One-Pot Bi-Enzymatic Cascade Synthesis of Novel Ganoderma Triterpenoid Saponins

Round 1

Reviewer 1 Report

See my comments in the attached file.

Comments for author File: Comments.pdf

Author Response

We are very grateful for your positive remarks and the helpful comments for improving our manuscript. The manuscript has been modified accordingly. Please see our point-to-point responses to your comments in the following.

1. “……Therefore, the authors should discuss in more depth the advantage of their approach in comparison to many other studies that rely on the use of glycosyltransferases to glycosylate triterpenoids.”

Response:

Thank you very much for the comment. We added the following paragraph in the Discussion section, Line 234-250, of the revised manuscript to discuss in detail the advantage of current approach compared to those in the previous studies using glycosyltransferase to glycosylate triterpenoids.

“There are several advantages of combining BtGT_16345 and Toruzyme in catalyzing glycosylation of triterpenoids. Firstly, most of microbial GTs proven to catalyze glycosylations of triterpenoids have less regioselectivity toward the triterpenoid substrates compared with the BtGT_16345 used in the present study. For examples, UGT109A1 from Bacillus subtilis catalyzes glycosylations of ginsenosides at either C-3, C-12, or C-20 [39-40]; BsYjiC from B. subtilis catalyzed glycosylations of ginsenosides at either C-3, C-6, C-12, or C-20 [41-42]. In contrast, BtGT_16345 from B. thuringiensis regioselectively catalyzed C-15 glycosylation of GAA among its multiple glycosylation sites [24]. Secondly, present and previous studies all show that Toruzyme® catalyzes chain-growth a-(1®4) glycosylation linkages toward the sugar acceptor of the saponins [26, 30-31]. Accordingly, combining BtGT_16345 and Toruzyme achieved the specific glycosylation of GAA with maltogenic, maltotriogenic, and so on, sugar moiety. Finally, an additional advantage coming from the combination of a retaining α-glycosidase (Toruzyme®) with a glycosyltransferase (BtGT_16345) adding the first sugar unit by a β bond is that it generates saponins resistant to a-glycosidase hence stable in the following reaction process. Therefore, the strategy could be applied to other triterpenoids in producing regioselectively polyglycosylated triterpenoid saponins.”

2. Lines 110, 124, 134, 274, 276, 280 and 288: The amount of enzyme is expressed as “10% enzyme” or “10% Toruzyme”. There is a lack of consistency in the use of this expression. Sometimes it is followed by “v/v” but it is not always the case. In line 276 it is written “10% (w/v) enzyme”. This way of stating the enzyme concentration is ambiguous. I suggest using conventional units, such as U/mL (activity measured against a standard substrate) or mg/mL (mg of enzyme).

Response:

In this study, the concentration of commercial Toruzyme® was 3 KNU (kilo novo units)/mL. Accordingly, we corrected all the description 10% (v/v) Toruzyme® to 0.3 KNU/ml Toruzyme® in the revised manuscript. The concentration of commercial a-amylase from A. oryzae (Sigma 10065) was 34 U/mg. We therefore rewrote 10% (w/v) as 3400 U/mL in Line 312 of the revised manuscript. For the commercial a-amylase from B. licheniformis (Sigma A3306), the concentration is 20000~60000 U/mL from the supplier. Thus, we rewrote the used concentration 10% (v/v) as ~4000 U/mL in Line 314 of the revised manuscript. Finally, the recombinant DgAS enzyme was purified then mass concentration determined in our previous study. Since there is no definite unit measured, we wrote the mass concentration applied in the reaction, 10 μg/mL, in the manuscript.

3. Line 152: Delete “estimate”. Mass spectrometry analysis provides an accurate value of the monoisotopic molecular weight.

Response:

We deleted “estimated” in the revised manuscript Line 155.

4. Line 156: The term dehydro-glucose is correct but rarely used, since it is well-known that the mechanism of glycosylation involves the loss of a water molecule. Use simply “glucose residue” instead.

Response:

It has been rewritten as suggested in the revised manuscript Line 159.

5. Line 198: Glycosylation might also affect absorption negatively. Therefore, I suggest rewriting the sentence to state “glycosylation may also improve bioavailability”.

Response:

We have rewritten as suggested in the revised manuscript Line 201.

6. Lines 320-321: The specific pulse sequences and parameters for the NMR experiments should be provided in order to ensure reproducibility.

Response:

The pulse sequences and parameters for this study were conducted by following the basic 1D and 2D program of Bruker Topspin 4.0. We thus rewrite the description to “Standard Bruker pulse sequences and parameters (Topspin program 4.0) were used for the NMR experiments.” in the revised manuscript Line 359.

