Metabolomics insights into the polyketide-lactones produced by Diaporthe caliensis sp. nov., an endophyte of the medicinal plant Otoba gracilipes

ABSTRACT Plants of the genus Otoba have been the basis for the treatment of tropical diseases in indigenous communities of countries like Colombia. Despite the lack of knowledge about their bioactive principles, endophytic fungi derived from medicinal plants are a prolific source of innovative chemistry. We systematically investigated the secondary metabolite production of a previously undescribed species of Diaporthe, herein introduced as Diaporthe caliensis sp. nov., using different metabolomics approaches together with classical chemical screening. To get an outline of the chemical space produced by this fungus, an exploratory molecular networking (MN) analysis was undertaken. A major molecular family was found to contain the known 10-membered lactone phomol (1), together with other putative congeners as compound 3. After isolation by preparative high-performance liquid chromatography, we confirmed phomol (1) as the main reason for the antimicrobial activity of the crude extract. The unknown absolute configuration of 1 was determined by the synthesis of α-methoxy-α-trifluoromethylphenylacetyl (MTPA)-esters and chemical degradation experiments. Moreover, caliensolides A (2) and B (3) were isolated, and their structures were elucidated as novel butenolides structurally unrelated to 1. Overall, the initial MN analysis incorrectly clustered compounds 1 and 3 within a single molecular family, despite evident differences in chemical structures and biosynthetic origin. Contrariwise, the unsupervised substructure discovery algorithm MS2LDA provided a deeper understanding of the fragmentation patterns and correctly clustered the polyketide-lactones produced by D. caliensis sp. nov. Our findings encourage the exploration of Colombian fungal diversity, which as demonstrated here could result in the discovery of new natural products. IMPORTANCE The integration of metabolomics-based approaches into the discovery pipeline has enabled improved mining and prioritization of prolific secondary metabolite producers such as endophytic fungi. However, relying on automated untargeted analysis tools might lead to misestimation of the chemical complexity harbored in these organisms. Our study emphasizes the importance of isolation and structure elucidation of the respective metabolites in addition to deep metabolome analysis for the correct interpretation of untargeted metabolomics approaches such as molecular networking. Additionally, it encourages the further exploration of endophytic fungi from traditional medicinal plants for the discovery of natural products.

1.In the Figure S19, where the authors show the cosine score adjustment, maybe it is worth to add the caveat about the limitation of this molecular network topology analysis without a robust support of the structural chemistry data.2. One suggestion for future work trying to induce the sporulation would be to try the Microcultivation technique.3.In the table S2 and S3, the concentration must to be consistent.This reviewer recommends µM units, as presented in table S3. 4. The inconsistency in the units for compounds concentration avoid to see the actual cytotoxicity effect of the phomol.Please note that, if the information provided in the table S2 are correct, then the statement in the biological activity session "compound 1 showed weak cytotoxic activity against the mammalian cell lines tested" and in the discussion session "weak cytotoxic effects against the human endocervical adenocarcinoma and mouse fibroblasts evaluated cell lines" do not holds.As presented by the reference number 34, phomolide C had activity against human tumor cell at 50 μg/mL close to the concentrations found in the present manuscript (~66 μg/mL) against the microorganisms.In molar concentration this 66 μg/mL would be equivalent to approximately 161 µM, which is way higher than the IC50 for the fibroblast cell line.The biological activity is due to high toxicity of phomol or specific activity against the microorganisms?This point needs a solid clarification.
Reviewer #2 (Comments for the Author): Charria-Girón et al. successfully isolated a novel endophyte named Diaporthe caliensis.They conducted secondary metabolomics to characterize the molecular features of the culture extract, which were subsequently analyzed using molecular networking techniques.In their study, they not only isolated a known compound, phomol (1), but also identified two new structurally distinct molecules named caliensolides A (2) and B (3).Furthermore, they determined the absolute stereochemistry of secondary alcohols in compounds 1 and 2 using Mosher's method.
While I find that some aspects of the manuscript might not align perfectly with the scope of the journal, the sections focusing on the isolation and elucidation of the compound structures were executed excellently and hold significant merit for potential publication.
1. Table 1 can be moved to the Supplementary Information (SI).Consequently, citation references within the table should also be transferred to the SI. 2. Figure 1 is referenced but not included in the manuscript.3.In Table S4, the current format is not suitable.Reevaluate the presentation style to include appropriate protein alignment formatting.Additionally, ensure that the table numbers follow the order of appearance.4. The excessive use of abbreviations can hinder readability.There are repeated instances of abbreviation use.When an abbreviation is introduced for the first time, spell it out completely. 5. Lines 96-109 are challenging to comprehend.Consider revising the presentation style to enhance clarity.6. Lines 110-123: Incorporating a photographic image of the culture would facilitate the understanding of the description.7. Lines 143-152: Including figure numbers would aid in understanding the description.

