Microbial Pyrrolnitrin: Natural Metabolite with Immense Practical Utility
Round 1
Reviewer 1 Report
This review focuses on the synthesis, activity and application of an important group of compounds, pyrrolnitrin and its derivatives. Authors described nearly every aspect of pyrrolnitrin, from the synthesis to downstream purification, from the activities to applications. However, this paper lacks focus, or in other words, putting too much effort on piling experimental data from others’ work without further analysis, and omitting the most promising part of current progress on pyrrolnitrin biosynthesis. The reviewer suggests a major revision before publication, and the major questions are as following:
1. From the abstract and introduction part, it seems that authors intend to claim that biosynthesis is preferred over the chemical synthesis. It is crucial to understand the pathway and genes when refers to biosynthesis. However, the gene cluster and genetic engineering related to pyrrolnitrin was not described in detail. PrnA was the first characterized flavin-dependent halogenase. Mutagenesis research showed the capability of converting the 7-tryptophan halogenase to the 5-tryptophan halogenase. The cofactor of PrnB was not determined, and the mechanism of this step remains debatable. The references should be cited for PrnB. PrnC is also a flavin-dependent halogenase, but it is quite different with PrnA. The mechanism of PrnC is not revealed. Those genes are the key for pyrrolnitrin biosynthesis, but authors use two paragraphs to introduce PrnF, which is substitutable by other flavin reductase such as Fre or RebF. Without clearly demonstrating the putative biosynthesis approaches, it is not easy to further push the biosynthesis into industry on the current basis of fermentation without genetic optimization.
2. This article listed 6 tables, while most of them are not necessary and fail to provide enough information. For example, Table 4 does not provide much useful information like purify, recover rate, resolution, etc. It is assumed that the data is from column chromatography, but authors should summarize and evaluate the effects of different systems, instead of list all the experimental data for readers to test them. Table 5 shows the retention time, which does not make much sense since the dead volume of columns and equipment, condition of columns vary a lot, not to mention some values of flow rate are missing. It does not worth a table to describe that information. On the contrary, several figures should be included. The structures of pyrrolnitrin and its derivatives, and the numbering should be listed (not limited to the structures listed in Table 2). Process of chemical synthesis and scheme of proposed biosynthesis route are preferred to be seen.
3. The conclusion part does not well summarize the paper, but bring in some uncertified statements, such as “Microbial systems tolerant to a wide range of organic solvents of industrial use may likely to have newer route of economic PRN biosynthesis”, or “application of halogenase from high yielding bacteria could help to overcome issues of regioselectivity, dependency on chemical”. Those are not mentioned previously and if possible, please specify how would those technique benefit the industry and how to solve the substrate specificity problems.
Minor questions:
4. Table 1 chlortetracycline appears twice, combine them.
5. Line 226, 60.50 mg/L.
6. Line 277, Line 280 C10H6N2O235Cl2
7. MS spectrum should show the m/z of C10H6N2O235Cl35Cl : C10H6N2O235Cl37Cl : C10H6N2O237Cl37Cl =9:3:1. Please confirm it.
8. Line285, Combine the HNMR data with line 289.
9. NMR data of PRN is mainly influenced by the solvent used and machine, not where the compounds come from, since they are purified. It does not make sense to list different chemical shifts from different producing sources of PRN, but the solvent and frequency of machine should be mentioned.
10. Line 297, 52˚
11. Line358 put spaces between “and” and compounds
12. Line 359 “have been”
Author Response
From the abstract and introduction part, it seems that authors intend to claim that biosynthesis is preferred over the chemical synthesis. It is crucial to understand the pathway and genes when refers to biosynthesis. However, the gene cluster and genetic engineering related to pyrrolnitrin was not described in detail. PrnA was the first characterized flavin-dependent halogenase. Mutagenesis research showed the capability of converting the 7-tryptophan halogenase to the 5-tryptophan halogenase. The cofactor of PrnB was not determined, and the mechanism of this step remains debatable. The references should be cited for PrnB. PrnC is also a flavin-dependent halogenase, but it is quite different with PrnA. The mechanism of PrnC is not revealed. Those genes are the key for pyrrolnitrin biosynthesis, but authors use two paragraphs to introduce PrnF, which is substitutable by other flavin reductase such as Fre or RebF. Without clearly demonstrating the putative biosynthesis approaches, it is not easy to further push the biosynthesis into industry on the current basis of fermentation without genetic optimization.
Author's Response : Whole abstract is rewritten taking into account the comments. It is now representing whole MS in more meaningful way. Looking into the critical suggestions and comments, we have put a separate section on "PRN Biosynthesis: the genes and enzymes" in order to discuss and describe the biosynthetic route, gene clusters and the enzyme characteristics that regulate PRN synthesis in microorganisms. The prnA,B,C,D genes are described taking into account the most updated references and the KEGG database analysis results. We have also included TWO fresh Figures (Fig 1 & 2) on the biosynthetic pathway of PRN and on the gene cluster organization in various species. The Figure 1 also possess the structure of all the compounds (from No 1 to the end product as well as the intermediates) which better explains the pathway and improves the understanding of the work. Now we believe that the new section has improved the quality of the MS very well.
