Fermentative Production of L-Theanine in Escherichia coli via the Construction of an Adenosine Triphosphate Regeneration System
Round 1
Reviewer 1 Report (Previous Reviewer 2)
The key results reported are the construction of an engineered E.coli strain for production of L-Theanine. The strain expresses recombinant γ-glutamylmethylamide synthetase (GMAS), polyphosphate kinase (PPK), alanine transaminase (BsAld), alanine decarboxylase (CsAlaDC), and glutamine permease (GNP1) enzymes, which mediate the L-Theanine synthetic pathway.
This manuscript is an improvement over the previously submitted version but still difficult to follow. Combining results and discussion, in addition to a subsequent discussion section, makes the paper difficult to read. The paper might be easier to read if all results not generated in this study were moved either earlier, into the introduction, or later, into the discussion sections of the paper.
Major comments
(1) The authors have not presented data showing if the Theanine produced is indeed sterio-pure L-Theanine or a mixture of L- and D-Theanine.
(2) The description of various methods of L-Theanine production in the introduction is difficult to follow. It is not clear which methods are cell-free enzymatic reactions and which are purifications of L-Theanine produced within engineered living cells. It is also not clear which (if any) additives were needed to support each of the various different reactions.
(3) Introduction lines 78-84: The authors jump to describing new approaches for ATP regeneration and conversion of pyruvate to ethylamine without discussing how (or if) these reactions were addressed in the methods described previously in the introduction.
(4) Results and discussion lines 149 – 150: Please describe the sources of the ethylamine and glutamate that become condensed to produce L-Theanine.
(5) Results and discussion lines 152 – 158: Were these results generated in the current study or in previously published work? It is not clear, if not generated in the current study please make this clear and consider moving to a different section.
(6) Results and discussion lines 162 – 173: It is not clear how these molecular docking results were used to inform the strategy for engineering the new strains; please explain what decisions were made differently compared to if the docking study results had not been available.
Minor comments
(1) Abstract line 12: Possible typo “Theanine is a highly representative non protein amino acid…”; should this read “Theanine is a highly represented non protein amino acid…”?
(2) Introduction line 65: It is not clear if the L-Theanine production method using engineered γ-glutamylcysteine synthetase (γ-GCS) is a second example of the glutamine-mediated pathway or something else.
(3) Introduction line 71: Abbreviation GGT is undefined (presumably it stands for γ-glutamyl transpeptidase, but this is unstated).
(4) Methods: the methods are difficult to follow and do not seem to contain enough information to repeat the work independently. Were ethylamine and glutamate supplemented into any of the reactions performed? If yes, which ones and at which concentrations?
(5) Results and discussion lines 148 - 149: Possible typo “pETDuet-1-gmas-ppk was transferred … to generate … FD04”; should this be strain “FD02”?
The grammar and spelling are generally fine, although there are several typos that should be addressed. However, the manuscript is difficult to read and, in many places, it is not clear what message the authors are setting out to communicate.
Author Response
Reviewer #1: The key results reported are the construction of an engineered E.coli strain for production of L-Theanine. The strain expresses recombinant γ-glutamylmethylamide synthetase (GMAS), polyphosphate kinase (PPK), alanine transaminase (BsAld), alanine decarboxylase (CsAlaDC), and glutamine permease (GNP1) enzymes, which mediate the L-Theanine synthetic pathway. This manuscript is an improvement over the previously submitted version but still difficult to follow. Combining results and discussion, in addition to a subsequent discussion section, makes the paper difficult to read. The paper might be easier to read if all results not generated in this study were moved either earlier, into the introduction, or later, into the discussion sections of the paper.
Comment 1: The authors have not presented data showing if the Theanine produced is indeed sterio-pure L-Theanine or a mixture of L- and D-Theanine.
Response: Thank you for your comments and suggestions. This is a very good question. In analysis methods, we have added a description as follows:
In line 158-160 on page 6, we have added this sentence:" For the analysis of L-theanine and D-theanine, the Chirex®3126 (D)-penicillamine LC column (4.6 × 250 mm, Phenomenex, USA) was used in HPLC system.”
In line 309-310 on page 10, we have added the following content:" All of the theanine was L-theanine. The optical purity of L-theanine was 100%.”
Comment 2: The description of various methods of L-Theanine production in the introduction is difficult to follow. It is not clear which methods are cell-free enzymatic reactions and which are purifications of L-Theanine produced within engineered living cells. It is also not clear which (if any) additives were needed to support each of the various different reactions.
Response: Thank you for your comments. We provide a clearer description of L-theanine production in introduction, making it easier for readers to understand the specific details of each method. Thank you again for your valuable suggestion.
In line 72-74 on page 3, "Fan et al. [24] achieved a high L-theanine production of 70.6 g/L by heterologously introducing GMAS from Paracoccus aminovorans in a 5-L bioreactor." was revised as “Fan et al. [24] achieved a high L-theanine production of 70.6 g/L by heterologously introducing GMAS from Paracoccus aminovorans in a 5-L bioreactor through fed-batch fermentation.”
In line 82-84 on page 3, " Li et al. [27] utilized a salt-tolerant mutant γ-glutamyl- transpeptidase variant (V319A/S437G) to enhance catalytic activity, increase the yield of L-theanine from 58% to 83%." was revised as “Li et al. [27] utilized salt tolerant mutants γ-glutamyl-transpeptidase variant (V319A/S437G) to enhance the enzyme catalytic activity, increasing the yield of L-theanine from 58% to 83% by purified enzyme.”
