Highly Efficient Synthesis of Glutathione via a Genetic Engineering Enzymatic Method Coupled with Yeast ATP Generation
Round 1
Reviewer 1 Report
In this paper, Chen et al. proposed a new method to increase the GSH production termed as “post addition CTAB method”, which not only effectively coupled the yeast energy system but also greatly reduced the feedback inhibition and resulted in high T-GSH production. They started with the evaluation of different surfactants showing CTAB treated yeast has the highest GSH production. They next investigated the volume concentration of yeast in the reaction medium, demonstrating that the larger yeast cell volume concentration yields higher GSH concentration when yeast cell volume concentration below 20%. They tested the proportion of yeast to permeabilizing agent revealing that 10:1 is the optimum permeabilizing proportion. With those conditions, they evaluated post-addition CTAB method, which used endogenous enzymes in yeast cells to synthesize GSH, followed by the addition of CTAB and exogenous enzyme to catalyze the synthesis of GSH. They demonstrated this new method resulted in higher efficient synthesizing of GSH. They further invested different carbon source and tested different oxidative stress agents aiming to maximize the GSH generation.
Several minor issues need to be fixed before publishing:
Loading control should be included in figure 1, also blank control without the inducer should be added for comparison. The protein band of glutathione synthetase should be labelled. In figure 1b, 14 °C has the highest expression to me instead of 26°C. In figure 5, it would be clearer to label HPLC peak corresponding compoundAuthor Response
Dear reviewer,
Thank you very much for your careful review and constructive suggestions with regard to our manuscript. The main corrections in the paper are as follows:
Response to comment: Blank control should be included in figure 1. Due to the limited time for graduation, I can't add more experiments. Thank you for your suggestion. It is very important. Because of your suggestion, I found the deficiencies in my current work. I will follow your suggestions to improve the scientific research level in future work. Response to comment: Labeling of protein bands. The protein bands of glutathione synthetase have been labeled in figure 1. Response to comment: In figure 1b, 14℃ has the highest expression. We are sorry for our incorrect description, and we have modified it. The recombinant protein has the highest expression at 26℃in in figure 1b. Response to comment: Labeling of HPLC peaks. In figure 5, we have labeled HPLC peak corresponding compound.
Special thanks to you for your good comments. We sincerely hope that you can give us more valuable Suggestions. We will try our best to improve our manuscript to meet with approval.
Best regards,
Chen Huang
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript is on a new reaction system to produce glutathione. Unfortunately, the manuscript is written in a complicated and unclear way. The reaction is done in two steps, but to me it has not become clear when the first step is considered finished (which criteria?), and where the substrates come from (endogenous, exogenous?).
The manuscript should be rewritten such that it becomes crystal clear what the authors have done, why they did it in this way, and which problems are now solved. I missed references to previous work, as well as a discussion of the current results with those of others.
The title of the manuscript is incomprehensible. Please, simplify the title.
Remove all abbreviations from the abstract.
Remove subjective statements (creative)
Rewrite the abstract to make it crystal clear what is new, how the synthesis is done, and when which compounds are added.
Below I give some language suggestions. However, I suggest the authors ask colleagues to help them to improve the clarity of the manuscript.
Line 9: -> A new, two-step reaction system has been developed to efficiently synthesize glutathione
Line 10: phase -> step
Line 10: -> glutamate and cysteine are condensed to glutamyl-cysteine by endogenous yeast enzymes inside the yeast cell, while consuming ATP.
Line 11: stage -> step
Line 12: permeabilizing agent(CTAB) -> permeabilizing agent CTAB
Line 12: to release a large amount of the intermediate product, glutamylcysteine -> to release the glutamyl-cysteine, upon which added glutathione synthase converts the glutamyl-cysteine and added glycine into glutathione. The ATP needed for this conversion is supplied by the permeabilized yeast cells. (?)
Line 16: that reduced -> that 2.1 g/L reduced glutathione is produced, and 17.5 g/L oxidized glutathione.
Line 26: replace “compound with … peptide bonds of” by “consisting of”
Line 30: activity[2], oxidation Glutathione -> activity[2]. Oxidized glutathione
Figure 1: indicate (with an arrow) where the glutathione synthetase is present. (The molecular weight is nowhere mentioned in the manuscript, I think). Why was the synthetase recombinantly expressed? This should be mentioned in the Introduction.
Line 196: founded -> found
Line 219: DNA maker -> DNA molecular weight markers (?)
Line 220: protein molecule Maker -> protein molecular weight markers (?)
