Metabolic Engineering of Escherichia coli for De Novo Biosynthesis of Mandelic Acid

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewers’ comments

In this study, Escherichia coli is engineered for the de novo biosynthesis of mandelic acid (MA), derived from an efficient hydroxymandelate synthase (HMAS) homolog from Actinosynnema mirum, identified by the authors for its high efficiency. Metabolic flux optimization and regulation of the shikimate pathway and aromatic amino acid metabolism were performed through targeted overexpression and CRISPR interference (CRISPRi)-mediated repression of selected genes. Overexpression of genes related to the abundance of phenylpyruvate - such as Phe^Afbr and Aro^Gfbr - was positively correlated with an increased MA production, with combinatorial strategies proving more effective than individual gene modifications. Regarding gene suppression, repression of genes involved in aromatic amino acid synthesis synthesis (trpR, trpE) and phenylpyruvate consumption (pykF) yielded the most significant improvements in MA production. For bioreactor-scale production, the authors performed high cell density cultivation using the most effective engineered strain combination, achieving a peak MA concentration of 62.81 mM (9.58 g/L).

Overall, the experimental approach is appropriate and well executed. Moreover, a visual inspection of the SDS-PAGE gels does not raise concerns about image manipulation. While the manuscript requires minor corrections, I believe it is a good fit for the journal pending revision. Therefore, I recommend minor revisions

Below are comments to specific lines:

L.17. de novo should be italicized

L.52. Please capitalize the word “Chemical”.

L.62-64. These lines appear to be duplicated - please revise accordingly.

L235. The text formatting appears inconsistent; the right margin is not justified - please correct.

L242. From the gels, HMAS4 appears to be more soluble than HMAS3. Why did you come to this conclusion? From the data presented, I can only conclude that HMAS3 is indeed the overall most “efficient” catalyst – both from MA yield and size wise. Could you clarify the basis for your conclusion regarding solubility?

L.263. The 1^st bioreactor conditions is 10 g/L glucose. However, in the Materials and Methods, its is only described that cultivation conditions were 20 g/L of glucose. Please clarify this discrepancy. Furthermore, given the influence of initial conditions on bioreactor performance, did the you compare 10 g/L and 20 g/L glucose concentrations experimentally, or was this choice based on prior literature/studies?

L.277. Referring to a 28-fold increase in production as a “limited effect” seems misleading, especially considering Table S3 shows raw MA yields of 85% and 65% from Phe^Afbr alone and the combination, respectively. I suggest rephrasing this to: “To further enhance MA production, combinatorial strategies were explored.”

L381. Again, formatting appears inconsistent; please ensure justified alignment.

L383. Saccharomyces cerevisiae should be italicized.

I also offer some comments below:

While testing gene repression combinations, why was the combination of tyrR and trpE not tested? It appears to me that aromatic amino acid synthesis appears to be a major competitor to MA biosynthesis and that these are major contributors to syphon resources from MA production.

Could you explain the rationale behind choosing the HMAS enzyme from Streptomyces coelicolor as a reference, since a lot of work had already been performed on a HMAS from the Actinomycetota Amycolatopsis orientalis – work which is in a cited in reference 20 (Reifenrath, M., & Boles, E. (2018))?

Regarding the bioreactors, while the ammonia is playing a key part in maintaining pH, it is also the strains only source of nitrogen. I would add a note of this in the Materials and Methods.

Author Response

Reviewer #1：

We sincerely thank Reviewer #1 for the positive and constructive evaluation of our manuscript. We are grateful for your recognition of the experimental design, metabolic engineering strategy, and bioreactor validation presented in this study. Your detailed comments and suggestions have been highly valuable in helping us improve the clarity, accuracy, and completeness of the manuscript. We have carefully addressed each point and revised the text accordingly. Our point-by-point responses are provided below.

 

Comment:

L.17. de novo should be italicized

Response:

Thank you for pointing this out. We have italicized de novo as recommended (Line 17).

 

Comment:

L.52. Please capitalize the word “Chemical”.

Response:

We have corrected the capitalization of “Chemical” in Line 52.

 

Comment:

L.62-64. These lines appear to be duplicated - please revise accordingly.

Response:

We appreciate the reviewer’s careful reading. The duplicated lines have been removed to ensure clarity and conciseness (Lines 64–65).

 

Comment:

L235. The text formatting appears inconsistent; the right margin is not justified - please correct.

Response:

Thank you for noting the formatting inconsistency. We have adjusted the alignment to ensure the text is fully justified at Line 254.

