Highly Effective, Regiospecific Hydrogenation of Methoxychalcone by Yarrowia lipolytica Enables Production of Food Sweeteners
Round 1
Reviewer 1 Report
The Authors of the manuscript aimed to obtain sweeteners - dihydrochalcones as a result of selective biohydrogenation of the double bond in a series of methoxychalcones. The proces was carried using 8 unconventional yeast strains. The highest seeteners productivity was observed in the culture of the Yarrowia lipolytica KCh 71 strain. In general, experiments were planned properly and explained in adequately. Obtained results were clearly presented. However, the manuscript requires some complementary infromation and some minor corrections before publication - all listed below.
LIST OF SUGGESTED CHANGES
Lines 57-58: The sentence: „In this work, we pay special attention to the study of the catalytic capacity of a strain of the species Yarrowia lipolytica” - is not consistent with the information provided in lines 82 – 87. There were tested different species: Rhodotorula rubra KCh 4, Yarrowia lipolytica KCh 71, Rhodotorula marina KCh 77, Rhodotorula rubra KCh 82, Candida viswanathii KCh 120, Rhodotorula glutinis KCh 242, Saccharomyces cerevisiae KCh 464) all with high regioselectivity during biotransformation. Authors mentioned about 8 strains while only 7 was shown here.
Line 120: spelling of the strain: „R. Glutinis KCh 242” and the same in case of Figure 1 title
Line 121: space after – (….. in the same ring)
Line 122: space after „… diametrically different”
Figure 1, Figure 2, Figure 3: no unit on y axis
Table 3: the substrate concentration unit should be standardized in the title and table (once mg/L and once g/L) - similarly when discussing the results (lines 152 – 154). The title of table 3 should be change to: „Conversion of selected substrates depending on their concentration and biotransformation time”. In tabel ther is no unit for yhe converion efficiency
Lines 187 – 190: the strain Candida parapsilosis KCh 909 was not listed in lines 82 – 87
Lines 193 – 194: what the authors mean by „Transplanted microorganisms”
Lines 235 – 273: Conclusions are too extensive. They resemble discussions of the results (some information can be linked to the results and discussion section). The conclusions should clearly summarize the obtained results and the observed relationships and set the directions for further research.
Author Response
We would like to thank the Reviewer whose comments have enabled us to improve the paper from its original version. We hope that our explanations and corrections will be satisfying and our paper will be finally accepted for publication.
Reviewer comments:
Reviewer #1 (Remarks to the Author):
The Authors of the manuscript aimed to obtain sweeteners - dihydrochalcones as a result of selective biohydrogenation of the double bond in a series of methoxychalcones. The proces was carried using 8 unconventional yeast strains. The highest seeteners productivity was observed in the culture of the Yarrowia lipolytica KCh 71 strain. In general, experiments were planned properly and explained in adequately. Obtained results were clearly presented. However, the manuscript requires some complementary infromation and some minor corrections before publication - all listed below.
LIST OF SUGGESTED CHANGES
Lines 57-58: The sentence: „In this work, we pay special attention to the study of the catalytic capacity of a strain of the species Yarrowia lipolytica” - is not consistent with the information provided in lines 82 – 87. There were tested different species: Rhodotorula rubra KCh 4, Yarrowia lipolytica KCh 71, Rhodotorula marina KCh 77, Rhodotorula rubra KCh 82, Candida viswanathii KCh 120, Rhodotorula glutinis KCh 242, Saccharomyces cerevisiae KCh 464) all with high regioselectivity during biotransformation. Authors mentioned about 8 strains while only 7 was shown here.
