Proteomic Analysis of the Effect of CaCl₂ and Sodium Citrate on Gentamicin Biosynthesis of Micromonospora echinospora SIPI-GM.01

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript from Yang et al. described a media optimization approach to improve gentamicin production in Micromonospora echinospora in combination with proteomics to explain the effects. This reviewer does not have any major concerns about the manuscript. The work seems to be appropriately done and the results are corroborated with experimental evidence. There are only two points authors should clarify in the manuscript:

1) It is unclear, why did the authors use CaCl2 and CH3COONa, as media improvement additives? Was it extrapolated from the previous unpublished results, or literature? Authors should clarify, otherwise it confuses the reader.

2) The method of the gentamicin quantification should be wrote clearer. For instance, authors should explain what is the potency? Why do they not use the concentration? I assume it is some historical method to measure gentamicin, but it is confusing for the majority of readers, which are used to the g, mg/L.

However, multiple technical errors exist in the text, and they should be addressed before the publication. Some of them are given below:

3 – Strain?

12 – Sentence is dubious, reads as C1a is synthetic, change: gentamicin C1a, a precursor of the high-efficacy and low toxicity synthetic antibiotic etimicin.

14 - … the influence of organic and inorganic salts on the gentamicin production was screened…

54 – Does (SF S1) belong to the beginning of the sentence or the end of the previous? Maybe say: The detailed scheme of gentamicin biosynthesis is given in SF S1.

56 – sisomicin and deoxystreptamine were not mentioned before. Explain in text what are these compounds.

68 – why capital in Etimicin?

71 - …C6' methyltransferase gene (gacD)…

72 - …overexpressed glycosyltransferase genes (specify) in the… Also sentence seems to be wrongly built, revise.

75 – genes in italics!

84-86 – this sentence is writing in nonscientific style, revise. “We” do not know, “we” assume using previously published results.

89 – In addition, the addition – rephrase.

91 – rephrase “we asked”.

119 – space: 37 °C, also check the manuscript for other such mistakes.

121 – you sometimes write ml, l, and sometimes mL, L – unify the style throughout the manuscript.

134-135 – references for the both methods?

139 – what is O phenyldehyde? You mean O-phthalaldehyde

136-141 – What is that done for?

142 – Explain why dubious “potency” is calculated instead of the concentration? Why don’t you calculate cumulative concentration of the whole complex?

156 – 96 h, check further.

157 – 8 Mol, more such mistakes further, check.

157 – 30 Min, check further, also sec

164 - 37◦C – write that properly

167 – space before -20, also check for other similar mistakes which often happen.

177-189 – too many spaces lost in this paragraph!!!

191-192 – M. echinospora.

193-196 – Site these resources!

200 – t

203 – Strange title. Enhancing the Gentamicin production by adding CaCl2 and Sodium Citrate to the cultivation medium.

240-241 – encourage is a wrong word, change.

245 – CaCl₂

268 – Explain what is the germ concentration? Explain why you do not have error bares here?

256 – I would avoid to call that a “significant improvement”, it is “observable” at best.

275 - processes [30]. – check you have similar mistakes multiple times in the manuscript.

352 - p-value. Small letter and italics, check in the whole paper.

473 – genes in italics!

Comments on the Quality of English Language

Language is OK, although authors shoud thoroughly proofread the manuscript for technical errors.

Author Response

1. Responses to Questions from Reviewer 1:

We appreciate the general assessment of the manuscript by the reviewers and are grateful for their suggestions, which are helpful in improving the quality of the present work and for our future studies. In the revised manuscript, all questions and suggestions from the reviewer #1 have been addressed. Because the manuscript has been revised and the text has been changed, in order to save time for the reviewer, the table below is provided to describe the changes.

Table. Changes in Text and Figures

Section	Lines	Changes
Title	3	Added the name of the strain
Abstract	12	Changed “gentamicin C1a, a precursor of the high-efficacy and low toxicity synthetic antibiotic etimicin” to “gentamicin C1a, a precursor for the synthesis of high-efficacy and low-toxicity antibiotic etimicin”
Abstract	14	Changed “organic and inorganic salts were screened to achieve the research goal” to “the influence of organic and inorganic salts on the gentamicin production was screened”
Introduction	54	Changed the description about SF S1 to “The detailed scheme of gentamicin biosynthesis is given in Figure 1.”
Introduction	68	Changed “Etimicin” to “etimicin”
Introduction	84-86	Rewrote the nonscientific statement into “In a previous study, it was found that changes in medium components, such as carbon source, nitrogen source, inorganic salt, organic salt, amino acid, vitamin, nucleoside, etc.”
Introduction	89	Rephrased “In addition” to “Besides”
Introduction	91	Rephrased “we asked” to “we aimed to determine”
Material and Methods	119; 164	Added space between “37” and “°C”
Material and Methods	157	Added the unit “g/mL” after “potency”
Material and Methods	121; 260; 261	Unified the style to “mL”
Material and Methods	134-135	Added references for both methods
Material and Methods	139	Modified “O phenyldehyde” to “O-phthalaldehyde”
Material and Methods	196	Modified “96 hours” to 96 h”
Material and Methods	157	Modified “8 M” to “8 Mol”
Material and Methods	156; 157; 158; 169; 171; 174	Modified “minutes” to “Min”
Material and Methods	167	Added space before -20
Material and Methods	177-189	Added spaces lost in this paragraph
Material and Methods	191-192	Revised “M. echinospora.”  in italics
Material and Methods	193-196	Sited relevant resources after GO, COG, KEGG, Pfam and iProX
Material and Methods	200	Revised “t” in italics
Material and Methods	203	Changed “ Enhancing the Gentamicin production by introducing CaCl2 and Sodium Citrate in Flask.” to “Enhancing the Gentamicin production by adding CaCl2 and Sodium Citrate to the cultivation medium.”
Material and Methods	240-241	Modified “encourage” to “contribute to”
Material and Methods	245	Revised “CaCl2” to “CaCl2”
Results and Discussion	256	Changed “significant” to “observable”
Results and Discussion	275; 289; 366; 367	Changed “processes” to “process”
Results and Discussion	325; 384; 400; 401; 421	Revised “P-value” to “p-value”
Results and Discussion	473	Revised genes in italics

