Up Front Unfolded Protein Response Combined with Early Protein Secretion Pathway Engineering in Yarrowia lipolytica to Attenuate ER Stress Caused by Enzyme Overproduction

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research presented in the manuscript concerns engineering Y. lipolytica cells toward more efficient production of recombinant proteins. To my assessment, the subject is of high importance and relevance. I found that subject very interesting and, therefore, agreed to revise the manuscript immediately upon receipt of the invitation.

Overall impression is positive, and the study deserves to be published. In particular, I appreciate the experiments in which combinations of different “helper” genes were tested for their recombinant-protein-synthesis-promoting effect. This is something new, which has not been addressed before in studies with Y. lipolytica (I’ll get back to this later in this revision). However, there are some serious issues that must be addressed before.

 

Major concerns:

1) The most serious problem is that the manuscript contains large passages of text that are identical to previously published papers. This must be amended – the data are original, but the text is repetitive and in some specific spots, the original paper is not cited. 

2) There is no evidence for any statistical analysis. It is particularly disturbing for the sets of data presented in Figures 3, 4, and 5, and the following interpretation. Statistical significance must be added. I suggest running the simplest HSD Tukey’s multiple comparison test at p 0.05. From the information provided by the Authors, it seems that an adequate number of repetitions have been conducted. Furthermore, considering the SD bars height and the number of repetitions used for preparing the average, it gives quite high confidence that the outcome will not be changed after statistical analysis (the best-producing strain will remain the best, and modifications resulting in no improvement, will remain without any significance), so the interpretation will remain the same. However, this must be proved by statistical analysis.

3) The Authors repeatedly state that in this study they “identified several potential ER chaperones and translocation components…” (or in other words; e.g. Lines:  15, 21, 398). However, these genes have been known previously. Moreover, they have been studied in the same context previously (see: doi.org/10.1016/j.btre.2021.e00669). So in fact, this study only provides an analysis of these genes’ impact on the specific recombinant proteins’ production. Please, correct the inaccurate statements.

 

Other relevant issues:

L 53 – 54: “narrowing its glycosylation patterns from mammalian cells" What does it mean? Please rephrase or clarify.

L 56: The accurate study to cite here is: 10.1007/s00253-018-8966-9

L 47 – 85: the whole section is full of inaccurate citations. For example, L 55: The accurate study to cite here is: 10.1007/s00253-018-8966-9, L 56: correct citation is the first study on copy number and protein synthesis is: 10.1007/BF00326302, L 73 – 75: citations 17 and 18 are irrelevant. There are studies on Y. lipolytica elegantly demonstrating these processes in the Y. lipolytica system. See studies by Beckerich JM. and Boisramé A…,

Fig.1 caption:  “Noteworthy, the co-translational translocation pathway has been suggested to be dominant and essential in Y. lipolytica.” They have not been “suggested”, but they have been proved - see the paper by: https://doi.org/10.1074/jbc.273.47.30903. Please correct.

L 118 – 119: the statement “However, the roles of these essential components on protein secretion in Y. lipolytica have not been studied so far” is not true, please refer to 10.1016/j.btre.2021.e00669...

Fig. 2 Please, address the arising question, why overexpression of B-gal elicited higher stress on Y. lipolytica cells?

L 161 – 163: “There results indicated that the determinant of protein product level is not limited at the transcriptional level but rather on translational level, more specifically, by the efficiency of protein synthesis machinery”. Please, confront this observation with 10.3390/ijms23073602 (Fig.2) and for example with: https://doi.org/10.1007/s00253-020-10937-w, where the linearity between transcription and synthesis of recombinant proteins in Y. lipolytica was extensively studied.

L 169: benchmark à hallmark

L 175 – 176” “HAC1 was suggested to play an essential role in regulation of the genes involved in cell cycle arrest, mitochondria and ribosomes operation, proteolysis and RNA metabolism modulation, etc. “ this sentence comes directly from 10.1016/j.btre.2023.e00801. Such phenomena (mitochondrial gene expression, cell cycle gene expression) have not been studied here. Please add the accurate reference.

L 184 and other: Ssa1p to Ssa4p are named Ssa5p to Ssa8p in Y. lipolytica. Please correct.

L 189: how many clones were tested?

L 201: I'm not convinced of the naming Ssas as "translocation component". They are cytosolic chaperones, involved not only involved in secretory proteins synthesis. Please consider.

L 211: “status” à correct to "folding competent status"

L 213: “better protein expression” à “higher protein synthesis”

L 226 – 227: “ Taken together, these results illustrate that increasing the activities of the non-predominant pathway of translocation may enhance its role as an alternative route targeting the nascent polypeptide to the ER lumen in yeast.” This statement does not match the preceding paragraph. This conclusion does not match the threat of this paragraph. This is a conclusion on Ssas (previous paragraph), and here - Srps were discussed. Please correct this.