Author Response
Dear reviewer,
Thank you very much for your careful review and constructive suggestions with regard to our manuscript. Based on your comment and request, we have made extensive modification on the original manuscript. The main corrections in the paper are as follows:
Response to comment: when the first step is considered finished. We are sorry that we didn't make it clear in the manuscript when the first step is finished. The first step of the reaction was completed for about 10 hours. Sufficient glutamyl-cysteine and ATP can be produced, which is most beneficial for the second step of glutathione synthesis catalyzed by exogenous enzymes, as shown in Figure S1. Response to the source of the substrate. The substrate comes from exogenous addition. In the first step of the reaction, three precursor amino acids were added. The enzyme reaction was also supplemented with three substrates in the second step. We have mentioned in 3.3 The reaction system of glutathione synthetase coupled with yeast energy production. Response to abstract. We have rewritten the abstract to make it crystal clear how the synthesis is done, when which compounds are added, and what is the main purpose and highlight of this research Response to abbreviations and subjective statements. We have removed all abbreviations from the abstract and remove some subjective statements. Response to language. Thank you very much for your language suggestions, we have corrected the errors you mentioned. Additionally, we undergo extensive English editing for the manuscript to improve the clarity of the text. Response to figure 1. Figure 1 has been modified. We have indicated where the glutathione synthetase is present with an arrow. The molecular weight has been mentioned in 3.2 construction of the pET28a-gshII recombinant plasmid. The reason why the synthetase was recombinantly expressed has been mentioned in the introduction. The expression of GSH synthase can be improved by inserting the glutathione synthase gene into the prokaryotic expression system to construct the recombinant strain through genetic engineering. Response to comment: The title of the manuscript is incomprehensible. We have modified the title of the manuscript to make it easier to understand. In addition, there is another title to be chosen: A new method for efficient synthesis of glutathione based on genetic engineering enzymatic method coupling yeast ATP generation. Our work aims to further increase the yield of glutathione synthesis based on the enzymatic method and coupling with yeast energy production. In the first step, glutamate and cysteine are condensed to glutamyl-cysteine by endogenous yeast enzymes inside the yeast cell. After 10 h reaction, CTAB was added to permeate the cell membrane to release a large amount of the intermediate product, glutamylcysteine. In the second step, exogenous enzymes were added to catalyze glutamyl-cysteine and glycine to synthesize glutathione. The ATP needed for this conversion is supplied by the permeabilized yeast cells of the glycolytic pathway. This method not only effectively couples yeast ATP generation, but also reduces the feedback inhibition of glutathione for the first-step enzymatic reaction. In this method, we found that the final yield of glutathione has been significantly increased.
Once again, thank you very much for your comments and suggestions. We sincerely hope that you can give us more valuable comments and suggestions. We will try our best to improve our manuscript to meet with approval.
Best regards,
Chen Huang
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The manuscript has greatly improved. I thank the authors for their careful work to improve the manuscript.
I have only a few minor textual corrections:
Line 21: subsequent the separation -> the subsequent separation
Line 28: condensed by peptide bonds with -> consisting of (it is not necessary to mention the peptide bonds, because that information is already implied in the word “tripeptide” (line 27)
Line 65: synthease -> synthetase
Author Response
Dear reviewer,
Thank you very much for your careful review and constructive suggestions with regard to our manuscript. Those comments are very helpful for us to revise and improve our paper. The main corrections in the paper are as follows:
Response to comment: Line 21: subsequent the separation -> the subsequent separation. We are sorry for our incorrect writing,we have modified “subsequent the separation”to “the subsequent separation”. Response to comment: Line 28: condensed by peptide bonds with -> consisting of. Thank you very much for your suggestions. we have used “consisting of” instead of “condensed by peptide bonds”. Response to comment: Line 65: synthease -> synthetase.We are sorry for our incorrect writing, we have modified “synthease” to “synthetase”.
Special thanks to you for your good comments. We sincerely hope that you can give us more valuable suggestions. We will try our best to improve our manuscript to meet with approval.
Best regards,
Chen Huang
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This manuscript describes a novel method for the synthesis of glutathione, based on the use of glutathione synthetase expressed in yeast, with an endogenous enzyme step followed by membrane permeabilization and an exogenous enzyme step.
The development of efficient methods for synthesis of glutatione could be of interest. However, this work is not presented in a suitable way to be published. Extensive editing of the manuscript should be carried out; as it is, relevant parts of the text are difficult to understand. Also, the methods are not adequately explained and relevant experimental details are missing (i.e. reaction conditions, reaction times, etc.). It is unclear why some of the experiments were carried out (i.e. oxidative stress). Also, the selection of the experimental conditions needs to be justified.
Sound discussion of the results is also lacking. The manuscript is eminently descriptive, and I would suggest the authors to try publication in a journal specifically focused on methods.
Reviewer 2 Report
The submitted paper describes a new method for efficient synthesis of glutathione based on genetic engineering enzymatic method coupling yeast ATP generation.
Unfortunately, I cannot recommend the publication of this paper in its present form due to the points raised below.
Major concerns:
- First of all, the paper needs extensive editing for language and style.
- The objective of the study should be rewritten to become more precise (Abstract and Introduction section).
- Materials and methods, 2.3. Reaction system of glutathione synthetase-coupled yeast energy: this section should be rewritten (style), the procedures should be described in details.
- Figure’s 4 captions do not correspond with presented data.
Minor concerns:
- Introduction, lines 33-34: references should be added to support information given in these lines.
- Materials and methods: source of Saccharomyces cerevisiae should be given.
- Abbreviations (e.g. CTAB) should be defined at first mention and used consistently thereafter.
- Line 109: ‘Results and discussion’ vs. ‘Result and discussion’.
- Results and discussion, lines 206-212: this part should be rewritten (style).
