Bioconversion of L-Tyrosine into p-Coumaric Acid by Tyrosine Ammonia-Lyase Heterologue of Rhodobacter sphaeroides Produced in Pseudomonas putida KT2440

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The current research investigated the possible heterologous production of TAL for the production of p-coumaric acid. However there are a lot of drawbacks to going further to the next steps. 

Introduction

- The English language must be improved throughout the manuscript

- the p- in p-coumaric acid should be italicized. Also, genes and E. coli (line 79) must be italicized 

- p-CA or pCA, please unify. Also, only the first time p-coumaric acid and then pCA or p-CA! 

- Line 34, what is the relation between food flavor with the antioxidant activity?

- the second paragraph (lines 42-47) needs extensive English editing, nothing is understood!

- The microorganism should be written with a full genus name, and then, the first letter is enough.

-  Short paragraphs can be merged

- the last paragraph in the introduction must describe the research's main objectives and what is the importance and consequences

Methodology

What were the cut enzymes?

Results

The figure legend of Figure 6 should be translated to English!

The Discussion should be either in one paragraph or to be written with the Results.

Really, I cannot go through it, it isn't easy to understand what has been written!

Comments on the Quality of English Language

The English must be improved!

Author Response

Por favor vea el archivo adjunto.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Carlos G. Calderón and co-authors explored the potential of genetically engineered Pseudomonas putida KT2440 to produce p-coumaric acid (pCA) and optimized the bioconversion conditions to enhance pCA yield. By optimizing the bioconversion conditions and extraction methods, the study significantly increased pCA bioconversion yield, providing valuable experimental data and theoretical basis for industrial production. However, there are still some issues to be addressed and minor errors to be corrected before publication:

 

1. According to the authors, pBTBX-2w/tal is a plasmid with arabinose-inducible promoter. I am wondering what the induction time point is? How many hours after cultivation? Or reached certain strain density?

2. Compared to the wild-type strain, how to determine that the KT-TAL strain successfully expressed TAL enzyme? Did the authors have evidence to prove that the TAL enzyme was indeed highly expressed? Is there any background expression of the TAL enzyme in the wild-type strain can work on pCA yield?

3. In the two-factor optimization experiment, why were pH and temperature chosen as factors? Would other factors such as reaction time, substrate concentration, etc, affect conversion efficiency?

4. There are many writing issues in the article:

- p-coumaric acid or p-hydroxycinnamic: Please standardize the name and use the abbreviation pCA after the first occurrence. Correct all instances of inconsistent terms like p-CA, etc.

- Please correct the writing of chemical names, such as (NH₄)2SO₄, NH4OH, H20, etc. Please check and correct throughout the text.

- Please unify units: ml or mL?.

5. Figure 1: Please label the marker size.

6. Figures 3 and 4: How many replicates in the experiment?

7. Figure 5: what is pH mean? Please correct “H2O” to “H₂O”.

8. Figure 6 caption: Please use English.

9. Figure 8: It is suggested to introduce this Figure in the introduction section.

Comments on the Quality of English Language

Please ensure all figure captions and text are in English.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks to the authors who made good efforts to improve the quality of the manuscript. Some points are still need further revisions. including

- the p in p-CA needs to be italicized throughout the abstract. Similarly, the p letter in the p-value for the significance test and pH should be italicized. It is also a small letter not capital. The same is applicable for figures also.

- The abstract is too general and the key findings must be mentioned.

- Too many keywords, please focus on a maximum of 6 words

- The introduction should be cut shorter. 46 References are too much!

- Use h instead of hours and h^-1 not h-1

- Table 1 title should be revised. There is nothing called abstract in the table title.

- The authors mentioned that 3 replicates were done for each experiment, however, the Figure legends mentioned 4!

-   The title of section 3.2.2. should be revised. It is H₂O not H₂0

- The English language seems to be revised only in the abstract and introduction. Unify the sentence used throughout the manuscript.

- Unify, Figure or Fig.

- Line 141, For 8 what?

- Protein purification using for example FPLC is recommended to investigate its composition and amino acid sequence, in addition to molecular weight. Also, comparison with authentic TAL with in vitro reaction should be done to assess the enzyme activity.

- Conclusion should be summarized and in one section to see the important results and future outlook.

Comments on the Quality of English Language

It needs still some editing.

Author Response

Comment 1. the p in p-CA needs to be italicized throughout the abstract. Similarly, the p letter in the p-value for the significance test and pH should be italicized. It is also a small letter not capital. The same is applicable for figures also.

This has been corrected in the manuscript. However, "pH" is correctly written, and it is not common to italicize it.

Comment 2. - The abstract is too general and the key findings must be mentioned.

Response: The abstract has been modified to include the key results.

Comment 3. - Too many keywords, please focus on a maximum of 6 words

Response: This has been modified.

Comment 4 - The introduction should be cut shorter. 46 References are too much!

Response: We believe that the references are adequate and support the work.

Comment 5 - Use h instead of hours and h^-1 not h-1

Response: This has been modified.

Comment 6- Table 1 title should be revised. There is nothing called abstract in the table title.

Response: This has been modified.

Comment 7- The authors mentioned that 3 replicates were done for each experiment, however, the Figure legends mentioned 4!

Response: This has been corrected.

Comment 8-  The title of section 3.2.2. should be revised. It is H₂O not H₂0

Response. This mistake has been corrected.

Comment 9- The English language seems to be revised only in the abstract and introduction. Unify the sentence used throughout the manuscript.

Comment 10- Unify, Figure or Fig.

Response: This has been corrected

Comment 11- Line 141, For 8 what?

 Response: This has been corrected to "for 8 h."

Comment 12- Protein purification using for example FPLC is recommended to investigate its composition and amino acid sequence, in addition to molecular weight. Also, comparison with authentic TAL with in vitro reaction should be done to assess the enzyme activity.

Response: Thank you for your observation. The sequence corresponds to the L-tyrosine enzyme and could be verified by sequencing in the future. In a thesis study, we evaluated the enzyme's kinetic parameters, such as the Kₘ using L-tyrosine as a substrate.

Comment 13- Conclusion should be summarized and in one section to see the important results and future outlook.

Response: This has been modified.