Enhanced Glutathione Production in Saccharomyces cerevisiae by High-Throughput Screening System Based on Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis
Round 1
Reviewer 1 Report
Comments and Suggestions for Authorsfermentation-3535835-peer-review-v1
This is interesting work on generation of mutants and furthermore exploring glutathione-producing properties of the strains. IN my opinion paper have potential and can be considered for publication, however, extensive revision will need to be provided, with special focus on discussion section of the manuscript.
Introduction is well structured and gives principal advantages of the further applied experimental approaches in generation of mutants and screening for the detections of metabolites of interests.
Please, provide information for suppliers of all the chemicals and equipment used in the study according to requirements form the journal. This information need to include the name of the company, address where city, state (in case of federal countries) in abbreviated form, and name of the country. Please, this information need to be provided when it is presented for the first time in the manuscript. In following occasions, only name of the company needs to be provided.
For additional analytical methods (section 2.4.) provide references for each of them.
Please, in entire manuscript, centrifugation conditions needs to be presented as g force, and not as rpm.
Please, provide more details for the experiment under section 2.4.3.
More details for the experiment under 2.4.4. and 2.4.5. needs to be provided.
Be sure that italics are used appropriately. See Ln 193 and etc.
Authors presented well with their results and provided comments on the observed fact. However, most of the comments will be positive if they can be provided with references that are in agreement with their observation, or even if the opposite results were observed. It is important in discussion of the results that authors will refer to available scientific sources in support of their observations.
As example, in section between lines 256 and 263, authors can mention that other authors as well was showing that microplate approach can be used instead HPLC and validated appropriate for both methods and give some information regarding positive and negative aspects for both methods. In fact, this was discussed further, however, no references were provided.
Authors have provided very detailed descriptions of their results, supported by illustrative material. However, discussion of the results obtained is very preliminary. Authors will need to improve discussion section, provide references in agreement with their observation; provide arguments and discuss the applications of obtained results.
The conclusion needs to be presented better. IN current way, this is just short summary of obtained results.
Author Response
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Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript describes the use of ARTP for strain mutagenesis and high throughput screening to produce higher level GSH producing strains. I think the focus of the paper is a bit off balance. Using ARTP has been published numerous times, at least for a decade or longer, so using it for strain improvement is not especially novel although it may not be as well known or commonly as other methods. The primary reason for this, I believe, is access to the needed instrumentation and not lack of awareness.
So given the methodology for strain improvement is not especially novel, the remaining thrust of this manuscript is to generate improved strains for producing GSH. The authors have achieved this end, albeit a fairly minor increase (15-20%). I am not sure this couldn't have also been achieved merely by optimization of growth conditions. Nevertheless the work was done well enough.
Given this, I think the overall theme of the manuscript should be modified to not be a test study for ARTP but rather for increasing GSH. This would just require some adjustments to the title and bit of rewriting in the introduction and conclusions. The bulk of the manuscript is actually not about ARTP.
Overall the manuscript is well written and clearly presented but I have a few minor errors that need correcting which are listed below by line number or similar.
Line 77 please introduce ethionine to the reader and why ethionine resistance is being used in the screen. This will not be clear to many readers.
91 change "of" to "in"
94-99 What volume was used in the ARTP mutagenesis? You give the concentration but not the volume so can not determine how many cells were mutagenized for the screening.
103 How many colonies were selected and innoculated?
115 Stains should be Strains I believe
117 do you mean identified or found instead of harvested?
151 server instead of sever
156 pellet instead of cluster
196 I think you mean Figure 2c, d (instead of 2b, c)
Fig 3c It is not clear to me what the samples are here. One of my concerns in this section and also in the section on lines 129-133 is whether or not there is a high background from the lysed cells at 305 nm used to measure. I don't doubt you get a nice standard curve in buffer, but I imagine there is high absorbance from the cell debris at this wavelength. I would like to see a standard curve done using a lysed cell sample and spike with varying concentrations of GSH to show linearity in the background of cell debris.
334-225 This does not make sense. Chemically reactive species by ARTP would be gone after a few generations. So you are not looking at the after effects of ARTP but genetic changes that modulate physiology.
348-353 this comparison does not really seem valid to me. The authors starting strain produces more GSH than they report for the engineered strain from other labs (SKSC222) so this suggests they are either using a different host strain or different growth conditions. This means that a comparison between their mutant and another labs mutant does not really have much validity.
393-400 Again this section implies the paper is about ARTP when it really isn't.
506 this reference is incomplete
Author Response
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Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
The manuscript concerns a topic involving beverages and novel techniques , plasma discharges, to overcome the usage of conventional chemicals. The article is well organized, whit good outcome. I would expect more details on the plasma source usage on this experiment, not only the reference of a previous work ( from 2015 ??, newer results??). Because from the manuscript title 'plasma' is present, than a paragraph concerning the plasma source should be inserted, for all the readers to understand better the results. This paragraph should contain besides the description of the device, the parameters used, along with a picture / schematic of the plasma source during experiments. A short / quick electro-optical diagnosis of the plasma also shoulb de inserted in the paragraph.
Whit such info added , than I believe that , taking the others referee sugestions as well, to be considered for publication.
Major revision.
Author Response
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Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsIn my opinion authors have improved the manuscript, taking in consideration questions and critics from the reviewers. And in my opinion paper can be suggested for publication.
Author Response
Thank you for your valuable insights and support.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors made the appropriate edits to the manuscript. I have no further significant concerns.
Author Response
Thank you for your valuable insights and support.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
The revised version of the manuscript is improved compared to the previous one. Some of the information/clarifications requested by the reviewers have been introduced into the text. However, in the paragraph describing the plasma source used in this study, graphical information regarding the type of applied voltage signal, and the discharge current, a global plasma emission spectrum, and possibly an estimate of the plasma power consumed should be introduced. After introducing this information, the paper can be considered for publication.
Minor revision.
Author Response
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