Heterologous Biosynthesis of Hyaluronic Acid Using a New Hyaluronic Acid Synthase Derived from the Probiotic Streptococcus thermophilus
Round 1
Reviewer 1 Report
Hyaluronic acid (HA) a is valuable multipurpose commodity. Its microbial production is hampered by the low effectivity of producer strains, their pathogenicity, or low capacity to form intermediates of HA pathway. To avoid these problems, the authors were expressing Streptomyces thermophilus hyaluronic acid synthase (Sthas) gene originating from low-yielding Streptomyces thermophilus in metabolically engineered Bacillus amyloliquefaciens strain. By manipulating precursor supply modules and the global regulatory factor, and optimizing fermentor conditions it was possible to increase the production of HAS significantly.
The manuscript is well written, with several minor faults.
Querries:
1) UDP dehydrogenase should read UDPG dehydrogenase in the whole manuscript
2) medium optimization: give the complete list of carbon sources tested
3 "carbon-derived“ sucrose, was used, what do you mean?
4) have you observed the carbon catabolite repression effect?
In conclusion, the paper brings valuable information on the possibility of bacterial HA production using heterologous expression of has gene in food-grade nonpathogenic bacteria.
Author Response
- UDP dehydrogenase should read UDPG dehydrogenase in the whole manuscript
Response: Thank you for your suggestion. We have revised the mistake according to the reviewer's comment in the revised manuscript. (Page 1, Line 18; Page 2, Line 71; Page 4, Line 108, 109; Page 5, Line 168; Page 6, Line 217; Page 8, Line 250; Page 9 Line 259, Line 262; Page 15, Line 443).
- Medium optimization: give the complete list of carbon sources tested.
Response: Thank you for your suggestion. We have listed information on the carbon sources tested in the supplementary material according to the reviewer's comment in the revised manuscript. (Figure S1).
- "Carbon-derived“ sucrose, was used, what do you mean?
Response: Thank you for your suggestion. We mean sucrose as a source of carbon and we have corrected the spelling mistake according to the reviewer's comment in the revised manuscript. (Page 4, Line 141).
- Have you observed the carbon catabolite repression effect?
Response: Thank you for your suggestion. Sorry, we did not observe the carbon catabolic repression effect.
Author Response File: 
Author Response.doc
Reviewer 2 Report
The report is in the attached file.
Comments for author File: 
Comments.pdf
Author Response
- Abstract – To remove the standard deviation data.
Response: Thank you for your suggestion. We have removed the standard deviation data in the abstract according to the reviewer's comment in the revised manuscript. (Page 1, Line 21).
- Page 4, lines 128-137 – Provide the composition of the optimized growth medium and cultivation conditions.
Response: Thank you for your suggestion. We have provided the composition of the optimized growing medium and conditions according to the reviewer's comment in the revised manuscript. (Page 4, Line 129-131).
- Page 4, lines 151-152 – Write a brief HPLC analysis condition.
Response: Thank you for your suggestion. We have added HPLC methods according to the reviewer's comment in the revised manuscript. (Page 5, Line 150-156).
- Page 4 - Include a description of the statistical analysis.
Response: Thank you for your suggestion. We have added statistical analysis according to the reviewer's comment in the revised manuscript. (Page 5, Line 162-163).
- Page 6, line 191 – Check the order of the Figure 2 and Figure 3.
Response: Thank you for your suggestion. We have changed the order of the Figure 2 and Figure 3 according to the reviewer's comment in the revised manuscript. (Page 6, Line 196, Line 207, Line 213, Line 216; Page 7, Line 232).
- Figure 4 - Give an explanation the various superscript letters in the same row (a, b, c, d, e, f).
Response: Thank you for your suggestion. We have added an explanation of the various superscript letters in the same row according to the reviewer's comment in the revised manuscript. (Page 9, Line 270-271).
- Why was the experiment conducted within 72 h? and not extended further? What factors limited the growth of the strain after 36 h? What other products have been synthesized during fermentation (HA production was low)? What is theoretical yield of HA from sucrose? Change the scales (from zero).
Response: Thank you for your suggestion.
