Efficient Asymmetric Synthesis of (S)-N-Boc-3-hydroxypiperidine by Coexpressing Ketoreductase and Glucose Dehydrogenase
Round 1
Reviewer 1 Report
In this paper, X. Gao et al. report on the synthesis of Boc-protected, enantiomerically pure (S)-3-hydroxypiperidine via enzymatic reduction of the respective ketone. The mentioned molecule is a known key building block for large-scale preparation of anticancer drug ibrutinib, and the presented method offer straightforward access to optically pure material (>99% ee) under optimized reaction conditions. In the light of nicely cited previous work e.g. by Xu and Chen groups, the novelty of this work relies on using ketoreductase and glucose dehydrogenase as co-catalytic system coupled in the same cell (E. coli). Despite limited access to the studied biocatalytic system and hence limited practical applications, the reported result is of some interest and can be published in the Journal.
Author Response
Special thanks to you for your good comments.
Reviewer 2 Report
The research Article, Efficient asymmetric synthesis of (S)-N-Boc-3-hydroxypiperidine by co-expressing ketoreductase and glucose dehydrogenase, by Gao and co-workers demonstrated a highly efficient biosynthesis of (S)-N-Boc-3-hydroxypiperidine by engineering an E coli strain to co-express the KRED and GDH under one single T7 promoter. The authors also optimized the catalysis conditions to obtain the most efficient substrate conversion. The paper reads well, and the experiments were designed and conducted rigorously. I only have a few comments and questions.
- Page 1, line 41, coenzyme should be coenzymes
- Page 3, line 87, are there any specific reasons for the authors to choose KRED from Canndida glabrata and GDH from Bacillus sp.? Potentially, if KRED and GDH from other organisms are chosen, is there a chance to further enhance the catalytic efficiency?
- Figure 1, panel a and b have never been mentioned in the context.
- The author chose T7 as the promoter in these experiments, which is a very strong promoter. Have the authors thought about other promoters? Possibly, another promoter or a combination of two different promoters could give a better activity balance?
- Figure 2, are these samples cell lysates or purified proteins? They looks like purified proteins.
Author Response
1. Page 1, line 41, coenzyme should be coenzymes.
Response: We are very sorry for our negligence, the “coenzyme” has been replaced with “coenzymes” (line 41).
2. Page 3, line 87, are there any specific reasons for the authors to choose KRED from Canndida glabrata and GDH from Bacillus sp.? Potentially, if KRED and GDH from other organisms are chosen, is there a chance to further enhance the catalytic efficiency?
Response: The KRED used in this work was the mutant (F92C/F94W) of wild-type enzyme from Canndida glabrata. On this basis, we are trying to further improve its catalytic efficiency via molecular modification. The GDH from Bacillus sp. used in this work exhibited nice catalytic ability, which has been used in the industrial production of chiral alcohol, such as (S)-CHBE. For the above reasons, these two enzymes were selected for this work.
If the protein expression level and catalytic ability of KRED and GDH from other organisms were better than the enzymes used in this work, the catalytic efficiency will be enhanced absolutely. This is a critical and meaningful suggestion, which will be carried out in our further research. Thanks for the reviewer’s suggestion.
3. Figure 1, panel a and b have never been mentioned in the context.
Response: Figure 1, panel a and b have been mentioned in line 325 and line 332, respectively.
4. The author chose T7 as the promoter in these experiments, which is a very strong promoter. Have the authors thought about other promoters? Possibly, another promoter or a combination of two different promoters could give a better activity balance?
Response: The pET vectors with T7 promoter have been widely used for the heterologous expression of enzymes in E. coli. Moreover, the high-density fermentation process of recombinant E. coli containing pET vector has been established, the OD600 can reached more than 200, and the production efficiency of recombinant enzymes was greatly improved. In view of this, we chose T7 as the promoter. In the further work, the constitutive promoter will be selected for the co-expression research. Thanks for the reviewer’s suggestion.
5. Figure 2, are these samples cell lysates or purified proteins? They look like purified proteins.
Response: The samples in figure 2 are cell lysates. After the optimization of codon and expression condition, KRED and GDH used in this work were well expressed in the soluble form. In order to compare the expression levels of the two proteins clearly, the cell lysates were diluted before SDS-PAGE analysis, resulting in the shallow bands of other proteins in the cell lysates.
Special thanks to you for your good comments.
Reviewer 3 Report
This paper describes the biocatalytic asymmetric synthesis of (S)- N-BOC 3-hydroxypiperidine by an optimized co-expressing ketoreductase and glucose dehydrogenase. The topic is of some interest expecially for industrial applications of this compound. In my opinion, the manuscript may be published after minor revisions of the text.
First, the authors should indicate the scale (amount of substrate) used in the experiments, which could affect conversion and yield of the product.
Line 70: "3-piperidine ketone" should be replaced with "3-piperidone or piperidinone"; Lines 95 and 120 (Scheme 1 and Figure 1): "Schematic representation" instead of "Schematic presentation"; line 256: "conversion was higher than" instead of "conversion was higher, which was higher than".
Finally, the conversion and optical purity were "determined" not "calculated". wherever this is mentioned in the text.
Author Response
1. First, the authors should indicate the scale (amount of substrate) used in the experiments, which could affect conversion and yield of the product.
Response: Thanks for the reviewer’s suggestion, we have added the scale (at 100 mL scale) in the main text (line 387).
2. Line 70: "3-piperidine ketone" should be replaced with "3-piperidone or piperidinone"; Lines 95 and 120 (Scheme 1 and Figure 1): "Schematic representation" instead of "Schematic presentation"; line 256: "conversion was higher than" instead of "conversion was higher, which was higher than".
Response: Thanks for the reviewer’s correction. "3-piperidine ketone" has been replaced with “3-piperidone” (line 70); "Schematic presentation" has been replaced with "Schematic representation" (line 95 and 120); "conversion was higher, which was higher than" has been replaced with "conversion was higher than" (line 256).
3. Finally, the conversion and optical purity were "determined" not "calculated". wherever this is mentioned in the text.
Response: Considering the reviewer’s suggestion, we have replaced the "calculated" with “determined”, when the conversion and optical purity was described in the text (line 157, 372, and 394).
Special thanks to you for your good comments.
