From the journal RSC Chemical Biology Peer review history

Berlin, October 26, 2020

Dear Dr Leeper:

Manuscript ID: CB-ART-09-2020-000173
TITLE: Use of a synthetic thioester substrate to demonstrate the presence of a novel intermediate in the biosynthesis of the red antibiotic prodigiosin

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

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Reviewer 1

In the manuscript by Couturier et al. some biosynthetic steps towards prodigiosin are described.
The red coloured antibiotic prodigiosin and its producer organisms do have a very long history, which dates back to the middle ages. In recent times it has become an important lead compound for the development of pharmaceuticals, e.g., anticancer compounds. More than 15 years ago, the groups of Leeper and Salmond proposed a biosynthetic scheme (ref. 3; please check page 1: “originally proposed” = ref. 2(?)), which is still valid for most of its part. Moreover, this proposal was fundamental for further studies, as the one by Dresen et al.
In the present manuscript Leeper, Salmond and coworkers precise their original proposal by showing that a thioester, shown for compound 13 (the true intermediate might be a CoA or ACP derivative), can restore production of prodigiosin in a pigD deletion mutant. Bioinformatic analysis and in vitro characterization of PigE complement the data, which are in agreement with the ‘new’ proposal (Figure 1).
Some major and/or minor comments:
The ‘new’ proposal was the reason, why Dresen et al. and Kasparyan et al. tested thioester 12 as a substrate of PigD and two related enzymes: HapD was even more efficient with 12 as acceptor substrate and gave full conversion.
Kasparyan et al. (2014): “This finding supports the notion that the characteristic substrate range of these enzymes is related to their physiological substrates, such as phosphopantetheinylated acyl carrier proteins or as coenzyme A-activated substrates. As noted above, PigD and HapD are assumed to be involved in the biosynthesis of prodigiosin(s). This could be the reason for their high biocatalytic activities towards the thioester 1f.”
Morover, in her PhD thesis Kasparyan already tested the CoA derivative as a substrate, which was not accepted by PigD. She concluded:
„Die Vermutung über ein an Coenzym A gebundenes physiologisches Akzeptorsubstrat wurde im Rahmen dieser Arbeit nicht bestätigt. Andererseits wiesen Experimente mit dem Akzeptorsubstrat mit der verkürzten Thioester Seitenkette (ohne (N)-Acetamidoeinheit) auf einen drastischen Abfall der Enzymaktivitäten hin. Somit ist zu vermuten, dass sich das physiologische Substrat an einem Pantothen-Arm tragenden Enzym aktivieren lässt, so wie es bei Fettsäure oder Polyketid-Biosynthese beschrieben ist.“
Hence, although the proposal is not entirely ‚new‘, the results described by Couturier are absolutely worth to be published. Nevertheless, the term ‘new’ might be adjusted and the missing literature (Kasparyan et al, 2014) cited.
Finally, regarding the (final) experiment with purified pigE and ornithine: Did the authors observe a decrease of thioester 13? Could it be that the imine formation, although intramolecular, needs some ‘support’?
Putative N-source / ornithine: why is arginine not mentioned as well?

Reviewer 2

The authors present a new detail concerning the start of the prodigiosin biosynthesis pathway. In a first step, the authors proved that the true starter of the biosynthesis pathway is not 2-octenal, but rather its thioester. Subsequently, they showed that the enzyme PigE is not simply a transaminase, but rather possesses a dual function, i.e. beside the transaminase activity it is able to reduce the thioester to its aldehyde.

In order to prove the above-mentioned functions, the authors synthesized the hypothesized substrates and employed knockout mutants and chemical complementation. Furthermore, upon bioinformatics, in vitro assays were conducted. All experiments are convincing and the manuscript is very well written. The results contribute to the overall understanding of the prodigiosin biosynthesis, foremost, if it shall be reconstituted in vitro. I appreciate that the authors invested again time and efforts to sort out this detail.

I have just some minor comments.

The authors use the designations HMB and HBC in Figure 1. To the best of my knowledge, these abbreviations are nowhere explained / written out. This is necessary for all those readers who are not so familiar with the topic.

Legend of Figure 4d
It would be helpful to state at which wavelength the chromatogram was taken and please label the peaks.

Overall, I would appreciate if the authors chose a more informative title of the manuscript that refers or emphasize more somehow details of the start (alternatively: the 1st two steps) of the biosynthesis are clarified. The current title does not reflect this fact and it will not be found in a literature search – at least not on the first sight.

ESI –Plasmid construction (pPigE)
Serratia sp. S39006 (de-italicize “sp. S39006”)

ESI –NMR data of 3-Acetyloctanal 4
HBQR => HMQC

ESI in general
I saw that the analyzed compounds were just investigated by low-resolution MS; In order to deduce or corroborate the sum formula I recommend to determine the high resolution data.

ESI 13C NMR spectra
Nearly all 13C NMR spectra need definitively to be phased correctly (no negative spikes, please).

