Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity

Roxana Portieles; Hongli Xu; Feng Chen; Jingyao Gao; Lihua Du; Xiangyou Gao; Carlos Borroto Nordelo; Qiulin Yue; Lin Zhao; Nayanci Portal Gonzalez; Ramon Santos Bermudez; Orlando Borrás-Hidalgo

doi:10.1371/journal.pone.0281175

Peer Review History

Original SubmissionJanuary 30, 2023
27 Feb 2023 Decision Letter - Awatif Abid Al-Judaibi, Editor PONE-D-23-01318Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity.PLOS ONE Dear Dr. Orlando Borras-Hidalgo, 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 March 15 2023, 11:59 p.m. 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Awatif Abid Al-Judaibi, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. 3. Thank you for stating the following financial disclosure: "the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) If any authors received a salary from any of your funders, please state which authors and which funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Financial Disclosure section: "the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." We note that one or more of the authors are employed by a commercial company: YOTABIO-ENGINEERING CO., LTD a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please respond by return email with an updated Funding Statement and Competing Interests Statement and we will change the online submission form on your behalf. 5. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: 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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: General comments: The study explores the genetic transformation of L. lactis subsp. lactis wild-type strain to increase nisin production. Specifically, the study focuses on the overexpression of the nisA and nisRK genes in L. lactis subsp. lactis wild-type strain and evaluates the impact of these transformations on cell growth, nisin production, and gene expression. The study also compares the nisin productivity of the original strain and the engineered strains, including those that underwent serial genetic re-transformations. The study also shows that the co-expression of these two bacteriocins results in extremely effective antibacterial action against M. luteus. Overall, the findings of the study demonstrate the potential of genetic engineering to enhance the production of nisin and provide insights into the regulation of the genes involved in nisin biosynthesis. In general, quality of paper in terms of study design, and discussion of the results is fairly good. Below are my comments and suggestions to help improve the quality of this manuscript before it could be considered for publication. 1. The study provides valuable insights into the culture conditions, preparation of recombinant strains, and gene expression strategies for the recombinant expression of nisin in Lactococcus lactis subsp. lactis. The study could be useful for further optimization of the expression of nisin and other antimicrobial peptides in Lactococcus lactis. 2. This study focused on increasing the expression of the nisA and nisRK genes to increase nisin production, but there may be other genes involved in nisin biosynthesis that could be targeted to further enhance production. Additional studies could investigate other genes involved in nisin production and their potential for increasing production. 3. There are few studies indicating the potential toxicity of nisin for topical use such as “In vitro assessment of skin sensitization, irritability and toxicity of bacteriocins and reuterin for possible topical applications”. Therefore authors should explore if there is any research/concern regarding the potential toxicity or allergenicity of nisin in human. 4. This study showed that co-expressing nisin A and nisin Z resulted in a potentiation of activity against M. luteus only. It would be interesting to conduct additional antibacterial tests to evaluate the activity against other bacterial strains. 5. Please update the paper with latest references. There is only one reference from year 2022, while no reference from year 2023. Reviewer #2: Nice presentation. Good and ineresting piece of work. the is a very significant piece of work. these type of work must be published, tablesand figure are well presented. usefull inovative work with industrial application. ****** 6. 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: Yes: Sidra Abbas Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: PlosONE-review.pdf https://doi.org/10.1371/journal.pone.0281175.r001
Revision 1
8 Mar 2023 Author Response Dear Prof. Dr. Awatif Abid Al-Judaibi, Academic Editor PLOS ONE Thank you for your consideration of our manuscript entitled "Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity." (PONE-D-23-01318). We have reviewed the comments provided by the reviewers and have thoroughly revised the manuscript. We found the comments helpful and believe that our revised manuscript represents a significant improvement over our initial submission. We would like to thank you and the referees for the excellent comments made to our manuscript, and we feel that the comments helped us obtain an improved version of our manuscript. We have attempted to address all comments from the reviewers. Enclosed is a revised version of the manuscript with all changes indicated in red font to facilitate the review process. Best regards, Prof. Dr. Orlando Borrás-Hidalgo Reviewers' comments: Reviewer #1: General comments: The study explores the genetic transformation of L. lactis subsp. lactis wild-type strain to increase nisin production. Specifically, the study focuses on the overexpression of the nisA and nisRK genes in L. lactis subsp. lactis wild-type strain and evaluates the impact of these transformations on cell growth, nisin production, and gene expression. The study also compares the nisin productivity of the original strain and the engineered strains, including those that underwent serial genetic re-transformations. The study also shows that the co-expression of these two bacteriocins results in extremely effective antibacterial action against M. luteus. Overall, the findings of the study demonstrate the potential of genetic engineering to enhance the production of nisin and provide insights into the regulation of the genes involved in nisin biosynthesis. In general, quality of paper in terms of study design, and discussion of the results is fairly good. Below are my comments and suggestions to help improve the quality of this manuscript before it could be considered for publication. Authors: We appreciated very much the opinion of the reviewer about the topic of our work. We are so thankful for the excellent revision and suggestions. These enhance significantly the quality of our manuscript. 