Comment 3: Introduction lines 78-84: The authors jump to describing new approaches for ATP regeneration and conversion of pyruvate to ethylamine without discussing how (or if) these reactions were addressed in the methods described previously in the introduction.
Response: Thank you for your comments. We have made some revisions in the introduction.
We have added the following content in line 89-92 on page 3："Sun et al. [29] isolated endophytic bacteria from tea plants, successfully producing L-theanine. Although the titer was only 31.875 μg/L, this indirectly suggests the existence of a microbial synthesis pathway for the production of theanine that does not add ethylamine.”
Comment 4: Results and discussion lines 149 – 150: Please describe the sources of the ethylamine and glutamate that become condensed to produce L-Theanine.
Response: Thank you for your comments. Regarding the sources of ethylamine and glutamate, we have provided additional explanations of these two substrates.
In line 171-172 on page 6, "Ethylamine and glutamate are condensed to produce L-theanine using GMAS." was revised as “Ethylamine (MERUER, Shanghai, China) and glutamate (Aladdin, Shanghai, China) are condensed to produce L-theanine using GMAS.”
Comment 5: Results and discussion lines 152 – 158: Were these results generated in the current study or in previously published work? It is not clear, if not generated in the current study please make this clear and consider moving to a different section.
Response: Thank you for your comments. These results are based on previously published research. We have explicitly mentioned this in the manuscript and have retained them in the Results and Discussion section. Here, I want to express the advantages of whole cell catalysis. The next paragraph describes the synthesis of L-theanine using whole cell catalysis. Here, we would like to express the advantages of whole-cell catalysis. The next paragraph describes the production of L-theanine by whole-cell catalysis.
Comment 6: Results and discussion lines 162 – 173: It is not clear how these molecular docking results were used to inform the strategy for engineering the new strains; please explain what decisions were made differently compared to if the docking study results had not been available.
Response: Thank you for your comments. The binding pattern diagram of L-gutamate molecule with GMAS protein was shown in Fig. 3. As shown in Fig. 3C and Fig. 3D, the docking results of GMAS and L-glutamate showed that L-glutamate formed seven hydrogen bonds with the side chain Trp189, Tyr191, Ser239, Arg317 and Arg312. In addition, the binding of GMAS with L-glutamate could be attributed to the hydrophobic interaction because the strong hydrophobic interactions of L-glutamate with His237 and Arg323 were formed. Molecular docking results showed that L-glutamate could bind to the hydrophobic cavity of GMAS due to the formation of hydrogen bonds and hydrophobic interactions with surrounding amino acid residues.
Due to the impact of GMAS catalytic efficiency, there is still room for further improvement in the production of L-theanine. Through molecular docking, we identified some key sites that affect catalytic activity of GMAS. In our next manuscript, we will use semi-rational design to perform saturation mutations on key sites to enhance the catalytic activity of GMAS and thereby increase L-theanine production.
Reviewer 2 Report (New Reviewer)
This research paper on the fermentative production of L-theanine in Escherichia coli by constructing an ATP regeneration system is well-structured and provides valuable insights into the biosynthesis of L-theanine. The paper is generally well-written and organized. However, ensuring consistent formatting, especially regarding enzyme names and abbreviations, can improve readability. In summary, this paper provides valuable insights into the production of L-theanine using Escherichia coli and an ATP regeneration system. Enhancing the abstract, discussing the implications of the results in more detail, and possibly including visual aids could further improve the clarity and impact of the paper.
Here are some comments and feedback:
· Title: The title effectively summarizes the main focus of the paper. It clearly states the subject matter and the approach taken in the study.
· Abstract: The abstract provides a concise overview of the study's objectives, methods, and results. However, it could be improved by including specific findings or conclusions. For instance, mentioning the percentage increase in L-theanine production achieved by overexpressing GNP1 in FD04 could make the abstract more informative.
· Introduction: The introduction effectively sets the context for the study by highlighting the importance of L-theanine and its increasing market demand. It would be beneficial to briefly introduce the significance of L-theanine and its applications in various industries.
· Methods: The paper describes the methodology and enzymes used for L-theanine production in a clear and organized manner. However, it would be helpful to provide more details about the experimental setup and conditions used in the shake flasks and bioreactor experiments.
· Results: The results section presents the key findings, including the L-theanine titers achieved in different strains. Tables or figures that visually represent the data have been scientifically present. Additionally, explaining the significance of the 14.7% increase in L-theanine production in FD04 after overexpressing GNP1 would provide context for readers.
· Discussion: The discussion section should delve deeper into the implications of the results. It could explore why the selected enzymes and ATP regeneration system were effective, as well as potential limitations or challenges encountered during the study.
· Authors are encouraged to separate the results and discussion section to enhance the readability to the scientific audience.
· 4. Discussion Line 296 should be replacing with Conclusion: The conclusion is well-structured and summarizes the main findings of the study effectively. It highlights the significance of constructing an ATP regeneration system and the potential for one-step fermentation methods using microorganisms. However, it could be strengthened by briefly discussing the broader implications of the research in terms of industrial applications or future directions for this field of study.
Minor editing of English language required
Author Response
Reviewer #2: This research paper on the fermentative production of L-theanine in Escherichia coli by constructing an ATP regeneration system is well-structured and provides valuable insights into the biosynthesis of L-theanine. The paper is generally well-written and organized. However, ensuring consistent formatting, especially regarding enzyme names and abbreviations, can improve readability. In summary, this paper provides valuable insights into the production of L-theanine using Escherichia coli and an ATP regeneration system. Enhancing the abstract, discussing the implications of the results in more detail, and possibly including visual aids could further improve the clarity and impact of the paper.