Response: changed according to the suggestion
Line 120: spelling of the strain: „R. Glutinis KCh 242” and the same in case of Figure 1 title
Response: changed according to the suggestion
Line 121: space after – (….. in the same ring)
Response: changed according to the suggestion
Line 122: space after „… diametrically different”
Response: changed according to the suggestion
Figure 1, Figure 2, Figure 3: no unit on y axis
Response: changed according to the suggestion
Table 3: the substrate concentration unit should be standardized in the title and table (once mg/L and once g/L) - similarly when discussing the results (lines 152 – 154). The title of table 3 should be change to: „Conversion of selected substrates depending on their concentration and biotransformation time”. In tabel ther is no unit for yhe converion efficiency
Response: changed according to the suggestion
Lines 187 – 190: the strain Candida parapsilosis KCh 909 was not listed in lines 82 – 87
Response: changed according to the suggestion and added above
Lines 193 – 194: what the authors mean by „Transplanted microorganisms”
Response: changed according to the suggestion, changed into Used microorganisms
Lines 235 – 273: Conclusions are too extensive. They resemble discussions of the results (some information can be linked to the results and discussion section). The conclusions should clearly summarize the obtained results and the observed relationships and set the directions for further research.
Response:
One paragraph, as recommended by the reviewer, was transferred to the discussion chapter. Thanks to this, conclusions are shorter and more clearly summarize the experiments described in this manuscript.
Reviewer 2 Report
The authors present a study of the reduction of a series of chalcone substrates using yeast strains. Although the substrates are established for ene-reductases, and work has been published these using recombinant enzymes, there is still room for experiments on wild-type microbiology as they may result in new enzymes for cloning and heterologous expression.
Abstract Line 11 suggest ‘number’ rather than ‘amount’
Abstract Line 15 – ‘hydrogenated at’
Abstract Line 16 ‘0.5 g L-1’ or ‘0.5 g /L’
Abstract Line 19 – ‘decreased’
Abstract Line 19 ‘The only exception was…’
Line 48 ‘Phloretin and..’
Line 53 – need a chemical scheme here that illustrates the general biotransformation reaction to be explored, otherwise the numbers in the Tables are meaningless and the reader is not clear what reactions are being performed
Line 120 ‘R glutinis’ also line 144
Line 121 ‘ring) is’
Throughout – notation – use d, min, s, h; also quantity_ space unit e.g. ‘5 mg’, ‘1 L’ etc.
Line 161 – Figure 2 is not satisfactory without error bars showing the triplicate reactions described
Conclusion – could the authors comment on how what enzymes within Yarrowia they think are catalysing the reaction, and how many of these might be contributing to the whole cell reaction?
Author Response
We would like to thank the Reviewer whose comments have enabled us to improve the paper from its original version. We hope that our explanations and corrections will be satisfying and our paper will be finally accepted for publication.
Reviewer #2 (Remarks to the Author):
The authors present a study of the reduction of a series of chalcone substrates using yeast strains. Although the substrates are established for ene-reductases, and work has been published these using recombinant enzymes, there is still room for experiments on wild-type microbiology as they may result in new enzymes for cloning and heterologous expression.
Abstract Line 11 suggest ‘number’ rather than ‘amount’
Response: changed according to the suggestion
Abstract Line 15 – ‘hydrogenated at’
Abstract Line 16 ‘0.5 g L-1’ or ‘0.5 g /L’
Abstract Line 19 – ‘decreased’
Abstract Line 19 ‘The only exception was…’
Line 48 ‘Phloretin and..’
Response: All changed according to the suggestion
Line 53 – need a chemical scheme here that illustrates the general biotransformation reaction to be explored, otherwise the numbers in the Tables are meaningless and the reader is not clear what reactions are being performer
Response: scheme was added, according to the suggestion
Line 120 ‘R glutinis’ also line 144
Line 121 ‘ring) is’
Throughout – notation – use d, min, s, h; also quantity_ space unit e.g. ‘5 mg’, ‘1 L’ etc.
Response: changed according to the suggestion
Line 161 – Figure 2 is not satisfactory without error bars showing the triplicate reactions described
Response: This diagram is only illustrative, more detailed data including repetitions are in Table 3.
Conclusion – could the authors comment on how what enzymes within Yarrowia they think are catalysing the reaction, and how many of these might be contributing to the whole cell reaction?
Response:
On the basis of the obtained results, it can be assumed that the enzyme catalyzing this process is ene-reductase and it is a constitutive enzyme. However, identification which group of enzymes catalyzes this reaction requires the use of molecular biology methods.
