Enhancing Caffeic Acid Production in Escherichia coli Through Heterologous Enzyme Combinations and Semi-Rational Design
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript presents a potentially valuable study on engineering HpaB to enhance microbial production of caffeic acid. However, several major concerns regarding the scientific narrative, methodological clarity, and data interpretation must be addressed before it can be considered for publication.
- The introduction needs to be revised. The introduction lacks sufficient background. A clearer reason should be provided: why was HpaB chosen as an important target for modification in this pathway? What evidence previously indicated that it was a potential speed limiting step?
- From shake flask cultivation to 5-L fermentation tank, but omitting key fermentation parameters. Details such as feeding strategy, dissolved oxygen control, induction time, and final cell density should be summarized to ensure the reproducibility of the reported high titer.
- Caffeic acid is a hydroxycinnamic acid. It's not phenylalaninein abstract.
- Briefly discuss the broader implications of this work, such as the potential of this engineered HpaB variant in synthesizing other valuable hydroxycinnamic acids or its applicability in more complex microbial communities.
- The language quality of the manuscript requires improvement to enhance clarity.
Author Response
Reviewer #1: This manuscript presents a potentially valuable study on engineering HpaB to enhance microbial production of caffeic acid. However, several major concerns regarding the scientific narrative, methodological clarity, and data interpretation must be addressed before it can be considered for publication.
Comment 1: The introduction needs to be revised. The introduction lacks sufficient background. A clearer reason should be provided: why was HpaB chosen as an important target for modification in this pathway? What evidence previously indicated that it was a potential speed limiting step?
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 2: From shake flask cultivation to 5-L fermentation tank, but omitting key fermentation parameters. Details such as feeding strategy, dissolved oxygen control, induction time, and final cell density should be summarized to ensure the reproducibility of the reported high titer.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 3: Caffeic acid is a hydroxycinnamic acid. It's not phenylalaninein abstract.
Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.
Comment 4: Briefly discuss the broader implications of this work, such as the potential of this engineered HpaB variant in synthesizing other valuable hydroxycinnamic acids or its applicability in more complex microbial communities.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 5: The language quality of the manuscript requires improvement to enhance clarity.
Response: Thank you for your comments. We have made revisions to the language of the manuscript.
Reviewer 2 Report
Comments and Suggestions for Authors- Line 80. There is no need to write the source and square brackets in italics.
- Line 99. You write ‘Firstly’ twice.
- Figure No. 1 contains a very low-quality dendrogram. The figure should either be enlarged, or the Picture should be resolved in some other way.
- I suggest adding a Statistical Analysis section to the Materials and Methods chapter, explaining the number of experimental replicates, the software used, and the statistical tests applied.
- The second part of Table 1 needs formatting. The spacing between the rows is too large.
- In Figures 4 and 5, you present mean values. I would recommend performing a one-way ANOVA test to assess statistical significance.
- In the Results and Discussion section, the results are discussed more extensively than the discussion itself. The discussion should be expanded, as there are few cited sources. A deeper comparison with studies conducted by other authors is lacking.
- In the Conclusions, expressions such as ‘we’ should be avoided, as this is formal scientific language. In addition, organism names such as E. coli should be written in italics.
- Review the font of the reference list, as it is different from the text, which is not how it should be.
Author Response
Reviewer #2:
Comment 1: Line 80. There is no need to write the source and square brackets in italics.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 2: Line 99. You write ‘Firstly’ twice.
Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.
Comment 3: Figure No. 1 contains a very low-quality dendrogram. The figure should either be enlarged, or the Picture should be resolved in some other way.
Response: Thank you for your comments. We have made modifications to Figure 1, presenting a clearer image.
Comment 4: I suggest adding a Statistical Analysis section to the Materials and Methods chapter, explaining the number of experimental replicates, the software used, and the statistical tests applied.
Response: Thank you for your comments. We have added a Statistical Analysis section to the Materials and Methods chapter based on your recommendation.