Here are some comments and feedback:
Comment 1: The title effectively summarizes the main focus of the paper. It clearly states the subject matter and the approach taken in the study.
Response: Thank you for your comments and affirmations.
Comment 2: The abstract provides a concise overview of the study's objectives, methods, and results. However, it could be improved by including specific findings or conclusions. For instance, mentioning the percentage increase in L-theanine production achieved by overexpressing GNP1 in FD04 could make the abstract more informative.
Response: Thank you for your comments. We have improved the conclusions and findings in the abstract. Thank you once again for your review and suggestions.
We have added the following content in line 19-21 on page 1 of the paper："Polyphosphate kinase from Escherichia coli was overexpressed in E. coli FD02, constructing an ATP regeneration system that increased the titer of L-theanine by 13.4% compared to E. coli FD01."
Comment 3: The introduction effectively sets the context for the study by highlighting the importance of L-theanine and its increasing market demand. It would be beneficial to briefly introduce the significance of L-theanine and its applications in various industries.
Response: Thank you for your comments and affirmations. We have made revisions in the introduction.
Comment 4: The paper describes the methodology and enzymes used for L-theanine production in a clear and organized manner. However, it would be helpful to provide more details about the experimental setup and conditions used in the shake flasks and bioreactor experiments.
Response: Thank you for your comments and suggestions. We have made revisions in the revised manuscript.
Comment 5: The results section presents the key findings, including the L-theanine titers achieved in different strains. Tables or figures that visually represent the data have been scientifically present.
Response: Thank you for your comments and affirmations.
Round 2
Reviewer 1 Report (Previous Reviewer 2)
The authors have not addressed the previous review comments:
Minor comment 1 “Abstract line 12: Possible typo “Theanine is a highly representative non protein amino acid…”; should this read “Theanine is a highly represented non protein amino acid…”?”
Please address this comment.
Major Comment 1: “The authors have not presented data showing if the Theanine produced is indeed sterio-pure L-Theanine or a mixture of L- and D-Theanine”
In the revised manuscript the authors have stated “The optical purity of L-theanine was 100%.” but have not presented data to support that statement. Please include supporting data.
Minor comment 5 “Results and discussion lines 148 - 149: Possible typo “pETDuet-1-gmas-ppk was transferred … to generate … FD04”; should this be strain “FD02”?
Please fix this typo.
Comment 2: “The description of various methods of L-Theanine production in the introduction is difficult to follow.”; Minor comment 2 & 3 “Introduction line 65: It is not clear if the L-Theanine production method using engineered γ-glutamylcysteine synthetase (γ-GCS) is a second example of the glutamine-mediated pathway or something else.”; “Introduction line 71: Abbreviation GGT is undefined (presumably it stands for γ-glutamyl transpeptidase, but this is unstated).”
The descriptions are still difficult to understand. What are the relationships between pathways mediated by gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase? Are these the same pathway or different? Are they both examples of the glutamine-mediated pathway?
Are all of the examples cited in the “glutamine-mediated pathway” section (72 – 89) cell-free reactions using purified recombinant enzymes, live-cell biosynthetic processes, or are some one and other the other (and which are which)?
Please rewrite this section to make it easier to understand.
Comment 4: “Results and discussion lines 149 – 150: Please describe the sources of the ethylamine and glutamate that become condensed to produce L-Theanine.”; Minor comment 4: “Methods: the methods are difficult to follow and do not seem to contain enough information to repeat the work independently. Were ethylamine and glutamate supplemented into any of the reactions performed? If yes, which ones and at which concentrations?”
The author’s response does not address the concern, apologies that my previous comment was not clear. The intended question was: what is the source of ethylamine and glutamate in the reaction? That is to say: how do the ethylamine and glutamate become introduced into the reaction, given that they are not mentioned in section 2.2 Cultivation Conditions, of the Materials and Methods section (lines 124 – 139). When were these reagents added, at what concentrations, if a researcher wanted to repeat the experiment what would they need to do?
Please rewrite the methods section explaining how each experiments which generated the reported results was performed.
Comment 6: “Results and discussion lines 162 – 173: It is not clear how these molecular docking results were used to inform the strategy for engineering the new strains”
The author’s reply that “Due to the impact of GMAS catalytic efficiency, there is still room for further improvement in the production of L-theanine. Through molecular docking, we identified some key sites that affect catalytic activity of GMAS. In our next manuscript, we will use semi-rational design to perform saturation mutations on key sites to enhance the catalytic activity of GMAS and thereby increase L-theanine production.”
Please say this in the manuscript.
New comment: Figure 4, the y-axis scales are different between graphs. This makes it very difficult to compare between them. Please redraw the graphs with the same y-axis scale in all panels.
The English language is OK from a grammar/spelling/punctuation point of view. However, the narrative manuscript is difficult to follow, the authors haven't fully described what they did or why.
