Enhancement of S-Adenosylmethionine-Dependent Methylation by Integrating Methanol Metabolism with 5-Methyl-Tetrahydrofolate Formation in Escherichia coli

Round 1

Reviewer 1 Report

This is a nice contribution in the field of biocatalysis. Particularly, it deals with the regeneration of SAM (S-adenosylmethionine), and offers a novel strategy by constructing a 5-methyl-THF regeneration system in Escherichia coli. The engineered biosystem is available for the production of a variety of methylated compounds.

The manuscript is clear and concise, well written. The results are complete and the conclusions are supported by the experiment. I did not detect flaws in this work, which nicely fits in Catalysts journal.

Author Response

Thank you very much for taking your precious time to review our manuscript.

Reviewer 2 Report

See attached file

Comments for author File: Comments.pdf

Author Response

Thank you very much for your valuable comments and suggestions.

Reviewer summary:
This manuscript presents a novel method to address SAM availability in biocatalytic systems by coupling CH3OH metabolism to 5-methyl-tetrahydrofolate formation. The study is well-designed and executed. The paper is well-written and figures and tables are clear and informative. However, I take issue with the authors making any claim referring to regeneration of 5-methyl-THF. Higher concentrations of methylated products are indeed observed under the appropriate conditions when CH3OH is added to the reaction. The increase is modest, possibly because transport of esculetin into the cell is rate-limiting. The authors failed to perform the critical experiment: addition of 13C-methanol followed by confirmation of the label at the site of methylation. In the absence of this experiment, the authors should not claim regeneration of 5-methyl-THF, particularly given the modest increase in methylated products. Finally, no mention is made of the impact of CH3OH and transient formation of formaldehyde has on the biocatalyst.

Our response:
Thank you for your valuable comment. As the reviewer pointed out, we did not perform ¹³C trace experiments due to the unavailability of LC-MS and NMR in our facility. Therefore, we agree to the reviewer’s opinion that it is difficult to claim that 5-methyl-THF is really regenerated in the methylation reactions. However, our experimental results show that introduction and/or enhancement of methanol metabolic pathway with 5-methyl-THF formation can increase the concentration of methylated compounds.

Therefore, we revised the assertive descriptions such as “5-methyl-THF is regenerating” and “the concentration of methylated compounds is improved by enhancing the regeneration of 5-methyl-THF” to the description supported by the experimental data such as “the concentration of methylated compounds is improved by the introduction of the methanol metabolic pathway for the formation of 5-methyl-THF” throughout the manuscript.

The points to be corrected are as follows:
Title, Lines 17-19, Lines 119-121, Lines 180-182, Lines 192-194, Lines 209-210, Lines 304-305, Lines 329-331, Lines 336-337, Lines 420-424, Lines 433-434, Figure Legend of Fig. S3.

Reviewer specific comments 1:
Line 1: The title should be changed to eliminate use of the word “regeneration”

Our response 1:
Thank you for your suggestion. As described above, it is difficult to prove whether 5-methyl-THF is regenerated or not from our experimental data. Therefore, we eliminated the word “regeneration”, and changed the title to “Enhancement of S-adenosylmethionine-dependent methylation by integrating methanol metabolism with 5-methyl-tetrahydrofolate formation in Escherichia coli”

Reviewer specific comments 2:
Line 4: tetrahydrofolate is misspelled

Our response 2:
Thank you for pointing out our mistake. We corrected the spelling of tetrahydrofolate (Line 4).

Reviewer specific comments 3:
Line 104: “we first performed deregulation of SAM biosynthesis by optimization of culture composition and by metabolic engineering…” Altering the culture medium is not “deregulation” of SAM biosynthesis. This sentence should be edited for clarity.

Our response 3:
Thank you for the valuable comment. SAM biosynthesis is negatively regulated by methionine and SAM in the medium components and our results suggested that the use of methionine free-medium facilitated the SAM biosynthesis and the methylation reactions. Therefore, we considered that omitting methionine from culture medium can deregulate SAM biosynthesis. However, this discussion was performed in the result section, not in introduction section, and thus the description “we first performed deregulation of SAM biosynthesis by optimization of culture composition” lacks clarity.

We corrected this sentence to “we first optimized culture composition and deregulated SAM biosynthesis by metabolic engineering of E. coli” in the revised manuscript (Lines 117-118).

Reviewer specific comments 4:
Line 170: “These results show that enhancement of 5-methyl-THF regeneration is an effective strategy to activate SAM regeneration.” See comments above regarding claims of regenerating 5-methyl-THF.

Our response 4:
Thank you for the valuable comment. As described above, whether 5-methyl-THF is regenerated or not is not supported by our experimental data. Therefore, we corrected this sentence to “These results show that integration of methanol metabolic pathway for 5-methyl-THF formation is an effective strategy to activate the SAM regeneration and SAM-dependent methylation reactions” in the revised manuscript (Lines 192-194).

Reviewer specific comments 5:
Line 287: “A remarkable increase (39%) was observed by the addition of 500 mM”. The large increase is due in part to the decrease in methylation by the corresponding cells lacking the B. methanolicus mdh gene. Since cofactor turnover is the best measure of a regeneration system, I would argue that an increase of 39% is less than remarkable.

Our response 5:
We apologize for misleading the reviewer. This description does not relate to the comparison of the methylation reaction between the omt single expression cells and omt-mdh co-expression cells. Instead, we described about the comparison of the methylation reaction using the omt-mdh co-expression cells with and without 500 mM methanol. Therefore, we considered that all of 39% increase is caused by the addition of 500 mM methanol.

To avoid such a confusion, we revised the sentence to “A clear increase in the concentration of the methylated compounds (39%) was observed by the addition of 500 mM methanol compared with that without methanol” in the revised manuscript (Lines 321-323).

Reviewer specific comments 6:
Line 347: italics font?

Our response 6:
Thank you for pointing out our mistake. We corrected font in this paragraph from italic to regular font in revised manuscript (Lines 389-401).

Reviewer specific comments 7:
Line 369: “wet cells of recombinant E. coli, 10 mg/L.” Is this value correct? Since the reaction was run on 1 mL scale, that means only 0.01 mg of cells were added to each reaction. Please clarify.

Our response 7:
Thank you for pointing out our mistake. We added the 10 mg/mL of wet cells of recombinant E. coli, not 10 mg/L. We corrected the unit of the cell concentration in the revised manuscript (Line 411).

Reviewer 3 Report

Dear Authors,

The manuscript by Kenji et.al described a novel pathway of E.coli host-derived S-Adenosylmethionine (SAM) regenerative system in which the expressed methanol dehydrogenase (MDH) catalyzes methanol to formaldehyde under NAD+/NADH reduction. The resulted formaldehyde spontaneously combined with THF and forms the 5, 10-methylene-THF, which finally turns to 5-methyl-THF under NADH mediated methylene-THF reductase (MetF) enzyme catalysis. Implications of these findings will be adopted for methylation of diverse biomolecules include DNA, protein, and secondary metabolites in many cellular processes. It is interesting work, present form of material can be published in ‘Catalyst’ journal without further revision.  

Thanks!

Author Response

Thank you very much for taking your precious time to review our manuscript.