoing competition amongst various microorganisms for resource-limited niches …" but we must remember that it can also impact our lives, mycotoxins in agricultural crops and food.
The manuscript is written in proper scientific language, and its individual chapters are coherent. I confirm that the research methods were chosen appropriately. The results are well documented, and their graphic presentation is of a very high standard. All this demonstrates the credibility of the obtained results.
As I have already mentioned, in my opinion, the results are important from both a scientific and an applied perspective.
I support the acceptance of the work for publication, but before it is officially accepted for publication, I have two questions for the authors:
1) Why were these fungal genera/species chosen and not others?
2) Why are the E-values and %ID values relatively low?
LANGUAGE
The manuscript is well structured, and the language is understandable and grammatically correct except for occasional occurrences of wrongly placed or missing commas and a few additional issues. Examples of missing or wrongly placed commas:
34 The phylogenetic placement of amplicons against a large set of reference polyketide synthases has allowed us to detect potential BDL-like compounds
=> The comma separates the subject from its verb. It should be removed.
47 scaffolds for the development of novel drugs (Newman & Cragg, 2020a). After the wealth of new drugs based on NPs introduced in the late 1980s, we have seen a slight decrease
=> The long introductory passage is correctly separated from the main sentence by commas, but the second comma after "1980" is missing.
61 biosynthesis. Combinations of different functional blocks, followed by heterologous expression (Xu et al., 2014a; Kim, Sengupta & Sim, 2021), are being tested to produce completely.
=> The insertion "followed by… Sim, 2021)" has to be separated from the main sentence by two commas; the second comma after "Sim, 2021)" is missing.
52 samples for desired compound activities, with possible variations such as the One Strain Many Compounds approach, often ended up in re-isolation of the already known compounds.
=> as above: a comma after "approach" is missing
Further minor language issues to consider:
47 scaffolds for the development of novel drugs
=> "for the development of…"
The adjective "novel" is used 13 times. Inflationary use of "novel" has often been criticized (e.g., BMJ (2015) 351:h6467; Nature 10.1038/nature.2015.19024). I advise the authors to check whether all uses of "novel" are justified by pointing out breakthrough developments that are qualitatively different from similar developments in the past. For instance, the design of degenerated primes amplifying conserved segments of another protein family is new but not novel because the strategy has been used for countless protein families in the past. Similarly, "the novel findings" on L519 should be replaced with "new findings" (without an article).
"Completely" in "Completely novel" (L66) is redundant
Section "Conclusion" is grammatically fine and understandable, but the language style is noticeably less smooth than the remaining text. If the authors wish to keep the language as concise and elegant as in the previous sections, I recommend checking the wording. Also check for missing commas and unjustified use of "novel". I recommend reconsidering whether passages like "As with every phylogenomic inquiry, based on a rapidly growing body of knowledge, one has to be prepared to reconsider the obtained results under new evidence", apart from sounding awkward, are suitable for the section Conclusion.
REFERENCES
All relevant studies are cited except one, which is important because it is one of a few studies that rigorously proved the role of a fungal RAL in antagonistic interactions by gene disruption:
Kosawang, C.; Karlsson, M.; Vélëz, H.; Rasmussen, P.H.; Collinge, D.B.; Jensen, B.; Jensen, D.F. Zearalenone detoxification by zearalenone hydrolase is important for the antagonistic ability of Clonostachys rosea against mycotoxigenic Fusarium graminearum. Fungal Biol. 2014, 118, 364–373.
Most citations are correctly placed, but there are two inaccuracies:
485 C3-C8 lactone congeners. While the hypothesis of role in virulence is viewed as less probable, both phytotoxic (Meepagala, Johnson & Duke, 2016) and antibacterial properties (Choi, Le & Kim, 2022) were previously noted for curvularin/dehydrocurvularin.
=> The sentence implies that both phytotoxic and antibacterial properties are potentially relevant for virulence, but this holds only for phytotoxicity. Antibacterial properties support the role of RALs in microbial competition, which is a different hypothesis that has been discussed in an earlier passage.
109 The resulting polyketides vary in bioactivity and constitute potent tools for competition between producing fungi and other organisms for the same ecological niches (Gaffoor et al., 111 2005; Gaffoor & Trail, 2006)(Rojas et al., 2020).
