Identification of Five Robust Novel Ene-Reductases from Thermophilic Fungi

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article “Discovery of five robust ene-reductases from thermophilic fungi” by Pedro Damada and Marco Fraaije is devoted to the structural and functional analysis of five new fungi ene-reductases. The authors had their sights on new ene-reductases from fungi genomes as the source of more thermostable and solvent-resistant enzymes. Five new thermostable ene-reductases were characterized biochemically and structurally using AlfaFold models.

The high percent match  (using iThenticate) deals with the published thesis of Pablo Damada “Discovery and biocatalytic exploration of fungal ene-reductases ”. The submitted manuscript is written using the material of this thesis.

Comments:

1.In Table S2 , Table 3 and Table 2 the values should be reported in the correct form (4.18 ± 0.01), showing the correct number of significant digites. An error value should contain only one significant digit. The rounding of the average should be corrected accordingly.  

2.Please, add a scheme of the catalyzed by ene-reductases reaction

3.The choice of the substrates for the substrate acceptance analysis should be explained in more detail.

4. The advantages of the new thermostable fungal ene-reductases should be demonstrates in bioconversion experiments. The reduction of R-carvone or any other substance should be conducted at high temperature and in the water-organic mixture.

 

5. The word “discovery ” in the title should be changed to something less pathetic.

Author Response

We thank the reviewer for the constructive comments. We have addressed them as can be read below, and resubmitted a revised version of the manuscript. Changes in the manuscript are indicated in yellow.

 

Comment 1: In Table S2, Table 3 and Table 2 the values should be reported in the correct form (4.18 ± 0.01), showing the correct number of significant digites. An error value should contain only one significant digit. The rounding of the average should be corrected accordingly.  

Response 1: We corrected the tables as suggested.

 

Comment 2: Please, add a scheme of the catalyzed by ene-reductases reaction

Response 2: As suggested, we added a scheme of the catalyzed reaction to the introduction section.

 

Comment 3: The choice of the substrates for the substrate acceptance analysis should be explained in more detail.

Response 3: As suggested, we added the information concerning the reasoning behind the substrate selection.

 

Comment 4: The advantages of the new thermostable fungal ene-reductases should be demonstrates in bioconversion experiments. The reduction of R-carvone or any other substance should be conducted at high temperature and in the water-organic mixture.

Response 4: This initial study has focused on disclosing the properties of these newly-identified reductases. Tus, we prioritized experiments to reveal their expressibilities, pH profile for activity, steady-state kinetics, (solvent & pH & thermal) stabilities, and substrate ranges. We also performed conversion of one substrate using all five reductases. Further studies, such as the suggested bioconversions at higher temperatures and with different substrates, can be pursued in future projects to provide additional insights into the potential of these thermostable fungal ene-reductases.

 

Comment 5: The word “discovery” in the title should be changed to something less pathetic.

Response 5: As suggested, we have replace ‘discovery’ by ‘identification’.

Reviewer 2 Report

Comments and Suggestions for Authors

The work presented for review concerns the search for new stereoselective ene-reductases. These are enzymes with potential industrial applications in stereoselective reduction reactions. The work is written in a clear manner, and contains all the necessary information both theoretical (introduction) and practical (materials and methods). The results are presented in a clear and readable manner. I have no major comments. Perhaps only the statement ”Considering that maleimide and p-benzoquinone were the substrates with relatively high activities,...."(line 218). It is generally accepted that it is the enzymes that show activity relative to the substrates and not vice versa.

 

 

Author Response

We thanks the reviewer for the positive evaluation of our manuscript.

Comment 1: Perhaps only the statement ”Considering that maleimide and p-benzoquinone were the substrates with relatively high activities,...."(line 218). It is generally accepted that it is the enzymes that show activity relative to the substrates and not vice versa.

Response 1: We rephrased the sentence as follows: 'Considering that the enzymes showed relatively high activity towards maleimide and p-benzoquinone...'"

Reviewer 3 Report

Comments and Suggestions for Authors

In this article, the authors explored fungi that can grow at temperatures of 50-55 °C and they expected that the enzymes produced and utilized by these fungi are relatively thermostable compared to ERs from microbes. Specifically, they focused on the predicted proteomes of the following thermophilic fungi for the discovery of Old Yellow Enzyme (OYE-type ERs): Aspergillus thermomutatus, Chaetomium thermophilium, Thermothielavioides terrestris, Lachancea thermotolerans, Ogataea polymorpha. Finally, they report the biocatalytic properties of the five respective OYEs,

and shown to be efficient in reducing one or more of the tested subtrates. CtOYE and OpOYE shown to be the most stable, with melting temperatures of >60 °C. This confirms that enzymes from thermophilic organisms is efficient in expanding the toolbox of available biocatalysts. AtOYE exhibits a rather broad substrate scope, being active on all tested compounds. TtOYE showed relatively high catalytic efficiencies. Using R-carvone, both enzymes, AtOYE and TtOYE, promoted almost the complete conversion into the product desired [(R,R)-diastereoisomer]. The data show that, with this newly identified set of OYEs, new robust biocatalysts have been identified with attractive biocatalytic properties.

Author Response

We thank the reviewer for the positive evaluation of our manuscript. We appreciate your recognition of the potential of the Old Yellow Enzymes (OYE-type ERs) derived from thermophilic fungi, particularly regarding their thermostability and broad substrate scope.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript now is ready for publication