(1) From 36 h to 72 h, the sucrose content in the medium is almost exhausted and there is not enough energy to support the growth of the thalli, so the cell density starts to de-crease and tends to stabilize. At the same time, bacterial metabolic activity is reduced and HA accumulation is slow, so fermentation stops at 72 h. And we have added relevant information according to the reviewer's comment in the revised manuscript. (Page 14, Line 424-427).
(2) During fermentation there is also a small amount of exopolysaccharide synthesis, which may utilize some of the energy substances, resulting in lower HA yield.
(3) The theoretical yield of HA from sucrose is 5.57 ± 0.11 g/L. (Page 14, Line 428).
(4) We have changed the vertical axis to sucrose concentration, and the scale from zero according to the reviewer's comment in the revised manuscript. (Figure 8).
Reviewer 3 Report
Hyaluronic acid (HA) is a polymer of repeating units of glucuronic acid and N-acetylglucosamine, which is in great demand in various industries. Various attempts have been made to develop a safe microorganism for the synthesis of HA. However, the HA titers obtained in these assays are generally lower than those obtained by natural, pathogenic producers. In recent years attention has been paid to ways to increase the length and titer of HA molecules in already developed strains. The manuscript presented for review describes the promising research on the genetically recombinant Bacillus amyloliquefaciens. I found the article to be generally well written and of interest to a wide range of scientists. However, it needs improvement before publishing. Below are my detailed comments.
1. In the Introduction section, in the lines 59-66, values ​​should be given, not just that the HA was produced in high or low yields. The reader should first find out what amounts of HA can be produced with the participation of microorganisms.
2. In the line 131: "Power"- what does it mean there?
3. In the line 140: Please provide the model and manufacturer of the 5L fermenter
4. In the Analytical method section, there should be described in details HPLC methodology applied for HA determination in the samples. Moreover, there should be also descriebed how was sugar content estimated.
5. In the 2. Materials and methods, there is no information on the statistical analysis. How many biological repeats, technical repeats?
6. In the Fig. 4 and Fig. 6, there are letters a - f, which are not explained.
7. There are too many words containing "ferment" in lines 400-409, please correct the language.
8. Please provide the reference/ references to the data described in the lines 431-439
Author Response
- In the Introduction section, in the lines 59-66, values hould be given, not just that the HA was produced in high or low yields. The reader should first find out what amounts of HA can be produced with the participation of microorganisms.
Response: Thank you for your suggestion. We have increased specific HA yield according to the reviewer's comment in the revised manuscript. (Page 2, Line 65).
- In the line 131: "Power"- what does it mean there?
Response: Thank you for your suggestion. We have corrected the spelling mistake according to the reviewer's comment in the revised manuscript. (Page 4, Line 130).
- In the line 140: Please provide the model and manufacturer of the 5L fermenter
Response: Thank you for your suggestion. We have provided the model and manufacturer of the 5L fermenter according to the reviewer's comment in the revised manuscript. (Page 4, Line 138-139).
- In the Analytical method section, there should be described in details HPLC methodology applied for HA determination in the samples. Moreover, there should be also descriebed how was sugar content estimated.
Response: Thank you for your suggestion. We have added HPLC methods and how estimate the sugar content according to the reviewer's comment in the revised manuscript. (Page 5, Line 151-156).
- In the 2. Materials and methods, there is no information on the statistical analysis. How many biological repeats, technical repeats?
Response: Thank you for your suggestion. We have added statistical analysis according to the reviewer's comment in the revised manuscript. (Page 5, Line 162-163).
- In the Fig. 4 and Fig. 6, there are letters a - f, which are not explained.
Response: Thank you for your suggestion. We have added an explanation of the letters a - f according to the reviewer's comment in the revised manuscript. (Page 9, Line 270-271; Page 11, Line 335-336).
- There are too many words containing "ferment" in lines 400-409, please correct the language.
Response: Thank you for your suggestion. We have corrected the language according to the reviewer's comment in the revised manuscript. (Page 13, Line 406-409; Page 14, Line 410-432).
- Please provide the reference/ references to the data described in the lines 431-439.
Response: Thank you for your suggestion. We have added the references according to the reviewer's comment in the revised manuscript. (Page 14, Line 441; Page 15, Line 445, Line 450).