Fig. 3 and Fig. 4: please show these data also in units normalized per biomass (specific activity = U/gDCW or U/OD) – otherwise it is not clear whether the effects observed are the result of the burden imposed by multiple proteins over-synthesis or the actual effect triggered by a specific chaperone. For such a type of study, it is a more accurate measure, as it also considers any differences resulting from growth disruption due to a given protein synthesis.

L 263 -264: It has been expected - since B-galp originates from E. coli, it should not contain disulfide bonds.

L 277 – 278: Please rather confront with the studies and findings for Y. lipolytica – which "demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth" from https://doi.org/10.3390/ijms23073602, or for example Table 1 in https://doi.org/10.1007/s00253-020-10937-w

L 296-298: As commonly known dimorphism is a highlight of severe stress. Regarding the relation between morphology and protein production capacity in Y. lipolytica. please see: 10.1002/yea.3499, stating: "… using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis."

Fig.5: considering that up to 5 proteins were co-overexpressed in specific strains, these data must ALSO be presented in normalized units (U/biomass). Apart from U/mL please add U/mg or U/OD

L 406 – 408: no data on growth restoration in the main manuscript - consider adding them, especially, if this aspect is raised in both the conclusions and in the highlights

 

In Table S3, b two different columns show results for YlGAL

Comments on the Quality of English Language

No significant English-language-use issues have been detected. 

Author Response

Major concerns:

• The most serious problem is that the manuscript contains large passages of text that are identical to previously published papers. This must be amended – the data are original, but the text is repetitive and in some specific spots, the original paper is not cited. 

Response: The authors thank the referee for this advice. We have rewritten the manuscript accordingly. We also performed a plagiarism detection and the similarity rate was lower than 3%. In line with this, we have added the proper citations.

• There is no evidence for any statistical analysis. It is particularly disturbing for the sets of data presented in Figures 3, 4, and 5, and the following interpretation. Statistical significance must be added. I suggest running the simplest HSD Tukey’s multiple comparison test at p 0.05. From the information provided by the Authors, it seems that an adequate number of repetitions have been conducted. Furthermore, considering the SD bars height and the number of repetitions used for preparing the average, it gives quite high confidence that the outcome will not be changed after statistical analysis (the best-producing strain will remain the best, and modifications resulting in no improvement, will remain without any significance), so the interpretation will remain the same. However, this must be proved by statistical analysis.

Response: This comment is highly appreciated. Following the reviewer's suggestion, we have added statistical analysis by two-tailed Student’s t-tests, wherein P values were given to show the statistical significance for the Figures 3, 4 and 5.

• The Authors repeatedly state that in this study they “identified several potential ER chaperones and translocation components…” (or in other words; e.g. Lines:  15, 21, 398). However, these genes have been known previously. Moreover, they have been studied in the same context previously (see: doi.org/10.1016/j.btre.2021.e00669). So in fact, this study only provides an analysis of these genes’ impact on the specific recombinant proteins’ production. Please, correct the inaccurate statements.

Response: We agree with the referee that some of these components such as SSA5 and SSA8 have been studies previously by Korpys-Woźniak and coauthors. Following the reviewer's suggestion, we have revised our manuscript accordingly.

Other relevant issues:

L 53 – 54: “narrowing its glycosylation patterns from mammalian cells" What does it mean? Please rephrase or clarify.

Response: Thanks to the referee for this comment. This was not clearly written and we have made the correction in our revised manuscript (Page 2, Line 54).

L 56: The accurate study to cite here is: 10.1007/s00253-018-8966-9

Response: Thanks to the referee for this comment. We did make a mistake for this citation. Following the reviewer's suggestion, we have added the correct citation in our revised manuscript (Page 5, Line 57).

L 47 – 85: the whole section is full of inaccurate citations. For example, L 55: The accurate study to cite here is: 10.1007/s00253-018-8966-9, L 56: correct citation is the first study on copy number and protein synthesis is: 10.1007/BF00326302, L 73 – 75: citations 17 and 18 are irrelevant. There are studies on Y. lipolytica elegantly demonstrating these processes in the Y. lipolytica system. See studies by Beckerich JM. and Boisramé A…,

Response: Thanks to the referee for this comment. We apologize again for these mistakes and the correct publications were cited in our revised manuscript (Reference No. 10, 12, 17, 18, 20).

Fig.1 caption:  “Noteworthy, the co-translational translocation pathway has been suggested to be dominant and essential in Y. lipolytica.” They have not been “suggested”, but they have been proved - see the paper by: https://doi.org/10.1074/jbc.273.47.30903. Please correct.

Response: Following the reviewer's suggestion, we have corrected the inaccurate statement on “the predominant role of co-translational translocation pathway” in Y. lipolytica in our revised manuscript (Page 3, Line 94).