1. The study provides valuable insights into the culture conditions, preparation of recombinant strains, and gene expression strategies for the recombinant expression of nisin in Lactococcus lactis subsp. lactis. The study could be useful for further optimization of the expression of nisin and other antimicrobial peptides in Lactococcus lactis. Authors: Thank for your suggestion. We are agreeing with the reviewer. Further optimization will be necessary to scale-up the use of these strains. We have included this information suggested by the reviewer in “Discussion” section as follows: “Additionally, optimization in fermenter conditions would be important to introduce these L. lactis strains genetically modified in a production flow.” 2. This study focused on increasing the expression of the nisA and nisRK genes to increase nisin production, but there may be other genes involved in nisin biosynthesis that could be targeted to further enhance production. Additional studies could investigate other genes involved in nisin production and their potential for increasing production. Authors: We are agreeing with the reviewer. There are an important group of genes involved in nisin biosynthesis that were not studied. The nisin biosynthesis is very complex, where eleven genes are involved. These genes have a key role in the different biosynthesis stage. Sometimes, it is not easy produce high quantities of nisin, because the bacterium itself regulate the expression to avoid the toxicity. So, in our case, the strain that we were using in the experiments had a low expression of nisA and nisRK genes, when the expression was evaluated by qPCR. That was the reason why we focused out aim in these genes. However, we agree with the reviewer that, other genes should be evaluated in the future. We have included this information suggested by the reviewer in “Discussion” section as follows: “Although, we focused on these genes. Other genes involved in the nisin biosynthetic could be evaluated in the future.” 3. There are few studies indicating the potential toxicity of nisin for topical use such as “In vitro assessment of skin sensitization, irritability and toxicity of bacteriocins and reuterin for possible topical applications”. Therefore, authors should explore if there is any research/concern regarding the potential toxicity or allergenicity of nisin in human. Authors: The paper recommended by the reviewer is really novelty and interesting at all. In fact, we have included some comment in the “Introduction” section as follow: “An interesting study provided important evidence that nisin Z could be also safely used topically as disinfectant at recommended concentrations without skin toxicity [20].” At present, nisin was the bacteriocin approved by United States Food and Drug Administration for use as a food preservative. The safety and efficacy of nisin as a food preservative have resulted in its widespread use throughout the world. 4. This study showed that co-expressing nisin A and nisin Z resulted in a potentiation of activity against M. luteus only. It would be interesting to conduct additional antibacterial tests to evaluate the activity against other bacterial strains. Authors: Good question. We agree with the reviewer that the evaluation activity against other bacterial strains would be interesting. However, we used the indicator microorganism Micrococcus luteus, which it is recommended as nisin-sensitive microorganisms in agar diffusion bioassay. We agree that we should evaluate additional bacterial strains. 5. Please update the paper with latest references. There is only one reference from year 2022, while no reference from year 2023. Authors: We agree with the reviewer. We have included latest references in the manuscript. Dai Z, Han L, Li Z, Gu M, Xiao Z, Lu F. Combination of Chitosan, Tea Polyphenols, and Nisin on the Bacterial Inhibition and Quality Maintenance of Plant-Based Meat. Foods. 2022; 11(10):1524. Maresca D, Mauriello G. Development of Antimicrobial Cellulose Nanofiber-Based Films Activated with Nisin for Food Packaging Applications. Foods. 2022; 11(19):3051. Wu M, Ma Y, Dou X, Zohaib Aslam M, Liu Y, Xia X, Yang S, Wang X, Qin X, Hirata T, Dong Q, Li Z. A review of potential antibacterial activities of nisin against Listeria monocytogenes: the combined use of nisin shows more advantages than single use. Food Res Int. 2023; 164:112363. Chen H, Ji PC, Qi YH, Chen SJ, Wang CY, Yang YJ, Zhao XY, Zhou JW. Inactivation of Pseudomonas aeruginosa biofilms by thymoquinone in combination with nisin. Front Microbiol. 2023; 13:1029412. Soltani S, Boutin Y, Couture F, Biron E, Subirade M, Fliss I. In vitro assessment of skin sensitization, irritability and toxicity of bacteriocins and reuterin for possible topical applications. Sci Rep. 2022; 12(1):4570. Khazaei Monfared Y, Mahmoudian M, Cecone C, Caldera F, Zakeri-Milani P, Matencio A, Trotta F. Stabilization and Anticancer Enhancing Activity of the Peptide Nisin by Cyclodextrin-Based Nanosponges against Colon and Breast Cancer Cells. Polymers (Basel). 2022; 14(3):594. Serrano I, Alhinho B, Cunha E, Tavares L, Trindade A, Oliveira M. Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections. Life (Basel). 2023; 13(2):504. Reviewer #2: Nice presentation. Good and interesting piece of work. This is a very significant piece of work. This type of work must be published, tables and figure are well presented. Useful innovative work with industrial application. Authors: We appreciated very much the opinion of the reviewer about the topic of our work. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0281175.r002
27 Mar 2023 Decision Letter - Awatif Abid Al-Judaibi, Editor Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity. PONE-D-23-01318R1 Dear Dr. Orlando Borras-Hidalgo, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Awatif Abid Al-Judaibi, PhD Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281175.r003
Formally Accepted
31 Mar 2023 Acceptance Letter - Awatif Abid Al-Judaibi, Editor PONE-D-23-01318R1 Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity. Dear Dr. Borras-Hidalgo: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Awatif Abid Al-Judaibi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281175.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .