Recent Advances in Natural Product Biosynthesis and Yield Improvement Strategies Using Yarrowia lipolytica

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submitted review “Recent advances in natural product biosynthesis and yield improvement strategies in Yarrowia lipolytica” makes a significant contribution to the generalization of research on Y. lipolytica, an important industrial microorganism. Along with other reviews on this topic, this manuscript summarizes a large number of studies conducted in recent years. The article is written in an easy-to-understand language.

Only a few minor inaccuracies can be noted.

1. Line 85. It should be “highlights” instead of “highlight”.
2. Lines 178 – 186. In the legend to Fig. 2, could you please mention the CrtE abbreviation as well.
3. Table 1. It should be “Licopene” instead of “lycopene”; “Linaloole #1, Linaloole #2”; “Itaconic acid #1, Itaconic acid #2” - for uniformity. Also, in this table, the references are evidently incorrect: could you please check the references 134,135,144, 145, 59, 146, 151, 153?
4. In the reference list, some references are incorrect, for example,

in Line 983, the reference 171 is incomplete (it should be Lee S, Kim P. Current Status and Applications of Adaptive Laboratory Evolution in Industrial Microorganisms. J Microbiol Biotechnol. 2020 Jun 28;30(6):793-803. doi: 10.4014/jmb.2003.03072);

in Line 913, the reference 137 is incomplete.

Author Response

Comments 1: The submitted review “Recent advances in natural product biosynthesis and yield improvement strategies in Yarrowia lipolytica” makes a significant contribution to the generalization of research on Y. lipolytica, an important industrial microorganism. Along with other reviews on this topic, this manuscript summarizes a large number of studies conducted in recent years. The article is written in an easy-to-understand language.

Response 1: We thank the reviewer for the positive comments on our manuscript and their helpful comments on how to improve.

Comments 2: Only a few minor inaccuracies can be noted.

Line 85. It should be “highlights” instead of “highlight”.

Response 2: Thank you; we have corrected this in line 85 / page 3

Comments 3: Lines 178 – 186. In the legend to Fig. 2, could you please mention the CrtE abbreviation as well.

Response 3: We have added the definition of CrtE to the figure legend (now figure 3). Page 7, lines 204-207.

Comments 4: Table 1. It should be “Licopene” instead of “lycopene”; “Linaloole #1, Linaloole #2”; “Itaconic acid #1, Itaconic acid #2” - for uniformity. Also, in this table, the references are evidently incorrect: could you please check the references 134,135,144, 145, 59, 146, 151, 153?

Response 4: Thank you for these good points. We note that Lycopene and Linalool are the standard spellings in American English and consequently we recommend retaining these spellings. We have included the numbering for Linalool and itaconic acid as suggested, and done likewise for the additional information recommended by reviewer 2. We have carefully checked all the references in the table against the revised version’s reference list.

Comment 5: In the reference list, some references are incorrect, for example,

in Line 983, the reference 171 is incomplete (it should be Lee S, Kim P. Current Status and Applications of Adaptive Laboratory Evolution in Industrial Microorganisms. J Microbiol Biotechnol. 2020 Jun 28;30(6):793-803. doi: 10.4014/jmb.2003.03072); in Line 913, the reference 137 is incomplete.

Response 5: We thank the reviewer for pointing these out. We have corrected these and carefully checked all the references for any inaccuracies or incompleteness.

Reviewer 2 Report

Comments and Suggestions for Authors

Please, see the attached document.

Comments for author File: Comments.pdf

Author Response

Comments 1: This is a well-organized review that provides a useful overview of natural product biosynthesis in Yarrowia lipolytica, covering lipids, alcohols, terpenoids, flavonoids, organic acids, and other chemicals, as well as yield improvement strategies. It fills a niche by proposing morphology engineering as a novel and underexplored avenue for yield optimization. However, the manuscript has several areas that could be strengthened, particularly given the rapidly evolving literature and the need for a more in-depth mechanistic discussion of the proposed morphology-engineering approach.

Response 1: We are grateful to the reviewer for the thoughtful review of our manuscript and suggestions for improvement. As we were asked to revise in a short time window, we have not fully implemented all the suggestions, particularly those that would significantly lengthen the review. However, we have added material to cover more briefly the reviewer’s suggestions.

 

Suggestions for Improvement

Comments 2: Expand Section 3.6 (Morphology Engineering) to at least 3–4 pages. Include:

1. A mechanistic framework linking morphology to yield (secretion, nutrient uptake, bioreactor performance, cell cycle, metabolic intensity).
2. Additional experimental examples beyond CLA4/MHY1 knockouts, including recent work on HDAC inhibitors and other transcription factor targets.
3. A table or figure systematically linking specific morphological states to predicted effects on different product classes.

Response 2: We agree that some extension to section 3.6 would be helpful. We have added significant further detail to how morphogenesis impacts industrial yield; and provided more experimental examples. These are found on pages 21-23, and lines 691-768 and 783-793.

Comments 3: Add a section or significantly expand Section 3.1 to cover advanced genetic and synthetic biology tools:

1. CRISPR-Cas9 optimization (codon optimization, sgRNA design, HR/NHEJ balance).
2. CRISPR-Cas12a/Cpf1 for multiplexed editing.
3. Base editing and prime editing for scarless modifications.
4. CRISPRi/CRISPRa for dynamic gene regulation.
5. Biosensors for metabolite-responsive pathway control.
6. Machine learning-guided promoter and sgRNA design.

Response 3: We appreciate that CRISPR based editing is very important for very important for Yarrowia manipulations. We have added an additional paragraph to section 3.1 to highlight the use of CRISPR and tools that are available specifically for Y. lipolytica (lines 461-473; page 16). However, we do feel that covering all the material suggested would significantly add to the length of the review and would still be substantially incomplete. We have therefore instead referenced recent specialist reviews and primers on CRISPR to provide readers with key references.

Comments 4: Consider incorporating computational/systems biology approaches:

1. Genome-scale metabolic models and FBA for target identification.
2. Multi-omics integration (transcriptomics, proteomics, metabolomics).
3. Machine learning for titer prediction and strain design.

Response 4: We agree that these are important topics, but as with point 2, we feel that a thorough consideration of each of these topics would make the manuscript much longer and lose some of the focus. We have briefly mentioned multi-omics approaches (lines 819-822, page 24) and added a paragraph on machine learning (lines 610-617, page 20), as this is perhaps the most relevant of these topics in context.

 

Comments 5: Update Table 1 revise the latest titers from the literature:

8. Naringenin: 8.3 g/L
9. Squalene: 51.2 g/L
10. β-Farnesene: 28.9 g/L
11. Astaxanthin: 2.8 g/L
12. Verify and correct all reference numbers in the table.

Response 6: We thank the reviewer for pointing out these updates to our table and have made the changes appropriately to Table 1. In fact we found that a recent article demonstrated a slightly higher titer for naringenin and have updated to this titer instead.

Medium Priority

Comments 6: Restructure Section 2 to reduce descriptive redundancy with Table 1. For each product class, focus the text on:

1. Key metabolic challenges specific to that class.
2. Comparative analysis of strategies (which approach was most effective and why).

Response 6: We appreciate the reviewer’s perspective here, and indeed in the first version of our manuscript, we ordered the material as suggested. However, a previous review recommended that we alter the structure in the way that we have done, particularly using Table 1 to move information out of the main text and improve the flow. Having written the manuscript both ways, we feel that the current strategy reads better (as reviewers 1 and 3 commented), and so we would respectfully prefer to keep the material ordered as it currently is.

Comments 7: Strengthen the Conclusion (Section 4):

1. Synthesize how morphology engineering complements metabolic engineering, ALE, and computational approaches.
2. Provide a prioritized research roadmap (short-term vs. long-term goals).
3. Discuss integration of multiple strategies (e.g., ALE + morphology engineering + dynamic regulation).
4. Address economic viability and scale-up challenges.

Response 7: We thank the reviewer for these good suggestions for strengthening the conclusion and have made these changes (lines 810-816, 822-825, and 827-831; pages 24-25).

Comments 8: Add discussion of organelle compartmentalization strategies beyond the brief mention in Section 3.4. Peroxisomal, mitochondrial, and lipid body targeting have emerged as powerful approaches. The connection between compartmentalization and morphology/cell architecture could strengthen the review's core thesis.