L 118 – 119: the statement “However, the roles of these essential components on protein secretion in Y. lipolytica have not been studied so far” is not true, please refer to 10.1016/j.btre.2021.e00669...

Response: Following the reviewer's suggestion, we have corrected the inaccurate statement in our revised manuscript (Page 3, Line 119-123).

Fig. 2 Please, address the arising question, why overexpression of B-gal elicited higher stress on Y. lipolytica cells?

Response: This comment is highly appreciated. B-gal is a 253 kDa protein, much larger than Lip2p. We speculate this is related with the natural property of the protein, such as structure and stability. Unfortunately, we don’t have direct evidence to prove this. In the further, we plan to study the relationship of ER stress with the expression of some of these known ‘different-to-express’ proteins.

L 161 – 163: “There results indicated that the determinant of protein product level is not limited at the transcriptional level but rather on translational level, more specifically, by the efficiency of protein synthesis machinery”. Please, confront this observation with 10.3390/ijms23073602 (Fig.2) and for example with: https://doi.org/10.1007/s00253-020-10937-w, where the linearity between transcription and synthesis of recombinant proteins in Y. lipolytica was extensively studied.

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have carefully read the two publications and we found the results are extremely interesting and are consistent with our observations. They were cited in our revised manuscript (Page 5, Line 57).

L 169: benchmark à hallmark

Response: Following the reviewer's suggestion, ‘benchmark’ was changed into ‘hallmark’ in our revised manuscript (Page 5, Line 173).

L 175 – 176” “HAC1 was suggested to play an essential role in regulation of the genes involved in cell cycle arrest, mitochondria and ribosomes operation, proteolysis and RNA metabolism modulation, etc. “ this sentence comes directly from 10.1016/j.btre.2023.e00801. Such phenomena (mitochondrial gene expression, cell cycle gene expression) have not been studied here. Please add the accurate reference.

Response: Thanks to the referee for this comment. After we carefully read the reference, we found there was a misunderstanding. The irrelative parts were removed and a reference was added in our revised manuscript (Page 6, Line 181).

L 184 and other: Ssa1p to Ssa4p are named Ssa5p to Ssa8p in Y. lipolytica. Please correct.

Response: Following the reviewer's suggestion, Ssa1p to Ssa4p was renamed to Ssa5p to Ssa8pin our revised manuscript (Page 5, Line 173).

L 189: how many clones were tested?

Response: For each construct, the first screening of the transformants were conducted for 10 independent clones. From these clones, we chose the ones showing the average expression level of the target protein among the 10 clones. From these clones (normally 6 to 7 out of the 10), we chose 3 of them as the representatives for the detailed analysis.

L 201: I'm not convinced of the naming Ssas as "translocation component". They are cytosolic chaperones, involved not only involved in secretory proteins synthesis. Please consider.

Response: This comment is highly appreciated. In fact, this is also our concerns. We kept this name as historically they were referred as Hsp70 chaperone involved in translocation as an essential component in S. cerevisiae in several studies. For instance, in reference doi: 10.1083/jcb.135.5.1229: “Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER.”, in reference doi: 10.1074/jbc.M116.761122: “Our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.”

L 211: “status” à correct to "folding competent status"

Response: Following the reviewer's suggestion, ‘status’ was changed into ‘folding competent status’ in our revised manuscript (Page 8, Line 214).

L 213: “better protein expression” à “higher protein synthesis”

Response: Following the reviewer's suggestion, ‘better protein expression’ was changed into ‘higher protein synthesis’ in our revised manuscript (Page 8, Line 216).

L 226 – 227: “ Taken together, these results illustrate that increasing the activities of the non-predominant pathway of translocation may enhance its role as an alternative route targeting the nascent polypeptide to the ER lumen in yeast.” This statement does not match the preceding paragraph. This conclusion does not match the threat of this paragraph. This is a conclusion on Ssas (previous paragraph), and here - Srps were discussed. Please correct this.

Response: Following the reviewer's suggestion, the statement “Taken together, these results illustrate that increasing the activities of the non-predominant pathway of translocation may enhance its role as an alternative route targeting the nascent polypeptide to the ER lumen in yeast.” was moved into the right location after the data interpretation of the effects of Ssaps in our revised manuscript (Page 8, Line 220-222).

Fig. 3 and Fig. 4: please show these data also in units normalized per biomass (specific activity = U/gDCW or U/OD) – otherwise it is not clear whether the effects observed are the result of the burden imposed by multiple proteins over-synthesis or the actual effect triggered by a specific chaperone. For such a type of study, it is a more accurate measure, as it also considers any differences resulting from growth disruption due to a given protein synthesis.

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added specific activity in unit of U/g-DCW in our revised manuscript.

L 263 -264: It has been expected - since B-galp originates from E. coli, it should not contain disulfide bonds.

