Comprehensive Transformation of Escherichia coli for Nicotinamide Mononucleotide Production
Round 1
Reviewer 1 Report
In the manuscript entitled “Comprehensive transformation of Escherichia coli for nicotinamide mononucleotide production”, Bi and collaborators describe the metabolic optimization of an E. coli strain for the production of NMN from glucose and nicotinamide. To do this, they overexpress genes involved in NMN synthesis and excretion, as well as in glucose conversion to phosphoribosyl pyrophosphate (PRPP), an essential intermediate in NMN synthesis from nicotinamide (NAM). At the same time, this E. coli strain is engineered to avoid AMP degradation, and to increase adenosine recycling to AMP, thus enhancing ATP accumulation, which serves as a substrate for PRPP formation. While the design described in the present study is similar to that described by Shoji et al (PMID: 33220420), Bi’s work includes a novelty, namely, a system that, in principle, should enable ATP accumulation. This system allowed the authors to obtain 1105 mg/liter of NMN after external addition of NAM and glucose.
Although the work is interesting and provides a new approach for NMN production in Escherichia coli, I still have some concerns (some of them requiring experimental evidence) that should be addressed before I can recommend this paper for publication in Catalysts:
1. The authors achieve a maximum yield of 1105 mg/liter of NMN. This yield, obtained with strain A6 is based on the addition of 10g glucose and 1g NAM per liter of culture medium. In the discussion, the authors mention that they, at some point, tested 2g per liter of NAM (line 287), but these data are not shown in the manuscript. The yield with strain A6 should be studied using different amounts of glucose and NAM and data should be shown.
2. For NMN production, glucose and NAM must be added to the culture medium. However, only NMN was measured in the supernatant. Glucose and NAM should also be determined in the extracellular medium, at least using the top strain (A6) in optimal conditions. This will help clarify if NAM and glucose are cleared from the medium or are still present as “contaminants” after NMN production.
3. About the maximum yield. If 1000 mg of NAM per liter is added to the culture medium. How can be the NMN yield of 1105 mg per liter? Is this because NAM is present in the culture medium? If so, the authors should clarify this in the text.
4. The discussion is missing an overall view of the different methods for NMN production that have been described, at least the most recent ones (PMIDs: 30115969, 35362650, 36869544 ;DOI: 10.1186/s40643-022-00514-6). The authors should compare these methods with their own one to clarify the advantages of having an engineered E.coli strain.
5. In lines 143-144, the authors state that the use of the NAM transporter Niap reduces NMN production by 12.1%. Are they referring to their own work or to somebody else’s? This should be clarified and, if their own results, these should be shown.
6. In line 40, the reference (ref. 6) is not correct, since it links to Marinescu’s article describing NMN production in E.coli.
7. Line 130. After “strain B1”, (Table 1) should be added to guide readers to the list of strains.
8. Line 139. After “strain B2”, (Table 1) should be added.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Summary
The manuscript by Bi et al. (separations- 2191764) describes the development of a nicotinamide monophosphate-overproducer E. coli strain. The authors efficiently perform genetic engineering manipulation including plasmid and CRISPR/Cas9 methodologies. The work comprises a total of nine gene manipulations and represents a substantial piece of work, specially taking into account they achieve progressive increased fold-changes towards the high added value product titers.
Data is correct and self-explanatory, even though some further clarifications are necessary to fully understand the methodology. As a matter of opinion, I have found myself uneasy while reading certain discussions that exceed in length and complexity the way they could have been tackled, e.g., the explanation on the ATP balance. Remarkably, the most important critic I think I must raise is that information is not properly allocated in the sections, for example some information is misplaced between results or discussion. I would also like to pose as a general comment that some references are misplaced. Finally, regarding the images, I would strongly encourage to put the phenotype under each bar. Nonetheless, the research conducted is comprehensive and conceptual point of view the experiment is straightforward and does not offer any doubts. Following, I try to include some generic suggestions and doubts that arise and that may need to be solved before publishing.
Specific Comments for the authors:
Abstract
All in all, the abstract is OK, but I find it a bit diffuse in some aspects that are detailed below. Most importantly, the authors should fully disclose they are using Nam supplementation.
Line 14: I would suggest not to dedicate so much space to explain the particular salutary effect of NMN in the abstract.
Line 15: I would clearly define there are both plasmid transformation and CRISPR-Cas9 strategies (using the long form in the abstract is unnecessary I guess).
Line 18: It would be fantastic under my opinion If the reader could have information stepwise of the titer associated to each modification.
Introduction:
Introduction seems to me a bit long and some rephrasing could be useful. Nonetheless, all information is pertinent and backs up the project conceptualization.
Line 58: if NR pathway is kind of dismissed here it makes no sense to perform the NRK transformation in the experimental.
Line 66: this is not purely correct, since both methodologies can be alternatively used, as the own authors do. Please, rephrase and just mention both strategies are used or which particular technology is used for each step. Keep the any further discussion for the discussion section.
