From the journal RSC Chemical Biology Peer review history

02-Sep-2023

Dear Dr Mast:

Manuscript ID: CB-ART-08-2023-000143
TITLE: <b>Mutasynthesis 2.0: Generation of novel pristinamycin derivatives by engineering the phenylglycine residue</b>

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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RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy, https://credit.niso.org/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines https://www.rsc.org/journals-books-databases/author-and-reviewer-hub/authors-information/responsibilities/ for more information.

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Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

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

Reviewer 1

Pristinamycin I is a cyclic hexapeptide that is widely used in clinical practice, particularly for the treatment of multi-drug resistant bacterial infections. However, resistance to pristinamycin has increased in recent years, necessitating the development of derivatives to overcome resistance. In this manuscript, the authors targeted the L-phenylglycine residue of pristinamycin I for modification by mutasynthesis and several halogenated pristinamycin derivatives were successfully produced. The derivatives were structurally confirmed and tested for antimicrobial activity. In addition, a whole-cell biotransformation approach using E. coli was established to produce these derivatives. The data in the manuscript are reliable and sufficient for publication.

Note that the authors refer to the methodology of this paper as "Mutasynthesis 2.0" in the title, which seems to overstate the significance of this study, and it is recommended that the term be deleted in title.

In addition, the Figure 1 is a typical mutasynthesis principle; while, the authors tried to highlight their improved version for "Mutasynthesis 2.0", so I strongly suggest that Figure 1 should be redesigned (or add the third part) to exhibit the difference between the typical mutasynthesis and the new level one.

Reviewer 2

Hennrich et al. have developed mutasynthetic methods to incorporate non-natural (halogenated) phenylglycine derivatives into pristinamycin I, which is active against Gram-positive pathogens (together with pristinamycin II). The work involves the development of a Streptomyces pristinaespiralis phenylglycine auxotrophic mutant strain to enable enrichment of the growth media with the desired Phg analogue. 4-Cl and 4-F analogues were successfully incorporated in isolatable yield and characterised by NMR and LCMS. Both analogues possess comparable antimicrobial activity (at best) compared to authentic pristinamycin I. An E. coli-derived 4F-Phg mutasynthon was also prepared via the enzymatic transformation of commercially available precursors to broaden the range of Phg variants that can be incorporated into pristinamycin I.

Conceptually, this work is excellent, and the experimental work is a solid contribution towards the goal of developing new antibiotics that can evade antimicrobial resistance mechanisms. New and effective biosynthetic methods for preparing analogues of antimicrobial compounds is needed, particularly given the difficulties in accessing these molecules (such as pristinamycin I) via chemical methods.

I recommend publication after minor corrections (and once an additional experiment is complete), after the below points have been addressed.

- The authors should better articulate the limitations of their biosynthetic methods. For example, how do the yields compare with native pristinamycin I?

- Regarding nomenclature, greater consistency is required (e.g. 4-fluoro or para-fluoro)

- 121: Would replacing “…involved in…” with “…essential for…’’ be better?

- 122: Would it make more sense to replace “…Pristinamycin I (1) is a natural compound…” with “…Pristinamycin is….”?

- Figure 2: Threonine is missing the carbonyl group. Also, the stereochemistry of its alpha carbon is absent (Thr has 2 chiral centres). More generally, it would be aesthetically pleasing to indicate the stereochemistry by inserting up or down bonds between the alpha and beta carbons, rather than between the alpha and carbonyl carbons. The latter point is only a suggestion.

- 168: Refer the reader to Figure 3A. Also, the structure of quinupristin should also be labelled as “2” in the image.

- 209-210: Why was there a need to obtain an S. pristinaespiralis mutasynthesis strain that produces pristinamycin I derivatives exclusively? They seem to be relatively easy to separate via HPLC. Or is crude material being used as well?

- 250-253: The stereochemistry of the Phg variants in the various pristinamycin I analogues is unclear, as the authors suggest, and it is worth noting that Phg has a strong propensity to racemise. I recommend conducting additional experiments to determine whether chemical racemisation is occurring prior to addition to the culture. The authors should prepare the Phg solution again and either functionalise the amino acid with Marfey’s Reagent and perform RP-HPLC analysis or determine its optical rotation (at least 3 replicates). Moreover, can retention times (RTs) of the ‘D-Phg’ pristinamycin analogue and authentic pristinamycin I be compared (using a slow gradient)? Perhaps the D-Phg analogue has a different RT (if it in fact has D-stereochemistry)?