Author Response
Our response is attached
Author Response File: 
Author Response.docx
Round 3
Reviewer 1 Report (Previous Reviewer 2)
Major Comment 1: “The authors have not presented data showing if the Theanine produced is indeed sterio-pure…”. Author’s reply: “… The Results of the L-theanine standard (A) and Samples (B) were showed in Fig. S1. [and] … Fig. S2…”
Please cite these figures in the manuscript, after the statement “All of the theanine was L-theanine. The optical purity of L-theanine was 100%.” (lines 310 -311)
Major comment 2: “The description of various methods of L-Theanine production in the introduction … are still difficult to understand.”. Author’s reply: “… In the section of the glutamine-mediated pathway in the introduction, the studies conducted by Zhang et al. [15] and Sun et al. [36] involved biosynthetic reactions using live cells, while Li et al.'s [34] research utilized a cell-free reaction with recombinant enzymes. Yang et al.'s [35] investigation employed recombinant enzymes in combination with carrier immobilization, also constituting a cell-free reaction.”
Please add the information (saying which were live cell biosynthetic processes, and which were cell-free reactions), into the manuscript.
Major comment 4: Methods unclear, especially concerning sources of ethylamine and glutamate in the reactions.
This comment is still not addressed. The authors have not explained what they did. The materials and methods section says cells were grown either in shake flasks, in tryptone, yeast extract media, with IPTG added part way through, then washed and froze them (lines 129 – 137); or in a bioreactor in glucose media without IPTG addition. There is no mention of ethylamine, glutamate, or any other reagents being added. There is no mention of what material was taken for subsequent HPLC analysis. Was it the entire reaction mix, including cells in suspension, was it the supernatant, or was it some other fraction?
The materials and methods section contains the sentence “For whole-cell biotransformation, E. coli wet cells were then harvested by centrifugation at 10,000 rpm (15 min, 4 °C) and washed for a minimum of 3 times and stored at -20 °C.” (lines 135 – 137). What do the authors mean by this? What is whole-cell biotransformation? What is the purpose? How is it performed?
The authors have added the sentences “At the initial stage of the synthesis reaction, ethylamine (MERUER, Shanghai, China) and glutamate (Aladdin, Shanghai, China) are introduced into the reaction system at a concentration of 200 mmol/L. They are condensed to produce L-theanine through the catalytic action of GMAS.”
What stage is the initial stage? Is it when the 20 mL culture is first set up (if so, why not just add those reagents to the list of growth media constituents)? Is it when the IPTG is added? Or is it at some point after the grown cells have been washed and frozen? If it is after the cells have been washed and frozen then there is an entire method missing, possibly “whole-cell biotransformation”?
What about FD03 strain cells, are both ethylamine and glutamate supplemented into those reactions? The authors have stated that the reason for introducing the BsAld and CsAlDC enzymes was to remove the need for exogenous ethylamine, so presumably not, but as currently written the manuscript says that ethylamine was added.
What about FD04 strain cells grown in the bioreactor? Is ethylamine supplemented into that reaction? Is glutamate supplemented into that reaction? Or is glucose sufficient to support the entire biosynthesis?
Please make this more clear in the manuscript.
English language needs proof reading and several minor corrections before publication
Author Response
Major Comment 1: The authors have not presented data showing if the Theanine produced is indeed sterio-pure…”. Author’s reply: “… The Results of the L-theanine standard (A) and Samples (B) were showed in Fig. S1. [and] … Fig. S2…
Please cite these figures in the manuscript, after the statement “All of the theanine was L-theanine. The optical purity of L-theanine was 100%.” (lines 310 -311)
Response: Thank you for your comments and suggestions. We have cited Fig. S1 and Fig. S2 in the manuscript, after the statement “All of the theanine was L-theanine. The optical purity of L-theanine was 100%.” (lines 310 -311)
In line 306-309 on page 10, "This suggests that the overexpression of the theanine transport protein enhanced the syn-thesis of L-theanine in E. coli FD04. All of the theanine was L-theanine. The optical purity of L-theanine was 100%." was revised as " These results indicated that the overexpression of the theanine transport protein enhanced the syn-thesis of L-theanine in E. coli FD04. All of the theanine was L-theanine. The optical purity of L-theanine was 100% (Fig.S1 and Fig.S2)."
Major Comment 2: The description of various methods of L-Theanine production in the introduction … are still difficult to understand.”. Author’s reply: “… In the section of the glutamine-mediated pathway in the introduction, the studies conducted by Zhang et al. [15] and Sun et al. [36] involved biosynthetic reactions using live cells, while Li et al.'s [34] research utilized a cell-free reaction with recombinant enzymes. Yang et al.'s [35] investigation employed recombinant enzymes in combination with carrier immobilization, also constituting a cell-free reaction.
Please add the information (saying which were live cell biosynthetic processes, and which were cell-free reactions), into the manuscript.
