Improved Production of Kynurenic Acid by Yarrowia lipolytica in Media Containing Different Honeys

Round 1

Reviewer 1 Report

The manuscript by Wróbel-Kwiatkowska et al. describes production of kynurenic acid by yeast Yarrowia lipolytica. The aim of the study is generally interesting, but the manuscript needs improvement.

Recommendations:

1. Language correction is recommended.
2. Data for biomass growth should be added.
3. Data for KYNA production on fructose medium should be added.
4. Table 1 should be corrected, now it looks like three independent tables.
5. 2 should be corrected, now it looks like unfinished draft.

Questions:

1. Is lipolytica able to accumulate KYNA from environment? If yes, how could you distinguish between synthesized KYNA in yeast cells and KYNA accumulated from honey?
2. Since honey is precious product is it reasonable to use it as a substrate? Is the yeast biomass obtained by cultivation with honey a value added product?

Author Response

Answers to Referees’ comments and suggestions:

Rev. 1.

1. The language correction was done.
2. The data for biomass in the cultures with chestnut honey was incorporated into the ms in the new version.
3. The data for KYNA production in medium containing fructose was added into new version of the ms: “In our previous study [11] it was found that cultivation of Yarrowia lipolytica S12 under identical laboratory condition in medium containing optimal concentration of fructose (40 g/L) resulted in KYNA content reaching 7.9 mg/L and 39.5 mg/kg CDW in medium and biomass, respectively.”
4. Table 1 (current Table 3) was corrected and statistical analysis was performed and added to the data.
5. Table 2 was improved according to the Reviewer’s suggestion.

Questions:

1. There is no data on the uptake and accumulation of kynurenic acid by yeast in the literature. Our data published previously (Wróbel-Kwiatkowska et al., 2020) and presented in the manuscript shows that kynurenic acid produced by yeast is present also in incubation medium. To demonstrated or exclude uptake of kynurenic acid by yeast, the labeled kynurenic acid should be utilized in the experiments. However it was not the aim of this study.    

(Wróbel-Kwiatkowska M, Turski W, Kocki T, Rakicka-Pustułka M, Rymowicz W. An efficient method for production of kynurenic acid by Yarrowia lipolytica. Yeast. 2020 Apr 24. doi: 10.1002/yea.3469)

2. Honey is in fact a precious product, but as we showed in the ms the production of KYNA in medium with chestnut honey is much more efficient when compared to the medium containing comparable amount of fructose. The reason for this phenomenon is unknown. It can be speculated that honey is a complex mixture of different components also from KYNA pathway, which may stimulate KYNA production.

Author Response File: Author Response.docx

Reviewer 2 Report

I have carefully read the paper entitled 'Improved production of kynurenic acid by Yarrowia lipolytica in the media containing different honeys' (sustainability-974006) and I found it interesting and worth to publish. 

The research deals with production of kynureic acid (KYNA) by the yeast Yarrovia lipolityca using dilutet honey and fructose media. Additionally, the Authors studied lipid and protein production by said yeast along with fatty acids and essential amino acid in the obtained biomass. The Authors found that chestnut honey is the highest KYNA yielding substrate for biosynthesis and most of the KYNA is accumulating in the biomass while some can be detected in the fermentation media. Those are valuable results showing a new way for utilizing Y. lipolityca yeast for production of therapeutical compounds of high value.

Production of valuable chemicals via biotechnological methods is a viable alternative to chemical synthesis of extraction from natural sources. Therefore I feel that the topic of this study is relevant to the journal's scope. 

In my opinion the research is well designed and the paper is written in comprehensible way.  I don't have any comments to the way the paper was written. However, I feel that unsatisfied with the lack of the determination of KYNA in the used media (i.e. honeys used for cultivation). This would give us the information about the actual production yields. The cited literature (position 9) says that KYNA levels in chestnut honey ranges ca. 0.13-0.6 mg/g. Therefore the concentration of media with 50 g/L honey can reach 6,5-30 mg/L. Of course the values are lower than those obtained in the paper, nevertheless actual prodution yields would improve the practical apsect of the results. One more thing to improve is the providing of the KYNA levels  obtained in synthetic fructose media. 

Besiedes that I feel that the paper could be published in its present form.

Author Response

Rev 2.

1. The data concerning the level of KYNA in used media was added into the new version of the ms and presented in Fig. 1A. Thus total content of KYNA in broths at the end of incubation and de novo produced KYNA by yeast was determined. To calculate de novo produced KYNA, content of KYNA in medium on day 0 (after addition of honey) was subtracted from total content of KYNA in medium at the end of incubation. It can be seen that amount of KYNA originated from honey was marginal in comparison to de novo produced KYNA with the exception of chestnut honey. Interestingly, production of KYNA by yeast was not inhibited by high amount of product in extracellular compartment.
2. The data for KYNA production in medium containing fructose was added into new version of the ms.

Author Response File: Author Response.docx

Reviewer 3 Report

this article is an interesting approach to KYNA production using Y lipolytica.

the introduction is short and did not explain the importance of the study, why they used honey as carbon source or why to try different types of honey, and the reason or necessity to improve the production. please include more information in the introduction  

the experimental design should be included and the statistical analysis supporting the selection of treatments should be also included.

the conclusion is not clear and should be rewriten

Author Response

Rev.3

1. The Introduction section was corrected and the importance of the study was explained.
2. Statistical analysis was performed and the data was incorporated into the new version of the article.
3. Conclusion chapter was completely rewritten, it is believed that in present form it is more clear.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for your resposnses.