Homologous Overexpression of Acyl-CoA Thioesterase 8 Enhanced Free Fatty Acid Accumulation in Oleaginous Fungus Mucor circinelloides WJ11
, Chunchi Rong 3,* and Huaiyuan Zhang 1,*
Round 1
Reviewer 1 Report
1. As a result of a failure, italics disappeared when writing the names of organisms. Lines: 18, 19, 33-34, 56, 57, 60, 61, 62, 65, 88, 91-92, 96, 105, 106, 110, 111, 116, 120, 127.
2. As a result of the failure, the subscripts are disappeared: lines 140, 141, 142, 151, 152 and formula 1.
3. Line 132. Approximately 106 spores of ACOT8. Probably still 106 spores of ACOT8?
4. Line 58. The trivial name “myristic acid” used for C14:0 acid, while the chemical names are used for all others mentioned fatty acids. It is better to use chemical name - tetradecanoic acid.
5. Lines 144-145. What filter was used to collect the biomass? Explain what method of determining the dry weight was used?
6. Lines 150-152 and formula 1 - The specific growth rate (μ) is expressed in (g/L). Figure S1.C - The specific growth rate (μ) is expressed in (/h). Did you mean (h-1)?
7. Line 240. The growth rate is mentioned, the data are presented in Figure S1.A, but there is nothing in the text of the article about the calculation of this value.
8. In fig. S1 presents data of lipid growth intensity (E), but however, lipids are not mentioned at all in methods section.
9. It is necessary to rewrite the section Total fatty acid (TFA) extraction and analysis of overexpressing strains.
10. At the beginning of the article, it is necessary to clarify what the authors mean by the term "total fatty acids" (TFA). Since, when describing the method of their isolation, they use the Folch method for lipids extraction, and then they isolated the fraction of free fatty acids from total lipids by preparative TLC in a system for neutral lipids and identify the main individual classes: TAG fraction, free fatty acids, 1,2- and 1,3-diglycerides, as well as monoglycerides. When describing the results, the authors (line 292) are talking about lipids. Data on lipid growth intensity (g/L/h) are also mentioned in Figure S1. What does it mean TFA? How the content (%) (apparently % from mycelium dry weight) was determine?
11. Figure 4. What is Ratio? Does it mean the content of individual lipids classes (% of sum)?
12. How the content of individual fractions was determined? It's not described in the methods section. In the results (lines 296-297) "The area of the gray scale peaks was obtained after processing the developed bands with the gray scale scanning software ImageJ was plotted to scale". Then why was the C15:0 internal standard was added at the stage of extraction?
13. The authors of the article subjected to transesterification for obtaining fatty acid methyl esters (FAME): the extract of total lipids obtained according to method of Folch (the results are presented in Table 1), resulting in total fatty acids (sum of fatty acids obtained from the conversion of total lipids, synthesized by the organism), as well as the isolated fraction of free fatty acids (the band isolated from the TLC plate; the results are presented in Table 2). It is necessary to added time and temperature of the reaction. And also indicate how the individual fatty acids were identified. The referenced article [24] does not contain this information.
14. Line 168. Not N-hexane, n-hexane.
15. Line 245. As shown in Figure S4 - check the figure number.
16. It is necessary the sameness of tables 1 and 2 names.
The content of individual fatty acids (% of sum) of TFA or FFA (respectively for table 1 and 2) of 314 Mc2075, McACOT8a-1, McACOT8b-2 and McACOT8c-2.
Author Response
Point-by-point responses to the reviewer’s comments on the manuscript
Manuscript ID: fermentation-2413629
Manuscript Title: Homologous overexpression of acyl-CoA thioesterase 8 enhanced free fatty acid accumulation in oleaginous fungus Mucor circinelloides WJ11.
The authors would like to thank the reviewers for their extremely efficient and earnest work on our manuscript. The reviewers’ comments and suggestions are all valuable and very helpful for revising and improving our manuscript. We have studied the comments carefully and have carefully revised our manuscript. The itemized list of changes and point-by-point responses to the comments were listed below.
Responses Reviewer 1
The authors would like to thank reviewer “1” for the helpful comments. We have incorporated the following reviewer’s specific comments in preparation for the revised version of the manuscript. The following is our response to each comment of the reviewer one by one.
