HOM2 Deletion by CRISPR-Cas9 in Saccharomyces cerevisiae for Decreasing Higher Alcohols in Whiskey
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study is about the genetic modification of Saccharomyces cerevisiae with CRISPR-Cas9 technology with the objective of fermenting whiskey with reduced higher alcohols concentration. The target gene knocked-out seems to be effective, and the author proved the decreasing of higher alcohol in comparison with the wild type strain. The study presents interesting results with a complete optimization in the parameters applied in fermentation. I believe the manuscript could be accepted after some corrections and considerations presented below.
Line 34 – You could alter “alcohos” to “alcohols”
Line 35 – You could remove the “definition” “(S. cerevisiae)”
Line 43 – Could you clarify which amino acids are affected by the alteration in HOM2 expression? You mention “corresponding amino acids”, but it’s not clear which specific amino acids are being referred to.
Line 93-94 – The sentence about “Saccharification procedure” could be improved.
Line 97 – This first sentence could be rewritten.
Line 132 – I suggest replacing “original strain” with the specific name of the strain.
Line 133 – You could briefly describe the conditions of the electrotransformation (equipment, potency, times...).
Line 151 – You could alter “Fermentation” to “fermentation”
Line 151-170 – These sections should be revised, as the entire Materials and Methods section is written in the past tense, yet these parts are currently in the present tense.
Line 162 – Add a space between the number and °P.
Line 167 - Add a space between the number and °P.
Line 206 (Figure 3) – Why in these gels do you have multiple amplicons in B and C? In B, what is the position 1 and 2?
Line 214-215 – The word “respectively” seems to not be necessary.
Line 225 – You must define what are “**” in this graphic legend.
Line 234 – In this section I have some suggestions. First, I believe that you should have the evaluation of every higher alcohol, so you could include this data in the material supplementary. Try not to start every paragraph with the same sentence. Clarify why you only presented the detailed data from isoamylol and n-propanol.
Line 256 (Figure 6) – In the legend describe what are the letters in the graphics and which statistical analysis you applied. In the graphic “C” alter the “y” axis from “N-propanol” to “n-propanol”.
Line 264 (Figure 7) – The “y” axis should present the correct unit. Since these values represent weight loss per a specific volume, the unit should be “g/XXX mL.”. In the “x” axis you could alter this by only presenting the numbers and adding the label “Time (h)”.
Line 267 – Your figure 7 is showing the time in “h”, so I suggest the text following the same unit, and not “4 days”.
Line 268 – 271 – Did you evaluate how much ethanol was produced by each strain in this assay? This could complement the data presented.
Line 294 (Figure 8) – Complement the legend with the description of the statistical analysis applied to this graphic. Also, add the strain utilized, time evaluated…
Line 310 (Figure 9) – This figure has the same problem as figure 8.
Line 317 and 342 (Table 2 and 3) – In these tables, you could add the strain utilized.
Line 346 – I believe that the correct is “Table 3”.
Line 364 – Why do you utilize the word “relative” in this context? The higher alcohol content is expressed in absolute units.
Line 373 (Figure 10) – I liked the graphic in “b”, that have the inner numbers with higher alcohols concentration. You could add this in graphics “a” and “c”. Also, you should add the strain utilized.
Line 384 – Add a space in “50L”.
Line 384-387 – I believe that you utilized the strain XF0-LH for this assay, correct? Did you evaluate the wild-type strain in the same condition? I was curious about the difference observed.
Line 402 (Figure 11) – You highlighted HOM2 in this figure, so you could highlight LEU1 as well.
Line 411 – Alter “path-ways” to “pathways”.
The authors should pay special attention to the figures, almost all figures need an improvement in the legend.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authorsin attached file
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
