Discovery and Heterologous Expression of Unspecific Peroxygenases

Round 1

Reviewer 1 Report

This manuscript describes the identification of new UPOs from different source of fungi. The authors' work is original and in my view, the paper is within the scope of Catalysts. There are several points that should be address by the authors before it can be publish. All points that need authors' attention are described as follows:

 

1. Line 53-55 - The substrate scope of UPOs already exceeds 400 described compounds [10], however, further chemo-, regio- and stereoselective reactions are desired, for which no wild-type or mutant UPO is yet available. Can u please elaborate more why further chemo-, regio- and stereoselective reactions are a desired attributes for UPOs?

2. Line 92 - Is it a Neighbor-joining tree or as mentioned in line 513 a maximum likelihood tree?

3. Line 274 - performing

4. In Figure 2, why there is no PaDa control?

5. Is there any relationship between the motif-activity-expression for both UPOs families? 

 

 

Author Response

We thank the reviewer for constructive criticism and we modified the manuscript according to the suggestions. A point-by-point response letter is attached.

Author Response File: Author Response.pdf

Reviewer 2 Report

The characterization and screening of peroxygenases are certainly promising in advancing the biocatalytic oxidation reactions. I found the work by Winkler is interesting, however, some minor issues need to be address.

1.    As mentioned in Line 61, more robust UPOs were desired. What do the authors mean exactly by “robust”?

2.    In Line 68, to my knowledge, the unspecific peroxygenase from Collariella virescens (pdb_7ZCL) had structure in 2022.

3.    In Figure 1, the labels were not clear in the Figure. The authors could improve the quality of the Figure so that it is easier to read.

4.    In lines 119-121, “Numbering of the conserved motifs in 3DM is as follow: the PCP motif is P31, C32 and P33, respectively, and the second motif was numbered E119, G120/H120, D121, S123, R186 and E193, respectively.” What did it mean? Did this represent family II UPO? And which sequence was the reference?

5.    In Table 1, what does the different colors mean? Also the number and the proteins columns are confusing. Please explain the Table 1 (and Table 2.)

6.    In line 152, it mentioned that 3DM positions 22 and 40 are most abundantly (>50%) occupied by proline and serine, respectively, therefore they were kept fixed. But in table 1, most Ala and Asn occupied the position 22 and position 24, respectively. Similar questions were also in other positions, so how to explain them?

7.    The introduction section of the article has several formatting issues. For example, the "Basidiomycota" and "Ascomycota" in the fifth sentence of the second paragraph on page 2 and the last sentence of the first paragraph on page 6, as well as the strain names "AaeUPO", "MroUPO", and "HspUPO"marked in Figure 1 need to be italicized.

8.    The introduction of the article can be carefully revised, and it feels that it is not so suitable for the content of the article.

9.    In the abscises of Figures 2 and 3, the suffix of UPO needs to be explained, it refers to the position in the 96-well plate or just a code name.

10. Why is one UPO shown three times in Figures 2 and 3?

11. In the introduction part, the citation of the in-situ generation of H2O2 is in wrong orders, for instance, Ref. 20 has nothing to do with photocatalysis. The authors are also advised to cite the state-of-the-art references. Please have a check of the references very carefully.

Author Response

We thank the reviewer for careful reading and valuable suggestions. Please find our point-by-point response attached.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

All comments have been addressed by the authors. I recommend acceptance of this manuscript.