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Review Spectrum 311399
The authors produced a valuable study evalua2ng the secondary metabolites from a new species of Diaporthe fungus, classified as D. caliense sp.nov.. and found two new metabolites named caliensolides A and B. The authors applied an interes2ng pipeline for the data analysis which highlight the solving problems abili2es of the group.The study could extract the informa2on even with the annota2on problems inherent to fungi metabolome databases.They solved the caliensolide A structure showing the determina2on to find the answers.Yet, they surpassed the limita2on of the FBMN approach adjus2ng the cosine score and added value using MS2LDA.That said, this reviewer has some minor ques2on and observa2ons: 1.In the Figure S19, where the authors show the cosine score adjustment, maybe it is worth to add the caveat about the limita2on of this molecular network topology analysis without a robust support of the structural chemistry data.2. One sugges2on for future work trying to induce the sporula2on would be to try the Microcul2va2on technique.3.In the table S2 and S3, the concentra2on must to be consistent.This reviewer recommends µM units, as presented in table S3. 4. The inconsistency in the units for compounds concentra2on avoid to see the actual cytotoxicity effect of the phomol.Please note that, if the informa2on provided in the table S2 are correct, then the statement in the biological ac2vity session "compound 1 showed weak cytotoxic ac2vity against the mammalian cell lines tested" and in the discussion session "weak cytotoxic effects against the human endocervical adenocarcinoma and mouse fibroblasts evaluated cell lines" do not holds.As presented by the reference number 34, phomolide C had ac2vity against human tumor cell at 50 μg/mL close to the concentra2ons found in the present manuscript (~66 μg/mL) against the microorganisms.In molar concentra2on this 66 μg/mL would be equivalent to approximately 161 µM, which is way higher than the IC50 for the fibroblast cell line.The biological ac2vity is due to high toxicity of phomol or specific ac2vity against the microorganisms?This point needs a solid clarifica2on.

Rebuttal letter (spectrum02746-23) Reviewer 1:
The authors produced a valuable study evaluating the secondary metabolites from a new species of Diaporthe fungus, classified as D. caliense sp.nov.. and found two new metabolites named caliensolides A and B. The authors applied an interesting pipeline for the data analysis which highlight the solving problems abilities of the group.The study could extract the information even with the annotation problems inherent to fungi metabolome databases.They solved the caliensolide A structure showing the determination to find the answers.Yet, they surpassed the limitation of the FBMN approach adjusting the cosine score and added value using MS2LDA.That said, this reviewer has some minor question and observations: 1.In the Figure S19, where the authors show the cosine score adjustment, maybe it is worth to add the caveat about the limitation of this molecular network topology analysis without a robust support of the structural chemistry data.
Thanks a lot for the suggestions, we have included in lines 292 and 293 a statement about this fact.
2. One suggestion for future work trying to induce the sporulation would be to try the Microcultivation technique.
So far, we are not familiar with this technique.However, the authors are grateful to reviewer #1 for his remark, and will try ths method in future studies.S2 and S3, the concentration must to be consistent.This reviewer recommends µM units, as presented in table S3.

Table S2 has been modified accordingly.
4. The inconsistency in the units for compounds concentration avoid to see the actual cytotoxicity effect of the phomol.Please note that, if the information provided in the table S2 are correct, then the statement in the biological activity session "compound 1 showed weak cytotoxic activity against the mammalian cell lines tested" and in the discussion session "weak cytotoxic effects against the human endocervical adenocarcinoma and mouse fibroblasts evaluated cell lines" do not holds.As presented by the reference number 34, phomolide C had activity against human tumor cell at 50 μg/mL close to the concentrations found in the present manuscript (~66 μg/mL) against the microorganisms.In molar concentration this 66 μg/mL would be equivalent to approximately 161 µM, which is way higher than the IC50 for the fibroblast cell line.The biological activity is due to high toxicity of phomol or specific activity against the microorganisms?This point needs a solid clarification.
Thanks for pointing out this issue!Indeed the MICs for phomol against the different microorganisms are at least 4 times higher than the obtained IC50s.We have changed the results accordingly and included an additional sentence in the discussion indicating the consequences of these findings.

Reviewer #2:
Charria-Girón et al. successfully isolated a novel endophyte named Diaporthe caliensis.They conducted secondary metabolomics to characterize the molecular features of the culture extract, which were subsequently analyzed using molecular networking techniques.In their study, they not only isolated a known compound, phomol (1), but also identified two new structurally distinct molecules named caliensolides A (2) and B (3).Furthermore, they determined the absolute stereochemistry of secondary alcohols in compounds 1 and 2 using Mosher's method.
While I find that some aspects of the manuscript might not align perfectly with the scope of the journal, the sections focusing on the isolation and elucidation of the compound structures were executed excellently and hold significant merit for potential publication.
1. Table 1 can be moved to the Supplementary Information (SI).Consequently, citation references within the table should also be transferred to the SI.
The table 1 was moved into the SI as suggested, same as the corresponding references.
2. Figure 1 is referenced but not included in the manuscript.S4, the current format is not suitable.Reevaluate the presentation style to include appropriate protein alignment formatting.Additionally, ensure that the table numbers follow the order of appearance.

In Table
Alignments are always deposit or provided in this fasta format.In fact, it is the only format compatible with all kind of programs used in the phylogenetic analysis.Therefore, the authors consider that no changes are required.Moreover, the phylogenetic analysis is based on sequence data, so it cannot be changed to a protein alignment formatting.Line 287: Change "1 and 2" to "2 and 3".Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.

The excessive use of abbreviations can
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Thank you for submitting your paper to Spectrum.Sincerely, Sacha Pidot Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

Figure 1
Figure 1 is referenced in the text in the line 81 and the consensus ML tree is included in the manuscript at the end of the text (line 855).
hinder readability.There are repeated instances of abbreviation use.When an abbreviation is introduced for the first time, spell it out completely.We have made the corresponding changes in the revised version 5. Lines 96-109 are challenging to comprehend.Consider revising the presentation style to enhance clarity.It is the established formatting to introduce a new species based on molecular data.Any other style could invalidate the new species based on the nomenclatural code.6. Lines 110-123: Incorporating a photographic image of the culture would facilitate the understanding of the description.Pictures of the colonies are provided in the new manuscript SI. 7. Lines 143-152: Including figure numbers would aid in understanding the description.

Figure
Figure numbers have been included in these paragraphs accordingly.