Isolation, Identification, and Characterization of an Acid-Tolerant Pichia kudriavzevii and Exploration of Its Acetic Acid Tolerance Mechanism

Round 1

Reviewer 1 Report

The paper describes the characterization of isolates of yeast strains purified from a waste product of Baijiu production, which is a Chinese liquor. One strain Y2 was identified as Pichia kudriavzevii by PCR amplification and phylogenetic tree analysis. This strain was then subjected biological and biochemical tests with the emphasis on determining its acid tolerance in the presence of various concentrations of acetic acids. Next, the authors measured the transcriptome profile of Y2 following treatment with either 4 g/l or 8 g/l acetic acid. GO enrichment analysis of DEGs indicated that genes involved in processes of the TCA oxidative phosphorylation were significantly upregulated in the strain exposed to the higher concentration of acetic acid, thus achieving higher tolerance to acetic acid stress.

The discussion is too long. It should compare the molecular mechanisms of tolerance and adaptation to acetic acid in Saccharomyces cerevisiae and Pichia spp.

 

Minor points:

P3, 130: a reference for ITS1 and ITS2 should be provided.

P4, 168: organisms should be replaced by strains.

P4, 190: … was diluted, spread on YPD solid medium and streaked on plates, …

P5, 209: define, ‘…Y5 was more active in growth.’

P6, 240-241: … acclimatization …

P7, 225-229: refer to section 2.3 on p3, 127 that the ITS1 and ITS2 primers were used for PCR amplification.

P7, Figure 3, labelling of x-axis: Time [h]

P8, 289-290: pre-ribosome ( ) and membrane ( ) fractions?

P10, 328: OSCP, full name (oligomycin sensitivity conferral protein (delta subunit of mitochondrial ATP synthase).

P15, 433: it should read interconversion of malate and oxaloacetate.

Figure 1, p5: legend (A). Small colonies?

Figure 1, p5: legend (B). Methylene Blue staining?

Figure 1, p5, 202: … morphology of strains Y2, Y4 and Y7, respectively.

Figure 2, p6: legend (A), 221: … lowercase letters indicating significance.

Font of Figures 5 and 6 labels are too small!

Figure 9, p13: the indicated organelles should be labelled.

Moderate editing.

Author Response

Q1: The discussion is too long. It should compare the molecular mechanisms of tolerance and adaptation to acetic acid in Saccharomyces cerevisiae and Pichia spp.

Response: We are very grateful to Reviewer 1 for the constructive suggestions on our manuscript. We have re-polished the manuscript and discussed the acid resistance mechanism of Saccharomyces cerevisiae on P12, lines 394-398, and then compared our research results on P12, lines 398-408.

 

Q2: P3, 130: a reference for ITS1 and ITS2 should be provided.

Response: We have provided additional reference [26] on P3, line 126.

 

Q3: P4, 168: organisms should be replaced by strains.

Response: Revised.

 

Q4: P4, 190: … was diluted, spread on YPD solid medium and streaked on plates, …

Response: Revised.

 

Q5: P5, 209: define, ‘…Y5 was more active in growth.’

Response: We have supplied the definition as your suggestion. (P5, Lines 202-203)

 

Q6: P6, 240-241: … acclimatization …

Response: Revised.

 

Q7: P7, 225-229: refer to section 2.3 on p3, 127 that the ITS1 and ITS2 primers were used for PCR amplification.

Response: Thanks for your kind suggestions. We have revised on P6, line 219.

 

Q8: P7, Figure 3, labelling of x-axis: Time [h]

Response: Revised.

 

Q9: P8, 289-290: pre-ribosome ( ) and membrane ( ) fractions?

Response: We have double checked the description of these two GOID and additionally provided the detail information as supplemental tables (Table S2, Table S3 and Table S4).

 

Q10: P10, 328: OSCP, full name (oligomycin sensitivity conferral protein (delta subunit of mitochondrial ATP synthase).

Response: Revised.