 

Comment:

L242. From the gels, HMAS4 appears to be more soluble than HMAS3. Why did you come to this conclusion? From the data presented, I can only conclude that HMAS3 is indeed the overall most “efficient” catalyst – both from MA yield and size wise. Could you clarify the basis for your conclusion regarding solubility?

Response:

We thank the reviewer for the valuable comment. We agree that the SDS-PAGE results do not support a definitive conclusion regarding the relative solubility between HMAS3 and HMAS4. We have clarified this point in the revised manuscript and explained that HMAS3 was selected for further study based on its higher MA titer under the tested conditions, rather than superior solubility. The revised paragraph can be found in Lines 240–252.

 

Comment:

L.263. The 1^st bioreactor conditions is 10 g/L glucose. However, in the Materials and Methods, its is only described that cultivation conditions were 20 g/L of glucose. Please clarify this discrepancy. Furthermore, given the influence of initial conditions on bioreactor performance, did the you compare 10 g/L and 20 g/L glucose concentrations experimentally, or was this choice based on prior literature/studies?

Response:

We thank the reviewer for this thoughtful comment and the opportunity to clarify. The 10 g/L glucose condition refers to shake flask-based whole-cell catalysis experiments, in which glucose was added as a fixed substrate to evaluate the effect of gene overexpression on MA production. These assays were designed to compare the relative performance of engineered strains under uniform conditions, and were not part of the bioreactor fermentation setup.

In contrast, the 20 g/L glucose mentioned in the Materials and Methods was used as the initial carbon source during high-cell-density fermentation in a bioreactor. This glucose was consumed during the cell growth phase, prior to IPTG induction. No MA production occurred at this stage. Therefore, the 10 g/L and 20 g/L glucose concentrations belong to two separate experimental contexts and were not directly compared. We have revised the text in the Results section (Lines 268–270) to clarify this distinction.

 

Comment:

L.277. Referring to a 28-fold increase in production as a “limited effect” seems misleading, especially considering Table S3 shows raw MA yields of 85% and 65% from Phe^Afbr alone and the combination, respectively. I suggest rephrasing this to: “To further enhance MA production, combinatorial strategies were explored.”

Response:

We thank the reviewer for the constructive suggestion and fully agree with the concern. We have revised the sentence accordingly to improve clarity and accuracy. The change has been made in Line 284 of the revised manuscript.

 

Comment:

L381. Again, formatting appears inconsistent; please ensure justified alignment.

Response:

Thank you for pointing this out. We have corrected the formatting of the entire paragraph containing Line 438 to ensure consistent justification and alignment throughout.

 

Comment:

L383. Saccharomyces cerevisiae should be italicized.

Response:

Thank you for the correction. Saccharomyces cerevisiae has been italicized as requested in Line 414 of the revised manuscript.

 

Comment:

While testing gene repression combinations, why was the combination of tyrR and trpE not tested? It appears to me that aromatic amino acid synthesis appears to be a major competitor to MA biosynthesis and that these are major contributors to syphon resources from MA production.

Response:

We thank the reviewer for this insightful comment. In fact, we did attempt the co-repression of tyrR and trpE. However, the resulting strain exhibited severely impaired growth and substantially prolonged cultivation time, making it unsuitable for further evaluation of MA production. Due to these limitations, this combination was not included in the main experimental comparison. We have now clarified this point in the revised manuscript (Lines 354–357).

 

Comment:

Could you explain the rationale behind choosing the HMAS enzyme from Streptomyces coelicolor as a reference, since a lot of work had already been performed on a HMAS from the Actinomycetota Amycolatopsis orientalis – work which is in a cited in reference 20 (Reifenrath, M., & Boles, E. (2018))?

Response:

We thank the reviewer for this valuable question. The HMAS enzyme from Streptomyces coelicolor was selected as the reference because it had already been constructed and tested in our laboratory during early exploratory work. However, its catalytic efficiency toward MA production was found to be suboptimal. Based on this observation, we used it as the query sequence to perform a BLAST search for related HMAS homologs via the EFI-EST platform, with the goal of identifying more efficient candidates. While the HMAS from Amycolatopsis orientalis has indeed been studied in previous literature (reference 20), we aimed to compare a broader set of homologs across different phylogenetic backgrounds. This rationale has now been clarified in the revised manuscript (Lines 212–215).

 

Comment:

Regarding the bioreactors, while the ammonia is playing a key part in maintaining pH, it is also the strains only source of nitrogen. I would add a note of this in the Materials and Methods.