Response 8: This is a good suggestion, and we have added some additional thoughts to section 3.4 to cover organelle compartmentalization more thoroughly (lines 593-609, pages 19-20).

Comments 9: Discuss substrate valorization and circular bioeconomy more explicitly in Section 3.5, linking to real-world industrial waste streams and cost reduction.

Response 9: This is another good suggestion. We have added material to section 3.5, highlighting some specific examples, and discussing the importance of techno-economic analysis (lines 635-642, 648-650, 652-657; pages 20-21).

Low Priority

Comments 10: Figure improvements:

1. The scale bars in Figure 1 need to be more readable.

Response 10: We have amended Figure 1 to make the scale bars easier to read.

Comments 11: Minor writing issues:

1. "dation-terpenoid synthesis" appears to be a typographical error (line 493 494).
2. "an significant proportion" → "a significant proportion" (line 522).
3. Some sentences are overly long; consider splitting for readability.
4. Ensure consistent formatting of units (g/L, mg/L, mg/g DCW).

Comments 11: We thank the reviewer for pointing these out and have amended as suggested. Particularly, we have reviewed the whole manuscript and split overly long sentences where possible. We have ensured uniformity of units where possible, except in cases where the original studies did not report their findings in a way that permits this.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript systematically reviews the recent advances in natural product biosynthesis and yield improvement strategies in Y. lipolytica. The manuscript is generally well-structured and covers a wide range of products and strategies. However, the inclusion of more detailed information is necessary before publication.

1. While the manuscript provides description of heterologous natural products synthesis in Y. lipolytica, it lacks concluding figures that summarize the key biosynthetic pathway for each class of compounds discussed. Please add such figures.
2. Lines 464 to 474: Including more details regarding methods A to H.
3. Section 3.6: Elucidate the mechanistic link between cellular morphogenesis and metabolic flux in Y. lipolytica.
4. Section 4: The part of prospect is thin. More perspectives that are relevant for natural biosynthesis in Y. lipolytica.
5. Line 136: “YALI0F18590g” should not be italicized and check the formatting and presentation of gene and protein names.
6. Line 201: This sentence should be “Another…flux of pathways by providing…”.
7. Line 587: In this sentence, “deletion” should be deleted.

 

Author Response

Comments 1: This manuscript systematically reviews the recent advances in natural product biosynthesis and yield improvement strategies in Y. lipolytica. The manuscript is generally well-structured and covers a wide range of products and strategies. However, the inclusion of more detailed information is necessary before publication.

Response 1: We thank the reviewer for their constructive and insightful review of the manuscript and for these good suggestions.

Comments 2: While the manuscript provides description of heterologous natural products synthesis in Y. lipolytica, it lacks concluding figures that summarize the key biosynthetic pathway for each class of compounds discussed. Please add such figures.

Response 2: This is a good suggestion. We have added additional figures for sections 2.1, 2.2, 2.4, and 2.5 (Figures 2 and 5).

Comments 3: Lines 464 to 474: Including more details regarding methods A to H.

Response 3: We have extended this section to add more details (lines 534-543, page 18).

Comments 4: Section 3.6: Elucidate the mechanistic link between cellular morphogenesis and metabolic flux in Y. lipolytica.

Response 4: Following the suggestions of reviewer 2, we have discussed the links between cell morphology and metabolism in more detail in section 3.6 (these are found on pages 21-23, and lines 691-768 and 783-793).

Comments 5: Section 4: The part of prospect is thin. More perspectives that are relevant for natural biosynthesis in Y. lipolytica.

Response 5: This is again in agreement with reviewer 2, and we have extended section 4 following both reviewers’ suggestions (lines 810-816, 822-825, and 827-831; pages 24-25).

Comments 6: Line 136: “YALI0F18590g” should not be italicized and check the formatting and presentation of gene and protein names.

Response 6: We have specifically changed the point that the reviewer suggested and checked other gene and protein names throughout the manuscript.

Comments 7: Line 201: This sentence should be “Another…flux of pathways by providing…”.

Line 587: In this sentence, “deletion” should be deleted.

Response 7: We have altered these two points as suggested (now lines 223 and 778).

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed the questions appropriately. I have no further questions.