 

Q11: P15, 433: it should read interconversion of malate and oxaloacetate.

Response: Revised.

 

Q12: Figure 1, p5: legend (A). Small colonies?

Response: Yes. It is smaller colonies.

 

Q13: Figure 1, p5: legend (B). Methylene Blue staining?

Response: We used Lactophenol Cotton Blue Stain and have added this description in Materials and methods Section and Figure legend (Line 111-112, 196).

 

Q14: Figure 1, p5, 202: … morphology of strains Y2, Y4 and Y7, respectively.

Response: Revised.

 

Q15: Figure 2, p6: legend (A), 221: … lowercase letters indicating significance.

Response: Revised.

 

Q16: Font of Figures 5 and 6 labels are too small!

Response: We have uploaded the vector images and tried to increase the image scale. Moreover, we have provided Supplemental Tables (S2-S7) for better understanding.

 

Q17: Figure 9, p13: the indicated organelles should be labelled.

Response: Revised.

 

Q18: Moderate editing.

Response: We are very grateful to Reviewer 1 for the constructive suggestions on our manuscript. We have re-polished the manuscript.

Reviewer 2 Report

GENERAL COMMENTS

The manuscript describes a study where the authors isolated a strain of Pichia kudriavzevii and investigated its underlying molecular mechanism of acetic acid tolerance. They used comparative transcriptomic analysis and claimed that their results had proven a possible mechanism of acid tolerance regulation in P. kudriavzevii, providing a theoretical basis for further investigation of the acid tolerance mechanism.

The work is interesting, the methodology is appropriate, and the conclusion well-supported. The contribution of the work would be appreciated if the possible mechanism of tolerance to acetic acid by yeast were more clearly enunciated in the conclusion section. It is suggested to include also in the abstract. The main concern is that the authors did not consider making (or not showing) acetic acid kinetics. It could reinforce the work and its conclusions. Is it too difficult to carry out for including it?

 

SPECIFIC COMMENTS

 

1. Line 26. The acronyms KEGG and DEG are less commonly used. Then, they must be defined before their first use in the text.
2. Line 29-33. Please, split this phrase into two clear sentences.
3. Line 45-48. Splitting this sentence into two sentences is recommended for better clarity.
4. Line 53-57. As in the last two comments, these lines' sentences should be shorter. It should be split into almost two sentences.
5. Line 120-121. The authors did not adjust pH values. When changing the pH of a solution (or culture medium), it is decided what pH value is desired, and this is obtained by adding the necessary amount of acid or base. But that's different from what the authors did here. Simply, they added acetic acid in knowing concentrations, and certain pH values were obtained, which are not stated in the text. Please correct the wording.
6. Line 121-122. Please inform how many hours the inoculum was incubated here.
7. Line 144-151. Were the cultures performed without replicates?
8. Line 164-165. Same comment as in Comment 5.
9. Line 207-211. Authors should report the incubation time at which OD readings were taken for each strain.
10. Line 227-229. Is it enough to have a 97.7% similarity in the sequencing of a DNA fragment to affirm that yeast has been unequivocally identified? Please go deeper and justify.
11. Line 239-240. Please review the wording since it seems that the same phrase is repeated.
12. Line 236-245. Fig. 1 shows that yeast growth was faster at an acetic acid concentration of 12 g/L than at 8 and 10 g/L. The authors do not comment on this obvious fact.
13. Line 246. The graph does not show error bars. Were the cultures performed without replicates?
14. Line 258-262. The description in Fig. 4A is very brief and superficial. Please improve it.
15. Line 331-332. Fig. 7 is unnecessary since it is a scheme that can be consulted in various classical texts. It is recommended to remove it or make a description in the text that fully justifies its inclusion in the manuscript.
16. Line 382-386. The information in this sentence must be incorporated into conclusions since it correlates the transcriptomic findings with the physiological aspects of the acetic acid resistance of the yeast. Please consider including it also into the abstract.
17. Line 405-409. The sentence seems incomplete. Four protein complexes were significantly… upregulated?