Response: Thank you for your comments and suggestions. We have revised the manuscript based on your suggestion as follows:
In line 79-94 on page 3, "Zhang et al. [15] cloned and expressed the γ-glutamyltranspeptidase from Bacillus amyloliquefaciens in B. subtilis. Finally, 190 mM of L-theanine was obtained by targeting muta-tions and promoter screening. Li et al. [34] utilized salt tolerant mutants γ-glutamyl-transpeptidase variant (V319A/S437G) to enhance the enzyme catalytic activity, increasing the yield of L-theanine from 58% to 83% by purified enzyme. Yang et al. [35] obtained (GGT) mutants with improved transpeptidation activity through high-throughput screening and immobilized GGT on a carrier treated with oxidized cellulose nanofiber. Over 70 g/L of L-theanine was accumulated within 18 h through fed-batch conversion. Table 1 presents strain types, engineered strategy, substrate types, titer and yield for L-theanine production. Sun et al. [36] isolated endophytic bacteria from tea plants, successfully producing L-theanine. Although the titer of L-theanine was only 31.875 μg/L, this indirectly suggests the existence of a microbial synthesis pathway for the production of L-theanine that does not add ethylamine. " was revised as " Zhang et al. [15] cloned and expressed the γ-glutamyltranspeptidase from Bacillus amyloliquefaciens in B. subtilis, conducting a biosynthetic reaction using live cells. Finally, 190 mM of L-theanine was obtained through a combination of mutagenesis and promoter screening. on the other hand, Li et al. [34] employed a cell-free reaction with purified enzymes, using salt-tolerant mutants γ-glutamyl-transpeptidase variant (V319A/S437G) to enhance enzyme catalytic activity, which led to a significant increase in the yield of L-theanine from 58% to 83%. Yang et al. [35] conducted a cell-free reaction combined with carrier immobilization by utilizing purified enzymes, which produced over 70 g/L of L-theanine within 18 h through a fed-batch conversion process. Sun et al. [36] isolated endophytic bacteria from tea plants and successfully produced L-theanine. Although the titer of L-theanine was only 31.875 μg/L, this indirectly suggests the existence of a microbial synthesis pathway for the production of L-theanine that does not require the addition of ethylamine. Table 1 presents strain types, engineered strategy, substrate types, titer and yield for the L-theanine production."
Major comment 4: Methods unclear, especially concerning sources of ethylamine and glutamate in the reactions.
This comment is still not addressed. The authors have not explained what they did. The materials and methods section says cells were grown either in shake flasks, in tryptone, yeast extract media, with IPTG added part way through, then washed and froze them (lines 129 – 137); or in a bioreactor in glucose media without IPTG addition. There is no mention of ethylamine, glutamate, or any other reagents being added. There is no mention of what material was taken for subsequent HPLC analysis. Was it the entire reaction mix, including cells in suspension, was it the supernatant, or was it some other fraction?
The materials and methods section contains the sentence “For whole-cell biotransformation, E. coli wet cells were then harvested by centrifugation at 10,000 rpm (15 min, 4 °C) and washed for a minimum of 3 times and stored at -20 °C.” (lines 135 – 137). What do the authors mean by this? What is whole-cell biotransformation? What is the purpose? How is it performed?
The authors have added the sentences “At the initial stage of the synthesis reaction, ethylamine (MERUER, Shanghai, China) and glutamate (Aladdin, Shanghai, China) are introduced into the reaction system at a concentration of 200 mmol/L. They are condensed to produce L-theanine through the catalytic action of GMAS.”
What stage is the initial stage? Is it when the 20 mL culture is first set up (if so, why not just add those reagents to the list of growth media constituents)? Is it when the IPTG is added? Or is it at some point after the grown cells have been washed and frozen? If it is after the cells have been washed and frozen then there is an entire method missing, possibly “whole-cell biotransformation”?
What about FD03 strain cells, are both ethylamine and glutamate supplemented into those reactions? The authors have stated that the reason for introducing the BsAld and CsAlDC enzymes was to remove the need for exogenous ethylamine, so presumably not, but as currently written the manuscript says that ethylamine was added.
What about FD04 strain cells grown in the bioreactor? Is ethylamine supplemented into that reaction? Is glutamate supplemented into that reaction? Or is glucose sufficient to support the entire biosynthesis?
Please make this more clear in the manuscript.
Response: Thank you for your comments and suggestions.
In line 170-173 on page 6, "After the OD600 reached 0.6, 0.5 mM IPTG was added, and culture was continued at 30 °C for 48 h. " was revised as "After the OD600 reached 0.6, 0.5 mM IPTG was added, at the same time, L-glutamate and ethylamine were added, and culture was continued at 30 °C for 48 h."
In line 254-256 on page 8, we described the added concentration of L-glutamate and ethylamine both were 200 mmol/L in subsequent experiments.
In both E. coli FD03 and FD04, the production of L-theanine does not require the addition of exogenous ethylamine and glutamate. Ethylamine is generated through the conversion of pyruvate by BsAld and CsAlaDC, while glutamate is from glucose though TCA cycle."
In line 141-143 on page 5, “For whole-cell biotransformation, E. coli wet cells were then harvested by centrifugation at 10,000 rpm (15 min, 4 °C) and washed for a minimum of 3 times and stored at -20 °C.” was revised as “After the OD600 reached 0.6, 0.5 mM IPTG was added, and culture was continued at 30 °C for 12 h. For whole-cell biotransformation of E. coli FD01 and FD02, E. coli wet cells were then harvested by centrifugation at 10,000 rpm (15 min, 4 °C) and washed for a minimum of 3 times and stored at -20 °C. Then the cells was added in 3 mL reaction mixture (OD600 = 20) containing 200 mM L-glutamate, 200 mM ethylamine, 10 mmol/L MgCl2·6H2O and 150 mmol/L (NaPO3)6.”
Round 4
Reviewer 1 Report (Previous Reviewer 2)
Many thanks to the author’s for making the requested changes:
Major Comment 1: The authors have replied: “In line 170-173 on page 6, … was revised as "After the OD600 reached 0.6, 0.5 mM IPTG was added, at the same time, L-glutamate and ethylamine were added, and culture was continued at 30 °C for 48 h.”
I cannot find this change in the manuscript. It is important information, please add it to the manuscript.