- Figure 4: It would be good to show the full HPLC chromatograms, rather than a narrow X-axis range. Presumably there are other products present in the ethyl acetate extract.

- Figures S2-S4: The LCMS data should be better presented. There is no real need to show the TIC spectrum. Probably no need for 2 UV spectra, 210 nm would be enough. Does 200-600 nm refer to the range for the UV-Vis (diode array) spectrum? The mass spectra are difficult to read (especially the peak labels). Please fix. Regarding Figure S2, presumably this is the crude material? If so, please indicate which peak the MS spectrum corresponds to.

- Figure S24: Please include the UV chromatogram at 210 nm. It is important to know the effectiveness or limitations of this strategy and how it compares to using Phg derivatives directly.

Response to Referee: 1

Thanks for the advice! We understand the objection. The headline has been changed to “Biotransformation-coupled mutasynthesis for the generation of novel pristinamycin derivatives by engineering the phenylglycine residue”. The wording “mutasynthesis 2.0” was removed throughout the text. Also Fig. 1 has been changed accordingly and the biotransformation step for mutasynthon supply is now included. Thanks for the valuable suggestion!

Response to Referee: 2

Thanks for the positive feedback!

- The authors should better articulate the limitations of their biosynthetic methods. For example, how do the yields compare with native pristinamycin I?

Indeed, we focused on the general feasibility of our mutasynthesis approach in this study rather than on production yields. Unfortunately, we cannot provide reliable data concerning the amount of produced PI derivatives. The first fermentations that were conducted to ascertain whether or not a given Phg-derivative was incorporated into PI, was measured by MS analysis but the samples did not deliver enough substance to calculate concentrations. Subsequent large-scale fermentations were pooled for extraction and purification without prior quantitative PI analysis. We can only provide production amounts of one of these large-scale fermentations that were analyzed by HPLC. These measurements concern the production of 6-fluoropristinamycin I, 6-chloropristinamycin I, and C-PI (natural pristinamycin I produced from S. pristinaespiralis ∆pglA∆snaE1 supplemented with L-Phg), which were produced in amounts of 1.52 µg/ml, 1.55 µg/ml, and 1.51 µg/ml, respectively. The data indicate an incorporation of the unnatural derivatives at almost the same efficiency as the natural L-Phg precursor. Furthermore, Fig. 4 shows that the production of C-PI can reach roughly half the amount produced by the WT strain, which with our fermentation conditions produced an average of about 5 µg/ml PI. The lower PI derivative production yields are more likely due to the production performance of the ∆pglA∆snaE1 mutant strain, which produced PI derivatives inconsistently, rather than that the acceptance of the halogen derivatives supplied would have been limiting. We added some explanatory sentences to the manuscript to address the query on the limitations of the biosynthesis method.

- Regarding nomenclature, greater consistency is required (e.g. 4-fluoro or para-fluoro)
Done. All “para” designations have been changed to “4-fluoro/chloro”.

- 121: Would replacing “…involved in…” with “…essential for…’’ be better?
Done.

- 122: Would it make more sense to replace “…Pristinamycin I (1) is a natural compound…” with “…Pristinamycin is….”?
Yes, this is correct and has been changed accordingly.

- Figure 2: Threonine is missing the carbonyl group. Also, the stereochemistry of its alpha carbon is absent (Thr has 2 chiral centres). More generally, it would be aesthetically pleasing to indicate the stereochemistry by inserting up or down bonds between the alpha and beta carbons, rather than between the alpha and carbonyl carbons. The latter point is only a suggestion.
Correct. Thanks for this attentive observation! The chemical structure in Figure 2 has been corrected.

- 168: Refer the reader to Figure 3A. Also, the structure of quinupristin should also be labelled as “2” in the image.
Done.

- 209-210: Why was there a need to obtain an S. pristinaespiralis mutasynthesis strain that produces pristinamycin I derivatives exclusively? They seem to be relatively easy to separate via HPLC. Or is crude material being used as well?
Pristinamycin I and pristinamycin II both have antibiotic activity. We aimed to create a mutasynthesis producer host, which has an antibiotic-free background so that we can directly test the mutasynthesis samples for bioactivity. Knocking out both, pglA and snaE1, led to loss of PI and PII production, respectively, so that, as a result of feeding with Phg derivatives, only PI derivatives are produced, which can be directly tested for bioactivity. In addition, analysis and purification of the mutasynthesis products is facilitated. We have now added another sentence in the text to clarify that:
“This should allow for PI derivative production in an antibiotic-free background so that the mutasynthesis samples can be directly tested for bioactivity.”