Referee 1
The ‘new’ proposal was the reason, why Dresen et al. and Kasparyan et al. tested thioester 12 as a substrate of PigD and two related enzymes: HapD was even more efficient with 12 as acceptor substrate and gave full conversion.
Kasparyan et al. (2014): “This finding supports the notion that the characteristic substrate range of these enzymes is related to their physiological substrates, such as phosphopantetheinylated acyl carrier proteins or as coenzyme A-activated substrates. As noted above, PigD and HapD are assumed to be involved in the biosynthesis of prodigiosin(s). This could be the reason for their high biocatalytic activities towards the thioester 1f.”
We have included a reference to Kasparyan et al. (2014) and the sentence
“Kasparyan et al. extended this work to some homologues of PigD and suggested that thioester 3 may be the true natural substrate of PigD.8 ”. This acknowledges that these revised initial steps have been proposed previously, though they have not been tested with the downstream enzymes before now.
Morover, in her PhD thesis Kasparyan already tested the CoA derivative as a substrate, which was not accepted by PigD. She concluded:
„Die Vermutung über ein an Coenzym A gebundenes physiologisches Akzeptorsubstrat wurde im Rahmen dieser Arbeit nicht bestätigt. Andererseits wiesen Experimente mit dem Akzeptorsubstrat mit der verkürzten Thioester Seitenkette (ohne (N)-Acetamidoeinheit) auf einen drastischen Abfall der Enzymaktivitäten hin. Somit ist zu vermuten, dass sich das physiologische Substrat an einem Pantothen-Arm tragenden Enzym aktivieren lässt, so wie es bei Fettsäure oder Polyketid-Biosynthese beschrieben ist.“
I don’t think this PhD thesis is publically available, so we cannot really refer to it. I don’t think the referee is suggesting we should but if the editor(s) think it should be included I would be happy to include it as a personal communication.
Hence, although the proposal is not entirely ‚new‘, the results described by Couturier are absolutely worth to be published. Nevertheless, the term ‘new’ might be adjusted and the missing literature (Kasparyan et al, 2014) cited.
We have changed “New proposal” to “Revised proposal” in Fig. 1. I think the changes detailed above make it clear it is not “new”.
Finally, regarding the (final) experiment with purified pigE and ornithine: Did the authors observe a decrease of thioester 13?
The final experiment was with pigE expressed in E. coli but not purified – the crude cell-lysate was used. Detection of thioester 13 and the product H2MAP 6 was only performed by LCMS and it is difficult to do this in a quantitative fashion, as the detection efficiencies for different compounds can be very different. Therefore we have not claimed that the level of thioester has decreased (even though looking at Fig. 6 it looks very likely that it has). Also, as we have shown that H2MAP 6 is produced from thioester 13, the observation of a strong peak for 6 implies that the level of 13 must have decreased.
Could it be that the imine formation, although intramolecular, needs some ‘support’?
We have synthesised the aminoketone precursor of H2MAP 6 and it cyclised to H2MAP 6 spontaneously under the reaction conditions (ref. 6). Similar formations of cyclic imines are well known, e.g. in the biosynthesis of proline and of the tropane alkaloids, and I have never seen any proposal that the cyclisation requires a catalyst. From both my experience as a chemist and my reading of the literature on biosynthesis, I think it highly unlikely that this cyclisation needs “some support”.
Putative N-source / ornithine: why is arginine not mentioned as well?
Yes, Arginine is not far behind Ornithine on the list of amino donors, so we have included it as another possible amino donor (page 3 last paragraph and page 4 last line before “Experimental”).
Referee 2
The authors use the designations HMB and HBC in Figure 1. To the best of my knowledge, these abbreviations are nowhere explained / written out. This is necessary for all those readers who are not so familiar with the topic.
The referee is quite correct and in fact it should be “HBM” and not “HMB” (ref. 3). Fig. 1 has been modified and the full names of the compounds included in the caption.
Legend of Figure 4d
It would be helpful to state at which wavelength the chromatogram was taken and please label the peaks.
This has been included in the caption: “(535 nm)” and the peaks have been labelled with the corresponding compound numbers “10” and “11”.
Overall, I would appreciate if the authors chose a more informative title of the manuscript that refers or emphasize more somehow details of the start (alternatively: the 1st two steps) of the biosynthesis are clarified. The current title does not reflect this fact and it will not be found in a literature search – at least not on the first sight.
The title has been modified to “Revision in the first steps of the biosynthesis of the red antibiotic prodigiosin: use of a synthetic thioester to validate a new intermediate”. I think this satisfies the referee’s concerns.
ESI –Plasmid construction (pPigE)
Serratia sp. S39006 (de-italicize “sp. S39006”)
Done
ESI –NMR data of 3-Acetyloctanal 4
HBQR => HMQC
We have replaced it with “HSQC and HMBC” as both techniques were used.
ESI in general
I saw that the analyzed compounds were just investigated by low-resolution MS; In order to deduce or corroborate the sum formula I recommend to determine the high resolution data.
This work was done more than two years ago. We have found as many of the compounds as we can and submitted them for high-resolution mass spectroscopy. Unfortunately two of the compounds were missing from the collection (either used up or lost) and two failed to give correct accurate masses (presumably because they have degraded) but the others have been included in the SI.
ESI 13C NMR spectra
Nearly all 13C NMR spectra need definitively to be phased correctly (no negative spikes, please).
Done.
Finally we have made changes to the references to make them conform to the Journal’s style. We have also included the (now available) volume, issue and page numbers for ref. 6. Following insertion of the new ref. 8 all the subsequent references have been renumbered in both the text and the list of references.

Berlin, January 1, 2021


Dear Dr Leeper:

Manuscript ID: CB-ART-09-2020-000173.R1
TITLE: Use of a synthetic thioester substrate to demonstrate the presence of a novel intermediate in the biosynthesis of the red antibiotic prodigiosin

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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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

Reviewer 2

The authors addressed all raised issues and responded sufficiently to all comments. Therefore, I would recommend the acceptation of the manuscript.

Reviewer 1

All comments by the reviewers have been acted upon, hence, I suggest publication of the manuscript.

Please check page 1, citation of ref 2: "proposed to be the starting point of the pathway.2" Should ref 3 be cited instead?