The manuscript wording must be enhanced since many sentences are too long, which must be split for a better understanding. There are only some examples in the specific comments. It is recommended that a native English speaker review the manuscript. 

Author Response

Q1: The contribution of the work would be appreciated if the possible mechanism of tolerance to acetic acid by yeast were more clearly enunciated in the conclusion section. It is suggested to include also in the abstract. The main concern is that the authors did not consider making (or not showing) acetic acid kinetics. It could reinforce the work and its conclusions. Is it too difficult to carry out for including it?

Response: We are very grateful to Reviewer 2 for the constructive suggestions on our manuscript. We have re-polished this manuscript and provided a more clearly explanation of the acid resistance mechanism of strain Y2 in the abstract and conclusion sections. We will conduct an in-depth study of acetic acid kinetics in our further work.

 

Q2: Line 26. The acronyms KEGG and DEG are less commonly used. Then, they must be defined before their first use in the text.

Response: Revised.

 

Q3: Line 29-33. Please, split this phrase into two clear sentences.

Response: Revised.

 

Q4: Line 45-48. Splitting this sentence into two sentences is recommended for better clarity.

Response: Revised.

 

Q5: Line 53-57. As in the last two comments, these lines' sentences should be shorter. It should be split into almost two sentences.

Response: Revised.

 

Q6: Line 120-121. The authors did not adjust pH values. When changing the pH of a solution (or culture medium), it is decided what pH value is desired, and this is obtained by adding the necessary amount of acid or base. But that's different from what the authors did here. Simply, they added acetic acid in knowing concentrations, and certain pH values were obtained, which are not stated in the text. Please correct the wording.

Response: Thanks for your kind suggestions. We have revised on P3, lines 117-118.

 

Q7: Line 121-122. Please inform how many hours the inoculum was incubated here.

Response: We have added this information on P3, line 118.

 

Q8: Line 144-151. Were the cultures performed without replicates?

Response: The cultures performed three replicates per group. We have revised it in MS.

 

Q9: Line 164-165. Same comment as in Comment 5.

Response: Revised.

 

Q10: Line 207-211. Authors should report the incubation time at which OD readings were taken for each strain.

Response: The incubation time for OD readings of each strain is 48 h. We have revised in Materials and Results Sections.

 

Q11: Line 227-229. Is it enough to have a 97.7% similarity in the sequencing of a DNA fragment to affirm that yeast has been unequivocally identified? Please go deeper and justify.

Response: We compared the sequence of strain Y2 with existing strains in NCBI (BLAST: Basic Local Alignment Search Tool) and reconstructed the phylogenetic tree. We have revised on P5, Figure 2C and P6, lines 222-223.

 

Q12: Line 239-240. Please review the wording since it seems that the same phrase is repeated.

Response: Revised.

 

Q13: Line 236-245. Fig. 1 shows that yeast growth was faster at an acetic acid concentration of 12 g/L than at 8 and 10 g/L. The authors do not comment on this obvious fact.

Response: Thank you for your kind correction! According to transcriptome data, ATP synthesis and metabolic activities were upregulated with higher acetic acid concentration. It was speculated that acetic acid might be the carbon source for this strain of Pichia kudriavzevii at the early growth stage. Therefore, the growth rate of 12 g/L is faster than that of 8 g/L and 10 g/L. We have revised it in Lines 231-234.

 

Q14: Line 246. The graph does not show error bars. Were the cultures performed without replicates?

Response: There are three replicates per group, and we have added error bars in Figure 3.

 

Q15: Line 258-262. The description in Fig. 4A is very brief and superficial. Please improve it.

Response: We have expanded this description (Line 257-260).

 

Q16: Line 331-332. Fig. 7 is unnecessary since it is a scheme that can be consulted in various classical texts. It is recommended to remove it or make a description in the text that fully justifies its inclusion in the manuscript.

Response: We have removed Figure 7 and 8.