- 250-253: The stereochemistry of the Phg variants in the various pristinamycin I analogues is unclear, as the authors suggest, and it is worth noting that Phg has a strong propensity to racemise. I recommend conducting additional experiments to determine whether chemical racemisation is occurring prior to addition to the culture. The authors should prepare the Phg solution again and either functionalise the amino acid with Marfey’s Reagent and perform RP-HPLC analysis or determine its optical rotation (at least 3 replicates). Moreover, can retention times (RTs) of the ‘D-Phg’ pristinamycin analogue and authentic pristinamycin I be compared (using a slow gradient)? Perhaps the D-Phg analogue has a different RT (if it in fact has D-stereochemistry)?
Thanks for the valuable advice. It is correct that we cannot state about the stereochemistry of the D-Phg mutasynthesis product. We gladly take note of the advice to the additional experiments. We looked if we had sample left over that we could analyze in this regard, however, there was nothing left. As mentioned above, the ∆pglA∆snaE1 strain produces the derivatives rather inconsistently and most likely it will not be possible to produce the corresponding PI derivative in sufficient amounts for these analysis in short time. Unfortunately, we were not able to functionalize the amino acid with Marfey’s reagent before the request for resubmission. What we can offer is that we delete the data What we can offer is that we do not include the D-Phg supplementation data and associated results in the manuscript, however, by doing so we would be concealing interesting information.

- Figure 4: It would be good to show the full HPLC chromatograms, rather than a narrow X-axis range. Presumably there are other products present in the ethyl acetate extract.
Thanks for the comment. We can understand the query of the reviewer. However, we think that the original illustration with the cutout of the spectrum is better suited to represent the key message: With Figure 4 we wanted to illustrate the verification of the mutasynthesis mutant - the restoration of PI production after L-Phg supplementation, as well as the restoration of bioactivity against B. subtilis. Therefore, the narrow part of the X-axis shown should provide all necessary information. To assess whether additional compounds are present in any given sample, the UV chromatogram at 210 nm is not sufficient. The full spectrum of measured UV chromatograms would have to be displayed, which may include additional peaks not correlated with pristinamycin production.To give you an impression how the full HPLC chromatogram at 210 nm looks like, we add the original chromatograms as an additional file for the reviewer.
- Figures S2-S4: The LCMS data should be better presented. There is no real need to show the TIC spectrum. Probably no need for 2 UV spectra, 210 nm would be enough. Does 200-600 nm refer to the range for the UV-Vis (diode array) spectrum? The mass spectra are difficult to read (especially the peak labels). Please fix. Regarding Figure S2, presumably this is the crude material? If so, please indicate which peak the MS spectrum corresponds to.
Fig. S2-S4 have been adapted and do not show the TIC spectra anymore. Only UV spectra at 210 nm are shown. After shortening of the figure, the axes should now be more readable. Relevant peak in Fig. S2 is now indicated by an arrow.

- Figure S24: Please include the UV chromatogram at 210 nm. It is important to know the effectiveness or limitations of this strategy and how it compares to using Phg derivatives directly.
Thanks for the hint! We have included the UV chromatogram in Figure 24. Furthermore, to address the comment on effectiveness and limitations of the method we have modified the figure in the way that we now show the data from the sample supplemented with 4-fluoro-Phg obtained by biotransformation and in addition also show the control sample from a culture supplemented with pure, commercially available 4-fluoro-Phg from the same fermentation experiment for comparison. It can be seen that the values of pristinamycin derivative production are comparable and the mutasynthesis approach is comparable when using biotransformation samples or commercially available mutasynthon. We added an additional explanatory sentence in the manuscript.

Thanks again for carefully reading and evaluating our manuscript!

19-Sep-2023

Dear Dr Mast:

Manuscript ID: CB-ART-08-2023-000143.R1
TITLE: <b>Biotransformation-coupled mutasynthesis for the generation of novel pristinamycin derivatives by engineering the phenylglycine residue</b>

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.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log in to the system. For your account security you should not share this link with others.)