=> None of the cited papers stated or hypothesized that these PKSs are tools for competition between their producers and other organisms in the ecological niche. The first study addressed the potential role of zearalenone in a number of functions, but EXCEPT in competition. The second study identified the genes for the synthesis of zearalenone, but it has not addressed its function. The third study revealed suppression of fungi colonizing wheat ears by Fusarium spp., but it has not revealed anything about the role of polyketides in this process because wild-type strains producing two dozen secondary metabolites were used. A statement about polyketides as "a potent tool for competition between the producing fungi and other organisms in the same ecological niche" requires the two references cited by the authors in this context earlier, and eventually also the paper by Kosawang et al. that I recommended to consider above.
DATA PRESENTATION
The quality of figures is adequate, and the data are provided in the Supplement Materials, including the pipeline in GitHub.
The design is logical and comprehensible.
Just a note to consider. The study focuses on two kinds of PKS, namely RALs and DALs, jointly called BDLs. The authors describe the differences between RALs and DALs (Figure 2) and mention that the genes are considered orthologs. On this background, they present a strategy for developing PCR markers for PKS genes involved. In this context, it seems appropriate to address whether PKS genes involved in RAL and DAL synthesis differ. I know that it was not the purpose of the study to identify canonical differences between PKSs encoding RALs and DALs, but perhaps the authors can say what they think about such differences, as they decided to assign sequence hits to the RALs or DALs clades merely based on their phylogenetic placements.
The methods are well documented, the results are sound, and the files in Suppl. Data allow the readers to check all conclusions on their own (which, admittedly, I have not attempted). Provide large dendrograms in Supp. Data is a necessary compromise of studies with large sequence sets.
Two PKSs are involved in the synthesis of each DAL. A question whether PKSs of the RAL and DAL clade differ and whether the differences occur in the starting HR-PKS or in the folding/terminating NR-PKS might exceed the scope of the study, but the authors could address it while discussing their findings.
The ancestral RALs clade and DALs clade in the current study are well separated (Fig. 6). One of the papers cited in the manuscript claims that the same HR-PKS and NR-PKS were involved in the synthesis of both a RAL and a DAL in a single organism (Viñas et al. 2025, by coincidence also Fig. 6). Can the authors resolve this apparent controversy, or at least address it in the Discussion?
All in all, this is a great study that will be very helpful for wet labs working on fungal BDLs. I think that the minor issue described in 1. Basic reporting should be addressed. The issues related to the differences between PKSs of RAL and DAL pathways are raised in 2. Experimental design and 3. Validity of the findings is merely a suggestion, and the validity of the work will not be diminished if the authors don't want or cannot address them.
This work considers a particular problem of fungal natural products analysis using a customized bioinformatics tool. Biosynthesis of diverse aromatic polyketides is a widespread adaptation among fungi.
The authors screened fungal isolates for the presence of highly reducing polyketide synthases involved in the biosynthesis of benzenediol lactones. The problems of degenerate sequence target search are discussed.
The article has a professional structure, well-illustrated by original figures.
The authors cultured environmental fungal isolates and sequenced their products. The next analysis of selected sequences was based on bioinformatics tools. The authors created a computer pipeline for the classification of sequences. The code is available at GitHub.
The results are shown for highly reducing polyketide synthases and homologs search.
All the experimental and validation steps are correct.
The conclusion is well stated. Though the problem of only a limited gene family (involved in the biosynthesis of benzenediol lactones) seems to be limited.
I'd recommend showing the effectiveness of the bioinformatics pipeline for other gene families.
I have some comments to fix.
First, the keyword list is not shown.
Please add keywords.
It is worth shortening the Abstract.
Need to discuss the applicability of the phylogenomic pipeline to other gene families.
Please update the phrase "Our analysis highlights both efficacy and difficulty of degenerate amplification."
The word 'degenerate' is not clear here.
Analysis of Natural Products as a potential drug is an important problem.
Add older references to works "in the late 1980s".
Change wording "A contributing variable was..." - it is not a variable, but rather an impact.
"Until 2015, more than 190 natural BDLs were identified..." - need to add details, and give references separately after this sentence (avoid bulk citation).
"Phylogenomic analysis has previously found extensive usage ..." - again, bulk citation. And the statement is too common. Rephrase.
In 'Materials and Methods'
'BLASTP, ' - give reference, version. There are too many BLAST realizations.
“downloaded from JGI/MycoCosm, Ensembl/Fungi or NCBI” – please give exact references, links to these data sources. Note the database release.
“(Thermo Fisher Scientific, Waltham, MA, USA)” – used several times, even in one sentence. Cite it once at the end of the sentence.
“are matched to the UniProt/SwissProt” - please show the database version.
“Our results (Figure 6) support DAL/cytosporone…” – please add wording ‘presence of’, ‘observation of’, or ‘evidence of’, after the word ‘support’. Or rephrase in another way.
“in-depth” – typo.
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