Two-Step Optimization for Improving Prodigiosin Production Using a Fermentation Medium for Serratia marcescens and an Extraction Process

Round 1

Reviewer 1 Report

Since PG has significant biomedical value, the authors optimized the culture medium components and PG extraction process from Serratia marcescens. The results would provide essential theoretical and experimental support for the industrial production of PG.

Some changes need to be made.

1.      For the references inserted in the main text, it should have a space; and the references should be added at the end of the sentences. Carefully check all the context.

2.      In the title, Serratia marcescens strain name should be italic.

3.      In the Tables, could you describe the meaning of 1, 0 and -1?

generally it's good.

Author Response

1. For the references inserted in the main text, it should have a space; and the references should be added at the end of the sentences. Carefully check all the context.

The authors’ answer: Thanks for your suggestion, we have corrected all the references in the original text.

2. In the title, Serratia marcescens strain name should be italic.

The authors’ answer: Thanks for your suggestion, we have made changes in the title.

3. In the Tables, could you describe the meaning of 1, 0 and -1?

The authors’ answer: Thanks for your suggestion. In the tables for the PB test, the path of steepest ascent method, and BBD design -1 stands for low level, 1 for high level, and 0 for medium level. The labels 1, 0 and -1 are not only concise, but also give a clearer idea of how the experiment was run.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study, the authors reported the improvement of prodigiosin production in Serratia marcescens via optimization of fermentation medium composition as well as extraction protocol. The authors used Box-Behnken design (BBD) to optimize the response surface of the fermentation medium and this strategy led to a 64-fold increase in prodigiosin production compared to the initial medium. Additionally, optimization of the response surface of the extraction process further improved the yield. While this is an interesting study, many areas in the manuscript need further clarification.

1)    With the medium recipe and extraction protocol reported here it’s worth checking out their utility for an engineered strain where prodigiosin production has already been improved. Did you authors test any engineered Serratia marcescens strain with their current medium recipe and extraction protocol?

2)     Production of prodigiosin in the control group is 25.03±2.13 mg/L. while the yield in Experimental group 2 is 2142.75±12.55 mg/L. In my calculation, it’s approximately an 85-fold increase. Could the authors explain how it’s a 106-fold increase?

3)     Please pay attention while reporting the units. Table 8: the unit should be changed to mg/L from g/L.

4)     The comparison (%) column in Table 8 is confusing. In both Experimental group 1 and Experimental group 2, the comparison should be made based on the control group.

5)     ‘Sun et al. [42] inserted a fusion fragment comprising the genes proC, serC, and metH, encoding for proline, serine, and methionine, into the CpxR gene….’ Please specify what those genes encode for. Is it biosynthesis or degradation of those amino acids?

Author Response

1. With the medium recipe and extraction protocol reported here it’s worth checking out their utility for an engineered strain where prodigiosin production has already been improved. Did you authors test any engineered Serratia marcescens strain with their current medium recipe and extraction protocol?

The authors’ answer: Thanks for your careful reading, we have not yet validated it with engineered strains to this day. However, our team has analyzed the whole genome of Serratia marcescens ZPG19 [1], and we will carry out genetic engineering modification on the strains and validate it based on your suggestion in the next step.

• Li, X.; Tan, X.; Zhang, J.; Zhang, J. Complete Genome Sequences of One Prodigiosin-Producing Serratia marcescens Strain ZPG19. Frontiers in Bioengineering and Biotechnology 2021, 9, doi:10.3389/fbioe.2021.665077.

2. Production of prodigiosin in the control group is 25.03±2.13 mg/L. while the yield in Experimental group 2 is 2142.75±12.55 mg/L. In my calculation, it’s approximately an 85-fold increase. Could the authors explain how it’s a 106-fold increase?

The authors’ answer: Thanks for your suggestion. We obtained 106-fold increase mistakenly due to my carelessness. We believe that your suggestion is more reasonable, now we have made corrections in the original text according to your suggestion.

3. Please pay attention while reporting the units. Table 8: the unit should be changed to mg/L from g/L.

The authors’ answer: Thanks for your careful reading, we have made the changes in Table 8.

4. The comparison (%) column in Table 8 is confusing. In both Experimental group 1 and Experimental group 2, the comparison should be made based on the control group.

The authors’ answer: Thank you for your careful scrutiny, we have amended the comparisons (%) in the table with the corrections made in the original text.

5. ‘Sun et al. [42] inserted a fusion fragment comprising the genes proC, serC, and metH, encoding for proline, serine, and methionine, into the CpxR gene….’ Please specify what those genes encode for. Is it biosynthesis or degradation of those amino acids?

The authors’ answer: Thank you for your careful reading, the reason for encoding these genes is because the authors found that In the ΔcpxR mutant strain, the transcription levels of the pig gene cluster and the genes involved in the pathways of PG precursors, such as proline, pyruvate, serine, methionine, and S-adenosyl methionine, were significantly increased, hence promoting the production of PG. It is the biosynthesis of these amino acids.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript is prepared clearly and concisely, but it needs an in-depth discussion, and at the moment it can not be accepted for publication in Fermentation. The authors focused mainly on the results of the two experiments and briefly described the obtained results along with the attached figures.

Minor suggestions:

- Latin names should be written in italics – lines 3, 16, 41 etc.

- Line 114 – Do you mean ammonium chloride (NH4Cl)? If yes, it should be corrected here and in the whole manuscript.

- Figure 2 is of very low quality.

Author Response

1. The manuscript is prepared clearly and concisely, but it needs an in-depth discussion.

The authors’ answer: Thanks very much for your professional evaluation of the thesis. As you said, our thesis is mainly a description of the experimental results and we failed to discuss it in depth, but the original purpose of our thesis is to optimize the yield of PG, and the highlight part of the thesis is the two-step optimization of the fermentation medium of Serratia marcescens and the extraction process of PG by response surface, including the results of the response surface and interactions among the factors are also analyzed, and the results of the response surface and interactions between the factors are also analyzed in the summary part. The final yield of PG is listed more clearly, which is in line with the theme of our manuscript.

We have revised the manuscript to the best of our ability in the Discussion section and marked it in yellow. We sincerely thank you for your enthusiastic work and hope that our corrections will be approved.

2. Latin names should be written in italics – lines 3, 16, 41 etc.

The authors’ answer: We apologize for our careless error and thank you for your criticism. Changes have been made in lines 3, 16 and 41 of the original text.

3. Line 114 – Do you mean ammonium chloride (NH4Cl)? If yes, it should be corrected here and in the whole manuscript.

作者回答：感谢您的仔细审查。我们已根据您的建议在第 114 行进行了更改。

4. 图 2 的质量非常低。

作者回答：感谢您的宝贵建议，培养基优化的单因素检验如图 2 所示。不同因素之间选择的药物数量存在一些差异，这不可避免地导致不同图片之间的不一致。然而，最佳选择结果如图2所示。我们希望得到您的认可。

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The manuscript has been revised and can be accepted in its present form.