Line 72: please refer the statement about the availability of Nam.
Line 82: please add reference about the non-availability of PRPP.
Line 94-105: last paragraph of the introduction, being usually assigned to describe the objectives of the work, seems to me a bit out of scope in this case. I would suggest to explain the reader which is the objective and very briefly the four strategies that are going to be used.
Line 106: add please al names of enzymes that are missing but present in E. coli, and distinguish heterologous/endogenous enzymes and modified/non-modified metabolic step with a legend.
Results:
In general, data in self-explanatory and I agree with the authors that too many detail regarding the construction of the plasmids should be allocated in Material and methods (giving though a necessary amount of information). However, the long discussion pertaining to each of the steps and why was it performed I think they should belong to the discussion part. Results section is not to refer to someone else’s work or justifying but to explain what has been observed. Therefore, I would suggest to keep any discussion which is not strictly an observable for the discussion or introduction in its defect, see comments below:
Line 119-125: the discussion about using CRISPR or plasmids can be kept for the Discussion section.
Line 145: pleas add phenotype under the bar.
Line 151-175: move please this to discussion.
Line 166: please state firstly which is the AEC value expected, this sentence is unfinished. Then, describe please exactly what the authors propose to manipulate: if varying the ratio or varying the absolute quantities of the pool because this is not a very clear explanation.
Line 176: more exactly deaminates adenosine to inosine, please use this more specific info.
Line 177: which is the name of this add KO-strain. How does it compare with the following strain in which A0 in which amn has been substitute by ado1. Both ado1 and amn are reversible enzymes therefore this info would reveal in establishing a ade-AMP or ado-AMP is advantageous
Line 194-199: move please to discussion.
Line 201: please show evidence.
Line 213: as previously mentioned, it seems to me simply an article structural incongruence to use references in the results.
Line 263: adding NRK does not provide synergistic effect since it is a orthogonal pathway, I guess. Furthermore, is the NR in E. coli ?
Discussion:
Discussion is heavily short-handed, and it would benefit from receiving some of the aspects discussed in results, as previously mentioned. Some other details below:
Line 272: I do not think the discussion should start talking about CRISPR, this is just a methodology that has been used but the paper should not be centred on it
Line 273: mention this paper as the cornerstone of CRISPR/Cas9 genome seems to me simply equally inaccurate and inadequate.
Line 288: this statement about Nam supplementation is not backed up by results, authors should only discussed on the pacts they present in the results section.
Line 293: why synergistic?
Line 298: please discussed which modifications slow down growth and which could be the reason. Nonetheless, the important value is the titer per L of culture, independently if there is more or less biomass so this discussion may be futile.
Line 300: pleas compare with the current production models of NMN.
Material and methods:
Information seems to me a bit chaotic, please add and reorganise as suggested. Avoid also please any discussion in the material and methods section and use the passive form.
Line 391: please state separately the methods used for the measurement of NMN and the measurement of cells.
Line 408: please mention you use Nam supplementation.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Summary
In this first round of revision the authors have thoroughly addressed and solved all questions raised. The text has also been reorganised in a more comprehensive structure for improved readability regarding canons in scientific literature. Except for some details explained below, to be take into account in my opinion before publication though, the article should be considered suitable for definitive processing.
Specific Comments for the authors:
Abstract
Line 15: I would mention here classical plasmid transformation has also been used, especially because later on the authors discuss how this methodology outperforms CRISPR-Cas approach.
Line 22-24: please rephrase, this “next” makes no sense to me.
Introduction:
No comments, it would help though to separate information for each of the engineering approaches in one paragraph.
Results:
Line 149: the explanation is unclear from my perspective. AEC is stable, but dynamic, so the authors mean that increasing any of the participant in the equilibrium – in this case adenosine and AMP, should raise the rest of related compounds. This is not trivial, but simple, and should be approached this way (in introduction though, in the corresponding paragraph).
Discussion:
The discussion has appropriately improved, through allocation of the content previously dispersed in the rest of the section.
There is only one main comment regarding the discussion of ATP level increase (Line 264-282). First to mention, that part of the discussion based on assumption previous to the work should appear in introduction. Most importantly, the discussion about AEC seems to me a bit unfocused:
AEC can vary in the cell, as the author mentioned this is well-known: “The AEC is a scalar index ranging between 0 and 1. When all adenine nucleotide pool is in form of AMP the energy charge (AEC) is zero, and the system is completely discharged (zero concentrations of ATP and ADP). With only ADP, the energy charge is 0.5. If all adenine nucleotide pool is in form of ATP the AEC is 1.” doi: 10.1371/journal.pone.0108676.
The authors claim in line 283 they want to reduce the AEC value and this may be confusing. Long story short, I think the reader would be satisfied with and explanation that clearly indicated that the engineering strategy targets the increase of AMP so the cell may react in second instance by raising the ATP absolute content and restoring and acceptable AEC ratio
Material and methods:
No further comments.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