2. This article listed 6 tables, while most of them are not necessary and fail to provide enough information. For example, Table 4 does not provide much useful information like purify, recover rate, resolution, etc. It is assumed that the data is from column chromatography, but authors should summarize and evaluate the effects of different systems, instead of list all the experimental data for readers to test them. Table 5 shows the retention time, which does not make much sense since the dead volume of columns and equipment, condition of columns vary a lot, not to mention some values of flow rate are missing. It does not worth a table to describe that information. On the contrary, several figures should be included. The structures of pyrrolnitrin and its derivatives, and the numbering should be listed (not limited to the structures listed in Table 2). Process of chemical synthesis and scheme of proposed biosynthesis route are preferred to be seen.
Author's Response : Taking into account the comments, we have deleted Table 4 and 5, and the content therein is briefly mentioned in the text along with complete references. Now, this seems to reduce the bulk of the MS.
3. The conclusion part does not well summarize the paper, but bring in some uncertified statements, such as “Microbial
systems tolerant to a wide range of organic solvents of industrial use
may likely to have newer route of economic PRN biosynthesis”, or
“application of halogenase from high yielding bacteria
could help to overcome issues of regioselectivity, dependency on
chemical”. Those are not mentioned previously and if possible, please
specify how would those technique benefit the industry and how to solve
the substrate specificity problems.
Author's Response : Conclusion part is fully revised and improved. The lines in comments "application of halogenase......." are removed and are replaced by some new futuristic facts that may improve PRN production and its implications.
4. Table 1 chlortetracycline appears twice, combine them.
Author's Response : Corrected
5. Line 226, 60.50 mg/L.
Author's Response : Corrected
6. Line 277, Line 280 C10H6N2O235Cl2
Author's Response : Corrected
7. MS spectrum should show the m/z of C10H6N2O235Cl35Cl : C10H6N2O235Cl37Cl : C10H6N2O237Cl37Cl =9:3:1. Please confirm it.
Author's Response : Corrected
8. Line285, Combine the HNMR data with line 289.
Author's Response : Corrected
9. NMR data of PRN is mainly influenced by the solvent used and
machine, not where the compounds come from, since they are purified. It
does not make sense to list different chemical shifts from different
producing sources of PRN, but the solvent and frequency of machine
should be mentioned.
Author's Response : Modified, Corrected
10. Line 297, 52˚
Author's Response : Corrected
11. Line358 put spaces between “and” and compounds
Author's Response : Corrected
12. Line 359 “have been”
Author's Response : Corrected
Besides above, almost all the sections were revised and new lines or work have been included therein to improve the understanding on the whole MS.
The REVISED MS is also being submitted.
Reviewer 2 Report
Dear Authors,
The manuscript ID: biomolecules-532749-v1 entitled “Microbial pyrrolnitrin: natural metabolite with immense practical utility” written by Shraddha Pawar, Ambalal Chaudhari, Ratna Prabha, Renu and Dhananjaya P. Singh contains a lot of valuable data on the microbial pyrrole halometabolite.
This manuscript is are very interesting and makes a some contribution to current knowledge about natural bioactive pyrrolnitrin from different subgroups of bacterial species. It is concise, generally well written, documented and organized.
I have very small suggestions in order to improve paper, which are the following:
· some of the information were collected and presented in the form of 5 tables, which should be harmonized in accordance with the authors' instructions;
· Table 3: Sr. no. 7. B. cepacian à B. cepacia;
· Lines 255 and 256: [63] and [64] à change the red text to black;
· Line 429: Microsporium àMicrosporum;
· some of the bibliographic items aren’t prepared according to the authors' instructions (e.g. they don’t have abbreviations; Line 542: Evolution, Medicine, and Public Health à Evol Med Public Health; Line 605: Helvetica Chimica Acta à Helv Chim Acta, etc…
I have no negative comments on this article. I think that this manuscript is valuable and worth publishing in Biomolecules.
With highest regards,
Author Response
· some of the information were collected and presented in the form of 5 tables, which should be harmonized in accordance with the authors' instructions;
Author's response : Table 5 is removed as per the suggestions of Reviewer 1. The data therein is compiled in brief in the text.
· Table 3: Sr. no. 7. B. cepacian à B. cepacia;
Author's response : Corrected
· Lines 255 and 256: [63] and [64] à change the red text to black;
Author's response : Corrected
· Line 429: Microsporium àMicrosporum;
Author's response : Corrected
· some of the bibliographic items aren’t prepared according to the authors' instructions (e.g. they don’t have abbreviations; Line 542: Evolution, Medicine, and Public Health à Evol Med Public Health; Line 605: Helvetica Chimica Acta à Helv Chim Acta, etc…
Author's response : Corrected
Round 2
Reviewer 1 Report
This revised version makes improvement compared to the previous one. The logic is sound and the focus is clear. The separate part of biosynthesis and modified conclusion meet the requirement of reviewer's suggestions. All the other minor errors are corrected. There are several minor issues needs to be double checked.
The Table 1 is not shown in full. Maybe change it to landscape will solve it.
In Figure 1, double check the structure of aminopyrrolnitrin and the step of PrnC.
Figure 2 is not necessary, but it is OK to be shown.
Author Response
Comment : The Table 1 is not shown in full. Maybe change it to landscape will solve it.
Response : Table 1 is modified. Certain columns are deleted and the content is arranged in single columns.
In Figure 1, double check the structure of aminopyrrolnitrin and the step of PrnC.
Response : Figure 1 is rechecked. The structure of aminopyrrolnitrin is corrected. also the PrnC step is checked and the name of the enzyme is corrected as per the MetaCyc database. Now this is final.
Figure 2 is not necessary, but it is OK to be shown
Response : Fig 2 is okay. May be decided for keeping in the text or to be deleted at your end.
Hope this will satisfy reviewer comments.