 

Q17: Line 382-386. The information in this sentence must be incorporated into conclusions since it correlates the transcriptomic findings with the physiological aspects of the acetic acid resistance of the yeast. Please consider including it also into the abstract.

Response: Thank you for your construction suggestions. We have refreshed the Abstract and Conclusion Section.

 

Q18: Line 405-409. The sentence seems incomplete. Four protein complexes were significantly… upregulated?

Response: Revised.

 

Q19: The manuscript wording must be enhanced since many sentences are too long, which must be split for a better understanding. There are only some examples in the specific comments. It is recommended that a native English speaker review the manuscript.

Response: We have made this manuscript re-polish by a native speaker.

Reviewer 3 Report

The study addresses an important knowledge gap by investigating the acid tolerance mechanism of Pichia kudriavzevii, a major non-Saccharomyces cerevisiae yeast in Chinese Baijiu brewing, which is known for its tolerance to acid, heat, and ethanol.

 

The isolation of a highly acetic acid-tolerant strain, Y2, from the yellow water of strong-flavored Baijiu brewing waste provides a valuable resource for studying the underlying molecular mechanisms of acetic acid tolerance in Pichia kudriavzevii.

 

The utilization of comparative transcriptomic analysis and growth experiments to investigate the molecular mechanisms associated with acetic acid tolerance demonstrates a comprehensive approach in understanding the physiological and genetic responses of Pichia kudriavzevii to acetic acid stress.

 

The identification of key pathways, such as Oxidative phosphorylation, Citrate cycle, Glycolysis/Gluconeogenesis, and Carbon metabolism, through KEGG pathway enrichment analysis provides insights into the metabolic adaptations that contribute to acid tolerance in Pichia kudriavzevii.

 

The differential expression of genes related to protein complexes involved in the electron transport chain, including NADH dehydrogenase, succinate dehydrogenase, cytochrome c reductase, cytochrome c oxidase, and F-type H+-transporting ATPases, suggests their significant role in the acetic acid tolerance of Pichia kudriavzevii, thus enhancing our understanding of the molecular mechanisms involved.

 

The findings of upregulated genes in the TCA cycle, such as phosphoenolpyruvate carboxykinase, citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase, provide valuable insights into the metabolic adaptations and energy generation processes that contribute to acetic acid tolerance in Pichia kudriavzevii.

 

The study results contribute to a possible mechanism of acid tolerance regulation in Pichia kudriavzevii, paving the way for further investigations and providing a solid theoretical basis for future research on the acid tolerance mechanisms of this yeast species.

 

Overall, this scientific manuscript significantly advances our understanding of the acid tolerance mechanism in Pichia kudriavzevii and lays the groundwork for further studies that can contribute to improved fermentation processes in Baijiu brewing and related industries.

However, The abstract lacks a clear statement of the objectives and the significance of the study. It would be helpful to explicitly state the research aim and the potential implications of understanding the acid tolerance mechanism in Pichia kudriavzevii.

 

 

The isolation of a highly acetic acid-tolerant strain, Y2, is mentioned, but there is limited information provided about the methodology used for isolation. Adding details about the isolation procedure would enhance the clarity and reproducibility of the study.

 

The abstract would benefit from including more specific information regarding the comparative transcriptomic analysis and growth experiments. Describing the methodologies employed and the specific results obtained would provide readers with a clearer understanding of the study's findings.

Author Response

Q1: The abstract lacks a clear statement of the objectives and the significance of the study. It would be helpful to explicitly state the research aim and the potential implications of understanding the acid tolerance mechanism in Pichia kudriavzevii.

Response: We are very grateful to Reviewer 3 for the constructive suggestions on our manuscript. We have modified the Abstract Section as your suggestion.

 

Q2: The isolation of a highly acetic acid-tolerant strain, Y2, is mentioned, but there is limited information provided about the methodology used for isolation. Adding details about the isolation procedure would enhance the clarity and reproducibility of the study.

Response: We have added details about the acid-tolerant yeast isolation procedure in Lines 108-120.