Increasing Ethanol Tolerance and Ethanol Production in an Industrial Fuel Ethanol Saccharomyces cerevisiae Strain

Round 1

Reviewer 1 Report

Fuel-ethanol production is challenging due to the stress ethanol imposes on Saccharomyces cerevisiae cells. Several studies have shown that overexpression of the tryptophan synthesis-related gene TRP1 or the stress transcription factor MSN2 is effective in enhancing tolerance to high ethanol concentrations in S.cerevisiae. However, most of these factors are being tested using experimental strains and it is still unknown if they will work in industrial strains. In this manuscript, the authors tested three common expression strategies for improving ethanol tolerance in the industrial fuel ethanol strain CAT-1, including overexpression of the TRP1 or MSN2 genes, or overexpression of a truncated version of the MSN2 gene. Under the industrial conditions commonly used in Brazilian distilleries, only strains overexpressing the truncated MSN2 gene allowed the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses.

However, I find the paper very difficult to read, especially the results section. Hence, I recommend this manuscript for the publication in Fermentation, after the following issues should be addressed.

Major concern

1. According to the logic of the manuscript, the subheadings should be added in the RESULT section for easier reading.

2. Normally, the growth of S.cerevisiae is measured at OD_600nm. However, OD_570nm was chosen for the test in this manuscript, and the reason for this choice should be explained or the test should be repeated at OD_600nm.

3. In fact, tryptophan derivatives (e.g. Melatonin) have been shown to inhibit catalase activity and reduce ROS accumulation. Therefore, the Intracellular oxidation level data of the CAT1-TRP1oe strain should be supplemented.

Minor concern

1. Figure 1 should be changed to a more visual line or curve chart.

2. The lowercase letters in Figure 5 should be added with more detailed notes for easier reading.

3. Line 76-77. Change "(22-26)" to "[22-26]"

4. Line 349. Change "VGH" to "VHG"

5. The format of the references needs to be modified in accordance with the journal's requirements. e.g. Line 461-463

6. The English of the manuscript needs to be carefully checked and improved.

Author Response

We would thank the reviewers for their comments/suggestions/criticisms, which we feel contributed to improve our manuscript. Detailed answers to the points raised by each reviewer are given below:

Answers to Reviewer # 1:

Fuel-ethanol production is challenging due to the stress ethanol imposes on Saccharomyces cerevisiae cells. Several studies have shown that overexpression of the tryptophan synthesis-related gene TRP1 or the stress transcription factor MSN2 is effective in enhancing tolerance to high ethanol concentrations in S. cerevisiae. However, most of these factors are being tested using experimental strains and it is still unknown if they will work in industrial strains. In this manuscript, the authors tested three common expression strategies for improving ethanol tolerance in the industrial fuel ethanol strain CAT-1, including overexpression of the TRP1 or MSN2 genes, or overexpression of a truncated version of the MSN2 gene. Under the industrial conditions commonly used in Brazilian distilleries, only strains overexpressing the truncated MSN2 gene allowed the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses.

However, I find the paper very difficult to read, especially the results section. Hence, I recommend this manuscript for the publication in Fermentation, after the following issues should be addressed.

Major concern

1. According to the logic of the manuscript, the subheadings should be added in the RESULT section for easier reading.

Answer: subheadings were added in the Results section

2. Normally, the growth of S.cerevisiae is measured at OD_600nm. However, OD_570nm was chosen for the test in this manuscript, and the reason for this choice should be explained or the test should be repeated at OD_600nm.

A: While many laboratories measure growth of yeasts at OD 600nm (especially in synthetic define media), we see less interference of the rich YP media (containing yeast extract and peptone) when we measure cell growth at OD 570nm, and this wave-length has been used by our group in SEVERAL previous publications to measure growth of S. cerevisiae and even other yeasts (please refer to: Appl. Environ. Microbiol. 74:1494-1501, 2008; FEMS Yeast Res. 9:1338-1342, 2009; J. Appl. Microbiol. 109:248-259, 2010; Antonie van Leeuwenhoek 103:421-431, 2013; Microbiol. Res. 179:12-19, 2015; Lett. Appl. Microbiol. 67:377-383, 2018; Arch. Microbiol. 202:1729-1739, 2020; as examples). This wave-length of 570nm has also been used to measure growth by other researchers working with S. cerevisiae (e.g. Mariani et al. Redox Rep. 246-254, 2008; Rolim et al. Braz. J. Med. Biol. Res. 36:829-837, 2003; Ferreira et al. Biochim. Biophys. Acta. 1335:40-50, 1997; Mansure et al. Biochim. Biophys. Acta. 1191:309-316, 1994; just to mention a few of them).