7. Line 330: What do the authors mean by “authentic samples”?

Response:

To be clear, we corrected the sentence as the following in Line 366-368 of the revised manuscript:

“The concentrations of the tested compounds were calculated based on their peak areas using calibration curves prepared with HPLC analyses of the same compounds dissolved in dimethyl sulfoxide (DMSO).”

8. Line 216-220: The text is hard to follow. I suggest rewriting the paragraph.

Response:

We rewrite the sentences as the following in the revised manuscript Line 220-225:

“In addition, previous studies have shown that Toruzyme® catalyzed a-(1®4) glycosylation linkage on the sugar acceptors of flavonoid glucoside [30] or steroidal saponins [26,31]. The result of the present study showing that Toruzyme® catalyzed a-(1®4) glycosylation linkage on the sugar acceptor of GAA-15-G (Figure 4) was consistent with the previous studies.”

9. Line 220-225: All the mentioned examples consist on the combination of different glycosidases for the synthesis of a valuable glycoconjugate. Examples of combining a glycosyltransferase and a glycosidase with the same purpose should be also added (if any).

Response:

After survey, we found a study combining a glycosyltransferase and a glycosidase in the glycosylation of resveratrol. We added the following sentence in the discussion section Line 230-231 of the revised manuscript.

“Shimoda et al. combined a plant glucosyltransferase and cyclodextrin glucanotransferaase to produce resveratrol glucosides [38].”

Reviewer 2 Report

I recomend the publication without any corrections.

Author Response

We sincerely thank you for accepting our work.

Reviewer 3 Report

Main comments

Several factors were assessed for increasing the conversion of GAA-15-G into GAA-G2, such as sugar concentration, pH, and temperature. However, it is not clear what type of experimental design was used. Also, a discussion about the interactions between different factors is missing.  

Protein accession code should be provided for BtGT_16345, for GenBank, or another public database.

Minor comments

Ganoderma” should be written in cursives “Ganoderma” 

The term “catalyze” seems misused in several parts of the manuscript, for example:

Line 93: ”catalyze GAA”.

Lines 95.96: “catalyze flavonoid 95 glucosides [30,34,35], steroidal saponins [26,31], and triterpenoid saponin ginsenoside [32];” 

Author Response

Thank you for the positive remarks and the helpful comments for improving our manuscript. Please see the following point-to-point response to the comments. Your positive consideration is highly appreciated.

1. Several factors were assessed for increasing the conversion of GAA-15-G into GAA-G2, such as sugar concentration, pH, and temperature. However, it is not clear what type of experimental design was used. Also, a discussion about the interactions between different factors is missing.

Response:

We added the following explanation of our experimental design in the Discussion section Line 251-265 of revised manuscript. Since several factors have been assessed in the literature, we designed our experiments accordingly. Thus, we did not determine the interactions between different factors in the present study.

“According to the literature [26-32], the reaction conditions for transglycosylation catalyzed by Toruzyme®, including various sugar donors, pH 5-7 and 50-60 °C, have been assessed. Here we designed our experiments seeking the optimal reaction conditions based on the literature. First, various sugar donors, including soluble starch, maltose, dextrin, and cyclodextrin, were tested in the reaction conducted by 0.3 KNU/ml of Toruzyme in 50 mM acetate buffer of pH 5, at 60 °C for 4 hr. Among these sugar donors, maltose gave the highest conversion. Therefore, different concentrations of maltose were conducted next in the same conditions mentioned above. Base on the result, the reaction condition with the best conversion using 20% (w/v) maltose was conducted except that the reaction temperatures were different. Finally, the reaction buffers with different pH values were tested in the optimized reaction condition established above. From the results (Figure 2), the optimal reaction conditions, pH 5 and reaction temperature 50 to 60 °C, are similar to the literature. The only one factor has not been assessed previously is that the maltose concentrations ranging 2-40% (w/v) affect significantly the reaction efficiency of Toruzyme®.”

2. Protein accession code should be provided for BtGT_16345, for GenBank, or another public database.

Response:

We added the genome accession number (GenBank BioProject accession no. PRJNA557365; Genome accession no. CP042270) and the protein accession number (NCBI Protein accession no. QFR29366) in the Materials and Methods section, Line 302, of the revised manuscript.

3. Ganoderma” should be written in cursives “Ganoderma”

Response:

All were rewritten as Italic font type.

4. The term “catalyze” seems misused in several parts of the manuscript, for example:

Line 93: ”catalyze GAA”.

Lines 95.96: “catalyze flavonoid 95 glucosides [30,34,35], steroidal saponins [26,31], and triterpenoid saponin ginsenoside [32];”

Response:

In Line 80, 99, 210, 211 of revised manuscript, the term “catalyze” was corrected as “catalyze glycosylation of”.

In Line 94, 102, 145, 158, 218 of revised manuscript, the term “catalyze” was corrected as “biotransform”.