Response: Following the reviewer's suggestion, the sentence was reformulated in our revised manuscript (Page 9, Line 267-269).

L 277 – 278: Please rather confront with the studies and findings for Y. lipolytica – which "demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth" from https://doi.org/10.3390/ijms23073602, or for example Table 1 in https://doi.org/10.1007/s00253-020-10937-w

Response: Following the reviewer's suggestion, two references were cited and discussed in our revised manuscript (Page 10, Line 283).

L 296-298: As commonly known dimorphism is a highlight of severe stress. Regarding the relation between morphology and protein production capacity in Y. lipolytica. please see: 10.1002/yea.3499, stating: "… using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis."

Response: Thanks to the referee for this comment. We have corrected this mis-interpretation of the results in our revised manuscript (Page 10, Line 302-309).

Fig.5: considering that up to 5 proteins were co-overexpressed in specific strains, these data must ALSO be presented in normalized units (U/biomass). Apart from U/mL please add U/mg or U/OD

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added specific activity in unit of U/g-DCW in our revised manuscript.

L 406 – 408: no data on growth restoration in the main manuscript - consider adding them, especially, if this aspect is raised in both the conclusions and in the highlights

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added the growth and glucose consumption data in our revised manuscript (Fig. 6).

In Table S3, b two different columns show results for YlGAL

 Response: Thanks to the referee for this comment. The first column was the wild type strain used as the control for comparison. This was corrected in our revised manuscript (Table S3).

Reviewer 2 Report

Comments and Suggestions for Authors

Improving proteins secretion during the production of recombinant proteins using yeast is an important element of this process affecting its final industrial use. Yarrowia lipolytica is a well-known protein expressing system, and the authors provided their original research article dealing with overexpressing some potential ER chaperones and translocation components, as well as, they studied effects of this proteins on the production of extracellular Lip2 lipase and β-galactosidase from E. coli using Y. lipolytica.

The major flaw of this paper is the lack of the statistical analysis. In my opinion, presented results in Figures should be analyzed statistically. Providing means and standard deviation is definitely not enough. I recommend providing ANOVA and any post-hoc test.

Minor sugestions:

- the reference list should be prepared according to the instructions for authors

- the citation style should be improved, i.e., number should be placed in brackets

- microorganisms names should be written in italics, the same for "p-" in p-nitrophenol and other chemicals

- figures 2-5 are of low quality

Author Response

Reviewer 2:

Improving proteins secretion during the production of recombinant proteins using yeast is an important element of this process affecting its final industrial use. Yarrowia lipolytica is a well-known protein expressing system, and the authors provided their original research article dealing with overexpressing some potential ER chaperones and translocation components, as well as, they studied effects of this proteins on the production of extracellular Lip2 lipase and β-galactosidase from E. coli using Y. lipolytica.

The major flaw of this paper is the lack of the statistical analysis. In my opinion, presented results in Figures should be analyzed statistically. Providing means and standard deviation is definitely not enough. I recommend providing ANOVA and any post-hoc test.

Response: This comment is highly appreciated. Following the reviewer's suggestion, we have added statistical analysis by two-tailed Student’s t-tests, wherein P values were given to show the statistical significance for the Figures 3, 4 and 5.

Minor sugestions:

- the reference list should be prepared according to the instructions for authors

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have prepared the reference list according the journal’s instructions.

- the citation style should be improved, i.e., number should be placed in brackets

Response: Following the reviewer's suggestion, the citation style of the manuscript was thoroughly checked and brackets were added to the citation numbers.

- microorganisms names should be written in italics, the same for "p-" in p-nitrophenol and other chemicals

Response: Following the reviewer's suggestion, the names of microorganism were written in italics as well as the specific letter as ‘p-’ in p-nitrophenol and other chemicals in our revised manuscript.

- figures 2-5 are of low quality

Response: Following the reviewer's suggestion, we have remade the figures and improved the resolution of image with high quality in our revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Recommendations are attached.

Comments for author File: Comments.pdf

Author Response

# Reviewer 3:

In this manuscript, genetic engineering of Y. lipolytica was carried out to improve production and secretion of overexpressed enzymes. The objective and results of this study are interesting but several points should be considered for improvement and clarity:

- Title and entire manuscript: Species names should be in italics

Response: Following the reviewer's suggestion, the names of microorganism were written in italics throughout our revised manuscript.

- Format of in-text references should be adjusted (currently number without spacing)

Response: Following the reviewer's suggestion, the citation style of the manuscript was thoroughly checked and brackets were added to the citation numbers.

- Figures are of very low resolution, please provide higher resolution images

Response: Following the reviewer's suggestion, we have remade the figures and improved the resolution of images with high quality in our revised manuscript.