In line 208-211 on page 7, “The assay values represent the average of three independent experiments, and the error bars represent standard errors. Statistical analysis was carried out by using student’s t-test (one-tailed; two-sample unequal variance; p = not significant (ns), *p < 0.05).” was added in the Materials and Methods chapter.
Comment 5: The second part of Table 1 needs formatting. The spacing between the rows is too large.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 6: In Figures 4 and 5, you present mean values. I would recommend performing a one-way ANOVA test to assess statistical significance.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 7: In the Results and Discussion section, the results are discussed more extensively than the discussion itself. The discussion should be expanded, as there are few cited sources. A deeper comparison with studies conducted by other authors is lacking.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 8: In the Conclusions, expressions such as ‘we’ should be avoided, as this is formal scientific language. In addition, organism names such as E. coli should be written in italics.
Response: Thank you for your comments. We have made revisions based on your recommendation.
In line 336-337 on page 11, "In conclusion, we have delineated a biotransformation system that employs Tal, phaB, and phaC to transform tyrosine into caffeic acid within E. coli.” was revised as “In conclusion, a biotransformation system that employs Tal, phaB, and phaC to transform tyrosine into caffeic acid within E. coli has been constructed.”
Comment 9: Review the font of the reference list, as it is different from the text, which is not how it should be.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis article explores the production of caffeic acid (CA) by fermentation using metabolic engineering and enzyme design. The authors present an innovative strategy for increasing CA titer based on a combination of heterologous enzymes and a semi-rational design of the key enzyme in the system, HpaB. The study is distinguished by its meticulously developed methods and compelling experimental evidence. Despite the stated objective, the presented study has several shortcomings that reduce the value and significance of the results.
1. The authors provide a fairly thorough justification for the proposed method, which significantly increases CA yield by improving the catalytic activity of the HpaB enzyme through a semi-rational design. However, the rationale for the selection of specific enzyme combinations appears insufficiently substantiated. Why these specific enzyme variants were chosen or why they proved effective remains unclear.
2. Only a few variants yielded satisfactory caffeic acid production, while most variants demonstrated extremely low efficiency. The lack of a clear explanation of the mechanisms of success and failure raises questions about the reliability of the proposed solutions. 3. The presented molecular docking results demonstrate the importance of several amino acid residues for substrate binding; however, the relationship between these residues and improved production has not been adequately demonstrated. The molecular models constructed by the authors contain predictions regarding the effects of specific mutations, but the lack of control over changes in the functionality of mutated proteins significantly reduces the scientific value of the results. Further verification of changes in the functional properties of mutated enzyme forms is required.
4. Although the authors cite previous studies conducted in the field of biotechnological production of caffeic acid, an in-depth comparative analysis and applicability assessment of previous technologies are lacking. A detailed comparative analysis of the achieved values ​​with those of modern caffeic acid production methods has not been conducted. The claim of superiority of the proposed approach requires serious support with numerical data.
5. The final conclusions sound overly ambitious, as the actual productivity gains are lower than stated expectations.
6. pp. 59–62 “…The existing production methods generally extract and purify caffeic acid from plants; however, the concentration of caffeic acid found within these plants is usually quite low, and the process of extracting and purifying caffeic acid from plants is complex [15], resulting in low efficiency….” It should be indicated within what concentrations the concentration of caffeic acid in plants fluctuates.
7. “2.5. Fermentation of caffeic acid in a 5-L bioreactor.” The brand and model of the bioreactor should be indicated.
8. Figure 3. There are no labels for A and B in the figure.
9. Figures 4 and 5 should be combined to make 4a and 4b.
10. pp. 318–319. “…Under the most favorable biotransformation conditions, a caffeic acid concentration of 2335.48 mg/L was achieved in a 5-L fermenter after 48 h….” What are the most favorable conditions? What characteristics do these conditions have?
