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Peer-Review Record

Microbial Biosynthesis of Antibacterial Chrysoeriol in Recombinant Escherichia coli and Bioactivity Assessment

Catalysts 2019, 9(2), 112; https://doi.org/10.3390/catal9020112
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Catalysts 2019, 9(2), 112; https://doi.org/10.3390/catal9020112
Received: 18 December 2018 / Revised: 21 January 2019 / Accepted: 22 January 2019 / Published: 24 January 2019
(This article belongs to the Section Biocatalysis)

Round 1

Reviewer 1 Report

The manuscript deals about the opportunity to obtain chrysoeriol through biotransformation by E. coli cells. According to the authors this is a novel and cheaper way to produce this compound. They describe the production and purification and finally the antimicrobial activity of chrysoeriol. There are some minor points which should be improved in the manuscript before publication:


1) In the introduction information is missing about the anti-CK2 activity of chrysoeriol which is much higher than for other flavonoids.

2) Fig. 5 is not explained, what can be seen on each plate?

3) The term "biotransformation" in line 248 should be "transformation".

4) Which is the appropriate antibiotic mentioned in line 258.

5) The authors should write MHA or MH agar, not MHA agar.

6) Method 4.4.3 is difficult to understand: in line 318 the culture reached 0.6, but in line 322 for inoculation an overnight culture was used. I guess that this is not the culture with OD 0.6?

Author Response

Reviewer#1

The manuscript deals about the opportunity to obtain chrysoeriol through biotransformation by E. coli cells. According to the authors this is a novel and cheaper way to produce this compound. They describe the production and purification and finally the antimicrobial activity of chrysoeriol. There are some minor points which should be improved in the manuscript before publication:

Response: Thank you so much for going through the manuscript.

In the introduction information is missing about the anti-CK2 activity of chrysoeriol which is much higher than for other flavonoids.

Response: We added information in the text, line 38-40 with suitable reference.

Fig. 5 is not explained, what can be seen on each plate?

Response: We added information about figure 5 in figure legend. The result is explained in L170-182.

The term "biotransformation" in line 248 should be "transformation".

Response: It is corrected.

Which is the appropriate antibiotic mentioned in line 258.

Response: The information is added.

The authors should write MHA or MH agar, not MHA agar.

Response: It is corrected throughout the manuscript.

Method 4.4.3 is difficult to understand: in line 318 the culture reached 0.6, but in line 322 for inoculation an overnight culture was used. I guess that this is not the culture with OD 0.6?

Response: It is corrected.


Author Response File: Author Response.pdf

Reviewer 2 Report

Please see attached docx!

Comments for author File: Comments.pdf

Author Response

Reviewer#2

The authors describe the transformation of the flavone luteolin to the 3’-O-methylated chrysoeriol by an engineered E. coli strain. The enzyme responsible for the transformation is a sugar O-methyltransferase that obviously has quite a broad substrate spectrum.The success of the biotransformation was analysed by HPLC, ESI-MS and NMR spectroscopy.

Furthermore, additional tests of anti-bacterial activity of chrysoeriol and related compounds are presented. The results shown are – in principal – interesting and, in my view suited for publication in Catalysts.

Response: Thank you so much for going through the manuscript.

However, major revisions are needed:

Major points:

61 – The authors should clarify, why feeding luteolin to E.coli is more economical and and ecofriendly than isolating chrysoeriol. Luteolin would also have to be isolated from plant material.

Response: The information is added with a suitable reference. L70-76.

Fig 2a, bottom – What are the compounds eluting at 3.5, 4, and 5.5min from the HPLC? These seem to show up only in the presence of SpnK. Can the authors exclude that these compounds contribute to the anti-bacterial activity? An analytical chromatogram of the purified chrysoeriol should be presented!

Response: The compounds eluting at aforementioned retention time (3.5, 4, and 5.5 min) are not the metabolites of luteolin. Compounds eluted at 3.5, and 4.0 min are also present in control E. coli culture. The peak appeared at 5.5 min is also in the level of noise. Moreover, we checked the UV-VIS and LC-MS of this peak which does not resemble any metabolite of luteolin. Thus, we excluded this peak. Importantly, this is eluted very early in the HPLC which could be any indigenous metabolite of E. coli produced during fermentation.

Since we purified the chrysoeriol from the fermentation broth using preparative-HPLC, there is no role of these metabolites in the antibacterial activity. As suggested by the reviewer, the analytical chromatogram showing the purified product (chrysoeriol) is presented in figure 2a.

  98-115 – The wording used to describe the NMR spectroscopic results is not appropriate! Please check carefully!

Response: Thank you so much for the suggestions. We have revised the result section thoroughly. L114-134.

115 – What is the result of the comparison with published data?