 

The following are our responses to the rest comments that aren’t listed in above table:

Q1: It is unclear, why did the authors use CaCl2 and CH3COONa, as media improvement additives? Was it extrapolated from the previous unpublished results, or literature? Authors should clarify, otherwise it confuses the reader.

Response: We feel sorry for the inconvenience brought to the reviewer. Considering that mineral elements are essential for the growth of organisms, which serve as the providers of metallic or non-metallic elements for cell growth, the components of biological cell, the regulators of cell osmotic pressure and the co-factors of enzymes, we screened some common mineral salts in the preliminary experiment. Results showed that CaCl₂ could increase the potency of gentamicin and the proportion of C1. We screened different kinds of organic acids and organic acid salts in shake flask culture for the optimization of fermentation process, and found that sodium citrate could not only provide energy for fermentation as the carbon source, but also played a role in up-regulating PH. Given that the alkaline environment was favorable for the synthesis of gentamicin, subsequent experiments were conducted on the basis of these two substances. Details are shown in Figure 3 as follows:

Figure 3. Effects of adding inorganic salt and organic acid salt to medium during fermentation process on the gentamicin production in flask culture. A. Relative potency of gentamicin after adding different types of inorganic salts to culture media. B. Relative potency of gentamicin after exposure to different types of inorganic salts. C. Effects of CaCl₂ concentrations on titers of gentamicin C complex and relative potency. D. Effects of sodium citrate concentrations on titers of gentamicin C complex and relative potency. E. Effects of added CaCl₂ and sodium citrate on titers of gentamicin C complex and C1a proportion. F. The chromatogram of each treatment group.

 

Q2: The method of the gentamicin quantification should be wrote clearer. For instance, authors should explain what is the potency? Why do they not use the concentration? I assume it is some historical method to measure gentamicin, but it is confusing for the majority of readers, which are used to the g, mg/L.

Response: Thank you for pointing out this problem in our manuscript. The potency is a historical method to measure the concentration of gentamicin. According to the revised content, we have added the units in the method section. The specific modifications were shown as follows:

The potency (mg/L) can be calculated based on gentamicin content from the HPLC assay using the following equation:

U=(Us×St)/Ss

where U is the potency of gentamicin in the test sample (mg/L), Us is the potency of gentamicin in the reference standard (mg/L), St is the peak area of gentamicin in the test sample, Ss is the peak area of gentamicin in the reference standard.

 

Q3: 136-141 – What is that done for?

Response: We gratefully appreciate for your comment.

This paragraph was written to illustrate how to derivatize the sample for further measurement using HPLC with UV detection.

We sincerely apologize for the mistake in the paragraph describing the derivatization process for the product. Upon reviewing our research, we realized that we made an error in the method description. In this paper, we actually employed the HPLC-ELSD assay protocol, which does not involve derivatization and allows for direct detection. We have now made the necessary revisions to adapt our protocol to the HPLC assay. The revised method description is as follows:

....

The fermentation broth was acidized to 2.0 by H₂SO₄ and kept still for 4 h, after which the broth was centrifuged (12,000 rpm × 5 min) at 12000 rpm for 5 min and then the supernatant was collected.

High-performance liquid chromatography (Agilent Technologies Co., Ltd., California, USA) with NanoMicro Unisil 5-120 C18 column (250 mm × 4.6 mm, 5 μm) was available for the measurement of gentamicin potency. The mobile phase at a flow rate of 0.8 mL/min was a 0.2 mol/L trifluoroacetic acid solution and methanol (96:4). The column temperature and injection volume were 55 °C and 20 μL, respectively. The evaporative light scattering detector (ELSD) was operated in cryogenic shunt mode with a drift tube temperature of 55 °C, a carrier gas flow rate of 2.5 L/min, a carrier gas of nitrogen, and a carrier gas pressure of 0.4 MPa. The potency (mg/L) can be calculated based on gentamicin content from the HPLC assay using the following equation:

U=(Us×St)/Ss

where U is the potency of gentamicin in the test sample (mg/L), Us is the potency of gentamicin in the reference standard (mg/L), St is the peak area of gentamicin in the test sample, Ss is the peak area of gentamicin in the reference standard.

...

Once again, we sincerely apologize for any confusion caused by the incorrect protocol in the initial version. We are grateful for the reviewer's support and guidance, which have greatly contributed to the improvement of our research.

 

Q4: 268 – Explain what is the germ concentration? Explain why you do not have error bares here?