Author Response
Reviewer #3: This article explores the production of caffeic acid (CA) by fermentation using metabolic engineering and enzyme design. The authors present an innovative strategy for increasing CA titer based on a combination of heterologous enzymes and a semi-rational design of the key enzyme in the system, HpaB. The study is distinguished by its meticulously developed methods and compelling experimental evidence. Despite the stated objective, the presented study has several shortcomings that reduce the value and significance of the results.
Comment 1: The authors provide a fairly thorough justification for the proposed method, which significantly increases CA yield by improving the catalytic activity of the HpaB enzyme through a semi-rational design. However, the rationale for the selection of specific enzyme combinations appears insufficiently substantiated. Why these specific enzyme variants were chosen or why they proved effective remains unclear.
Response: Thank you for your comments. We have made revisions based on your recommendation. As shown in Fig. 3B, the docking results of EchpaB and р-coumaric acid showed that р-coumaric acid established five hydrogen bonds with the side chains of R113, Y117, H155, S210 and Y461. Furthermore, the interaction between EchpaB and р-coumaric acid is likely due to hydrophobic forces, as evidenced by the formation of robust hydrophobic interactions between р-coumaric acid and the residues L93, Y132, Y143, and R145. Therefore, in our experiment, we selected Y117 and S210 as candidate sites for mutation selection. As expected, the Y117 and S210 sites have a significant impact on caffeic acid synthesis.
Comment 2: Only a few variants yielded satisfactory caffeic acid production, while most variants demonstrated extremely low efficiency. The lack of a clear explanation of the mechanisms of success and failure raises questions about the reliability of the proposed solutions.
Response: Thank you for your comments. We have made some revisions in the introduction. Only a few variants are capable of producing satisfactory caffeic acid, while the efficiency of most variants is extremely low. This is often the case when conducting mutation experiments on enzymes, where only a few variants are able to produce satisfactory caffeic acid, while the majority have extremely low efficiency. For example, Pang et al. first constructed a mutant library of Pseudomonas aeruginosa lipoxygenase through semi-rational design to enhance the catalytic activity of lipoxygenase [37]. Only a few mutants are able to produce satisfactory efficiency, while the majority of mutants exhibit extremely low efficiency.
[37] Pang C. P.; Liu S.; Zhang G. Q.; Zhou J. W.; Du G. C.; Chen J. Improving the catalytic efficiency of Pseudomonas aeruginosa lipoxygenase by semi-rational design. 2023, 162, 110120.
Comment 3: The presented molecular docking results demonstrate the importance of several amino acid residues for substrate binding; however, the relationship between these residues and improved production has not been adequately demonstrated. The molecular models constructed by the authors contain predictions regarding the effects of specific mutations, but the lack of control over changes in the functionality of mutated proteins significantly reduces the scientific value of the results. Further verification of changes in the functional properties of mutated enzyme forms is required.
Response: Thank you for your comments. We have made revisions based on your recommendation. As shown in Fig. 3B, the docking results of EchpaB and р-coumaric acid showed that р-coumaric acid established five hydrogen bonds with the side chains of R113, Y117, H155, S210 and Y461. Furthermore, the interaction between EchpaB and р-coumaric acid is likely due to hydrophobic forces, as evidenced by the formation of robust hydrophobic interactions between р-coumaric acid and the residues L93, Y132, Y143, and R145. Therefore, in our experiment, we selected Y117 and S210 as candidate sites for mutation selection. As expected, the Y117 and S210 sites have a significant impact on caffeic acid synthesis. The discussion on the changes in functional characteristics of mutated enzyme forms will be elaborated in the next article.
Comment 4: Although the authors cite previous studies conducted in the field of biotechnological production of caffeic acid, an in-depth comparative analysis and applicability assessment of previous technologies are lacking. A detailed comparative analysis of the achieved values with those of modern caffeic acid production methods has not been conducted. The claim of superiority of the proposed approach requires serious support with numerical data.
Response: Thank you for your comments. We have deleted this keyword in the revised manuscript. References 16-23 and 35 are all typical examples of producing caffeic acid using biotechnology. At the same time, we have also added some discussions.
