From the journal RSC Chemical Biology Peer review history

10-Jan-2021

Dear Dr Owen:

Manuscript ID: CB-ART-12-2020-000228
TITLE: Metathramycin, a new bioactive aureolic acid discovered by heterologous expression of a metagenome derived biosynthetic pathway.

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

************

Reviewer 1

Article ID: RSC CB-ART-12-2020-000228

Comments:
Scientists have drawn inspiration from natural products for the development of new drugs; the diverse bioactivities natural products display also make them useful molecular tools in basic research. As traditional culture-based methods experiencing a “high rediscovery rate” problem, we desperately need new methods for natural product discovery. The current manuscript reports the discovery of a mithramycin congener (mithralog) that shows potent anticancer activity via a novel culture-independent method. In my view this work would be of significant interest to researchers in many fields, including natural product discovery, bioinformatics, microbiology, and medicinal chemistry.

The authors started from a New Zealand soil library and used PCR to identify ketosynthase (KS) genes homologous to that in a mithramycin gene cluster. They then fished out two cosmid containing all relevant biosynthetic genes and reconstructed the complete biosynthetic gene cluster, which was heterologously expressed in Streptomyces albus. HPLC purification of the heterologous expression culture led to the discovery of the precursor of new mithralog, which was tested against microorganisms (MIC) and structurally characterized by a combination of NMR and MS/MS. The authors were able to convert the precursor to the final natural product by co-overexpressing two putative biosynthetic enzymes and collect enough material for anticancer activity testing (IC50).

This work is a tour de force and I highly recommend acceptance / publication. Detail experimental methods and structural characterizations have already been presented; I see only a few minor changes necessary (see below).

Suggested corrections:
1. “Figure 1” was not cited anywhere in the text.
2. Page 2, line 7. “Another method for uncovering novel chemistry is the discovery of new biosynthetic pathways from environmental microbes (Newman and Cragg, 2016).” This is THE review that catalogs natural product discoveries, but it seems odd to be cited here. It is an excellent review that should be cited somewhere in the article.
3. Page 2, line 9. While the Brady lab has done some very nice work, the authors should cite work from other labs as well. They are not the only lab working on metagenomic approaches for natural product discovery.
4. Page 2, line 10. “… providing access to the biosynthetic capacity of microbes independent of our ability to culture these organisms …”
5. Page 2, line 23. “… eDNA was extracted, purified, and cloned …” (missed a comma)
6. Page 2, line 34. “Notably, however, the individual amino acid …” (missed a comma)
7. Page 3, line 3. “… and in a single transformation all emergent colonies (12) analysed …”
8. Page 5, line 46. “… C47H68O21 calcd. 967.4180,  = 0.6 ppm).”
9. Page 5, line 50. “… the amount isolated from 7 L of culture was insufficient to allow the use of standard quantification methods.”
10. Figure 2 legend. 1) “Compound 5 is the major metabolite peak (specify retention time).” 2) Specify based on which peak was the inset spectrum obtained? (I understand that with the same aromatic core these compounds all have very similar UV absorption pattern, but one should still specify).
11. Figure 4 legend. The putative MS fragments should not be named 1 to 6, which already referred to various precursor and mithralogs in Figure 1.
12. Table 2. Add a column to show metathramycin IC50.
13. Figure S1 is exactly the same as Figure 1. No need to show it again.

Reviewer 2

The present manuscript describes the discovery of a new aureolic acid metabolite from metagenomic DNA through an interdisciplinary approach. The authors present convincing and solid work that includes the screening of metagenomic DNA, heterologous expression of a yeast-reconstituted gene cluster in which the content of two overlapping cosmids is joined, isolation and structural elucidation of premetathramycin and metathramycin, bioactivity assays.

The low yields of metathramycin obtained mean that NMR data of the compound could not be generated. However, MS/MS fragmentation data and comparison of the spectra with those of the closely-related mithramycin, allow the authors to confidently predict the planar structure of metathramycin.

Overall, I think that this manuscript is suitable for publication and may only require minor proofreading for typos, e.g. page 7, line 43 and page 9, line 14: “(Figure S3.).” -> no full stop required inside the brackets.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

We thank the reviewers and the editor for their assessment of our manuscript and the comments they have provided. We have addressed each of the minor points outlined by the reviewers. The changes we have made are outlined in the point-by-point response we have uploaded.

Dear referees,

Thank you for taking the time to review our manuscript. We have incorporated each of the suggestions made, as outlined in the point-by-point response below.

Best regards,

Jeremy Owen

Referee: 1

Suggested corrections:
1. “Figure 1” was not cited anywhere in the text.
We have added appropriate figure references to the main text.
2. Page 2, line 7. “Another method for uncovering novel chemistry is the discovery of new biosynthetic pathways from environmental microbes (Newman and Cragg, 2016).” This is THE review that catalogs natural product discoveries, but it seems odd to be cited here. It is an excellent review that should be cited somewhere in the article.
We have amended the text and moved this citation.
3. Page 2, line 9. While the Brady lab has done some very nice work, the authors should cite work from other labs as well. They are not the only lab working on metagenomic approaches for natural product discovery.
References for examples of research from additional lab groups have been added.
4. Page 2, line 10. “… providing access to the biosynthetic capacity of microbes independent of our ability to culture these organisms …”
Added “our”.
5. Page 2, line 23. “… eDNA was extracted, purified, and cloned …” (missed a comma)
Added comma.
6. Page 2, line 34. “Notably, however, the individual amino acid …” (missed a comma)
Added comma.
7. Page 3, line 3. “… and in a single transformation all emergent colonies (12) analysed …”
This sentence has been amended as suggested.
8. Page 5, line 46. “… C47H68O21 calcd. 967.4180, = 0.6 ppm).”
Added “ppm”.
9. Page 5, line 50. “… the amount isolated from 7 L of culture was insufficient to allow the use of standard quantification methods.”
Added “was”.
10. Figure 2 legend. 1) “Compound 5 is the major metabolite peak (specify retention time).” 2) Specify based on which peak was the inset spectrum obtained? (I understand that with the same aromatic core these compounds all have very similar UV absorption pattern, but one should still specify).
Retention time has been added to the inset panel in Figure 2.
11. Figure 4 legend. The putative MS fragments should not be named 1 to 6, which already referred to various precursor and mithralogs in Figure 1.
Fragment labels have been changed to i – vi
12. Table 2. Add a column to show metathramycin IC50.
This column has been added to table 2.
13. Figure S1 is exactly the same as Figure 1. No need to show it again.
Figure S1 has been removed and the numbering updated.


Referee: 2


Overall, I think that this manuscript is suitable for publication and may only require minor proofreading for typos, e.g. page 7, line 43 and page 9, line 14: “(Figure S3.).” -> no full stop required inside the brackets.
We have proofed the article and corrected minor errors.

21-Jan-2021

Dear Dr Owen:

Manuscript ID: CB-ART-12-2020-000228.R1
TITLE: Metathramycin, a new bioactive aureolic acid discovered by heterologous expression of a metagenome derived biosynthetic pathway.

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

Reviewer 2

The authors have addressed all reviewers' comments, hence the article should be accepted for publication in its present form.