Membrane Engineering for Carotenoid Production in Escherichia coli
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe aim of this study is to evaluate the impact of different promoter strengths on the chromosomal integration of the IUP pathway in E. coli strains and compare the specific yield of lycopene. This study presents interesting findings; however, it is essential to delineate the practical implications of the research, specifically the real-world applications of the findings. The study design is methodologically sound, and the results are described accurately, with appropriate references to the existing literature. Nonetheless, the conclusions would benefit from a clear articulation of the study's innovative aspects and its practical significance.
Author Response
Comment 1:
The aim of this study is to evaluate the impact of different promoter strengths on the chromosomal integration of the IUP pathway in E. coli strains and compare the specific yield of lycopene. This study presents interesting findings; however, it is essential to delineate the practical implications of the research, specifically the real-world applications of the findings. The study design is methodologically sound, and the results are described accurately, with appropriate references to the existing literature. Nonetheless, the conclusions would benefit from a clear articulation of the study's innovative aspects and its practical significance.
Response: Thank you for your positive review of our manuscript. We agree that real-world applications were not well articulated in the manuscript and therefore we have added the following to the introduction and conclusions to situate the practical aspects of this work.
Regarding chemical vs. biological synthesis: "However, carotenoid production using biomanufacturing processes is still more costly than chemical synthesis due to low productivities and this limits uptake of these natural products [3]."
Regarding the insertion of the IUP pathway: "This improvement is important as these strains no longer require antibiotics for plasmid maintenance, which is costly at large scale. Furthermore, the high lycopene yield obtained in this work used minimal salt media, another significant cost savings during at large scale production. "
Reviewer 2 Report
Comments and Suggestions for AuthorsImpact of the Study: The manuscript would benefit from a more explicit discussion of the overall impact of this work on scientific advancement. Please elaborate on how this study contributes to the broader field of carotenoid production & its application.
Manuscript Format: The current format of the manuscript does not follow the standard structure. The appropriate sequence should be:
- Introduction
- Materials & Methods
- Results & Discussion
- Conclusion
Please revise the manuscript to reflect this correct format.
Keywords: Ensure that the first letter of each keyword is capitalized to align with standard manuscript formatting guidelines.
Conclusions: The conclusions section needs significant revision. It should emphasize the significance and impact of the study, highlighting future research directions and how the findings could influence the field.
Literature Comparison: To provide more context, consider including a table or paragraph that compares your findings with similar studies reported in the literature. This will allow readers to better understand how your work fits within existing research.
Metabolism Diagram: Include a diagram that visually depicts the metabolic pathways for lipid and carotenoid production across different strains. Highlight both positive and negative examples of strains used in this study to enhance clarity.
Figure Formatting: In Figure 6, panels A, B, and C should be referenced before the title of the figure caption. Please adjust the formatting accordingly.
Author Response
Comment 1: Impact of the Study: The manuscript would benefit from a more explicit discussion of the overall impact of this work on scientific advancement. Please elaborate on how this study contributes to the broader field of carotenoid production & its application.
Response: Thank you for your comments. We agree that a few more details about the context of carotenoid production could be more explicitly stated. We have added the following to the introduction:
"However, carotenoid production using biomanufacturing processes is still more costly than chemical synthesis due to low productivities and this limits uptake of these natural products by end-users [2]. As the first pigmented product in the carotenoid biosynthesis pathway, lycopene has received significant attention to increase specific yield and productivity of this molecule [4]. In E. coli, yields have varied between 5-200 mg/g and largely depend on the genetic modifications, the type of cultivation used (batch, fed-batch), and the media employed (rich, waste feedstocks, minimal salt media) [4]."
Comment 2: Manuscript Format: The current format of the manuscript does not follow the standard structure. The appropriate sequence should be:
- Introduction
- Materials & Methods
- Results & Discussion
- Conclusion
Please revise the manuscript to reflect this correct format.
Response: The journal manager has advised that the format is correct and should be left as is. Thank you.
Comment 3: Keywords: Ensure that the first letter of each keyword is capitalized to align with standard manuscript formatting guidelines.
Response: the keywords have been capitalized. Thank you.
Comment 4: Conclusions: The conclusions section needs significant revision. It should emphasize the significance and impact of the study, highlighting future research directions and how the findings could influence the field.
Response: The importance of this work was added to the conclusion to put this work in the greater context as recommended:
"This improvement is important as these strains no longer require antibiotics for plasmid maintenance, which is costly at large scale. Furthermore, the high lycopene yield obtained in this work used minimal salt media, another significant cost savings during at large scale production. "
Comment 5: Literature Comparison: To provide more context, consider including a table or paragraph that compares your findings with similar studies reported in the literature. This will allow readers to better understand how your work fits within existing research.
Response: There are a number of review articles that already summarize recent work in table form. In order to avoid duplication of this work, a recent review is cited in the introduction. The discussion focuses on those studies that use the IUP pathway for carotenoid production, and those that focus on membrane engineering as they are most relevant. The following discussion was added to expand this section:
"Insertion of 13 copies of the IUP pathway using modulated ribosome binding sites (RBS) into the genome of E. coli saw a 1.78-fold increase in lycopene yield, similar to this study, where a single copy increased lycopene specific yield by 2-fold [18]. Furthermore, the lycopene titre achieved in this study was produced using a minimal salt media. Although higher lycopene titres can be achieved using LB or other rich medias, these media are much more expensive that minimal salt media like the M9 used in this study. This is particularly important when considering the economic feasibility of producing carotenoids using biomanufacturing processes."
Comment 6: Metabolism Diagram: Include a diagram that visually depicts the metabolic pathways for lipid and carotenoid production across different strains. Highlight both positive and negative examples of strains used in this study to enhance clarity.
Response: The metabolic steps in carotenoid production have been added to Figure 1 to align with the plasmid maps and the caption has been altered as follows:
"B. Biosynthesis of lycopene and β-carotene from the universal isoprenoid precursors IPP and DMAPP from the endogenous MEP pathway or the artificial IUP pathway. "
Comment 7: Figure Formatting: In Figure 6, panels A, B, and C should be referenced before the title of the figure caption. Please adjust the formatting accordingly.
Response: The journal manager has advised that the format is correct and should be left as is. Thank you.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAll my previous comments have been addressed. As a result, the manuscript can be accepted.