In line 313-316 on page 10, Tian et al. Identified and characterized a novel phenolic acid-responsive transcription factor CarR, and engineered it into a p-coumaric acid biosensor. Finally, the engineered strain CA8 achieved a caffeic acid titer of 9.61 g/L in a 5-L bioreactor [30].
[30] Tian, D.G.; Qin, Z.; Liu, W.L.; Caiyin Q.; Li, W.G.; Zhao, G.R.; Qiao, J.J. Engineering a biosensor based high-throughput screening platform for high-yield caffeic acid production in Escherichia coli. Metabolic engineering 2026, 93, 128-144.
Comment 5: The final conclusions sound overly ambitious, as the actual productivity gains are lower than stated expectations.
Response: Thank you for your comments. We have made revisions based on your recommendation. There is still a certain distance to industrialization, and further optimization is needed.
Comment 6: pp. 59–62 “…The existing production methods generally extract and purify caffeic acid from plants; however, the concentration of caffeic acid found within these plants is usually quite low, and the process of extracting and purifying caffeic acid from plants is complex [15], resulting in low efficiency….” It should be indicated within what concentrations the concentration of caffeic acid in plants fluctuates.
Response: Thank you for your comments. We have made revisions based on your recommendation.
In line 43-45 on page 1, “The existing production methods generally extract and purify caffeic acid from plants, however, the concentration of caffeic acid found within these plants is usually quite low, and the process of extracting and purifying caffeic acid from plants is complex [15], resulting in low efficiency.” was revised as “The existing production methods generally extract and purify caffeic acid from plants, however, the concentration of caffeic acid found within these plants is usually quite low (0.131%), and the process of extracting and purifying caffeic acid from plants is complex [15], resulting in low efficiency.”
Comment 7: “2.5. Fermentation of caffeic acid in a 5-L bioreactor.” The brand and model of the bioreactor should be indicated.
Response: Thank you for your comments. We have made revisions based on your recommendation.
In line 43-45 on page 1, “Fermentation of the engineered strain for caffeic acid production was carried out in a 5-L bioreactor.” was revised as “Fermentation of the engineered strain for caffeic acid production was carried out in a 5-L bioreactor (BG-LabSeries 5L, Shanghai Baotech. Engineering Co., Ltd.).”
Comment 8: Figure 3. There are no labels for A and B in the figure.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 9: Figures 4 and 5 should be combined to make 4a and 4b.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 10: pp. 318–319. “…Under the most favorable biotransformation conditions, a caffeic acid concentration of 2335.48 mg/L was achieved in a 5-L fermenter after 48 h….” What are the most favorable conditions? What characteristics do these conditions have?
Response: Thank you for your comments. We have made revisions based on your recommendation. The most favorable conditions here refer to those for biotransformation in a 5 L fermenter.
In line 183-194 on page 6, “The inoculation proportions of seed culture were 6 % in the fermentor. The composition of the growth medium included 40 grams per liter of glucose, 7.5 g/L K2HPO4·3H2O, 1.6 g/L (NH4)2SO4, 1.6 g/L MgSO4·7H2O, 0.00756 g/L FeSO4·7H2O, 2 g/L citric acid, 0.02 g/L Na2SO4, 0.0064 g/L ZnSO4, 0.0006 g/L Cu2SO4·5H2O, 0.004 g/L CoCl2·6H2O, and 100 μg/mL Amp. The medium's pH was set to 6.8 by titrating with NH3·H2O throughout the biotransformation procedure. The temperature was maintained at 37 °C with an air flow rate of 2 liters per minute. Upon the optical density at 600 nm (OD600) reaching approximately 15, the fermenter's temperature was adjusted to 30 °C. Concurrently, 0.5 mM IPTG and 10 g/L of tyrosine were introduced to the culture to induce enzyme expression and the synthesis of caffeic acid. Samples were collected at defined intervals, and the remaining tyrosine, p-coumaric acid, and caffeic acid concentrations were quantified via HPLC analysis. Analytical methods. ”