Major Comment 2: The authors have replied “In both E. coli FD03 and FD04, the production of L-theanine does not require the addition of exogenous ethylamine and glutamate. Ethylamine is generated through the conversion of pyruvate by BsAld and CsAlaDC, while glutamate is from glucose though TCA cycle."
This is important information, please say this in the manuscript.
Proof-reading and editing to fix minor errors in English grammar required before final version is published
Author Response
Many thanks to the author’s for making the requested changes:
Major Comment 1: The authors have replied: “In line 170-173 on page 6, … was revised as "After the OD600 reached 0.6, 0.5 mM IPTG was added, at the same time, L-glutamate and ethylamine were added, and culture was continued at 30 °C for 48 h.”
I cannot find this change in the manuscript. It is important information, please add it to the manuscript.
Response: Thank you for your comments and suggestions. We have revised the manuscript based on your suggestion.
In line 139-145 on page 5, “After the OD600 reached 0.6, 0.5 mM IPTG was added, at the same time, L-glutamate and ethylamine were added, and culture was continued at 30 °C for 12 h. For whole-cell biotransformation of E. coli FD01 and FD02, E. coli wet cells were then harvested by centrifugation at 10,000 rpm (15 min, 4 °C) and washed for a minimum of 3 times and stored at -20 °C. Then the cells was added in 3 mL reaction mixture (OD600 = 20) containing 200 mM L-glutamate, 200 mM ethylamine, 10 mmol/L MgCl2·6H2O and 150 mmol/L (NaPO3)6.”
Major Comment 2: The authors have replied “In both E. coli FD03 and FD04, the production of L-theanine does not require the addition of exogenous ethylamine and glutamate. Ethylamine is generated through the conversion of pyruvate by BsAld and CsAlaDC, while glutamate is from glucose though TCA cycle."
This is important information, please say this in the manuscript.
Response: Thank you for your comments and suggestions. We have revised the manuscript based on your suggestion.
In line 145-148 on page 5, “In both E. coli FD03 and FD04, the production of L-theanine does not require the addition of exogenous ethylamine and glutamate. Ethylamine is generated through the conversion of pyruvate by BsAld and CsAlaDC, while glutamate is from glucose though TCA cycle.” has been added in the revisd manuscript.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This manuscript by Cao et al. dealt with improving L-theanine production in Escherichia coli by constructing an ATP regeneration system and overexpressing a transporter. The authors were successful to show the production of L-theanine and its improvement. However, the experiments were poorly designed. The data analysis and data presentation were very poor. Authors just pinpoint the optimal condition (temperature, IPTG concentration and substrate concentration) required for the production of L-theanine, running the same experiments multiple time using the different recombinant strains. Authors completely failed to discuss and analyze the results based on the progressive improvement of L-theanine (Titer, yield). Thus, the reviewer thought this paper is not appropriate at all for the publication in this Journal “Fermentation”.
Moderate english editing is needed.
Author Response
Thank you for your comments and suggestions. We have revised our manuscript according to your comments. At the same time, molecular docking studies are added in this revised manuscript.
Reviewer 2 Report
The authors report construction and testing of an E.coli strain expressing five exogenous proteins to promote production of L-theanine. The exogenous proteins are: gamma- glutamylmethylamide synthetase (GMAS), phosphorylase kinase (PPK), alanine transaminase (BsAld), alanine decarboxylase (CsAlaDC) and a glutamine permease (GNP1). Open reading frames encoding each of these proteins were inserted into an expression plasmid (called pETDuet-1-gmas-ppk-bsAld-csAlaDC-gnp1) and this plasmid was transformed into BL21(DE3) E.coli. The resultant strain was named FD04.
GMAS and PPK expression was intended to create an ATP regeneration system within the cells.
bsALD and csAlaDC expression was intended to drive synthesis of L-theanine within the cells.
The intention behind GNP1 expression is not clear from the manuscript but may have been to promote L-theanine secretion into the growth medium.
Overall, the paper is extremely difficult to read. It is not clear what the authors did in this study, how this relates to the previous work cited, or how the various examples from previous work relate to each other.
Major comments:
(1) The authors should explain the intention behind transforming the cells to express GNP1. Was it to promote L-theanine secretion into the growth medium to make subsequent purification easier (by removing the need for a cell lysis step)? In the introduction, lines 88-90, the authors state “a glutamine permease … was overexpressed … for enhancing the rate of L-theanine transport.” Was the intended transport into or out of the cells? Why was a higher rate of transport needed for the system?
(2) Materials and Methods: the authors should include the methods used for production of L-theanine in the 1L bioreactor system, reported in Results section 3.5.
(3) Materials and Methods: the authors should describe the culture conditions for L-theanine production from each of the various strains; for example, were glutamate and ethylamine supplemented into the growth media, was glucose supplemented into the media?
(4) Materials and Methods: the authors should describe what samples were analysed for L-theanine, L-glutamate, and glucose content, was it supernatant, whole culture mix, or something else? How were the samples processed before HPLC?
(5) The authors should explain why they needed to introduce GMAS and PPK to regenerate ATP and how they established if those enzymes were indeed expressed and how they established if the steady state cellular ATP concentrations were indeed higher as a result.
(6) Results and discussion, line 145; the authors state “only inexpensive glutamate and ethylamine are required”. When are they required and how much? Also, how does this align with the later statement “Ethylamine is an essential starting material for the synthesis of L-theanine. However its high cost and toxicity pose significant negative impacts on human health and the natural environment” (lines 202-204).
