Metabolic Engineering for the Biosynthesis of Pentalenene in the Rapidly Growing Bacterium Vibrio natriegens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript by Hu et al. presents the metabolic engineering of Vibrio natriegens for the biosynthesis of pentalenene. The study demonstrates the feasibility of using V. natriegens as a production host, employing a straightforward experimental design to achieve proof-of-concept results. While the foundational concept is promising, substantial revisions are required to improve the overall clarity and scientific rigor of the manuscript. First, the data analysis requires greater depth and robustness to support the conclusions drawn. Second, the manuscript is hindered by numerous grammatical issues, unbalanced sentence structures, and typographical errors, which collectively impede the readability and coherence of the narrative. A comprehensive revision of the text is strongly recommended to enhance the cohesion, precision, and scientific presentation of this work.

Specific Comments:

Comment 1:

The manuscript would benefit from a clearer explanation of the rationale behind the temperature optimization experiments. Elucidating the physiological or metabolic considerations that prompted this investigation—particularly in relation to pentalenene biosynthesis—would provide important context for interpreting the results and understanding how temperature may affect enzyme activity, pathway flux, or host metabolism.

Comment 2:

The selection of different media formulations should be better justified. In particular, the authors should discuss the observed increase in pentalenene production in the PPB medium. An explanation of the compositional or environmental characteristics of PPB medium that may have contributed to this enhancement—such as carbon source availability, nitrogen content, or trace elements—would be valuable.

Comment 3:

The manuscript currently lacks an in-depth analysis of the transcriptomic dataset. Further exploration of differentially expressed genes is recommended to identify potential regulatory or metabolic bottlenecks affecting pentalenene production. Such analysis could provide mechanistic insights into limitations in flux distribution, cofactor availability, or regulatory interference that constrain the pathway’s efficiency.

Comment 4:

If possible, the authors are encouraged to include a comparative analysis of pentalenene production levels between V. natriegens and other microbial hosts. This would contextualize the performance of the engineered strain and highlight its advantages or limitations relative to established production systems.

Comments on the Quality of English Language

English need to improve.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The work fermentation-3561525 is devoted to the biosynthesis of pentalenene using genetically modified bacteria Vibrio natriegens. The work is relevant, novel and has practical significance. The strengths include a 52-fold increase in the efficiency of pentalenene biosynthesis.

Comments

1) Pentalenene standards were prepared in the authors' laboratory. How? And how can one confirm that this is arbitration?

2) The conclusions (lines 70-85) should be removed from the introduction.

3) It is not clear from sections 2.4 and 2.5 which antibiotics were added to the nutrient media and in what quantities. What were these nutrient media?

4) The first reference in the work is number 17. I believe that the list of references should be given in the order of mention.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The paper is acceptable for publication.

Comments on the Quality of English Language

The english language is fine.