- Legend for Fig.2 lacks some important information. First, it should be stated that TEF, 4UTEF etc. indicate the promoter type used in the construct for overexpression. Second, information about “+Hac1” is completely missing. Presumably, this refers to co-overexpression of intron-less HAC1 mRNA (“spliced mHac1”), but this should be clearly stated. A construct used for “spliced” mHac1 expression is not described in the methods section or in table S2. The information given is for HAC1 cloned from genomic DNA. As such it wouldn’t be intron free. The information about which construct was used should be checked and updated.

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added further information to clarify the constructs in our revised manuscript (Page 5, Line 166-170, Table 1 and Table S2, as well as in Method section Line 359-362).

• Line 58: “However, only 16% of more than 150 r-proteins expressed using Y. lipolytica, can be produced at a bioreactor scale.” This statement could be explained in greater detail. Are there altogether >150 (published?) attempts to produce recombinant protein in Y. lipolytica and was upscale unsuccessful in the majority of cases? Please clarify

Response: Following the reviewer's suggestion, the statement “However, only 16% of more than 150 r-proteins expressed using Y. lipolytica, can be produced at a bioreactor scale.” was clarified in our revised manuscript (Page 2, Line 59-60).

• Line 141: “pTEF<p4UASTEF<p8UASTEF<2×p8UASTEF” The different promoters used should be briefly mentioned, as only cryptic names are given. It should become clear, where these elements were obtained from and what their difference is.

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added further information to clarify the constructs pTEF, p4UASTEF, p8UASTEF, 2×p8UASTEF in our revised manuscript (Page 4, Line 143-147).

 

• Line 161: “There results indicated that the determinant of protein product level is not limited at the transcriptional level but rather on translational level, more specifically, by the efficiency of protein synthesis machinery.” This statement should be modified, as not only limitation in protein synthesis but also in secretion could be responsible. “There” should read “These”

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, the statement “There results indicated that the determinant of protein product level is not limited at the transcriptional level but rather on translational level, more specifically, by the efficiency of protein synthesis machinery.” was modified in our revised manuscript (Page 6, Line 177-179).

• Line 169: “spliced mHac1”: As stated above, additional information is needed, does this refer to intron-less HAC1? If yes, details for construct generation are missing.

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added further information to clarify the constructs in our revised manuscript (Page 5, Line 166-170, Table 1 and Table S2, as well as in Method section Line 359-362).

• Line 171: “Therefore, HAC1 overexpression enhanced secretion of the correctly folded target protein as a result of upfront UPR.” Enhanced secretion was detected, the mechanism is speculative. It should be clarified what “upfront UPR” is.

Response: Thanks to the referee for this comment. This was a mistake. Following the reviewer's suggestion, we have clarified the statement “Therefore, HAC1 overexpression enhanced secretion of the correctly folded target protein as a result of upfront UPR.” in our revised manuscript (Page 6, Line 188-190).

• Line 173: “… consistent with previously studies” should read “previous”

Response: Following the reviewer's suggestion, “previously” was changed into “previous” in our revised manuscript.

• Line 175-178: This part has no connection to the results presented, should be removed

Response: Following the reviewer's suggestion, the mentioned part was irrelative, it was removed from our revised manuscript (Page 6, Line 192-195).

• Line 189: “did not obvious influence” should read “obviously”

Response: Following the reviewer's suggestion, “obvious” was changed into “obviously” in our revised manuscript.

• Line 220: “Surprisingly, over-expression of core co-translocation components Srp14p and Srp54p did not show a clear impact on the secretion of the expressed r-proteins (Fig. 3).” In this case, no definitive conclusions should be made without further confirmation of protein overproduction. It can’t be excluded that the constructs used do not lead to functional overexpression of the proteins Srp14 and Srp54, or other proteins for which the overexpression constructs didn’t show an effect on the secretion of r-proteins. This should be either experimentally verified or conclusions about absence of effects softened

Response: This comment is highly appreciated. We agree with the referee that further evidence is needed to draw such conclusions. Following the reviewer's suggestion, as we cannot rule out these possibilities, the mentioned part was modified in our revised manuscript (Page 7, Line 233-242).

• 3.4. mRNA analysis: RT-qPCR is used in this study for mRNA quantification. Please add primer sequences for qlip2-f/qlip2-r, qlacZ-f/qlacZ-r and qhac1-f/qhac1-r. In addition, the reference gene used is not specified. Please indicate which gene was used and add primer sequences for this, too.

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added further information regarding the primers and reference gene used in our revised manuscript (Method section Line 396-399).

• 3.5 Measurement of enzyme activity: Since the enzyme activities were compared between different strains (carrying different expression constructs) some kind of normalization should have been done to account for possible differences in biomass or cell density in the different strains.

Response: This comment is highly appreciated. Following the reviewer's suggestion, we have added specific activity in unit of U/g-DCW in our revised manuscript.