Response: Thank for your comments. The method of measuring germ concentrations was added in the manuscript, which was described as follows: The fermentation broth with a volume of 10 mL was centrifuged at 7000 rpm for 15 min and then the volume of supernatant was measured. The germ concentration (%) can be calculated by using the following equation: Cg=(Vt-Vs)/Vt*100, where Cg is the germ concentration (%), Vt is the total volume of fermentation broth (mL), Vs is the volume of supernatant(mL). As for the problem of error bars, we overlooked that error bars weren’t shown in the graph. Therefore, we have added error bars to make the revision.

 

Q5: 56 – sisomicin and deoxystreptamine were not mentioned before. Explain in text what are these compounds.

71 - …C6' methyltransferase gene (gacD)…

72 - …overexpressed glycosyltransferase genes (specify) in the… Also sentence seems to be wrongly built, revise.

75 – genes in italics!

Response: Thank you so much for your careful check. Considering that the introduction was too long, we re-wrote to reduce this part. Lengthy sentences were simplified to be more readable. Statements about synthetic steps and examples of developing strains that produce high yields of single-component C1a by employing genetic engineering techniques have been deleted. It’s noted that statements listed above have been deleted.

 

We trust that the modifications made in the manuscript and the above responses meet your and the reviewers’ expectations. We look forward to hearing from you and to publication of this manuscript. Thank you for your consideration.

 

Sincerely,

Ping Yang, Ph.D.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled (Proteomic analysis of the effect of CaCl₂ and sodium citrate on gentamicin biosynthesis of Micromonospora echinospora) by Yang et al discussed the effect of CaCl₂ and sodium citrate on the gentamicin biosynthesis of Micromonospora echinospora through label-free proteomics to produce high level of gentamicin. Authors also reported and determined the mechanisms responsible for the effect.

The work and idea are good and can be accepted after covering these issues.

1. English language and grammatical mistakes should be corrected through all MS

2-     Its better the authors draw the structures of gentamicin C1a and gentamicin C1a as figure I in the first. It will be better if figures S1 and S2 be added in the MS.

3-    Introduction is too long, please try to reduce it.

4-    All gene names should be italicized.

5-    In lines 134 and 135, reference should be added.

6-    Cities, countries, and manufacturers for all equipment should be added.

7-    All figures need improvement regarding their readability and the labels are unclear. The ligands in each figure are unreadable so, please increase font size. Resolution of Fig. 3 should be increased, Fig. 4 a and b, and Fig. 5, a and b.

8-    LC-MS/MS analysis chromatogram should be added with labels on each peak.

9-    Future perspectives of this work should be added to the conclusion.

 

 

 

 

 

Comments on the Quality of English Language

English language and grammatical mistakes should be corrected through all MS.

Author Response

Responses to Questions from Reviewer 2:

We appreciate the general assessment of the manuscript by the reviewer and are grateful for their suggestions, which are helpful in improving the quality of the present work and for our future studies. In the revised manuscript, all questions and suggestions from the reviewer #3 have been addressed. Because the manuscript has been revised and the text has been changed, in order to save time for the reviewer, the table below is provided to describe the changes.

 

Q1: English language and grammatical mistakes should be corrected through all MS

Response: We are very sorry for our negligence of grammar. We have checked the manuscript carefully and corrected grammatical mistakes one by one.

 

Q2: Its better the authors draw the structures of gentamicin C1a and gentamicin C1a as figure I in the first. It will be better if figures S1 and S2 be added in the MS.

Response: Thank you for the above suggestions. We have shown the structures of gentamicin C1a in figure 1 and added figures S1, S2 in the MS. The modifications are as follows:

Figure 1. The biosynthetic pathway of gentamicin. 2-DOS: 2-deoxystreptamine. A2: gentamicin A2. 6’-DOX: 6′-dehydro-6′-oxo-gentamicin X2.

Figure 2. Structure of gentamicin C complex (A) and Etimicin (B).

 

Q3: Introduction is too long, please try to reduce it.

Response: We have re-written to reduce this part according to the suggestion. Lengthy sentences were simplified to be more readable. Statements about synthetic steps and examples of developing strains that produce high yields of single-component C1a by employing genetic engineering techniques have been deleted. The modified introduction can be checked in the MS.

 

Q4: All gene names should be italicized.

Response: We are very sorry for our incorrect writing, and we have revised all gene names to be italicized. The adjustments are listed below:

Table S2. Differentially expressed proteins potentially related to gentamicin biosynthesis.