Response:

We thank the reviewer for this helpful observation. We agree that ammonia water served not only to control pH but also as an important nitrogen source during fermentation. This clarification has been added to the Materials and Methods section (Lines 192–194) to improve completeness and reproducibility.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript explores the production of mandelic acid. While the study presents potentially significant findings, particularly in the selection and implementation of enzymes, the manuscript would benefit from a substantial revision to improve its clarity, scientific depth, and coherence. The discussion is currently superficial and lacks critical interpretation of the results. Moreover, many grammatical errors and inconsistencies in terminology detract from the overall readability and professionalism of the work. A major revision is required to elevate the manuscript to the standards expected for publication.

Comment 1: The identity of the strain used in this study is not explicitly stated; it is recommended that the authors adopt a systematic strain-naming convention (e.g., E. coli Strain 1, E. coli 2, etc.) reflecting the number and type of genetic modifications introduced.

Comment 2: In most production profile figures, the Y-axis is labeled as "MA yield," but the unit is given as "mM." This raises ambiguity regarding whether the authors are reporting yield or titer. The authors are recommended to revise the labeling for clarity.

Comment 3: In section 3.1,  It is unclear whether the same E. coli strain was used for both the bioconversion experiments and the protein expression analyses. The authors are advised to clarify this point for consistency and reproducibility.

Comment 4: The rationale for targeting trpA and trpE in the L-tryptophan biosynthesis pathway is not clearly explained. Moreover, it remains unclear why a combinatorial approach involving both genes was not explored. The authors are encouraged to provide justification for their gene targeting strategy and clarify whether combinatorial constructs were tested.

Comment 5: When glucose is used as a carbon source, the production of byproducts is highly dependent on aeration conditions. The manuscript does not address this aspect, nor does it present any byproduct profiling data. The authors should justify why the byproduct formation pathways were not deleted or modulated and discuss this in the context of improving carbon flux toward the desired product.

Comment 6: For the bioreactor experiments, the manuscript would benefit from the inclusion of quantitative metrics such as yield and productivity. These data are essential for evaluating the performance and potential scalability of the engineered pathway. Furthermore, these should be compared with values reported in previous studies to highlight the advantages of the engineered strain. In addition to this the authors should also explain the rationale behind the temperature reduction implemented during the bioreactor cultivation.

Comments on the Quality of English Language

English must be improved.

Author Response

Reviewer #2：

We sincerely thank Reviewer #2 for the thorough and constructive review of our manuscript. We appreciate your recognition of the potential significance of our findings, as well as your thoughtful suggestions regarding clarity, scientific depth, and consistency in terminology. In response to your comments, we have carefully revised the manuscript to improve the coherence of the discussion, enhance the interpretation of results, and correct language-related issues. Your feedback has been invaluable in helping us strengthen the overall quality and presentation of the study. Below, we provide detailed point-by-point responses to each of your comments.

 

Comment 1:

The identity of the strain used in this study is not explicitly stated; it is recommended that the authors adopt a systematic strain-naming convention (e.g., E. coli Strain 1, E. coli 2, etc.) reflecting the number and type of genetic modifications introduced.

Response:

We thank the reviewer for this valuable suggestion. In response, we have adopted a systematic strain-naming convention throughout the manuscript. Each engineered strain is now assigned a unique identifier (MA01–MA29) based on its genetic modifications and stage of construction. A detailed summary of all strains and their corresponding genotypes has been added to the supplementary materials (Table S1). These strain names have also been incorporated consistently in the main text and figure legends to improve clarity and traceability.

 

Comment 2:

In most production profile figures, the Y-axis is labeled as "MA yield," but the unit is given as "mM." This raises ambiguity regarding whether the authors are reporting yield or titer. The authors are recommended to revise the labeling for clarity.

Response:

We thank the reviewer for pointing out this important issue. As correctly noted, the unit “mM” refers to the final concentration of MA in the reaction system, and thus “titer” is the appropriate term. We have revised all relevant figure labels, axis titles, and instances in the main text to use “MA titer” instead of “MA yield.” These corrections have been highlighted in the revised manuscript for clarity.

 

Comment 3:

In section 3.1, It is unclear whether the same E. coli strain was used for both the bioconversion experiments and the protein expression analyses. The authors are advised to clarify this point for consistency and reproducibility.

Response:

We thank the reviewer for raising this important point. We confirm that the same E. coli strains were used for both protein expression analyses (SDS-PAGE) and whole-cell catalysis experiments. All samples were obtained from identically prepared cultures of E. coli BW25113 harboring the corresponding plasmids, under the same induction conditions. We have clarified this point in the revised manuscript (Lines 241–243) to ensure consistency and reproducibility.