• Line 348: “primers listed in Table S2 (Supplementary file 1).” Table S2 contains only plasmids. Table S1 lists primers used for cloning, but lacks other primers used (RT-PCR)

Response: Thanks to the referee for this comment. This was a mistake. It has been revised in our manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

In this manuscript, genetic engineering of Y. lipolytica was carried out to improve production and secretion of overexpressed enzymes. The objective and results of this study are interesting but several points should be considered for improvement and clarity:

 

·         Title and entire manuscript: Species names should be in italics

·         Format of in-text references should be adjusted (currently number without spacing)

·         Figures are of very low resolution, please provide higher resolution images

·         Legend for Fig.2 lacks some important information. First, it should be stated that TEF, 4UTEF etc. indicate the promoter type used in the construct for overexpression. Second, information about “+Hac1” is completely missing. Presumably, this refers to co-overexpression of intron-less HAC1 mRNA (“spliced mHac1”), but this should be clearly stated. A construct used for “spliced” mHac1 expression is not described in the methods section or in table S2. The information given is for HAC1 cloned from genomic DNA. As such it wouldn’t be intron free. The information about which construct was used should be checked and updated.  

·         Line 58: “However, only 16% of more than 150 r-proteins expressed using Y. lipolytica, can be produced at a bioreactor scale.” This statement could be explained in greater detail. Are there altogether >150 (published?) attempts to produce recombinant protein in Y. lipolytica and was upscale unsuccessful in the majority of cases? Please clarify

·         Line 141: “pTEF<p4UASTEF<p8UASTEF<2×p8UASTEF” The different promoters used should be briefly mentioned, as only cryptic names are given. It should become clear, where these elements were obtained from and what their difference is.

·         Line 161: “There results indicated that the determinant of protein product level is not limited at the transcriptional level but rather on translational level, more specifically, by the efficiency of protein synthesis machinery.” This statement should be modified, as not only limitation in protein synthesis but also in secretion could be responsible. “There” should read “These”

·         Line 169: “spliced mHac1”: As stated above, additional information is needed, does this refer to intron-less HAC1? If yes, details for construct generation are missing.

·         Line 171: “Therefore, HAC1 overexpression enhanced secretion of the correctly folded target protein as a result of upfront UPR.” Enhanced secretion was detected, the mechanism is speculative. It should be clarified what “upfront UPR” is.

·         Line 173: “… consistent with previously studies” should read “previous”

·         Line 175-178: This part has no connection to the results presented, should be removed

·         Line 189: “did not obvious influence” should read “obviously”

·         Line 220: “Surprisingly, over-expression of core co-translocation components Srp14p and Srp54p did not show a clear impact on the secretion of the expressed r-proteins (Fig. 3).” In this case, no definitive conclusions should be made without further confirmation of protein overproduction. It can’t be excluded that the constructs used do not lead to functional overexpression of the proteins Srp14 and Srp54, or other proteins for which the overexpression constructs didn’t show an effect on the secretion of r-proteins. This should be either experimentally verified or conclusions about absence of effects softened

·         3.4. mRNA analysis: RT-qPCR is used in this study for mRNA quantification. Please add primer sequences for qlip2-f/qlip2-r, qlacZ-f/qlacZ-r and qhac1-f/qhac1-r. In addition, the reference gene used is not specified. Please indicate which gene was used and add primer sequences for this, too.

·         3.5 Measurement of enzyme activity: Since the enzyme activities were compared between different strains (carrying different expression constructs) some kind of normalization should have been done to account for possible differences in biomass or cell density in the different strains.

·         Line 348: “primers listed in Table S2 (Supplementary file 1).” Table S2 contains only plasmids. Table S1 lists primers used for cloning, but lacks other primers used (RT-PCR)

Comments on the Quality of English Language

Some minor spelling errors are indicated in the comments for authors. The manuscript should be checked for additional errors.

Author Response

# Reviewer 4

In this manuscript authors engineered ER chaperones and translocation system in the yeast Yarrowia lipolytica in order to improve its efficiency in producing recombinant proteins. They started with identification of several potential ER chaperones and translocation components that showed positive effects on production of recombinant proteins in S. cerevisiae background, with presumption that during the protein overproduction, the accumulation of unfolded and misfolded proteins causes ER stress and cell dysfunction. Their results showed that improving the activities of the SRP-independent translocation pathway boosted the production of the model proteins. However, the impact of ER chaperones was shown to be protein dependent, while the nucleotide exchange factor Sls1p shown common positive impact.

