Development of SynBio Tools for Pseudomonas chlororaphis: A Versatile Non-Pathogenic Bacterium Host

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Main comments

   This manuscript has developed and characterized the molecular tool boxes to test in P. chlororaphis. Author has tested the inducible and auto-inducible promoters in report gene expression study. Further, violacein biosynthetic pathway was constructed and produced violacein. The motivation of the project does not reflects in the experiments and data presented in this manuscript. Despite the attempt to advance synbio tools for P. chlororaphis, this manuscript lacks several experiments to justify the title of the manuscript. Author need to undergo major revisions in this manuscript.

Questions

1.     The introduction part in the manuscript was poorly written. For example Page 1, line 39 says “According to (3), one of…”. This statement has no explaination. Many such poorly written statements should be addressed in the manuscript

2.     Statements such as “Some Organisms” “Some members” are mentioned in this introduction without giving details about the statements. Author should be more specific and explain the examples

3.     Did author measured the RFU for blank (Media with no cells) in Figure 1? If not, author should show the reading for blank and normalize the blank reading with the sample reading to plot the Figure 1. In addition, please report the OD of the cell at different induction concentrations used in the Figure 1.

4.     In page 4, line 114 the author starts the sentence with “According to references [24,25]….”. It is the author responsibility to give brief about the cited paper in their manuscirpt. Not the reviewers task to look for the details in the cited paper. Author clearly need to rewrite the manuscript

5.     The movitation of this manuscript starts with engineering P. chlororaphis for the production of pesticides. However, author did not tested the biosynthetic pathway for pesticides production. Previous litratutes such as Ling Li, et al., 2020; Kaiquan Liu et al., 2021 has directly demonstrated the production of pesticides from P. chlororaphis

6.     In Figure 4, bioreactor data only show the violacein production. Please show other metabolic profiles such as Glucose consumption, Citrate accumulation, OD, pH. In addition, can author explain why the produce hampered at 20 h to 30 h of time profile?

Comments on the Quality of English Language

English quality is poor. Author should read the guidelines and rewrite the manuscript. Some sentences appears to be copied from previous published manuscript with slight change.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript by Bello-González et al. discussed about the development of expression vector for Pseudomonas chlororaphis. The experimental design is good. However, several works are needed to polish the overall quality of paper.

 

Major:                                                  

Comment 1: Authors stated that they have developed 6 different expression vectors (3 inducible by C6-AHL and other 3 inducible by IPTG). No information was provided how these vectors were different from each other: (i) among 6 vectors, (ii) among 3 C6-AHL inducible vector itself (iii) among 3-IPTG inducible vectors itself. Authors are suggested to provide the table summarizing the properties of each vectors so that similarity and differences between these vectors can be easily understood.

Comment 2: While developing the new expression vectors for P. chlororaphis, only C6-AHL and IPTG inducible promoter were chosen for the study. Why other promoters were not tested? If possible authors are suggested to provide information regarding to this.

Comment 3: In this study, two reporter genes (YFP and GFP) were selected to check the feasibility of the developed expression vectors. However, the results of only YFP expression were shown. Authors are also suggested to show the result of GFP expression or discuss the reason why GFP expression profile were not shown.

In Line 107-109, authors stated the leaky expression of AHL inducible promoter, but no result was provided showing the expression level of YFP without addition of C6-AHL in fig 1. Authors are recommended to show the results without addition of C6-AHL. In addition, authors are also suggested to show the error bars in figures and indicate how many times the experiment was repeated in figure legends.

Comment 4: In Line 118-122, authors stated that the presence of two analogous of luxR genes, named phzI and csaI. With deletion of phzI alone, auto induction of C6-AHL inducible promoter was completely eliminated. What about deletion of csaI alone? Authors are also suggested to show the result of csaI deletion also for better understanding the role of these two analogous genes.

Furthermore, deletion of phzI lowered the expression of YFP comparing to wild type with addition of inducer C6-AHL. Authors are suggested to discuss the possible reason behind this.

Comment 5: Authors showed that C6-AHL inducible promoter is much stronger than IPTG inducible promoter using GFP as reporter protein and violacein as target product. Authors are suggested to compare and discuss more in detail on the strength of C6-AHL inducible promoter comparing the IPTG-inducible promoter, rather than just stating violacein production or GFP expression.

Comment 6: Authors are also suggested to show the cell growth profile in fig 2, 3 and 4.

Comment 7: Authors are encouraged to discuss advantages and limitation of this study.

Minor:

Comment 1: In Fig 2C, there are two color indication for wild type (no induction). Authors are suggested to clarify it.

Comment 2: Authors are suggested to make the Y-axis unit (RFU) scale same in fig 1 and 2.

Comment 3: Authors are suggested to write the name of organism in italics throughout the manuscript.

Comment 4: Authors are suggested to mention the donor name of P. chlororaphis ATCC 9446 ΔphzI.

Comment 5: Authors are suggested to indicate the flask volume size and media volume used in methodology section.

Comment 6: Authors are suggested to rewrite the electroporation method more clearly indicating the time for electric pulse and how regeneration was done.

Comment 7: There was no supplementary data provided. Authors are suggested to provide it.

 

Comments on the Quality of English Language

Moderate editing is needed.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for an extensive writing revision done in the manuscript. However, there are several room in the manuscript for the author to be improved in terms of experimental design in their future projects. 

Comments on the Quality of English Language

English quality has been fairly improved in the new revised version. However, manuscript writing still be improved, especially when making a sentence. 

Reviewer 2 Report

Comments and Suggestions for Authors

Authors addressed the reviewers question properly and modified the paper accordingly. The paper is acceptable for publication.

Comments on the Quality of English Language

Minor editing is needed