Biosynthesis of 4-hydroxybenzylideneacetone by Whole-Cell Escherichia coli
Round 1
Reviewer 1 Report
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Comments for author File: 
Comments.pdf
Author Response
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Reviewer 2 Report
This paper reports research in the biosynthesis space, but is, overall, of quite limited scope. To bring this study to a level where publishing might be motivated the investigations also have to include findings from operating the biotransformation under a continuous regime (= flow chemistry principles). As the work currently stands, even with the substantial improvements in performance (yield) reported by the group compared to prior art, it has to be expanded to also include running the reaction under flow conditions with the relevant enzymes applied in an immobilized form. Otherwise, the protocol outlined in the paper is only advancing the technology incrementally from an overall point of view.
Besides these comments, several remarks have been noted in the amended version of the original article (see attachemnt). A general recommendation to the authors is to be much more diligent with their proof-reading, which, no doubt, could eliminate several of the flaws spottedin the text!
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The authors successfully used E. coli BL21(DE3) expressing DERA to convert 4-HBD and acetone to 4-HBA with good yield. However, I have few questions about their experiments.
1. The title is whole-cell E. coli. However, the authors used dead cells. I do not think that these experiments belongs to whole-cell biocatalyst, which is lives cells. Especially, the reaction temperature was 60 degree. I suspect that E. coli cells broke and released overexpressed DERA to catalyze the reaction. Can you clarify this point?
2. Line 74-76: I do not understand why you claimed "significantly increased amount of soluble DERA". In Figure 2A, the left one is empty vector and the right one is E. coli containing DERA overexpression plasmid. It is reasonable to see its overexpression.
3. In figure 2A, I am assuming the samples were crude lysate. But why does the SDA-PAGE not show other E. coli endogenous protein bands?
4. In Figure 2B, what does "N" mean? Add its information.
5. In Figure 3A, I suggest to add one negative control (E. coli containing empty vector at 60 degree).
6. Line 132: "4-HBD dropped to 88.5 mM". In Figure 4, the 4-HBD concentration is about 25 mM. Furthermore, adjust the y-axis of Figure 4 to starts with 0 not -50. At 24 h, there are two points, so it is better to write the information in the figure caption.
7. Add reaction temperature in Figure 3B and 3C caption.
8. There is no figures HPLC or calibration curves for analytical information. You can add them in SI.
9. Line 82: 4-nitrobenzaldehyde instead of 4-ni-trobenzaldehyde
10. Line 188: the numbers of KH2PO4 and K2HPO4 should be subscript.
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
The title of the manuscript Biosynthesis of p-Hydroxy benzylideneacetone by Whole-Cell Escherichia coli draft is good.
1. Recommended to author, Check all Figures it’s not clear structures and images.
2. Clearly abbreviate all shortcut points.
3. Include more reference. Check overall manuscript Spellings, commas and sentence formations,
4. Author should look for literature to understand the major characterizations.
Recommendation: Agree to Publish in Catalysts after minor revisions noted.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
My questions/comments/concerns were well addressed. The only part I still don't agree with is the equation for conversion. To my knowledge it should be
Conversion=mol of consumed 4HBD/mol of initial 4HBDx100%
Author Response
Reviewer 1
1. My questions/comments/concerns were well addressed. The only part I still don't agree with is the equation for conversion. To my knowledge it should be
Conversion=mol of consumed 4HBD/mol of initial 4HBDx100%
A1. The conversion equation has been revised in line 238 of the revised manuscript, and the relevant data has been recalculated and revised, thank you.
Reviewer 2 Report
The paper has taken one step in the right direction, but is still not in a publishable form. Thus, some of the points stated in the previous refereeing report have remained unaddressed, For example the structure of the ultimate target compound raspberry ketone is still missing and the citations have not been presented properly using the agreed abbreviated journal names.
The group claims to be planning to investigate the performance of this transformation under flow mode, but do not share any details at this stage, This is not acceptable and instead at least some preliminary thoughts and ideas have to be included to make it worthwhile publishing this otherwise far from finished story. This would also bring it to a level which would make it more interesting to the reader.
The recommendation is to carefully revise the manuscript again ad expand the topic as described above before resubmitting. In the current version the content does not suffice for acceptance!
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The authors have answered all my questions. I would suggest that the journal accept its current form. However, it would be better if the microscopic images of before and after reactions could be added with short description into the manuscript.
Author Response
Reviewer 3
- The authors have answered all my questions. I would suggest that the journal accept its current form. However, it would be better if the microscopic images of before and after reactions could be added with short description into the manuscript.
A1. Thanks for your comment. We have added relevant brief descriptions to lines 114-116 of the revised manuscript and placed the SEM results in the Supplementary Material.