Response: We compared our 1H-NMR, 13C-NMR spectra values (chemical shift, nature of the spectra, coupling constant value) with published data which shows exactly similar values. Thus, we confirmed the assignments of protons and carbons in respective 1D-NMR analyses.

=> All points regarding Fig. 3 / NMR assignment have to be carefully checked and explained!

We revisited the 1H-NMR spectrum and newly analyzed data are presented in the revised version of the manuscript. We also cross-checked these NMR data with previously published NMR spectra (Park et al., 2007; Magn. Reson. Chem 45; 1072-1075.)
Fig. 3 – An HSQC should be presented, at least in the SI!

Response: The HSQC is added in figure 4A along with HMBC showing a correlation of proton of methoxy-carbon with carbon of methyl group.

Fig. 3 – Are H-2’ and H-6’ really isochronous?

Response: We found 2’ and 6’ not isochronous as they appeared 6’-H (7.57, d, J= 2.19 Hz and 2’-H as singlet). Data are presented in Table 1 and the newly analyzed 1H-NMR spectrum is placed in Figure 3A.
Fig. 3 – Are both, H-2’ and H6’ really doublets with the same, large coupling constant?

Response: No. Please see the answer above. We corrected these data. Thank you so much for pointing out these mistakes.

Fig. 3 – Is H-5’ really a singlet?

Response: H-5’ is found to be doublet with J= 8.33 Hz. This data is added in Table 1 and figure 3A.

Fig. 3 – Does H-3 really display this large coupling constant?

Response: H-3 appeared singlet.

Fig. 4 – How about the HMBC correlation to C-4’? Both correlations should be presented, at least in the SI, to support the correct assignment of 3’-O-methylation!

Response: The newly added figure 4 shows full HMBC analysis. The cross peak showing a correlation of methoxy protons and C-3’ is shown by the arrow.


164-171 – Photos of the dilution series should be presented, at least in the SI.

Response: Photos of the dilution series are added in the supporting information as suggested by the reviewer.


Minor points:

48 – MRSA, first occurrence => add methicillin-resistant

Response: Corrected.


Fig. 3 – The numbering scheme of flavones would be helpful for many readers!

Response: The numbering is shown in the structure in figure 3.


Fig. 6 – In addition to different colors easily distinguishable symbols should be used!

Response: The symbols are used to distinguish data in figure 6. The old figure is replaced with new figure.


In addition, even as a non-native speaker I can identify numerous language issues. Therefore, the manuscript should be carefully checked in this regard.

Response: We also proofread the manuscript from a professional English editor.


Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Please, find my comments in the uploaded docx.

Comments for author File: Comments.pdf

Author Response

 Report on Revision #1

The manuscript has been improved significantly. However, there are still issues.

Major points:

- Fig 4A: The authors have to comment on the small signal upfield of the methoxy protons. From this, the assignment of C-4’ appears to be incorrect. Please explain the correlation between C-4’ and the proton signal adjacent to OCH3 (to the left, at ca. 3.85ppm)!

 

Response: We replaced previous figure 4 A with new figure showing zoomed view of HMBC correlation. The full HMBC is placed in SI (Figure S1) as suggested by the reviewer.

In zoomed HMBC, we can see a cross peak as reviewer pointed. But, this cross peak does not have any correlation with C-4’ and proton of O-CH3 as reviewer suspected. A dashed line is placed showing correlation (which can be seen if figure is zoomed carefully). This cross peak is downfield of C-4’ and upfield of OCH3 proton. Thus, from this we can clearly say this could be a peak from impurity in the sample. In 1H-NMR and 13C-NMR as well, we can see minor impurity which we excluded.

- Fig 4A: The presentation of the HMBC is inappropriate! The gaps in boths axes make it very difficult to interpret and to assess the real quality. It would be sufficient to show a zoom including both correlations, H3C/C-3’ and H3C/C-4’ in the main text, and then show the full spectrum, without any exclusions/gaps, in the SI.

 

Response: The full HMBC is placed in SI (Figure S2) as suggested by the reviewer.

 

- Fig 4B: Also in the HSQC, there is an additional set of signals of ca. 10% intensity relative to the signals of the main compound. Any idea what this is? Could it be the 4’-methoxy or the 3’,4’-dimethoxy isomer?

Response: We placed zoomed HSQC data in figure 4B. We can’t see any correlation of this cross peak with C-3’ or C-4’ and O-CH3. As mentioned above, this could be from the impurity in the sample. The full spectrum is presented in SI (Figure S2) as suggested by the reviewer.

Minor points:

- There are still significant language issues! E.g.:

- L115: replace “spectra/spectrum” by “peaks/peak” or “signals/signal”! This applies to several other instances, as well.

Response: The changes have been made as suggested by the reviewer.

 

- In total, the amended NMR part (L115-L138) does not seem to have been proof read by a native speaker.

Response: Grammatical and other minor corrections have been made.


Author Response File: Author Response.pdf

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