(7) It is not clear how strains FD01 and FD02 were able to synthesise L-theanine without expressing bsALD and csAlaDC.
(8) It is not clear why the authors optomised for temperature and IPTG concentration in strains FD01 and FD02, which do not contain the L-theanine synthetic pathway enzymes, rather than in strain FD04, which does.
(9) The authors should explain how they determined if the each of the exogenous proteins was expressed, at what level, and whether it was active when expressed in the strain.
(10)Figure 3: Graphs are not labelled to show which graph for which strain. Please add labels.
(11)Figure 3: Graphs have inconsistent scales on the y-axis. This makes comparison difficult. Please use the same scale for all graphs.
(12)Figure 4: Why was L-theanine production so much lower from cells expressing bsALD and csAlaDC (intended to support its synthesis) than seen previously from the strain that did not express those enzymes?
(13)Results section 3.4, lines 251 – 252: authors state “L-theanine produced by engineered E. coli strain FD03 was approximately 251 mg/L, while E. coli FD04 achieved a maximum titer of 383 mg/L”. These numbers are different from the information shown in figure 3 (FD03 production lower than 200 mg/L) and figure 4 (FD04 production also lower than 200 mg/L). Please explain the apparent discrepancy.
Minor comments
(14)Figure 1: Labels in inner circle are incorrectly placed “Cosmetic products” and “Food additive” labels are in the wrong places.
(15)Introduction, lines 48-49: Mismatched units in two consecutive sentences; 2020 market output value stated as over $50 million, whereas in the next sentence the market projection for 20254 is stated in tons (1200 tons) rather than as value. This makes comparison difficult. It would be better to state past and future market sizes in the same units.
(16)Introduction, lines 65-66: Mismatched units for comparison in two consecutive sentences; Yang et al L-theanine yield stated as mmol/L/h, whereas in the next sentence Fan et al yield stated as g/L. Please use the same units for each, to allow comparison.
(17)Introduction, line 88: States the reported system operates “…without additional addition of ethylamine.” However, this is the first time that addition of ethylamine has been mentioned. Please included a few sentences in the introduction explaining which of the previously mentioned systems require addition of ethylamine and why it is advantageous for a system to operate without needing this addition (for example, is ethylamine an especially expensive additive?).
(18)Results and discussion, lines 176-179: the sentences “On one hand, excessive IPTG can competitively bind to repressor proteins, leading to the transcriptional initiation of target proteins, which may not be favorable for protein expression. On the other hand, high IPTG concentrations can promote the formation of inclusion bodies, affecting protein quality.” are confusing. It is not clear what the authors are intending to communicate here.
(19)Results section 3.3 De novo biosynthesis of L-theanine by engineered E. coli FD03, lines 201 – 216: It is not clear how the information on these lines relates to results from this study. Is this in the right place in the manuscript? As written, it is confusing.
(20)Figures 3 and 4: Why were the 12 and 24 h timepoints selected? What were the conclusions from the having these data?
(21)Discussion lines 296 -298: This text is from the instructions to authors, please delete.
The English language are fine.
Author Response
Comment 1: The authors should explain the intention behind transforming the cells to express GNP1. Was it to promote L-theanine secretion into the growth medium to make subsequent purification easier (by removing the need for a cell lysis step)? In the introduction, lines 88-90, the authors state “a glutamine permease … was overexpressed … for enhancing the rate of L-theanine transport.” Was the intended transport into or out of the cells? Why was a higher rate of transport needed for the system?
Response: Thank you for your comments and suggestions.
In lines 85-88 on page 3, we have modified as follows:
Moreover, a glutamine permease (GNP1) from Saccharomyces cereviside was overexpressed in E. coli FD04 for increasing the rate of L-theanine transport out of cells, thereby increasing L-theanine production.
Comment 2: Materials and Methods: the authors should include the methods used for production of L-theanine in the 1L bioreactor system, reported in Results section 3.5.
Response: Thank you for your comments and suggestions.
In lines 124-130 on page 5, we have added the methods used for production of L-theanine in the 1 L bioreactor system as follows:
The production of L-theanine in remombinant E. coli FD04 was conducted in a 1-L bioreactor. The composition of the medium includes 40 g/L 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, 150 mmol/L (NaPO3)6, and 100 μg/mL ampicillin. The pH was maintained at 6.8 with NH3·H2O. The rotational speed and temperature were 220 rpm and 30°C, respectively.
Comment 3: Materials and Methods: the authors should describe the culture conditions for L-theanine production from each of the various strains; for example, were glutamate and ethylamine supplemented into the growth media, was glucose supplemented into the media?
Response: Thank you for your comments and suggestions. We have described the culture conditions in the revised manuscript.
Comment 4: Materials and Methods: the authors should describe what samples were analysed for L-theanine, L-glutamate, and glucose content, was it supernatant, whole culture mix, or something else? How were the samples processed before HPLC?
Response: Thank you for your comments and suggestions. The processing method for the sample was described in reference 35. We have revised the manuscript.
Comment 5: The authors should explain why they needed to introduce GMAS and PPK to regenerate ATP and how they established if those enzymes were indeed expressed and how they established if the steady state cellular ATP concentrations were indeed higher as a result.
Response: Thank you for your comments and suggestions. Because GMAS uses glutamate and ethylamine as substrates to synthesize L-theanine, which requires consume ATP (Fig. 1). If ATP is added in vitro, it is expensive. PPK can convert ADP into ATP. We conducted the SDS analysis and confirmed the successful expression of GMAS and PPK.
