Uncovering Novel Pathways for Enhancing Hyaluronan Synthesis in Recombinant Lactococcus lactis: Genome-Scale Metabolic Modeling and Experimental Validation
Round 1
Reviewer 1 Report
This paper worked out by Abinaya Badri etc. talked about the engineering production of
Hyaluronan in recombinant Lactococcus 3 lactis。 I have few comments on this manuscript.
1. For a better understanding by other readers, a brief description should be provided about why Lactococcus lactis was chosen as the expression host. Is there any report using other hosts and what is the advantages by using L. lactis?
2. Please carefully revise this manuscript to make sure all the titer numbers using the same significant figures. 2 g/l should be 2.00 for 3 significant figures.
Author Response
Reviewer: 1
This paper worked out by Abinaya Badri etc. talked about the engineering production of Hyaluronan in recombinant Lactococcus lactis.I have few comments on this manuscript.
Reviewer 1; Comment 1: . For a better understanding by other readers, a brief description should be provided about why Lactococcus lactis was chosen as the expression host. Is there any report using other hosts and what is the advantages by using L. lactis?
Author Response 1: Thank you for your feedback and your time to review this work. We had mentioned briefly in lines 61-63 some of the advantages of using L. lactis as the host. However, according to the suggestion, we have now included more information about the choice of the strain in the edited version §1; lines 61-68. We have also listed other recombinant hosts that are being developed with appropriate citations in lines 51-52.
Reviewer 1; Comment 2: . Please carefully revise this manuscript to make sure all the titer numbers using the same significant figures. 2 g/l should be 2.00 for 3 significant figures.
Author Response 2: Thank you for this observation to improve the manuscript. We have now revised the manuscript to make sure all data values have the same number (3) of significant figures.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript describes the combined application of genome modeling and experimental validation to identify potential ways to improve the natural flux leading to Hyaluronan (HA) in Lactococcus lactis. The study is generally of good quality, although some minor edits are required to improve the quality of data presentation and the text. I suggest making the following edits - 1. Lines 63-65 - not contiguous and doesn't make sense in the current form; 2. Figure 2- schematic needs to be better explained in the figure legend (i.e., what doe the different types of arrows mean?); 3. Figure 2- it may be useful to draw in the hypothetical model for how Inosine to Ribose fits into the entire schematic of HA production (using dotted lines?); 4 - Line 80- Flahaut model = one described by Vos and co-workers (ref. 26)?; 5 - Lines 88-90- too short to stand alone as a paragraph by itself, needs to be combined with the next paragraph; 6 - Figure 3 - "E" (presumably referring to the product) is not labeled in the actual schematic whereas it is described in the figure legend; 7 - Lines 201 to 212 - The Type-IIB targets need to be explained better. The term "Exchange fluxes" is used in line 209 but only explained in line 210. In general, the Type IIB targets can be better explained; Line 219 - it should be figure 8 and not 7; 8 - Fig 8 - Again the legend does not accurately describe what is illustrated, because I don't see a blue dotted line; 9 - The PDF conversion likely resulted in the introduction of several spurious characters in several figures, this may need to be addressed in the final manuscript; 10 - Figure A.2 - There is a peak with at RT=~23 min that is not mentioned about; 11- Reaction names are within quotes in table A.1, but not so in tables B.1 and B.3; same kind of discrepancy with capitalization of the enzymes in the tables.
Author Response
Reviewer 2: The manuscript describes the combined application of genome modeling and experimental validation to identify potential ways to improve the natural flux leading to Hyaluronan (HA) in Lactococcus lactis. The study is generally of good quality, although some minor edits are required to improve the quality of data presentation and the text. I suggest making the following edits
Reviewer 2; Comment 1: - . Lines 63-65 - not contiguous and doesn't make sense in the current form;
Author Response 1: Thank you for your feedback. We have now rephrased the lines 63-65 (new edited version lines 69-71), to deliver the point more precisely.
Reviewer 2; Comment 2: . Figure 2- schematic needs to be better explained in the figure legend (i.e., what does the different types of arrows mean?);
Author Response 2: Thank you for your feedback. The solid arrows represent the HA pathway reactions and striped arrows represent competing pathways that take metabolic intermediates away from HA biosynthesis. We have now updated the figure legend (lines 59-60) to include this information.
Reviewer 2; Comment 3: . Figure 2- it may be useful to draw in the hypothetical model for how Inosine to Ribose fits into the entire schematic of HA production (using dotted lines?);
Author Response 3: Thank you for this idea. We have not included that information in this pathway figure since the purpose of fig. 2 was to show proven traditional pathway associated with HA. Conversely, we have not proven the path taken by inosine to HA production. However, our model does predict some ways in which the inosine could go to HA. These are part of our sub-network figure A.1 in the supporting information file. As per your suggestion, we have now highlighted the potential inosine paths in that figure with details in the legend so as to enable the reader to visualize it.