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You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

All RSC Chemical Biology articles are published under an open access model, and the appropriate article processing charge (APC) will apply. Details of the APC and discounted rates can be found at https://www.rsc.org/journals-books-databases/about-journals/rsc-chemical-biology/#CB-charges.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy, https://credit.niso.org/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines https://www.rsc.org/journals-books-databases/author-and-reviewer-hub/authors-information/responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

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

Reviewer 2

123-124: Perhaps also delete “…a natural compound…” as it is two compounds.

Figure 2: Presumably the second image is marked for deletion in track changes?

255-256: I strongly encourage the authors conduct some polarimetry experiments on D-Phg, L-Phg and DL-Phg (value should be zero), incubated in 1 N NaOH (these instruments are common in chemistry departments). It should only take half a day and the result would provide greater clarity on whether D-Phg is incorporated or not. In my view, it would be surprising (and significant) if D-Phg is in fact incorporated, especially given that the analogue in question is also biologically active against B. subtilis (as per Table 1). If this extra work is conducted, then it would significantly improve this paper.

Figure S2: It would be good to rerun this at a higher concentration (or with a larger injection volume) if you have more sample. If not, then it is okay.

Referee: 2

Reviewer comment: 123-124: Perhaps also delete “…a natural compound…” as it is two compounds.
Reply: Correct. Has been changed to “is a natural compound mixture”

Reviewer comment: Figure 2: Presumably the second image is marked for deletion in track changes?
Reply: Exactly, this was still the tracked changes mode and is deleted in the final version.

Reviewer comment: 255-256: I strongly encourage the authors conduct some polarimetry experiments on D-Phg, L-Phg and DL-Phg (value should be zero), incubated in 1 N NaOH (these instruments are common in chemistry departments). It should only take half a day and the result would provide greater clarity on whether D-Phg is incorporated or not. In my view, it would be surprising (and significant) if D-Phg is in fact incorporated, especially given that the analogue in question is also biologically active against B. subtilis (as per Table 1). If this extra work is conducted, then it would significantly improve this paper.

Reply: Following the suggestions of the reviewer, we performed the requested experiments. We have conducted polarimetry experiments with D-Phg and L-Phg in 1 N NaOH, however, the rotation value practically did not change (see raw data below). Therefore, racemization is unlikely under these conditions, but this does not preclude racemization in the organism.
We also did a feeding study again. Here we were able to detect peak with m/z 867 Da, which had a different retention time than pristinamycin I. We also confirmed this with a co-injection. However, this peak referred to a different elemental composition / molecular formula as we proved with high resolution HPLCMS spectra! Thus, we could not confirm incorporation of D-Phg into PI. However, this result also of limited value. Due to the unstable production behavior of the mutant, this experiment would have to be performed in multiple replicates and coupled to bioassays.
Finally, there is indication from previous studies that incorporation of D-Phg is unlikely. In a chemoenzymatic approach to generate pristinamycin derivatives it was observed that the thioesterase domain of SnbDE can only accept L-Phg but not D-Phg (Mahlert et al., 2005).

Raw data of polarimetric analyses:
L-Phg α= + 0.218
D-Phg α= - 0,223

after 2.5 h
L-Phg α= + 0.213
D-Phg α= - 0,232

Calculated values

L-Phg [α]20D ◦ 109 (c 2 mg/mL, 1 N NaOH)
D-Phg [α]20D ◦ - 112 (c 2 mg/mL, 1 N NaOH)

After 2.5 h
L-Phg [α]20D ◦ 107 (c 2 mg/mL, 1 N NaOH)
D-Phg [α]20D ◦ - 116 (c 2 mg/mL, 1 N NaOH)

We have also adapted a sentence in the abstract, which addressed the incorporation of D-Phg.

Reviewer comment: Figure S2: It would be good to rerun this at a higher concentration (or with a larger injection volume) if you have more sample. If not, then it is okay.
Reply: We welcome the advice. Unfortunately, we have no sample material left, as we have spent everything for analyses, e.g. bioassays.

06-Oct-2023

Dear Dr Mast:

Manuscript ID: CB-ART-08-2023-000143.R2
TITLE: <b>Biotransformation-coupled mutasynthesis for the generation of novel pristinamycin derivatives by engineering the phenylglycine residue</b>

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Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 2

I am satisfied with the additional experiments/explanations the authors have conducted/provided to investigate whether D-Phg was incorporated into PI. I congratulate the team on their work and recommend that this manuscript be accepted for publication.

Line 267: delete the extra bracket.