Comment 6: Results and discussion, line 145; the authors state “only inexpensive glutamate and ethylamine are required”. When are they required and how much? Also, how does this align with the later statement “Ethylamine is an essential starting material for the synthesis of L-theanine. However its high cost and toxicity pose significant negative impacts on human health and the natural environment” (lines 202-204).
Response: Thank you for your comments and suggestions. We have revised these sentences in the revised manuscript.
Comment 7: It is not clear how strains FD01 and FD02 were able to synthesise L-theanine without expressing bsALD and csAlaDC.
Response: Thank you for your comments and suggestions. As seen in Fig. 2, it can be seen that the purpose of expressing bsAld and csAlaDC is to provide ethylamine. So engineered E. coli FD01 and FD02 can use glutamic acid and ethylamine as substrates to synthesize L-theanine.
Comment 8: It is not clear why the authors optomised for temperature and IPTG concentration in strains FD01 and FD02, which do not contain the L-theanine synthetic pathway enzymes, rather than in strain FD04, which does.
Response: Thank you for your comments and suggestions. We also optimized the temperature and IPTG concentration in FD04 in Fig. 6.
Comment 9: The authors should explain how they determined if the each of the exogenous proteins was expressed, at what level, and whether it was active when expressed in the strain.
Response: Thank you for your comments and suggestions. We conducted the SDS analysis and confirmed the successful expression.
Comment 10: Figure 3: Graphs are not labelled to show which graph for which strain. Please add labels.
Response: Thank you for your comments and suggestions. We have revised the manuscript.
Comment 11: Figure 4: Why was L-theanine production so much lower from cells expressing bsALD and csAlaDC (intended to support its synthesis) than seen previously from the strain that did not express those enzymes?
Response: Thank you for your comments and suggestions. FD03 with the expression of bsALD and csAlaDC use glucose as substrate to produce L-theanine. FD01 and FD02 without the expression of bsALD and csAlaDC use glutamate and ethylamine as substrates to produce L-theanine. The pathway from glucose to theanine is very long.
Comment 12: Results section 3.4, lines 251 – 252: authors state “L-theanine produced by engineered E. coli strain FD03 was approximately 251 mg/L, while E. coli FD04 achieved a maximum titer of 383 mg/L”. These numbers are different from the information shown in figure 3 (FD03 production lower than 200 mg/L) and figure 4 (FD04 production also lower than 200 mg/L). Please explain the apparent discrepancy.
Response: Thank you for your comments and suggestions. Fig. 5 and Fig. 6 showed the data optimized for temperature and IPTG concentration, without including the data for optimized glucose concentration (shown in Fig. S1). 383 mg/L was showed in Fig. S1.
Comment 13: Figure 1: Labels in inner circle are incorrectly placed “Cosmetic products” and “Food additive” labels are in the wrong places.
Response: Thank you for your comments and suggestions.
Comment 14: Introduction, lines 48-49: Mismatched units in two consecutive sentences; 2020 market output value stated as over $50 million, whereas in the next sentence the market projection for 20254 is stated in tons (1200 tons) rather than as value. This makes comparison difficult. It would be better to state past and future market sizes in the same units.
Response: Thank you for your comments and suggestions. According to your suggestion, we have revised the manuscript.
In 2020, the market output value of L-theanine surpassed $50 million [13]. The theanine market is expected to be worth more than $60 million by 2025.
Comment 15: Introduction, lines 65-66: Mismatched units for comparison in two consecutive sentences; Yang et al L-theanine yield stated as mmol/L/h, whereas in the next sentence Fan et al yield stated as g/L. Please use the same units for each, to allow comparison.
Response: Thank you for your comments and suggestions. According to your suggestion, we have revised the manuscript.
Yang et al. [23] screened a γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays. By optimizing protein expression and reaction conditions, 34.49 g/L of L-theanine was successfully produced. Fan et al. [24] achieved a high L-theanine production of 70.6 g/L by heterologously introducing GMAS from Paracoccus aminovorans in a 5-L bioreactor.
Comment 16: Introduction, line 88: States the reported system operates “…without additional addition of ethylamine.” However, this is the first time that addition of ethylamine has been mentioned. Please included a few sentences in the introduction explaining which of the previously mentioned systems require addition of ethylamine and why it is advantageous for a system to operate without needing this addition (for example, is ethylamine an especially expensive additive?).
Response: Thank you for your comments and suggestions. As seen in Fig. 2, if glutamate and ethylamine are directly used as substrates, ethylamine needs to be added. If glucose is used as the substrate, ethylamine does not need to be added. (As seen in Fig. 2, it can be seen that the purpose of expressing bsAld and csAlaDC is to provide ethylamine.)
Comment 17: Results and discussion, lines 176-179: the sentences “On one hand, excessive IPTG can competitively bind to repressor proteins, leading to the transcriptional initiation of target proteins, which may not be favorable for protein expression. On the other hand, high IPTG concentrations can promote the formation of inclusion bodies, affecting protein quality.” are confusing. It is not clear what the authors are intending to communicate here.
Response: Thank you for your comments and suggestions. This sentence explains why we need to optimize the concentration of IPTG, as high concentrations of IPTG are detrimental.
Comment 18: Discussion lines 296 -298: This text is from the instructions to authors, please delete.
Response: Thank you for your comments and suggestions. Sorry, we made such a mistake. We have revised the manuscript.