3. In fact, tryptophan derivatives (e.g. Melatonin) have been shown to inhibit catalase activity and reduce ROS accumulation. Therefore, the Intracellular oxidation level data of the CAT1-TRP1oe strain should be supplemented.

A: The point raised by the reviewer is interesting! We had measured the intracellular oxidation level of the CAT1-TRP1oe strain under ethanol stress, and it had a high level of ROS that did not differ from the parental CAT-1 strain (see new Figure 4 and lines 305-307 of the “revised with tracking changes” file). The yeast S. cerevisiae can synthesize melatonin from tryptophan, but this trait is very variable among yeast strains and growth conditions (see Rodriguez-Naranjo et al. J. Pineal Res. 53:219-224, 2012; Fernández-Cruz et al. Food Chem. 217:431-437, 2017; Morcillo-Parra et al. Microorganisms 8:853, 2020). For example, no melatonin was synthesized when yeast cells were grown in YPD medium containing even high concentrations (5x) of tryptophan. While melatonin supplementation can indeed protect the yeast cells under ethanol stress (Sunyer-Figueres et al. Antioxidants 10:1735, 2021), again the protective effect of melatonin was dependent on the yeast strain, melatonin concentration, and growth phase. Thus, further studies are needed to confirm the protective action of melatonin and the mechanisms that trigger its synthesis from tryptophan in yeasts.

Minor concern

1. Figure 1 should be changed to a more visual line or curve chart.

A: Figure 1 was changed (with smaller symbols, colors, and lines), thus we think it improved its visual presentation.

2. The lowercase letters in Figure 5 should be added with more detailed notes for easier reading.

A: The legend of the figure was changed, and now the meaning of the lowercase letters is explained as: “Within each cycle, bars with different lowercase letters indicate significant differences between strains (p <0.05).” (see lines 355-356) We hope that now is more clear….

3. Line 76-77. Change "(22-26)" to "[22-26]"

A: Corrected as indicated (see line 77).

4. Line 349. Change "VGH" to "VHG"

A: Corrected. (now line 361)

5. The format of the references needs to be modified in accordance with the journal's requirements. e.g. Line 461-463

A: All references have been corrected to the journal’s style.

6. The English of the manuscript needs to be carefully checked and improved.

A: We have checked the English of the manuscript. We hope that now is better!

Reviewer 2 Report

The authors describe increasing ethanol tolerance and ethanol production of S. cerevisiae.

Minor comments;

 

In Fig. 5, a, b, c, and ab are presented on the white, grey, dark grey, and black bars. What do those mean? Please describe the meanings of a, b, c, and ab in the caption of Fig. 5.

Author Response

We would thank the reviewers for their comments/suggestions/criticisms, which we feel contributed to improve our manuscript. Detailed answers to the points raised by each reviewer are given below:

Answers to Reviewer # 2:

The authors describe increasing ethanol tolerance and ethanol production of S. cerevisiae.

Minor comments;

In Fig. 5, a, b, c, and ab are presented on the white, grey, dark grey, and black bars. What do those mean? Please describe the meanings of a, b, c, and ab in the caption of Fig. 5.

Answer: As explained in the Figure legend and in the insert present in Fig. 5A, the different colors are the different strains used. The legend of the figure was changed to better explain the lowercase letters, and now the meaning of those lowercase letters is explained as: “Within each cycle, bars with different lowercase letters indicate significant differences between strains (p <0.05).” We hope that now is more clear…. (see lines 355-356 of the “revised with tracking changes” file)

Reviewer 3 Report

In this manuscript, the authors try to elevate ethanol tolerance of Saccharomyces cerevisiae by overexpression of TRP1, MSN2, or Truncated MSN2 gene. The highlight of this manuscript is the use of industrial yeasts, and verification that overexpression of truncated version of the MSN2 can increase yeast ethanol tolerance and performance at industrial conditions. However, limited novelty is available in this manuscript.

1, all the three strategies have been identified in many other studies, the authors only use their strains to replicate the result. How about co-overexpression of MSN2 and TRP1? The authors should explain more in the manuscript.

2, the figures are misunderstanding, and some are unclear. Figure 1, why OD570nm only 0.6? The authors did not indicate this in the manuscript. Figure 5, what does a and b in each bar mean? The figures are hard to understand.

3, the gene expression level should be verified by transcriptomic data, and the transcriptomic data can provide global expression information. The qPCR result can provide limited information, and the effects of other genes can’t be indicated.

