Evaluation of Long-Term Fermentation Performance with Engineered Saccharomyces cerevisiae Strains

Round 1

Reviewer 1 Report

Dear Sirs. Genetic stability of industrialy employed yeast  strains is very important parameter of strains and results in efficiency losses during the time. It is very important to evaluate phenomenon of changes in this stability. My overall rating for Your manuscript is very high but I suggest that 2-3 sentences with less specialized language, more popular attitude, should be added to elucidate why yECFP assay is so important and was chosen by You as a core assay for the eperimets described in the manuscript, and that is the reason why I think that quality of the methods and results presentation may be improved. Authors use very specialistic language and as a results it narrows the amount of the people able to fully evaulate Your manuscript, however for the industrial biotechnologists strains stability is known as the weak spot of the technology, which usually is improved only by the limiting of number of generations between strain obtaining and main bioprocess (usually this is obtained by storage the freshly obtained and tested strains by freezing or lyophilization) I think that 2-3 sentences in the conclusion should be added how, the end-user of the strain -  biotechnologist may use the results of Your experiments. After You will add these elucidation I think that Your paper should be published.

Author Response

I am pleased to hear that the reviewer thinks high of this manuscript and understands the necessity of robust microbial strains for industrial bioprocesses. We believe to have addressed the following comments appropriately.

• Add 2-3 sentences with less specialized language, more popular attitude, to elucidate why yECFP assay is so important and was chosen by us as a core assay for the experiments: First, the abstract has been rewritten to clarify the role of the reporter gene yECFP. Second, the final paragraph of the Introduction was expanded with 2 additional sentences on the yECFP assay, and a small restructuring took place to emphasize the usefulness of yECFP pertaining to the newly proposed parameters.
• Add 2-3 sentences in the Conclusions on how the end-user of the strain (biotechnologist) may use the results of your experiments: In accordance with the fifth comment of Reviewer 2, a Conclusions section was added. An emphasis on the practical application of the results presented in this manuscript, is given.

Reviewer 2 Report

The presented material seems interesting and requires partial revision.

The authors need to make minor corrections to improve the article.

Comments are provided below.

1. Form a generalized scientific goal of the research, indicate it in the abstract of the article

2. For all raw materials used, indicate the city of origin in addition to the country, as well as the origin of all reagents.

3. Give a more complete description of the research methods

4. The authors should give a more detailed description of the strains used

5. In conclusion, it is necessary to indicate possible options for the practical application of the results obtained.

6. The list of references should be increased to 50 sources for 2010-2022

Moderate English editing required

Author Response

We appreciate the reviewer’s comments and are happy to clarify his/hers suggestions.

• Form a generalized scientific goal of the research, indicate it in the abstract of the article: To accommodate with the first comment of reviewer 2, which was in part also given by Reviewer 1, we have divided the Abstract into Background, Methods, Results and Conclusions. Additionally, the abstract was rewritten to clarify the goal of this study, as highlighted in the revised manuscript.
• For all raw materials used, indicate the city of origin in addition to the country, as well as the origin of all reagents: Missing details are now provided in Section 1 Materials.
• Give a more complete description of the research methods: Additional information on the research methods has been providing in Section 4 Strain construction.
• The authors should give a more detailed description of the strains used: The structure of the first paragraph has been changed a little to focus on the strains. Also, the chromosomes on which the integration regions are located, have been provided to facilitate interpretation of the inter- and intrachromosomal robustness conclusions. Extra information on why retrotransposon sites were targeted, is given. The authors would also like to note that the genomic sequence of every strain is present in the Supplementary Materials.
• In conclusion, it is necessary to indicate possible options for the practical application of the results obtained: In accordance with the second comment of Reviewer 1, a Conclusions section was added. An emphasis on the practical application of the results presented in this manuscript, is given.
• The list of references should be increased to 50 sources for 2010-2022: The results presented in this paper are a first of their kind and thus little literature is present to link them to. Nevertheless, we have added additional references to the Introduction, Results and Discussion. However, in total we finish with 42 references compared to the initial 23 references.
• Moderate English editing required: Several changes have been made, and highlighted, throughout the text.