- As a result of a failure, italics disappeared when writing the names of organisms. Lines: 18, 19, 33-34, 56, 57, 60, 61, 62, 65, 88, 91-92, 96, 105, 106, 110, 111, 116, 120, 127.
Response: We are sorry that we did not check the content. We have modified the above problem and highlighted it in yellow in introduction. We have checked all text of the manucript carefully.
- As a result of the failure, the subscripts are disappeared: lines 140, 141, 142, 151, 152 and formula 1.
Response: We are very grateful to you for reading and reviewing this manuscript, and we have changed this and highlighted it in yellow. Thank you again for your review.
- Line 132. Approximately 106 spores of ACOT8. Probably still 106 spores of ACOT8?
Response: We are very grateful to you for reading and reviewing this manuscript, and we are sorry that we did not set the upper corner label, we have modified it in “106”.
- Line 58. The trivial name “myristic acid” used for C14:0 acid, while the chemical names are used for all others mentioned fatty acids. It is better to use chemical name - tetradecanoic acid.
Response: We are very grateful to you for reading and reviewing this manuscript, and we are very willing to modify the error from “myristic acid” into “tetradecanoic acid” in line 58 of the manuscipt.
- Lines 144-145. What filter was used to collect the biomass? Explain what method of determining the dry weight was used?
Response: We are very grateful to you for reading and reviewing this manuscript, in this experiment, we used decompression Buchner funnel to collect the mycelium of fermentation product, and determined the CDW of mycelium by differential weight method. The above explanation was also described in lines 145-149 of the manuscript.
- Lines 150-152 and formula 1 - The specific growth rate (μ) is expressed in (g/L). Figure S1.C - The specific growth rate (μ) is expressed in (/h). Did you mean (h-1)?
Response: We are sorry that these two places have not been carefully checked. We have changed the units in formula 1 into “/h”.
- Line 240. The growth rate is mentioned, the data are presented in Figure S1.A, but there is nothing in the text of the article about the calculation of this value.
Response: We are very grateful to you for reading and reviewing this manuscript, and we are very willing to accept your suggestions. The sentence “The ratio of CDW to time yields the growth rate.” was added in lines 151-152 of the manuscript.
- In fig. S1 presents data of lipid growth intensity (E), but however, lipids are not mentioned at all in methods section.
Response: We are sorry that the growth intensity of fatty acids should be here. Due to our negligence, there is ambiguity. We have made modifications in the supplementary document from “lipid growth intensity (E)” into “TFA growth intensity (E)”.
- 9. It is necessary to rewrite the section Total fatty acid (TFA) extraction and analysis of overexpressing strains.
Response: Thank you very much for your careful review of this manuscript, and we have rewritten this content in lines 155-174. Thank you again!
- At the beginning of the article, it is necessary to clarify what the authors mean by the term "total fatty acids" (TFA). Since, when describing the method of their isolation, they use the Folch method for lipids extraction, and then they isolated the fraction of free fatty acids from total lipids by preparative TLC in a system for neutral lipids and identify the main individual classes: TAG fraction, free fatty acids, 1,2- and 1,3-diglycerides, as well as monoglycerides. When describing the results, the authors (line 292) are talking about lipids. Data on lipid growth intensity (g/L/h) are also mentioned in Figure S1. What does it mean TFA? How the content (%) (apparently % from mycelium dry weight) was determine?
Response: Thank you very much for reviewing the manucript. We are sorry that we did not clearly describe the extraction of “lipids” and “TFA”. Based on this, we rewrote this part in lines 155 to 174 of the manuscript, in addition, the “lipid growth intensity” in the supplementary document was also modified into “TFA growth intensity”.
- Figure 4. What is Ratio? Does it mean the content of individual lipids classes (% of sum)?
Response: Thank you very much for your review of this manuscript. Ratio refers to the proportion of each component in the total lipid.
- How the content of individual fractions was determined? It's not described in the methods section. In the results (lines 296-297) "The area of the gray scale peaks was obtained after processing the developed bands with the gray scale scanning software ImageJ was plotted to scale". Then why was the C15:0 internal standard was added at the stage of extraction?