Manuscript is written well and there is no need for language grammar and syntax corrections. However, the graphic quality of the figures is poor - text and labels on the images are blurry, and marks for different proteins in figure legends are uneven. In general - protein ˝names˝ in the text and in the figure legends are uneven – for some proteins ˝p˝ is added, and for some is not, even for the same protein in different parts of the text (e.g. Kar2 or Kar2p – sometimes is written in one way and sometimes in another). Authors should made it even throughout the manuscript. As far as I know this ˝p˝ on the end of the protein name are generally not in use anymore. However, both ways are ok with me, but use it uniformly for all proteins and everywhere in the manuscript.

Response: Thanks to the referee for this comment. We have checked the manuscript thoroughly to make the name of the proteins are uniformed. Also, we have remade all the figures and improved the resolution of images with high quality in our revised manuscript.

Furthermore, authors should explain abbreviations and symbols when first time used (e.g. what is μmax??) in text. For data shown in supplementary materials I suggest that author give short summary in the text as well, so reader can have general idea without looking for supplementary materials.

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, we have added the explanations for the abbreviations and symbols in our revised manuscript. In addition, to help the readers to better understand the work, further description was added in supplementary file 1.

Text formatting is strange for reference numbers – the numbers are sticked to the words in front of them, what makes text hard to read and confuses at first glance (e.g. what is enzyme1 ???) .

Response: Following the reviewer's suggestion, the citation style of the manuscript was thoroughly checked and brackets were added to the citation numbers.

In line 106- 107 I suppose that there is a typo in sentence ˝For instance, studies have shown that overexpressing SRP proteins such as SPR14, the essential…..˝ - I guess that this should be Srp14 instead SPR14??

Response: Thanks to the referee for this comment. We apologize for this mistake and this was corrected in our revised manuscript (Line 108).

However, the most important remark is that, as a reader, I would like for authors to have more discussion of the results instead of just stating facts that some mutation resulted in x-fold higher activity of recombinant protein – what are authors ideas about why did they get such results? This is especially interesting when different results are obtained for two model proteins. What might be the cause for that? The manuscript in general is of average quality, and results contribute to general knowledge in the field and I would accept it for publication after these minor improvements mentioned above.

Response: This comment is highly appreciated. Following the reviewer's suggestion, we have rewritten the discussion section, and also presented more data (Figure 6). The revised parts are marked in red changes in our manuscript.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been corrected in regard to many raised points. However, still some points remained.

Major concerns:

Regarding statistical analysis of the results. I’ll first refer to the data presented in Figure 3 (but the same concerns the other figures presenting numerical data). I really don’t understand what was the key for comparison of activity by SSa5 and Ssa6, but not the two with the control strain (this statement is based on the bars showing statistical significance). Why the statistical significance for the key achievement (Ssa8 and Ssb1) vs control was not calculated and not shown (no bars or symbols notifying comparisons between the control and the ssa8/ssb1). Likewise, why statistical analysis was not conducted for the other interesting and important point - no effect by SRPs overexpression?

The same problem concerns data presented in Figure 4. Statistical significance was not calculated for comparisons that were discussed above.

Regarding data presented in Figures 5 and 6.

In Fig.5 for the strain with pNPGase+ssa6+ssb1+sls1, as shown in Figure 6 growth was severely impeded, so why the parameter of activity (U/mL) and specific activity (U/mg-dcw) are not different. This is the case that can be tracked. What about the other? Why in Fig. 6 growth was shown only for selected strains?

 

Regarding Figure 6. - it was inserted without any reasonable reference nor presentation. For example, it should be referred to in lines 292 and 306. In addition the data regarding growth shown here, do not correspond with those shown Figure 5 (specific activity).

 

Other remarks

L 15 – still the Authors claim that they have “we identified several potential ER chaperones and translocation components.” Which is not true. These genes have been known previously and their function, or even impact on r-proteins synthesis was studied earlier.

 

L 80 “interchangeable” should be changed to “partly overlapping”. ESPECIALLY when confronted with the outcomes presented in this and previous papers - the effects driven by different SSa was different

 

L 142 “was” should be changed to “were”

 

L 143 – 146 and elsewhere, please modify naming native pTEF as “normal” – it should be termed “native”; likewise, the promoters with UAS – they represent “hybrid” promoters

Author Response

Major concerns:

Regarding statistical analysis of the results. I’ll first refer to the data presented in Figure 3 (but the same concerns the other figures presenting numerical data). I really don’t understand what was the key for comparison of activity by SSa5 and Ssa6, but not the two with the control strain (this statement is based on the bars showing statistical significance). Why the statistical significance for the key achievement (Ssa8 and Ssb1) vs control was not calculated and not shown (no bars or symbols notifying comparisons between the control and the ssa8/ssb1). Likewise, why statistical analysis was not conducted for the other interesting and important point - no effect by SRPs overexpression?

Response: Thanks to the referee for this comment. We attempted to only show the results with statistical significance that were not easily estimated by eyes. For the results where the difference was obvious or there was no significant difference, the p values were not given. Obviously, the way of data interpretation was still confusing. Therefore, following the reviewer's suggestion, we have showed all statistical analysis in our revised manuscript.