Pathway	Uniprot AC	Gene name	Protein name	Fold changes*		p-value*
				CaCl2	Sodium Citrate	CaCl2	Sodium Citrate
Gentamicin biosynthesis	A0A0K0K513	genD1	Putative gentamicin oxidoreductase/methyltransferase	(-)2.17	-	0.0034	-
	A0A0K0K515	genB4	Putative gentamicin aminotransferase IV	ND	(-)3.66	ND	0.0279
	A0A0K0K516	genD2	Putative gentamicin oxidoreductase	ND	ND	ND	ND
	A0A0K0K518	genB3	Putative gentamicin aminotransferase III	-	(-)8.47	-	0.001
	A0A0K0K520	genX	Putative gentamicin production protein	-	-	-	-
	A0A0K0K522	genV	Putative gentamicin exporter	-	-	-	-
	A0A0K0K523	genS2	Putative gentamicin aminotransferase II	-	(-)5.31	-	0.0316
	A0A0K0K525	genU	Putative gentamicin production protein	-	ND	-	ND
	A0A0K0K526	genK	Putative gentamicin C-methyltransferase	(-)12.36	-	0.045	-
	A0A0K0K527	genM2	Putatative gentamicin glycosyltransferase II	ND	ND	ND	ND
	A0A0K0K528	genB1	Gentamicin (Hexosaminyl-6-) aminotransferase I	-	-	-	-
	A0A0K0K532	genI	Putative gentamicin exporter	(+)4.04	-	0.0007	-
	A0A0K0K535	genQ	Gentamicin hexosamiyl-6'-dehydrogenase	-	-	-	-
	A0A0K0K6A4	genB2	Putative gentamicin aminotransferase II	(-)3.20	-	0.0427	-
	A0A0K0K6B9	genH	Putative gentamicin exporter	-	(+)2.74	-	0.0005
Glycolytic pathway1	A0A1C4YCI0	pfk	Phosphofructokinase	(-)4.31	(-)5.14	0.0166	0.0024
	A0A1C4YHB7	pk	Pyruvate kinase	-	(-)4.45	-	0.0014
TCA pathway1	A0A1C5A2F4	cs	Citrate synthase	-	(-)2.46	-	0.0405

 

Q5: In lines 134 and 135, reference should be added.

Response: Thank you so much for your careful check, and we have added references for both methods. The specific revisions made in the manuscript are as follows:

The reducing sugar was determined by Fehling's reagent method[26] and the amino nitrogen was measured by using the formaldehyde oxidation method[27].

 

Q6: Cities, countries, and manufacturers for all equipment should be added.

Response: We have checked all equipments and made sure cities, countries, and manufacturers have been added such as fermentor(Shanghai Auzone Bio-engineering equipment Co., Ltd., Shanghai, China), High-performance liquid chromatography (Agilent Technologies Co., Ltd., California, USA), EASY-nLC 1000 system (Thermo Fisher Scientific, Waltham Mass, USA), timsTOF Pro2mass spectrometer (Bruker, Karlsruhe, Germany).

 

Q7: All figures need improvement regarding their readability and the labels are unclear. The ligands in each figure are unreadable so, please increase font size. Resolution of Fig. 3 should be increased, Fig. 4 a and b, and Fig. 5, a and b.

Response: We feel sorry for the inconvenience brought to the reviewer. To improve the readability, we have increased the font size in all pictures to make the labels clear. Besides, the resolutions of all pictures have been increased. The modified figures are given below:

 

Q8: LC-MS/MS analysis chromatogram should be added with labels on each peak.

Response: Thank you so much for your careful check. Labels have been added on each peak in LC-MS/MS analysis chromatogram. The modified figures are given below:

 

Figure 3. Effects of adding inorganic salt and organic acid salt to medium during fermentation process on the gentamicin production in flask culture. A. Relative potency of gentamicin after adding different types of inorganic salts to culture media. B. Relative potency of gentamicin after exposure to different types of inorganic salts. C. Effects of CaCl₂ concentrations on titers of gentamicin C complex and relative potency. D. Effects of sodium citrate concentrations on titers of gentamicin C complex and relative potency. E. Effects of added CaCl₂ and sodium citrate on titers of gentamicin C complex and C1a proportion. F. The chromatogram of each treatment group.

 

Q9: Future perspectives of this work should be added to the conclusion.

Response: Thank you for your rigorous consideration. We have re-written this part according to your suggestion. As follows:

....

This paper explored the impact of CaCl₂ and sodium citrate on the gentamicin biosynthesis of M. echinospora through label-free proteomics. The findings of the study demonstrated that CaCl₂ and sodium citrate exerted modulatory effects on gentamicin synthesis by perturbing the metabolic process of M. echinospora. CaCl₂ disrupted methylation events in the biosynthesis of gentamicin through the inhibition of vitamin B12 synthesis, leading to an augmentation in the relative proportion of gentamicin C1a. Conversely, sodium citrate impeded primary metabolism by regulating carbohydrate metabolism, thereby facilitating the production of gentamicin secondary metabolites. This process innovation establishes a foundation for the simultaneous production of single-component gentamicin C1a and multi-component gentamicin. It has the potential to serve as a framework for creating other intricate antimicrobial drug formulations. The study demonstrates the effectiveness of using sodium citrate in the biosynthesis of secondary metabolites, resulting in more efficient production methods. Additionally, the knowledge acquired from this investigation could advance the development of pharmaceutical manufacturing technologies, particularly in producing multi-component drugs. Producers have the ability to refine production methods for cultivating multi-component antimicrobials, increase the proportion of valuable single components in multi-component drugs, and accomplish the simultaneous production of single-component and multi-component pharmaceuticals. As a result, this can enhance efficiency and reduce costs.

In summary, the study's findings identify promising directions for future antimicrobial drug biosynthesis research and pharmaceutical production applications, particularly in complex antimicrobial formulations. These advancements may catalyze the development of multi-component drug biosynthesis in the evolution of pharmaceutical production technologies.

 

We trust that the modifications made in the manuscript and the above responses meet your and the reviewers’ expectations. We look forward to hearing from you and to publication of this manuscript. Thank you for your consideration.