Response: We are very grateful to you for reading and reviewing this manuscript. We put "The area of the gray scale peaks was obtained after processing the developed bands with the gray scale scanning software ImageJ was plotted to scale" in the method for description. C15:0 was added in the determination of fatty acid composition as an internal standard to calculate the proportion of each fatty acid.
- The authors of the article subjected to transesterification for obtaining fatty acid methyl esters (FAME): the extract of total lipids obtained according to method of Folch (the results are presented in Table 1), resulting in total fatty acids (sum of fatty acids obtained from the conversion of total lipids, synthesized by the organism), as well as the isolated fraction of free fatty acids (the band isolated from the TLC plate; the results are presented in Table 2). It is necessary to added time and temperature of the reaction. And also indicate how the individual fatty acids were identified. The referenced article [24] does not contain this information.
Response: Thank you very much for your detailed review of the paper. We have checked the references of code 24 again. In page 7 of the literature has a corresponding description "The resultant fatty acid methyl esters were extracted with n-hexane and were analyzed by GC equipped a 30 m×0.32 mm DB-Waxetr column with 0.25 µm film thickness. The program was as follows: 120°C for 3 min, ramp to 200°C at 5°C per min, ramp to 220°C at 4°C per min, hold 2 min."Although the literature does not describe the gas phase of TLC products, but it's actually the same method. The details are shown in the following figure.
- Line 168. Not N-hexane, n-hexane.
Response: We are very grateful to you for reading and reviewing this manuscript, and we are very willing to accept your suggestions. We have changed this into n-hexane.
- Line 245. As shown in Figure S4 - check the figure number.
Response: Thank you very much for reading and reviewing this manuscript, and we are very willing to accept your suggestions. We have modified it to Figure S4D in line 252 of the manuscript.
- It is necessary the sameness of tables 1 and 2 names.The content of individual fatty acids (% of sum) of TFA or FFA (respectively for table 1 and 2) of 314 Mc2075, McACOT8a-1, McACOT8b-2 and McACOT8c-2.
Response: We are very grateful to you for reading and reviewing this manuscript, and we are very willing to accept your suggestions. We have agreed on the questions in Table 1 and Table 2. Thank you again!
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Introduction Names of organisms should be written in italics.
L124：The source of pMAT2075 should be indicated.
Figure2：The values with Statistical significance are marked with symbols.
Discussion、Table3 Redundant descriptions of Acyl-ACP thioesterases from other species obscure the essential discussion.
Author Response
Point-by-point responses to the reviewer’s comments on the manuscript
Manuscript ID: fermentation-2413629
Manuscript Title: Homologous overexpression of acyl-CoA thioesterase 8 enhanced free fatty acid accumulation in oleaginous fungus Mucor circinelloides WJ11.
The authors would like to thank the reviewers for their extremely efficient and earnest work on our manuscript. The reviewers’ comments and suggestions are all valuable and very helpful for revising and improving our manuscript. We have studied the comments carefully and have carefully revised our manuscript. The itemized list of changes and point-by-point responses to the comments were listed below.
Responses Reviewer 2
The authors would like to thank reviewer “2” for the helpful comments. We have incorporated the following reviewer’s specific comments in preparation for the revised version of the manuscript. The following is our response to each comment of the reviewer one by one.
This research focused on the function of acyl-CoA thioesterase derived from oleaginous fungus, Mucor circinelloides WJ11. The authors invstigated the effect of over expression of acyl-CoA thioesterase genes on growth, fatty acid content and fatty acid composition. The result is clear and valuable. However, The discussion is redundant and unclear. My evaluation is that the paper is publishable with major revision. Listed below are my specific comments.
Response: We are very grateful to you for reading and reviewing this manuscript, and we are very willing to accept your suggestions.
- Introduction Names of organisms should be written in italics.
Response: We are very grateful to you for reading and reviewing this manuscript, and we have reviewed the full text and italicized all the species names.
- L124：The source of pMAT2075 should be indicated.
Response: Thank you very much for your review of the manuscript. We have labeled the source of pMAT2075, a plasmid constructed and preserved by our research group, which was also described in the manuscript and highlighted in blue in lines 124-125.
- Figure2：The values with Statistical significance are marked with symbols.