The same problem concerns data presented in Figure 4. Statistical significance was not calculated for comparisons that were discussed above.

Response: Thanks to the referee for this comment. We have uploaded an incomplete figure and sorry for the mistake.

Regarding data presented in Figures 5 and 6.

In Fig.5 for the strain with pNPGase+ssa6+ssb1+sls1, as shown in Figure 6 growth was severely impeded, so why the parameter of activity (U/mL) and specific activity (U/mg-dcw) are not different. This is the case that can be tracked. What about the other? Why in Fig. 6 growth was shown only for selected strains?

Response: Thanks to the referee for this comment. The strain pNPGase+ssa6+ssb1+sls1 exhibited a volumetric activity of 1320±30 U/ml. During its fermentation, 37 g-DCW/L was achieved. Therefore, a specific activity of 35.7±0.6 U/mg-DCW was obtained (≈1320 divided by 37). I guess this was a little bit confusion as the results of specific activity was indicated by the external right Y-axis, while the volumetric activity was shown by the left Y-axis, please note that the scale of the two axes were not the same. Regarding the growth data, we showed that most relevant and representative results given the large number of the strains constructed in this work. The rest results can be found in supplementary file Table S3. We don’t see the point to show repetitive growth curves as one cannot see any difference as they were overlapping. In this case, only the growth rate and biomass yield were demonstrated.

Regarding Figure 6. - it was inserted without any reasonable reference nor presentation. For example, it should be referred to in lines 292 and 306. In addition the data regarding growth shown here, do not correspond with those shown Figure 5 (specific activity).

Response: Thanks to the referee for this comment. Following the reviewer's suggestion, the figure 6 was interpreted in our revised manuscript. Regarding the specific activity, please see the response above.

Other remarks

L 15 – still the Authors claim that they have “we identified several potential ER chaperones and translocation components.” Which is not true. These genes have been known previously and their function, or even impact on r-proteins synthesis was studied earlier.

Response: Following the reviewer's suggestion, we have modified the relevant part in our revised manuscript.

L 80 “interchangeable” should be changed to “partly overlapping”. ESPECIALLY when confronted with the outcomes presented in this and previous papers - the effects driven by different SSa was different

Response: Following the reviewer's suggestion, we have changed “interchangeable” into “partially overlapping” in our revised manuscript.

L 142 “was” should be changed to “were”

Response: Following the reviewer's suggestion, we have changed “was” into “were” in our revised manuscript.

L 143 – 146 and elsewhere, please modify naming native pTEF as “normal” – it should be termed “native”; likewise, the promoters with UAS – they represent “hybrid” promoters

Response: Following the reviewer's suggestion, we have changed “normal” into “native” and the promoters with UAS into “hybrid” promoters in our revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been revised according to my comments and suggestions.

Author Response

Many thanks to the reviewer for the valuable comments！

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have adequately responded to specific points raised and the manuscript as well as the figures significantly improved. I noticed some need to correct wording in the newly added text passages (see below). Double checking of all the added/changed passages is recommended.

 

Line 59: “Although more than 150 r-proteins have been reported can be expressed using  Y. lipolytica, only 16% of them reached to produce at a bioreactor scale [1].“ Check and correct wording (remove “have been reported”)

Line 144: “p4UASTEF and p8UASTEF represent expressing the genes under TEF promoter combined with 4 and 8 tandem copies of UAS, respectively.” For correct wording, some insertion after “represent” is needed. For example “…represent constructs expressing…”

Line 367: “To expressed the spliced mHAC1…“ should read „To express the …“

Comments on the Quality of English Language

check newly added text passages

Author Response

The authors have adequately responded to specific points raised and the manuscript as well as the figures significantly improved. I noticed some need to correct wording in the newly added text passages (see below). Double checking of all the added/changed passages is recommended.

Response: Following the reviewer's suggestion, we have read the manuscript carefully to correct the grammar and wording issues in our revised manuscript.

Line 59: “Although more than 150 r-proteins have been reported can be expressed using  Y. lipolytica, only 16% of them reached to produce at a bioreactor scale [1].“ Check and correct wording (remove “have been reported”)

Response: Following the reviewer's suggestion, we have removed “have been reported” in our revised manuscript.

Line 144: “p4UASTEF and p8UASTEF represent expressing the genes under TEF promoter combined with 4 and 8 tandem copies of UAS, respectively.” For correct wording, some insertion after “represent” is needed. For example “…represent constructs expressing…”

Response: Following the reviewer's suggestion, we have corrected the relevant part in our revised manuscript.

Line 367: “To expressed the spliced mHAC1…“ should read „To express the …“

Response: Following the reviewer's suggestion, we have corrected the relevant part in our revised manuscript.