 

Sincerely,

Ping Yang, Ph.D.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript by Yang et al. dealt with the effect of calcium chloride and citrate on production of gentamicin by Micromonospora echinospora. The authors did the proteomic analysis to show the overall effect of calcium chloride and citrate addition on cellular and gentamycin metabolism. Paper possesses good amount of contents and information. With that being said, some work still needed to be polished to improve the overall quality of paper. Furthermore, there are many unbalanced sentences, grammatical errors and typos giving a bumpy flow to the reading. The authors are strongly encouraged to rewrite the whole manuscript for a cohesive and concise presentation of this interesting work. Thus, the reviewer thought this paper need moderate revision before considering for the publication in Fermentation.

Major Comments:

Comment 1: Authors showed the positive effect of calcium chloride or sodium citrate addition on the production of gentamycin in shake flask experiment. In addition, proteomic analysis was performed to reveal the detail mechanism. However, the combinatorial effect of both calcium chloride and sodium citrate addition together were not shown. In opposite, the bioreactor experiment was performed only with combinatorial additional of both calcium chloride and sodium citrate. Authors are suggested to clarify the reason and discuss why different experiment sets were designed: sole additional for shake flask culture and proteomic analysis, combinatorial addition for bioreactor experiment. It will be very interesting to see whether combinatorial addition further enhance the gentamycin production or not; and how it affects the metabolism.

Comment 2: Author showed the titer of C1, C1a, C2, C2a significantly improved with addition of calcium chloride or sodium citrate. Authors are suggested to compare and discuss the differences in improvement of C1, C1a, C2, C2a. In addition, authors are also encouraged to discuss why focus was made only C1a.

Comment 3: Authors are suggested to provide the table showing the obtained titer, yield and productivity of gentamycin and/or its component obtained during bioreactor experiment. In addition, authors are also suggested to compare the results with other studies.

Comment 4: One query, whether production of gentamycin could be improved with use of glucose or glycerol as sole carbon sources, yeast extract as nitrogen source instead of starch, flour and soybean powder.

Comment 5: Fig 2A is very messy. Authors are suggested to separate the figure into two parts for clear presentation.

Fig 4E is missing. Authors are suggested to rectify the mistakes.

Comment 6: Authors are suggested to transfer the table 1 to supplementary and indicate the all changes mentioned (in table 1) in figure 7.

Minor Comments:

Comment 1: Line 121: authors are rewrite “triangular shake flask” as flask only.

Comment 2: Authors are suggested to provide references for Line 134-135.

Comment 3: Authors are suggested to correct typos in Line 164.

Comment 4: Authors are suggested to make the unit of centrifugation uniform, either rpm or g.

Comment 5: Authors are suggested to write the name of organism in italics.

Comment 6: Authors are suggested to provide the full name of abbreviation used in figures.

Comment 7: In Line 203, authors are suggested to rewrite “flask” to “shake flask culture”.

Comment 7: Authors are suggested to make the scale of both Y and X-axis uniform in all figures.

Comment 8: Authors are suggested to rewrite “fed batch” to “fed batch without calcium chloride and sodium citrate”.

Comment 9: Authors are suggested to indicate fold change is whether increase or decrease in fig 7.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Responses to Questions from Reviewer 3:

We appreciate the general assessment of the manuscript by the reviewers and are grateful for their suggestions, which are helpful in improving the quality of the present work and for our future studies. In the revised manuscript, all questions and suggestions from the reviewer #2 have been addressed. Because the manuscript has been revised and the text has been changed, in order to save time for the reviewer, the table below is provided to describe the changes.

Table. Changes in Text and Figures

Section	Lines	Changes
Material and Methods	121	Rewrote “triangular shake flask” as “flask”
Material and Methods	134-135	Added references for both methods
Material and Methods	164	Corrected the style of 37 °C
Material and Methods	159; 169	Unified the unit of centrifugation to rpm
Material and Methods	193	Rewrote the name of the organism in italics
Results and Discussion	203	Changed “Enhancing the Gentamicin production by introducing CaCl2 and Sodium Citrate in Flask.” to “Enhancing the Gentamicin production by adding CaCl2 and Sodium Citrate to the cultivation medium.”

 

The following are our responses to the rest comments that aren’t listed in above table:

Q1: Authors showed the positive effect of calcium chloride or sodium citrate addition on the production of gentamycin in shake flask experiment. In addition, proteomic analysis was performed to reveal the detail mechanism. However, the combinatorial effect of both calcium chloride and sodium citrate addition together were not shown. In opposite, the bioreactor experiment was performed only with combinatorial additional of both calcium chloride and sodium citrate. Authors are suggested to clarify the reason and discuss why different experiment sets were designed: sole additional for shake flask culture and proteomic analysis, combinatorial addition for bioreactor experiment. It will be very interesting to see whether combinatorial addition further enhance the gentamycin production or not; and how it affects the metabolism.

Response: Thank you for your valuable suggestions. In this manuscript, our primary focus was to optimize fermentation conditions to enhance fermentation efficiency and increase the proportion of C1a fraction. To achieve this, we employed a combination of calcium chloride and sodium citrate during fermenter fermentation. We also included results from shake flask fermentations to complement the findings from the combined calcium chloride and sodium citrate experiments.

Furthermore, our aim was to investigate the effects of calcium chloride and sodium citrate on gentamicin fermentation results and associated metabolic processes. To elucidate the individual effects of these compounds, we conducted shaking flask fermentations and proteomics studies to analyze their respective impacts on metabolism.

The potential impacts of combinatorial addition of calcium chloride and sodium citrate on metabolism will be the subject of further investigations in future studies.

The specific revisions made in the manuscript are as follows:

....

