The Synthesis of Ginsenoside Compound K Using a Surface-Displayed β-Glycosidase Whole-Cell Catalyst

Round 1

Reviewer 1 Report

The conventional approach to synthesizing ginsenoside CK involves enzymatic conversion but the enzyme purification include a lot of effort and expense, and morover enzymes are prone to inactivation and additionally  whole cell catalysis suffers from inefficiency due to limited cell permeability, researchers in this paper tryed to manage these challenges by using a YiaT protein as an anchoring motif, establishing a surface display system for β-glycosidase Bgp3. This system they used as a whole cell catalyst for the efficient synthesis of ginsenoside CK. The obtained results are well presented and in general whole manuscript is well written. I have just few suggestions. I dont know is it just in this peer Review format or not, but Figure quality should be better. Now it looks blurry. Also, Conclusion miss some points, should be better conected to the results. And maybe in conclusion or in the sections 2.6 and 2.7 you should add one sentence explaning why the ginsenoside CK conversion decrease when its production is still growing. 

Just a small check on articles that are missing.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript reports the synthesis of a ginsenoside, based on a beta-glycosidase whole cell biocatalyst obtained through a surface display methodology. After optimizing protein expression, a series of experimental parameters of the biotransformation was screened and 81.8 % conversion towards the target ginsenoside was obtained. Even if glycosidase-catalysed synthesis of ginsenoside compound K has been previously reported (ref. 10-15), the herein reported surface display approach is experimentally advantageous in comparison with application of intracellular glycosidases.

There is a series of points to be cleared, completed and improved:

1) In Material and Methods, nothing is stated about ginsenoside substrate (source, composition). This should be included to facilitate reproducibility of the reported work.

2) “Conversion rate”: This term is widely employed through the manuscript. As it is presented, it is not a rate, but  conversion. Please revise the whole manuscript (abstract, text, tables) and replace it by “conversion”.

3) At which substrate concentration and catalytic temperature were conducted the biotransformations shown in Figure 4c? This should be included somewhere in the corresponding paragraph (lines 179-187).

4) For clarity reasons, in Figures 3b, 4c, 5, 6 and 7 legends, please detail the fixed experimental parameters of the reported biotransformation (temperature, substrate concentration, etc), so that the figures are self-explaining.

5) About ginsenoside CK:

a) It would be good to include somewhere its chemical structure.

b) In Material and Methods, nothing is stated about the source of CK standard, it would be useful to include it.

6) Regarding Material and Methods:

a) Section 3.5 (Ginsenoside CK synthesis):

a-1: It is stated: “… reaction was carried out at 37 °C…” (line 290). On the one hand, this is not the most satisfactory catalytic temperature found (30 °C); one the other hand it would be much clearer and representative of the reported work to replace “… at 37 °C…” by “… reaction was carried out at the assayed temperature and ginsenoside substrate concentration”. Afterwards, section 3.8 could be then integrated into section 3.5 and disappear as a separated section.

Moreover, a brief protocol detailing most satisfactory conditions conducting to the best conversion to ginsenoside CK, connecting with most satisfactory result given in the abstract should be added.

a-2: Section 3.8:  “at 37 °C” (line 345). Is it 37 °C or 30 °C?

a-3: It would be important to add a preparative protocol (preparative scale) for the synthesis of ginsenoside CK.

b) The protocol detailed in section 3.4 corresponds to 0.2 mM IPTG, but in the reported work a series of concentration of the inducer was assayed.

c) Please include and detail the experimental parameters corresponding to the biotransformation conducted to assay reusability (section 3.9).

7) Supplementary material: Please detail the experimental conditions of the biotransformation from which the sample (Figure S3, chromatogram (b)) was obtained.

8) PPD (line 48): please define (indicate protopanaxadiol between brackets).

9) Please check carefully the list of references. Article titles are sometimes provided with only the first letter in capital letter, sometimes with all the words of the title written with initial capital letter. Journal names have been sometimes abbreviated, sometimes not; moreover, some words of journal names have not been written with initial capital letter.  

The English of the manuscript is to be revised, some points are detailed:

"to synthesizing" (lines 11-12) is to be replaced by "to synthesize"; "for efficiently synthesizing ginsenoside" (lines 16-17), by "for efficient synthesis of ginsenoside"; "This study had" (line 22), by "This study has"; "...-glycosidase BG-1 converts" (line 45), by "...-glycosidase BG-1 to convert"; "as a catalyst for synthesizing ginsenoside" (line 58), by "as a catalyst for the synthesis of ginsenoside"; "for screening" (line 58), by "to screen"; "at the concentration of IPTG is 0.5 mM" (lines 155-156) by "at a concentration of IPTG of 0.5 mM" or "when the concentration of IPTG is 0.5 mM"; "we must" (line 163) by "we had to" (line 163); "with 15..." (line 346), by "at 15..."; "was obtained for" (line 356) by "was obtained as".

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The new submitted version and the response letter have been carefully prepared, so that the manuscript has been considerably improved.

Now I have just the following comments:

1) In new section 2.8, line 246, it is stated that the reaction went “to completion”. I think that this expression is misleading and should be removed, because it means 100 % conversion, but in Figure 7 conversion at 18 h is about 90 %, a value which is in agreement with best conversion attained in the submitted work.

2) Did you carry out work up of the scaled biotransformation reported in section 2.8, in order to isolate ginsenoside CK? I encourage the authors to include this in the manuscript because preparative biotransformations giving an isolated target product (with the corresponding attained isolated yield) are crucial to appreciate industrial potential. If not, take this comment into account by planning and reporting future research.

3) As far as I can read, the reaction time of 18 h (line 246) is the only reported reaction time involving the biotransformations of the manuscript. I appreciate that legends of Figures 3b, 4c, 5, 6, 7 and 8 are now detailed and self-explaining. I would be important to add in these legends the reaction time corresponding to the results presented in the figures. By doing this, please also add the reaction time of the biotransformation detailed in the abstract (lines 18-21).  

Author Response

Please see the attachment.

Author Response File: Author Response.pdf