Reviewer 2; Comment 4: - Line 80- Flahaut model = one described by Vos and co-workers (ref. 26)?;
Author Response 4: Thank you for pointing this out. Yes, this was the same model as ref. #26. We have now clarified it in the text as well as re-cited that reference (new version ref#32).
Reviewer 2; Comment 5: 5 - Lines 88-90- too short to stand alone as a paragraph by itself, needs to be combined with the next paragraph;
Author Response 5: We have now combined this with the next paragraph (new version lines 97-107).
Reviewer 2; Comment 6: 6 - Figure 3 - "E" (presumably referring to the product) is not labeled in the actual schematic whereas it is described in the figure legend;
Author Response 6: Thank you for pointing this out. We have now removed that reference from fig. 3 legend.
Reviewer 2; Comment 7: 7 - Lines 201 to 212 - The Type-IIB targets need to be explained better. The term "Exchange fluxes" is used in line 209 but only explained in line 210. In general, the Type IIB targets can be better explained; Line 219 - it should be figure 8 and not 7;
Author Response 7: Line 360 has now been updated to say Fig. 8 instead of 7.
Reviewer 2; Comment 8: 8 - Fig 8 - Again the legend does not accurately describe what is illustrated, because I don't see a blue dotted line;
Author Response 8: Thank you for this observation. As you mentioned, the blue dotted line is behind the axis and is hence not visible. According to your suggestion, we are now using the marker type to describe these lines (red and blue square markers). The legend has been accordingly updated.
Reviewer 2; Comment 9: 9 - The PDF conversion likely resulted in the introduction of several spurious characters in several figures, this may need to be addressed in the final manuscript;
Author Response 9: We have now verified the retention of figure quality upon converting to pdf.
Reviewer 2; Comment 10: 10 - Figure A.2 - There is a peak with at RT=~23 min that is not mentioned about;
Author Response 10: We appreciate your comment. We have not investigated the broad peak cluster at ~23min in our chromatogram. We suspect that it could be some impurities.
Reviewer 2; Comment 11: 11- Reaction names are within quotes in table A.1, but not so in tables B.1 and B.3; same kind of discrepancy with capitalization of the enzymes in the tables.
Author Response 11: We have now removed these discrepancies by following one single format for enzyme and reaction names.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript by Badri et al. use a genome-scale model (GEM) to account for the entire metabolic network of the cell while predicting strategies to improve HA production. They analyze the metabolic network of Lactococcus lactis adapted to produce HA and identify non-conventional strategies to enhance HA flux. They also identified an alternate route for enhancement of HA synthesis, originating from the nucleoside inosine, that can function in parallel with the traditionally known route from glucose. Adopting this strategy resulted in a 2.8-fold increase in HA yield. This work, for the first time, reported the use of a nucleoside to enhance HA production. The study is interesting but there are few concerns from my side.
The language need to be improvised with help of English expert.
The author should analyse the intracellular and extracellular metabolites/biosynthetic intermediates for supporting their studies.
The energy balance NAD/NADH and NADP/NADPH are crucial bottle necks in metabolic engineering. So, authors should also include such energy balance information in their models for better predictions.
Author Response
Reviewer 3:
The manuscript by Badri et al. use a genome-scale model (GEM) to account for the entire metabolic network of the cell while predicting strategies to improve HA production. They analyze the metabolic network of Lactococcus lactis adapted to produce HA and identify non-conventional strategies to enhance HA flux. They also identified an alternate route for enhancement of HA synthesis, originating from the nucleoside inosine, that can function in parallel with the traditionally known route from glucose. Adopting this strategy resulted in a 2.8-fold increase in HA yield. This work, for the first time, reported the use of a nucleoside to enhance HA production. The study is interesting but there are few concerns from my side.
Reviewer 3; Comment 1: The language need to be improvised with help of English expert.
Author Response 1: We have verified the correctness of the language used in this manuscript to the best of our abilities.
Reviewer 3; Comment 2: The author should analyse the intracellular and extracellular metabolites/biosynthetic intermediates for supporting their studies.
Author Response 2: We appreciate your suggestion. However, our study only aims to demonstrate that the inosine supplementation strategy results in higher hyaluronic acid levels and yields. We believe that measuring intracellular metabolites to prove the path taken is beyond the scope of this manuscript. Since we do not make any actual claims about intracellular metabolites here, we feel that data might not be vital to support our current assertions.
Reviewer 3; Comment 3: The energy balance NAD/NADH and NADP/NADPH are crucial bottle necks in metabolic engineering. So, authors should also include such energy balance information in their models for better predictions.
Author Response 3: Thank you for this feedback. We agree that redox energy cofactor balance is crucial in cellular metabolism. Our models, like any other flux balance models, generate flux distributions with steady state assumption. Hence all intracellular metabolites (including NAD(P), NAD(P)H) in our model do not accumulate. In other words, they are naturally balanced without bias to one state.. However, these, will be incorporated in future genome-scale models. We have emphasized this aspect in the last paragraph of the Discussion section.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
I am satisfied with answers to comments and the revised version of artile.Hhence, I recommed to accept the current version of manuscript.