We observed that the addition of CaCl₂ resulted in a significant increase in the proportion of gentamicin C1a. In addition, gentamicin C2 and C1 showed an increase compared to the control. Conversely, the presence of citric acid notably increased the quantity of individual components (gentamicin C1a, C2, C2a, C1) of gentamicin, although its effect on the proportion of gentamicin C1a was not as significant as that of CaCl₂ (Figure 3E, F). Considering our goal to enhance the proportion of high-value single component gentamicin C1a and overall gentamicin production, we simultaneously added both calcium chloride and sodium citrate. The results showed that compared to the group with only sodium citrate added, only gentamicin C1a showed an increase, while the other components showed no significant difference, further indicating that calcium chloride primarily enhances the proportion of gentamicin C1a (Figure 3E, F). These findings suggest that both CaCl₂ and sodium citrate additions increase gentamicin levels and the percentage of gentamicin C1a in shake flask fermentations.

Figure 3. Effects of adding inorganic salt and organic acid salt to medium during fermentation process on the gentamicin production in flask culture. A. Relative potency of gentamicin after adding different types of inorganic salts to culture media. B. Relative potency of gentamicin after exposure to different types of inorganic salts. C. Effects of CaCl₂ concentrations on titers of gentamicin C complex and relative potency. D. Effects of sodium citrate concentrations on titers of gentamicin C complex and relative potency. E. Effects of added CaCl₂ and sodium citrate on titers of gentamicin C complex and C1a proportion. F. The chromatogram of each treatment group.

 

In this research, we employed a label-free quantitative proteomics strategy to examine the underlying mechanisms behind the stimulatory effects of sodium citrate on gentamicin biosynthesis and the role of calcium chloride in promoting the production of C1a in M. echinospora. We analyzed differentially expressed proteins (DEPs) in M. echinospora under different treatment conditions.

...

 

Q2: Author showed the titer of C1, C1a, C2, C2a significantly improved with addition of calcium chloride or sodium citrate. Authors are suggested to compare and discuss the differences in improvement of C1, C1a, C2, C2a. In addition, authors are also encouraged to discuss why focus was made only C1a.

Response: Thank you for the above suggestions. We have discussed this in the Results and Discussion sections of the manuscript as follows:

...

We observed that the addition of CaCl₂ resulted in a significant increase in the proportion of gentamicin C1a. In addition, gentamicin C2 and C1 showed an increase compared to the control. Conversely, the presence of citric acid notably increased the quantity of individual components (gentamicin C1a, C2, C2a, C1) of gentamicin, although its effect on the proportion of gentamicin C1a was not as significant as that of CaCl₂ (Figure 3E, F). Considering our goal to enhance the proportion of high-value single component gentamicin C1a and overall gentamicin production, we simultaneously added both calcium chloride and sodium citrate. The results showed that compared to the group with only sodium citrate added, only gentamicin C1a showed an increase, while the other components showed no significant difference, further indicating that calcium chloride primarily enhances the proportion of gentamicin C1a (Figure 3E, F). These findings suggest that both CaCl₂ and sodium citrate additions increase gentamicin levels and the percentage of gentamicin C1a in shake flask fermentations.

...

The discussion of why we focus only on C1a has been discussed in detail in the Introduction section. As follows:

...

However, side effects such as ototoxicity and nephrotoxicity, common to aminoglycoside antibiotics, have limited the use of gentamicin[15]. A new broad-spectrum antibiotic, etimicin, that effectively fights against most gram-positive and gram-negative bacteria, can remedy these issues[16,17]. Etimicin is an aminoglycoside antibacterial with gentamicin C1a as its "mother nucleus", i.e. an ethyl group is introduced at the N1 position of gentamicin C1a (Figure 2). It is the first antimicrobial drug with independent intellectual property rights in China and was introduced to the Chinese market in 1999. It is widely used in China due to its high antimicrobial activity, broad antimicrobial spectrum, and low incidence of side effects[18]. The industrial production of etimicin requires a significant quantity of high-purity gentamicin C1a. As a result, many researchers have employed genetic engineering techniques to develop strains that produce high yields of single-component C1a[6,19-21].

...

 

Q3: Authors are suggested to provide the table showing the obtained titer, yield and productivity of gentamycin and/or its component obtained during bioreactor experiment. In addition, authors are also suggested to compare the results with other studies.

Response: We appreciate your valuable suggestion. Considering the reviewer's suggestion, we have provided the table showing the obtained yield of gentamicin and its component C1a obtained during the bioreactor experiment. Supplemented as follows：

...

In the presence of 0.1% CaCl₂ and 0.3% sodium citrate, the production of gentamicin increased to 2398 μg/mL, showing an 11.5% improvement compared to the control group without these additives (2150 μg/mL). This yield is also significantly higher than the highest reported yield in existing literature (approximately 1800 μg/mL)[30] (Figure 2A, B, C, Table S1). Moreover, the proportion of gentamicin C1a in the total gentamicin production also significantly increased, from 38% in the control group to 42% (Figure 2D, Table S1).

Table S1. Effect of CaCl₂ and sodium citrate on the fermentation process of gentamicin biosynthesis in M. echinospora.

