Enhanced production of l-fuculose by Escherichia coli engineered via genome-scale metabolic modeling

Gun-Hwi Yeon; Du-Kyeong Kang; Hyun-Jin Koo; Daewon Go; Jungyeon Kim; Bong Hyun Sung

doi:10.1371/journal.pone.0349374

Peer Review History

Original SubmissionNovember 9, 2025
25 Feb 2026 Decision Letter - Jian Xu, Editor PONE-D-25-60454 Enhanced production of l-fuculose by Escherichia coli engineered via genome-scale metabolic modeling PLOS One Dear Dr. Kim, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Apr 11 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following financial disclosure: “This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (RS-2025-23282972, NRF-2022M3J5A1056169, 2021M3A9I5023254, 2019R1A2C1090726, and 2018M3A9H3024746); a National Research Council of Science & Technology grant (No. CAP20024-200) funded by the Korean government (MSIT); the Research Initiative Program of KRIBB; and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Technology Commercialization Support Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (RS-2024-00401586).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. 5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Additional Editor Comments: The reviewer has identified several major issues in this study that require substantial revision at this stage. Additionally, the Editor has the following concerns and requests to help the authors further improve their manuscript: 1) Please provide growth curves and microscopic images (e.g., confocal microscopy) for all generated strains alongside the appropriate control samples. 2) How were the knockout (KO) and knock-in (KI) strains verified? Any PCR validation data must be included, either as a main or supplemental figure. Furthermore, evidence supporting the overexpression of fucA should also be provided. 3) The discussion section is currently unsatisfactory and requires more careful revision. Overstatements should be strictly avoided and conclusions must be supported by the results presented in this study. [Note: HTML markup is below. Please do not edit.] Reviewer's Responses to Questions -->Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. --> Reviewer #1: Yes ******** -->2. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: Yes ****** -->3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes ****** -->4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes ****** -->5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)--> Reviewer #1: Authros focus on constructing an L-fuculose producing strain by deleting fucI, fucK, tpiA, fucO, and aldA and overexpressing fucA. The final engineered strain produced 50.25 mg/L of l-fuculose via 120-h cultivation. Some questions and suggestions are listed as follows. (1) It is suggested to show the dynamic production of l-fuculose during the fermentation. (2) Considerable acetate was accumulated. Discussion is required. (3) AldA was deleted to block conversion of LAD to lactate. Was accumulation of lactate detected before and after aldA deletion? (4) fucA::T7, what’s the meaning of T7 here? Which promoter was applied to express fucA? (5) It is suggested to perform the fermentation in a bioreactor to further indicate the significance of this study. ****** -->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0349374.r001
Revision 1
22 Apr 2026 Author Response Response to Reviewers’ Comments Dear Editor and Reviewer, We sincerely thank the editor and reviewer for their careful evaluation and constructive comments. We have revised the manuscript accordingly and believe that these changes have significantly improved the clarity and quality of our work. Below, we provide a point-by-point response to all comments. <Editorial Comment> [Comments] Comment 1. Please provide growth curves and microscopic images (e.g., confocal microscopy) for all generated strains alongside the appropriate control samples. Response: We thank the editor for this valuable suggestion. Growth curves for all engineered strains have been included in the revised manuscript (Fig. 2 and 3). Regarding microscopic analysis, while such data may provide additional insights into cellular morphology, the genetic modifications introduced in this study were not expected to induce significant morphological changes. As the primary focus of this study was on metabolic flux redistribution and L-fuculose production, and growth characteristics were already evaluated through growth curves, we believe that the current dataset sufficiently supports our conclusions. Microscopic analysis may be considered in future studies. [Revision: Figs. 2 and 3] Comment 2. How were the knockout (KO) and knock-in (KI) strains verified? Any PCR validation data must be included. Furthermore, evidence supporting the overexpression of fucA should also be provided. Response: We thank the editor for this important comment. The knockout strains were verified by PCR during strain construction, as described in the Methods section (Construction of mutants). In addition, evidence supporting the overexpression of fucA has been added to the revised manuscript. As shown in Fig. S1, SDS-PAGE analysis revealed a prominent band at the expected molecular weight of FucA following IPTG induction, confirming successful overexpression. [Revision: Fig. S1] Comment 3. The discussion section is currently unsatisfactory and requires more careful revision. Overstatements should be strictly avoided and conclusions must be supported by the results presented in this study. Response: We thank the editor for this valuable comment. The Discussion section has been carefully revised to avoid overstatements and to ensure that all conclusions are strictly supported by the experimental results. The language has been moderated throughout to better reflect