Response: We are very grateful to you for reading and reviewing this manuscript, and we are very willing to accept your suggestions. We have added significance analysis to Figure 2 and written the correlation analysis method on the legend and highlighted it in blue.
- Discussion、Table3 Redundant descriptions of Acyl-ACP thioesterases from other species obscure the essential discussion. Since this is a study focused on Acyl-CoA thioesterases, the majority of the descriptions of Acyl-ACP thioesterases should be removed and the discussion should be stated clearly and concisely.
Response: Thank you very much for your review of the paper. We have deleted the ACP part and sorted out the discussion part. Thank you for your careful review of the manuscript!
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Thank you for all the corrections and additions.
But two questions has not answer.
Namely:
1. About reference 24 - Where conditions for separating a mixture of FAMEs on a column (Gas Chromatography Conditions) are mentioned. But the question was about the conditions for fatty acids methyl esters preparation (transesterification reaction), which were further analyzed by the gas chromatography. It is necessary to added time and temperature of this reaction. Lines 158-159 - 10% (w/w) methanol hydrochloride was added. Did the FAMEs formed immediately at room temperature?
2. And another question was about individual FAs identification.
How it was determined that the peak on the chromatogram or compound with a given retention time corresponds to a methyl ester of one or another fatty acid?
Author Response
Point-by-point responses to the reviewer’s comments on the manuscript
Manuscript ID: fermentation-2413629
Manuscript Title: Homologous overexpression of acyl-CoA thioesterase 8 enhanced free fatty acid accumulation in oleaginous fungus Mucor circinelloides WJ11.
The authors would like to thank the reviewers for their extremely efficient and earnest work on our manuscript. The reviewers’ comments and suggestions are all valuable and very helpful for revising and improving our manuscript. We have studied the comments carefully and have carefully revised our manuscript. The itemized list of changes and point-by-point responses to the comments were listed below.
Responses Reviewer 1
The authors would like to thank reviewer “1” for the helpful comments. We have incorporated the following reviewer’s specific comments in preparation for the revised version of the manuscript. The following is our response to each comment of the reviewer one by one.
Thank you for all the corrections and additions.
But two questions has not answer.
Namely:
- About reference 24 - Where conditions for separating a mixture of FAMEs on a column (Gas Chromatography Conditions) are mentioned. But the question was about the conditions for fatty acids methyl esters preparation (transesterification reaction), which were further analyzed by the gas chromatography. It is necessary to added time and temperature of this reaction. Lines 158-159 - 10% (w/w) methanol hydrochloride was added. Did the FAMEs formed immediately at room temperature?
Response: We apologize for not describing this problem in detail. Methyl ester was prepared in a water bath at 60°C for 3 h after methanol hydrochloride was added. We have added the reaction time and temperature of methyl ester in lines 158-159 and line 167 of the manuscript in green highlight.
- And another question was about individual FAs identification.
How it was determined that the peak on the chromatogram or compound with a given retention time corresponds to a methyl ester of one or another fatty acid?
Response: Thank you very much for your review of the manuscript. By adding the standard fatty acid to determine a peak corresponding to a particular fatty acid, the concentration of a component in the sample can be calculated by the chromatographic peak area. We have added it in lines 171-172 of the manuscript.
Reviewer 2 Report
The authors properly fulfilled my demands. No further comments.
Author Response
Point-by-point responses to the reviewer’s comments on the manuscript
Manuscript ID: fermentation-2413629
Manuscript Title: Homologous overexpression of acyl-CoA thioesterase 8 enhanced free fatty acid accumulation in oleaginous fungus Mucor circinelloides WJ11.
The authors would like to thank the reviewers for their extremely efficient and earnest work on our manuscript. The reviewers’ comments and suggestions are all valuable and very helpful for revising and improving our manuscript. We have studied the comments carefully and have carefully revised our manuscript. The itemized list of changes and point-by-point responses to the comments were listed below.
Responses Reviewer 2
The authors would like to thank reviewer “2” for the helpful comments. We have incorporated the following reviewer’s specific comments in preparation for the revised version of the manuscript. The following is our response to each comment of the reviewer one by one.
The authors properly fulfilled my demands. No further comments.
Response: Thank you for your patient review of the manuscript.
Author Response File: Author Response.pdf