Time（h）	Fermentation Potency (mg/L) *10^2		C1 Ratio (100%)		C1a Ratio (100%)		C2a Ratio (100%)		C2 Ratio (100%)
	CK	CS	CK	CS	CK	CS	CK	CS	CK	CS
0	2.50±0.12	2.70±0.13	19.11±0.87	15.32±0.76	55.00±1.33	57.23±0.33	9.88±0.38	10.23±0.09	16.01±0.08	17.22±0.52
12	3.90±0.13	4.20±0.16	25.00±0.26	20.33±0.42	32.03±1.21	39.96±0.21	13.88±1.99	12.98±0.27	29.09±1.04	26.73±0.48
24	6.30±0.16	7.00±0.13	18.22±1.68	17.24±0.95	52.12±1.10	57.98±0.10	11.43±0.2	12.4±0.31	18.23±0.78	12.38±1.16
36	9.10±0.17	10.10±0.14	21.45±1.87	17.36±0.69	50.98±1.50	53.23±0.50	11.25±1.43	12.54±0.83	16.32±1.8	16.87±1.02
48	11.50±0.13	12.90±0.15	22.11±0.22	18.23±0.31	49.13±1.80	52.00±0.80	11.87±0.85	13.21±1.51	16.89±0.73	16.56±1.02
60	13.90±0.15	15.20±0.16	23.44±1.66	19.34±1.22	47.22±2.10	49.43±0.18	12.77±1.18	13.45±0.52	16.57±0.74	17.78±1.56
72	15.80±0.17	17.58±0.15	24.10±0.76	21.45±0.56	46.11±2.33	48.13±0.53	13.22±1.69	14.12±0.91	16.57±0.12	16.3±0.94
84	18.20±0.09	19.00±0.10	24.31±0.69	20.21±1.11	42.96±1.50	45.15±0.50	13.87±1.74	15.3±1.05	18.86±0.93	19.34±1.66
96	19.60±0.11	20.90±0.12	24.11±0.64	23.66±1.07	41.32±1.70	45.27±0.70	14.12±1.66	16.33±1.5	20.45±0.60	14.74±1.87
108	20.70±0.13	23.10±0.14	25.23±0.49	26.57±0.09	39.40±1.22	43.34±0.72	14.24±0.77	16.57±0.31	21.13±0.04	13.52±0.324
120	21.50±0.50	23.98±0.34	25.34±1.67	25.77±1.17	38.33±1.80	42.43±0.80	15.33±0.83	16.96±1.44	21±0.70	14.84±1.81

CK denotes fed batch without calcium chloride and sodium citrate; CS denotes fed batch with calcium chloride and sodium citrate; Three biological replicates were performed, and the data were presented as mean ± SD.

...

 

Q4: One query, whether production of gentamycin could be improved with use of glucose or glycerol as sole carbon sources, yeast extract as nitrogen source instead of starch, flour and soybean powder.

Response: Thank you for your question, our answer is that the production of gentamycin couldn’t be improved with use of glucose or glycerol as sole carbon sources, yeast extract as nitrogen source instead of starch, flour and soybean powder, and the yield of gentamicin will be very low. The glucose, glycerol, and yeast extracts were screened during the previous experiments with single carbon and single nitrogen sources, and their individual use was not conducive to the synthesis of gentamicin.

 

Q5: Fig 2A is very messy. Authors are suggested to separate the figure into two parts for clear presentation.

Response: Thank you for your suggestion. We have re-written this part according to your suggestion. The modifications are as follows:

Figure 2. Effects of adding 0.1% CaCl₂ and 0.3% sodium citrate in fermentor culture by the fed-batch method on fermentation indicators and production of gentamicin. Comparisons between two processes in the presence and absence of 0.1% CaCl₂ and 0.3% sodium citrate in terms of pH, total carbonhydrate, reducing sugar, filtration rate(A), amino nitrogen, dissolved oxygen, germ concentration(B), fermentation potency(C) and C1a ratio(D).

 

Q6: Fig 4E is missing. Authors are suggested to rectify the mistakes.

Response: Thank you so much for your careful check. We are very sorry for our negligence. The corrections are as follows:

Figure 4. The differentially expressed proteins (DEPs) in different treatment groups. A. Volcano plot of DEPs in the Sodium citrate and control groups; B. Volcano plot of DEPs in the CaCl₂ and control groups; C. Numbers of differentially expressed proteins in different treatment groups. D. Hierarchical clustering analysis of the DEPs between the Sodium citrate, CaCl₂, and control groups.

 

Q7: Authors are suggested to transfer the table 1 to supplementary and indicate the all changes mentioned (in table 1) in figure 7.

Response: Thank you for the above suggestions. We have adjusted the section based on your suggestions. The adjustments are listed below:

1. We have transferred the table 1 to supplementary. As follows:

Table S2. Differentially expressed proteins potentially related to gentamicin biosynthesis.