the scope and limitations of the study. [Revision: Lines 249-254] Compared to Saccharomyces cerevisiae (used in a previous study), which produced only 1.55 mg/L of L-fuculose [1], the Fuc 4 strain achieved more than a 30-fold improvement, suggesting the potential of E. coli as a host for L-fuculose biosynthesis. Although aldA deletion was intended to block the conversion of LAD to lactate, lactate levels were not directly quantified in this study. Therefore, the contribution of aldA deletion to reducing lactate formation remains to be further validated. <Reviewer 1> [Comments] Comment 1. It is suggested to show the dynamic production of L-fuculose during the fermentation. Response: We thank the reviewer for this helpful suggestion. The time-dependent production of L-fuculose during fermentation has now been incorporated into the revised manuscript and is presented in Fig. 3. Corresponding descriptions have been added in the Results section. [Revision: Lines 203-207; Fig. 3] Fig 3. L-fuculose production during fermentation and confirmation by mass spectrometry. (A) L-Fuculose production by engineered E. coli strains (Fuc 1–4). Data are presented as mean ± standard deviation. (B) Mass spectrum of L-fuculose produced by the engineered strain. (C) Mass spectrum of the analytical L-fuculose standard. LAD was rapidly depleted within the first 12 h, whereas 1,2-PDO began to accumulate from 12 h, with its concentration increasing gradually until the end of fermentation. L-fuculose production was detected after 12 h and gradually increased throughout fermentation, reaching 38.92 ± 5.19 mg/L (Fig 3A). GC/MS analysis of the culture supernatant confirmed the presence of L-fuculose (Fig 3B, C). Comment 2. Considerable acetate was accumulated. Discussion is required. Response: We thank the reviewer for this important comment. A discussion on acetate accumulation has been added to the revised manuscript. We now describe that acetate accumulation likely reflects overflow metabolism in E. coli under high carbon flux conditions and may reduce carbon availability for L-fuculose biosynthesis. [Revision: Lines 258-261] In addition, the accumulation of acetate observed during fermentation likely reflects overflow metabolism in E. coli under high carbon flux conditions, which can divert carbon away from the desired product and reduce L-fuculose production efficiency [18]. Comment 3. AldA was deleted to block conversion of LAD to lactate. Was accumulation of lactate detected before and after aldA deletion? Response: We thank the reviewer for this insightful comment. Although aldA deletion was intended to block the conversion of LAD to lactate, lactate levels were not directly quantified in this study. This limitation has been acknowledged in the revised manuscript, and future studies will be required to further evaluate the contribution of aldA deletion to lactate reduction. [Revision: Lines 251-254] Although aldA deletion was intended to block the conversion of LAD to lactate, lactate levels were not directly quantified in this study. Therefore, the contribution of aldA deletion to reducing lactate formation remains to be further validated. Comment 4. fucA::T7, what’s the meaning of T7 here? Which promoter was applied to express fucA? Response: We thank the reviewer for this comment. The description has been clarified in the revised manuscript to indicate that fucA was expressed under the control of the T7 promoter. [Revision: Lines 196-200] In addition, fucA was overexpressed under the control of the T7 promoter to catalyze the aldol condensation of LAD and DHAP to L-fuculose-1-phosphate. Overexpression of fucA was confirmed by SDS-PAGE analysis following IPTG induction (Fig. S1), showing a distinct band at the expected molecular weight. The resulting strain, designated Fuc 1 (ΔfucI ΔfucK, fucA overexpression), was cultivated in glucose–LAD medium for 96 h. Comment 5. It is suggested to perform the fermentation in a bioreactor to further indicate the significance of this study. Response: We thank the reviewer for this valuable suggestion. While the present study was conducted in shake-flask cultures, we have added a statement in the Discussion section indicating that future studies involving bioreactor-scale fermentation will be important to evaluate scalability and industrial applicability. [Revision: Lines 268-271] Although this study was performed in shake-flask cultures, future studies involving bioreactor-scale fermentation will be important to validate the scalability and industrial relevance of the proposed strategy. Attachments Attachment Submitted filename: 260331_Response to Reviewer Comments.docx https://doi.org/10.1371/journal.pone.0349374.r002
29 Apr 2026 Decision Letter - Jian Xu, Editor Enhanced production of l-fuculose by Escherichia coli engineered via genome-scale metabolic modeling PONE-D-25-60454R1 Dear Dr. Kim, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jian Xu, Ph.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions -->Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.--> Reviewer #1: All comments have been addressed ******** -->2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. --> Reviewer #1: Yes ****** -->3. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: Yes ****** -->4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes ****** -->5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes ****** -->6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)--> Reviewer #1: The manuscript has been well revised according to the comments. I recommend that it should be accepted. ****** -->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.--> Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0349374.r003
Formally Accepted
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