 

Comment 4:

The rationale for targeting trpA and trpE in the L-tryptophan biosynthesis pathway is not clearly explained. Moreover, it remains unclear why a combinatorial approach involving both genes was not explored. The authors are encouraged to provide justification for their gene targeting strategy and clarify whether combinatorial constructs were tested.

Response:

We appreciate the reviewer’s thoughtful comments. Both trpA and trpE were selected as CRISPRi targets due to their involvement in L-tryptophan biosynthesis, a pathway that competes with MA production for chorismate-derived precursors. Specifically, trpE encodes anthranilate synthase, which catalyzes the first committed step converting chorismate into anthranilate, thus diverting precursors directly from the MA biosynthetic route. trpA encodes tryptophan synthase subunit alpha, functioning at a downstream step in the pathway, and its repression was intended to more directly limit L-tryptophan synthesis.

In our screening experiments, only trpE repression significantly enhanced MA production, while trpA repression showed little to no improvement. To maintain cellular metabolic balance and avoid unnecessary genetic perturbations, we decided not to pursue combinatorial repression strategies involving genes with limited single-target effects.

Our overall CRISPRi strategy focused on combining only those targets that individually showed notable enhancement of MA titer. As such, combinatorial repression experiments were performed with trpE, pykF, and tyrR, which yielded synergistic improvements and demonstrated the most promising regulation points. These clarifications have been added to the revised manuscript (Lines 344-350).

 

Comment 5:

When glucose is used as a carbon source, the production of byproducts is highly dependent on aeration conditions. The manuscript does not address this aspect, nor does it present any byproduct profiling data. The authors should justify why the byproduct formation pathways were not deleted or modulated and discuss this in the context of improving carbon flux toward the desired product.

Response:

We thank the reviewer for raising this important point. We fully agree that byproduct formation, especially under glucose-fed conditions, is closely associated with aeration and nutrient supply strategies. In our bioreactor experiments, we implemented a tightly regulated aeration and feeding protocol to suppress overflow metabolism. After induction, sterile air was supplied at 3 vvm, and dissolved oxygen (DO) was maintained 10%-30% via cascade agitation control. To avoid excessive carbon input, a pulsed fed-batch strategy was applied, in which a concentrated glucose solution (500 g/L) was added at a fixed rate, guided by real-time DO feedback. These conditions were designed to favor efficient glucose utilization while minimizing carbon flux into unwanted pathways. The details of this strategy have been added to the revised manuscript (Lines 390–397).

We also acknowledge that this study did not involve genetic modifications targeting the acetate branch or other byproduct routes, as our main focus was on reinforcing the MA biosynthetic pathway. Given that the current titer has not yet reached a level where carbon loss to byproducts becomes a major limiting factor, we prioritized pathway enhancement in this work. Nonetheless, we agree that overflow metabolism will become increasingly important to address as titers increase, and we have discussed this direction in Lines 421–428 of the revised manuscript.

 

Comment 6:

For the bioreactor experiments, the manuscript would benefit from the inclusion of quantitative metrics such as yield and productivity. These data are essential for evaluating the performance and potential scalability of the engineered pathway. Furthermore, these should be compared with values reported in previous studies to highlight the advantages of the engineered strain. In addition to this the authors should also explain the rationale behind the temperature reduction implemented during the bioreactor cultivation.

Response:

We thank the reviewer for this insightful suggestion. To address the comment, we have revised the manuscript to include key quantitative parameters describing bioreactor performance, including the final yield, overall volumetric productivity, and maximum instantaneous productivity. These values are now provided in Lines 404–408 of the revised manuscript.

In addition, we have added a direct comparison between our final MA titer and that of previously reported microbial production systems (engineered Saccharomyces cerevisiae) to better contextualize the advantages of our engineered strain. This comparison has been included in Line 414 of the revised manuscript.

Moreover, in response to the reviewer’s request regarding the temperature shift strategy, we have added an explanation immediately following the temperature reduction description in Lines 385–388. Specifically, we clarified that the reduction to 30°C was intended to minimize the metabolic burden on cells and promote proper folding of heterologously expressed enzymes, thereby enhancing MA production.

We believe these additions improve the clarity and completeness of the bioreactor section and better demonstrate the scalability and performance of our engineered system.

 

Comment:

English must be improved.

Response:

We thank the reviewer for the suggestion. We have thoroughly reviewed the manuscript to correct grammatical errors and improve clarity. Key sentences have been refined for readability and precision. We believe the revised version meets the language standards of the journal, but we remain open to further revisions if needed.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The paper is acceptable for publication.