Pathway	Uniprot AC	Gene name	Protein name	Fold changes*		p-value*
				CaCl2	Sodium Citrate	CaCl2	Sodium Citrate
Gentamicin biosynthesis	A0A0K0K513	genD1	Putative gentamicin oxidoreductase/methyltransferase	(-)2.17	-	0.0034	-
	A0A0K0K515	genB4	Putative gentamicin aminotransferase IV	ND	(-)3.66	ND	0.0279
	A0A0K0K516	genD2	Putative gentamicin oxidoreductase	ND	ND	ND	ND
	A0A0K0K518	genB3	Putative gentamicin aminotransferase III	-	(-)8.47	-	0.001
	A0A0K0K520	genX	Putative gentamicin production protein	-	-	-	-
	A0A0K0K522	genV	Putative gentamicin exporter	-	-	-	-
	A0A0K0K523	genS2	Putative gentamicin aminotransferase II	-	(-)5.31	-	0.0316
	A0A0K0K525	genU	Putative gentamicin production protein	-	ND	-	ND
	A0A0K0K526	genK	Putative gentamicin C-methyltransferase	(-)12.36	-	0.045	-
	A0A0K0K527	genM2	Putatative gentamicin glycosyltransferase II	ND	ND	ND	ND
	A0A0K0K528	genB1	Gentamicin (Hexosaminyl-6-) aminotransferase I	-	-	-	-
	A0A0K0K532	genI	Putative gentamicin exporter	(+)4.04	-	0.0007	-
	A0A0K0K535	genQ	Gentamicin hexosamiyl-6'-dehydrogenase	-	-	-	-
	A0A0K0K6A4	genB2	Putative gentamicin aminotransferase II	(-)3.20	-	0.0427	-
	A0A0K0K6B9	genH	Putative gentamicin exporter	-	(+)2.74	-	0.0005
Glycolytic pathway1	A0A1C4YCI0	pfk	Phosphofructokinase	(-)4.31	(-)5.14	0.0166	0.0024
	A0A1C4YHB7	pk	Pyruvate kinase	-	(-)4.45	-	0.0014
TCA pathway1	A0A1C5A2F4	cs	Citrate synthase	-	(-)2.46	-	0.0405

* The fold changes with (+) stand for up-regulated proteins and those with (-) stand for down-regulated proteins. The symbol of “-” stands for non-differentially expressed protein and “ND” stands for non-detected protein.

¹ Only key rate-limiting enzymes in the glycolytic and TCA pathways are shown here. Other differential expressed proteins are shown in Supplementary Figures S4, S5.

2. Considering the simplicity of the chart, we have shown the partially important changes proteins in Figure 7. As follows:

Figure 7. The general changes in gentamicin biosynthesis pathway by CaCl₂ and sodium citrate in Micromonospora echinospora. Blue represents changed protein by sodium citrate. Green represents changed protein by CaCl₂. The number in the brackets represents the fold change. Proteins with (+) fold changes represent up-regulated proteins, while those with (-) fold changes represent down-regulated proteins. EMP: Embden-Meyerhof pathway, glycolytic pathway. TCA: tricarboxylic acid cycle. 2-DOI: 2-deoxy-scyllo-inosose. X2: gentamicin X2. 6’-DOX: 6′-dehydro-6′-oxo-gentamicin X2.

 

Q8: Authors are suggested to provide the full name of abbreviation used in figures.

Response: Thank you so much for your careful check. We have provided the full names of the abbreviations in the figure in the figure legend, which are added below:

...

Figure 7. The general changes in gentamicin biosynthesis pathway by CaCl₂ and sodium citrate in Micromonospora echinospora. Blue represents changed protein by sodium citrate. Green represents changed protein by CaCl₂. The number in the brackets represents the fold change. Proteins with (+) fold changes represent up-regulated proteins, while those with (-) fold changes represent down-regulated proteins. EMP: Embden-Meyerhof pathway, glycolytic pathway. TCA: tricarboxylic acid cycle. 2-DOI: 2-deoxy-scyllo-inosose. X2: gentamicin X2. 6’-DOX: 6′-dehydro-6′-oxo-gentamicin X2.

...

 

Q9: Authors are suggested to make the scale of both Y and X-axis uniform in all figures.

Response: Thank you for your rigorous consideration. We have adjusted all figures according to your suggestion.

 

Q10: Authors are suggested to rewrite “fed batch” to “fed batch without calcium chloride and sodium citrate”.

Response: Thank you for your nice suggestion. We have re-written this part according to your suggestion. The modifications are as follows:

Figure 2. Effects of adding 0.1% CaCl₂ and 0.3% sodium citrate in fermentor culture by the fed-batch method on fermentation indicators and production of gentamicin. Comparisons between two processes in the presence and absence of 0.1% CaCl₂ and 0.3% sodium citrate in terms of pH, total carbonhydrate, reducing sugar, filtration rate(A), amino nitrogen, dissolved oxygen, germ concentration(B), fermentation potency(C) and C1a ratio(D).

 

Q11: Authors are suggested to indicate fold change is whether increase or decrease in fig 7.

Response: Thank you for your rigorous advice. We have used （+） or（-）to indicate whether fold change is increase or decrease, which are added below:

Figure 7. The general changes in gentamicin biosynthesis pathway by CaCl₂ and sodium citrate in Micromonospora echinospora. Blue represents changed protein by sodium citrate. Green represents changed protein by CaCl₂. The number in the brackets represents the fold change. Proteins with (+) fold changes represent up-regulated proteins, while those with (-) fold changes represent down-regulated proteins. EMP: Embden-Meyerhof pathway, glycolytic pathway. TCA: tricarboxylic acid cycle. 2-DOI: 2-deoxy-scyllo-inosose. X2: gentamicin X2. 6’-DOX: 6′-dehydro-6′-oxo-gentamicin X2.

 

We trust that the modifications made in the manuscript and the above responses meet your and the reviewers’ expectations. We look forward to hearing from you and to publication of this manuscript. Thank you for your consideration.

 

Sincerely,

Ping Yang, Ph.D.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The MS can be accepted. The authors did the required comments.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors reponsed the reviewers query properly and modified the manuscript accordingly. The manuscript is acceptablef for publication.

Comments on the Quality of English Language

Minor editing is needed