Using Omics Techniques to Analyze the Effects of Gene Mutations and Culture Conditions on the Synthesis of β-Carotene in Pantoea dispersa
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments
Line 70: In this study, we identified Pantoea dispersa as capable of synthesizing β-carotenoids.
- It is suggested: In this study, we identify a bacteria (Pantoea dispersa) capable of synthesizing….
Line 74: Pantoea dispersa change to P. dispersa. Homogenize throughout the manuscript.
Line 80: provide more information (name) of the laboratory where the strain was kept.
Line 81-82. How the mutagenesis was carried out, give more details of the process.
Line 83: It is suggested to change seed medium to "the cultivation base medium".
- Improve the writing of the following sentence: All experiments were conducted in 250 ml flasks with an oscillating motion, containing 100 ml of culture medium. Mention the rpm used for agitation of the culture.
Why did they determine bacterial growth by dry weight and not by optical density?
Line 101: change 12,000 rpm to x g.
Section 2.2. Mention the measurement technique (HPLC), because they only measure the column used.
- The authors performed RT-qPCR, not RT-PCR, there is confusion between techniques, please correct.
- Change qRT-PCR to RT-qPCR.
- More detail remains to be given on how they quantified the expression, whether they used syber green or Taqman probes, and the size of the amplicons.
- In supplementary table S1 and S2, they should write the full name of the gene and cellular function of the protein they encode. It is not clear which is the first forward and/or reverse.
- The authors do not mention anything regarding the method of DNA extraction for sequencing. Add information.
Line 157: define DMAPP.
- Add the retention time of Beta-carotene (fig. 1a).
- How the authors quantified the concentration of Beta-carotene. They made a standard curve, what concentrations they used, this because the mAU obtained in the sample (Fig. 1b) is very low compared to the control.
- In graph 1(d), what does LB and LB-C mean?. Also change Titer.
- There is no sense of the sentence (lines 156-159), for the title of section 3.1. The authors should remove or change figure 1(c) in another section.
- Placing Fig. 5a in section 3.3 is confusing, it is suggested that the authors modify the order. It does not even seem to correspond to that figure, but rather to Fig. 4a. Furthermore, the legends of fig. 3 do not correspond to the figures.
- Line 204: mention the two affected amino acids and the position.
- The authors reveal that there is a 33.74% decrease in the phosphogluconate dehydrogenase activity of strain 14P9 compared to MSC14. However, they do not perform some type of statistical analysis to see if the strains are really significantly different, they must perform a t-student.
- Despite showing the structure between strains, the zoom that would correspond to Fig. 3C does not explain it, and it is less understandable without the correct legend.
- The names of the genes indicated in section 3.3 (and the rest of the manuscript) should be written in italics. Also, describe the names the first time you mention them.
Line 276: remove a point.
-The trxC gene from strain MSC14 contains introns? Why did they generate cDNA?
Line 336-337. The following sentence "The successful construction of the overexpression strain was confirmed through electrophoresis using different primers (Figure 6-a)", does not correspond to the indicated figure. Please correct.
Line 338: Change qRT-PCR to RT-qPCR
Graph 6 (a). vertical axis correct "chang" by change
- In graph 6 (and previous ones), the authors show bars, but do not mention what they refer to, is it standard deviation? Furthermore, they indicate (P<0.05), but do not show or mention any type of statistical analysis.
How do the authors explain that beta-carotene production is similar between strain 14P9 and MSC14 (trxC)?
It is recommended to improve the design of Table S5
Comments on the Quality of English LanguageModerate editing of English language required
Author Response
We agree with and benefit from the issues you have raised. We have made changes in response to your questions and suggestions. Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this manuscript Liu and Cui present a study regarding the optimization of β-carotene synthesis by selected strains of Pantoea dispersa. The authors attempt to identify molecular factors that may relate to increased β-carotene production in the P. dispersa strains utilized: for this purpose they set up genomic and transcriptomics analyses.
The manuscript is very well written., presenting clear and sound data. There are some issues that the authors need to address before the manuscript can be considered for publication.
- Paragraph 2.3: please specify the wavelengths of the UV rays employed, as well as the irradiation times. Also, explain the rationale behind plating on media containing diphenylamine.
- Paragraph 2.8: please specify the transformation method used.
- Figure 2: please explain in the legend the significance of the red arrows in a/b.
- Figure 3: there is no correspondence between pictures a-d and legend.
- Figure 6 and text: it is not clear why the authors chose to overexpress the trxC gene only in MSC14 strain. It would be interested to see if transforming 14P9 with the trxC plasmid would also result in increased levels of trxC expression. Besides, the influence of trxC overexpression on the level of β-carotene production should be tested in 14P9-overexpressing strain, too.
Comments on the Quality of English LanguageThe English language is fine, although some minimal editing is necessary. For example X axis title of Figure 2b; Figure 6a: "Fold change" instead of "Fold chang"; Figure 6: Y axis name can be shortened to "Strain", etc.
Author Response
We agree with and benefit from the issues you have raised. We have made changes in response to your questions and suggestions. Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors responded to main reviewer's concerns.
Minor issues:
- line 149: replace "CaCl" with "CaCl2"
lines 152-153: no space within restriction enzyme names, e.g., "BamHI", not "BamH I"
- line 155: the strains denomination must ne non-italic, e.g., MSC14 or 14P9, not MSC14 or 14P9
Author Response
Once again, we would like to express our gratitude for your comments on our manuscript, which were very conscientious and attentive. We have read your review carefully and we feel very ashamed. Because of our negligence and did not find the errors you raised, we have carefully revised and made further checks. Your rigorous attitude towards scientific research is worth learning for our whole life, and we will definitely be more serious in our future studies and research. We would like to express our respect and gratitude to you again. We have corrected the minor issues you mentioned, and the revised version is uploaded, please review it. Wish you good work and happy life!