4, the authors need to carefully check the manuscript, such as line 262, ‘Thus, and?’

Author Response

We would thank the reviewers for their comments/suggestions/criticisms, which we feel contributed to improve our manuscript. Detailed answers to the points raised by each reviewer are given below:

Answers to Reviewer # 3:

In this manuscript, the authors try to elevate ethanol tolerance of Saccharomyces cerevisiae by overexpression of TRP1, MSN2, or Truncated MSN2 gene. The highlight of this manuscript is the use of industrial yeasts, and verification that overexpression of truncated version of the MSN2 can increase yeast ethanol tolerance and performance at industrial conditions. However, limited novelty is available in this manuscript.

1, all the three strategies have been identified in many other studies, the authors only use their strains to replicate the result. How about co-overexpression of MSN2 and TRP1? The authors should explain more in the manuscript.

Answer: We do agree that combining the overexpression of two or more genes can bring new results, and as suggested we include a statement calling the attention to this in the Discussion section: “While the three overexpression strategies were tested individually in the industrial yeast strain, it would be interesting to verify if combining two of them simultaneously (e.g. TRP1 and MNS2 overexpression) would improve the fermentation performance of the industrial strain.” Now in lines 398-401 of the “revised with tracking changes” file.

2, the figures are misunderstanding, and some are unclear. Figure 1, why OD570nm only 0.6? The authors did not indicate this in the manuscript. Figure 5, what does a and b in each bar mean? The figures are hard to understand.

A: Under the conditions used to monitor growth by several strains and medium conditions, 100 uL of medium in 96-well plates, the final OD of the cultures reaches a maximum of 0.8-0.9, as is the case of strains CAT-1 (Fig. 1A) and strain CAT1-TRP1oe (Fig. 1B) in the absence of ethanol (see also Figure 2B), and was even lower (ODmax. 0.6) for the two other yeast strains, as commented in lines 294-297 of the “revised with tracking changes” file. The reviewer can see our previous publications (e.g. Genome Res. 19:2271-2278, 2009; J. Appl. Microbiol. 109:248-259, 2010; Lett. Appl. Microbiol. 67:377-383, 2018) were growth of several yeast strains was also determined with 100 uL of medium in 96-well plates, and in all cases the OD reaches a maximum of 0.7-0.9 when the cells reach the stationary phase of growth. The OD or absorbance of a solution depends on the path/length of the liquid were the light will pass through to be measured (e.g. is 1 cm in a glass cuvette). With 100 uL in each well the maximum OD is 0.9, but other publications used, for example, 200 uL of medium in 96-well plates and the maximum OD was 1.2-1.5 (Hung et al. A simple and inexpensive quantitative technique for determining chemical sensitivity in Saccharomyces cerevisiae. Sci. Rep. 8:11919, 2018), and a maximum OD of 2.0 with 250 uL (Bisquert et al. Front. Microbiol. 9:318, 2018), or even 350 uL of medium (Weiss et al. High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae. Anal. Biochem. 327:23-34, 2004). However, these higher volumes require special formats of 96-well plates. We have made a comment on this (“Under the conditions used (100 μL of medium in 96-well plates) the maximum OD_570nm measured when the cells reach the stationary phase of growth is 0.8-0.9 [37].”) in the Materials and Methods section (lines 177-179).

Regarding Figure 5, the legend of the figure was changed to better explain the lowercase letters, and now the meaning of those lowercase letters is explained as: “Within each cycle, bars with different lowercase letters indicate significant differences between strains (p <0.05).” We hope that now is more clear…. (lines 355-356)

3, the gene expression level should be verified by transcriptomic data, and the transcriptomic data can provide global expression information. The qPCR result can provide limited information, and the effects of other genes can’t be indicated.

A: We agree with the reviewer that transcrptomic data would provide more detailed information on what is happening with the overexpression of the genes in the industrial strain, including the analysis under the different stressful conditions used. However, this will be a whole new project requiring not only funding and appropriate resources (microarrays, RNA-seq, etc.), but also extensive bioinformatic analysis to identify the differently expressed genes, associated GO-terms, etc., which certainly will take months (or even a year) to be achieved. We certainly need to include such analysis in our future projects. We used qPCR in our present manuscript just to confirm the overexpression of the corresponding genes in each strategy implemented in the industrial strain.

4, the authors need to carefully check the manuscript, such as line 262, ‘Thus, and?’

A: We have carefully checked the manuscript. We hope that now is better!

 

Round 2

Reviewer 3 Report

I am still not satified with this article.