Practical Insights and Emerging Trends for Strategic Cloning of Large Biosynthetic Gene Clusters from Bacteria

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Line 26-30
The definition is clear. To better motivate why “selective advantage” matters, consider adding one concrete example (e.g., competitive inhibition, signaling/communication, metal chelation, or defense), so the concept doesn’t remain purely general.

Line 72-75
“These methods are versatile, enabling high-throughput screening” - this is not consistently true across the approaches listed. BAC/cosmid libraries can support high-throughput screening, but targeted TAR/CATCH-style capture is often lower-throughput and more design-intensive. Consider narrowing the statement (e.g., to library-based methods) or specifying which approaches are high-throughput.

The point about “challenges in maintaining fragment integrity for very large clusters” is important. Consider briefly stating what drives integrity loss (e.g., DNA shearing during extraction/handling, off-target cutting, recombination instability, repetitive elements) and clarifying which approaches are most affected.

Line 76-80
The description of genome-integrated preconditioning is clear and well motivated. Consider stating the key advantage up front: capture occurs 'in vivo' after engineered flanking elements are installed, which can improve fidelity and stability for very large BGCs.

 Line 106-109
Replace “when the size of the sequence is unsure” with “when the sequence size is uncertain.” Also, in many cases the 'boundaries' (not just size) are uncertain-consider revising to “when BGC boundaries and size are uncertain.”

Line 144-147
Consider revising “an excellent choice for discovery of BGCs of unknown size” to “unknown size and/or boundaries.” Also, “intact BGC’s” should be “intact BGCs” (no apostrophe).

Line 474-481
Please avoid stacking “expected to”/“will likely” unless supported by citations or explicitly framed as hypotheses. A more cautious tone (e.g., “may,” “could,” “suggests”) would be more appropriate here.

Line 501-505
This section is important but currently reads generic. Since the review focuses on cloning large BGCs and heterologous expression, consider adding domain-specific ethics/safety points such as: potential transfer of resistance determinants, toxin/secondary metabolite hazard risk, containment and biosafety considerations for engineered production strains, and responsible sharing/access controls for higher-risk sequences.

Author Response

Comment 1: 

Line 26-30
The definition is clear. To better motivate why “selective advantage” matters, consider adding one concrete example (e.g., competitive inhibition, signaling/communication, metal chelation, or defense), so the concept doesn’t remain purely general.

Response 1:

Thank you for the comment. We added ‘For example, many antibiotics enable competitive inhibition by suppressing the growth of rival microorganisms [2], while siderophores chelate iron to secure vital nutrients in resource-limited niches [3].’ (Line 28-30). All following citations and references were updated as well.

Comment 2:

Line 72-75
“These methods are versatile, enabling high-throughput screening” - this is not consistently true across the approaches listed. BAC/cosmid libraries can support high-throughput screening, but targeted TAR/CATCH-style capture is often lower-throughput and more design-intensive. Consider narrowing the statement (e.g., to library-based methods) or specifying which approaches are high-throughput.

The point about “challenges in maintaining fragment integrity for very large clusters” is important. Consider briefly stating what drives integrity loss (e.g., DNA shearing during extraction/handling, off-target cutting, recombination instability, repetitive elements) and clarifying which approaches are most affected.

Response 2:

Thank you for the comment.

We moved high-throughput screening to the description of library-based approaches as ‘Library-based approaches using BAC or cosmid vectors fall under this group, as they rely on fragment release followed by vector integration and support high-throughput screening of large clone sets.’ (Line 71-72)

Also, the sentence mentioned in the reviewer’s comment was edited as ‘These methods are versatile, enabling high-throughput screening, but a key challenge across these methods is maintaining fragment integrity for very large clusters (>100 kb) [18], which can be compromised by DNA shearing during extraction and handling, off-target cleavage by nucleases, recombination instability, and the presence of repeti-tive elements. These issues are most pronounced in workflows involving physical fragmentation or CRISPR-based excision, whereas library-based approaches mitigate some risks by relying on partial digestion and stable vector systems.’ (Line 76-83)

Comment 3:

Line 76-80
The description of genome-integrated preconditioning is clear and well motivated. Consider stating the key advantage up front: capture occurs 'in vivo' after engineered flanking elements are installed, which can improve fidelity and stability for very large BGCs.

Response 3:

Thank you for the comment. We replaced the beginning sentences of the paragraph to ‘Genome-integrated preconditioning methods offer a key advantage: capture occurs in vivo after engineered flanking elements are installed, which greatly improves fidelity and structural stability for very large BGCs. These methods involve inserting essential genetic elements, such as site-specific recombination sequences (e.g., attP/attB for in-tegrases or loxP for Cre recombinase) and vector backbones, into the donor genome flanking the target cluster.’ (Line 85-90)

Comment 4:

Line 106-109
Replace “when the size of the sequence is unsure” with “when the sequence size is uncertain.” Also, in many cases the 'boundaries' (not just size) are uncertain-consider revising to “when BGC boundaries and size are uncertain.”

Response 4:

Thank you for the comment. We have edited the manuscript as suggested (Line 120-121).

Comment 5:

Line 144-147
Consider revising “an excellent choice for discovery of BGCs of unknown size” to “unknown size and/or boundaries.” Also, “intact BGC’s” should be “intact BGCs” (no apostrophe).

Response 5:

Thank you for the comment. We have edited the manuscript as suggested (Line 158-159).

Comment 6:

Line 474-481
Please avoid stacking “expected to”/“will likely” unless supported by citations or explicitly framed as hypotheses. A more cautious tone (e.g., “may,” “could,” “suggests”) would be more appropriate here.

Response 6:

Thank you for the comment. We have edited the manuscript as suggested (Line 492-499).

Comment 7:

Line 501-505
This section is important but currently reads generic. Since the review focuses on cloning large BGCs and heterologous expression, consider adding domain-specific ethics/safety points such as: potential transfer of resistance determinants, toxin/secondary metabolite hazard risk, containment and biosafety considerations for engineered production strains, and responsible sharing/access controls for higher-risk sequences.

Response 7:

Thank you very much for the comment. We added ‘In the specific context of cloning large BGCs, additional considerations arise: the po-tential transfer of antimicrobial resistance determinants or virulence associated genes embedded within BGCs [84]; risks associated with expressing toxins, cytotoxic metabo-lites, or otherwise hazardous secondary metabolites in laboratory or industrial hosts [85]; and the need for stringent containment practices when engineering high yield production strains [86]. Furthermore, responsible sequence sharing and access controls are essential for higher risk clusters, particularly those encoding potent bioactive compounds [87].’ as suggested (Line 522-529).

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is a very well-researched, comprehensive review of the field of cloning large biosynthetic clusters. It covers and analyzes current strategies in this area. Most of the citations are from the period 2015-2026. The review is clearly written and well illustrated. This review provides outlines on how next-generation strategies will accelerate heterologous expression, natural product discovery, and sustainable biomanufacturing. Review could be useful for a wide audience of researchers specializing in development of new industrial strains of microorganisms.

As for my opinion review is ready for publication.

 

Author Response

Comment:

This manuscript is a very well-researched, comprehensive review of the field of cloning large biosynthetic clusters. It covers and analyzes current strategies in this area. Most of the citations are from the period 2015-2026. The review is clearly written and well illustrated. This review provides outlines on how next-generation strategies will accelerate heterologous expression, natural product discovery, and sustainable biomanufacturing. Review could be useful for a wide audience of researchers specializing in development of new industrial strains of microorganisms.

As for my opinion review is ready for publication.

Response:

Thank you very much for your comments. We really appreciate it.