Biosynthesis of Guanidinoacetate by Bacillus subtilis Whole-Cell Catalysis
Round 1
Reviewer 1 Report
I think the graphics can be improved in quality but otherwise I think it is a good job.
Author Response
Q1: I think the graphics can be improved in quality but otherwise I think it is a good job.
Response: Thanks for your constructive comment. We have improved the figures in the manuscript.
Reviewer 2 Report
Even in the case of GM E.Coli, if a strain without endotoxin production is used, the toxicity problem can be solved, so why use GRAS?
It is desirable to use the abbreviation of the same gene, such as AgaT, agat, or ATAT.
I would like to present the statistically processed data in a graph or figure.
In line 131, green fluorescence was applied to strains B1-2, B1-3, and B1-4 to obtain strains of B2-1, B2-2, B2-3, and B2-4. By the way, I am curious about the generated result of B2-1.
Fig. 3 In a and b, B3-7 is expressed in the same strain B. subtilis 168, but there is a large difference in expression level. In a, the difference was 0.062 g/L and in b, 0.18 g/L. Explain why.
Author Response
Q1: Even in the case of GM E. coli, if a strain without endotoxin production is used, the toxicity problem can be solved, so why use GRAS?
Response: First, the GARS bacterium B. subtilis has long been used as production host for industrial and food enzymes and nutraceuticals, which should be potentially more favorable for GAA production as food additives. Second, compared with E. coli, B. subtilis is less susceptible for phage infection, which should be more suitable for industrial bioproduction. Therefore, B. subtilis is selected as production host.
Q2: It is desirable to use the abbreviation of the same gene, such as AgaT, agat, or ATAT.
Response: The same abbreviation was used for the same gene in the revised manuscript.
Q3: I would like to present the statistically processed data in a graph or figure.
Response: Thanks for your comment. We have supplemented statistically processed data in the manuscript (Figure 2 and Figure 3).
Q4: In line 131, green fluorescence was applied to strains B1-2, B1-3, and B1-4 to obtain strains of B2-1, B2-2, B2-3, and B2-4. By the way, I am curious about the generated result of B2-1.
Response: Thanks for your comment. There is an error in the manuscript, which I have corrected in the second table of Table1 (in line 88). We used plasmid pHT01 as the expression vector to express agaT under the control of a strong promoter PlytR. Then the plasmid was transformed into wild-type B. subtilis 168, and strains B1-1 was obtained. Then, on the basis of strains B1-1, we attached a green fluorescent protein (GFP) after agaT by a short linker, and strains B2-1 were obtained. And
both B1-2 and B2-2 are under the control of the P43 promoter; both B1-3 and B2-3 are under the control of the P333 promoter; both B1-4 and B2-4 are under the control of the P566 promoter. However, so far, we have not detected the production of GAA. We characterized the expression intensity of agaT under the control of different promoters according to the relative fluorescence intensity, which is shown in Figure 2b.
Q5: Fig. 3 In a and b, B3-7 is expressed in the same strain B. subtilis 168, but there is a large difference in expression level. In a, the difference was 0.062 g/L and in b, 0.18 g/L. Explain why.
Response: This is due to differences in the concentration of the substrates, as well as differences in the OD600 values of B. subtilis during whole-cell transformation. In Figure 3a, we added substrates with an initial concentration of 5 mM arginine (0.871 g/L arginine) and 60 mM glycine (0.504 g/L glycine). And during the whole-cell transformation, the cells were resuspended in a 5 ml reaction system to form a cell suspension (OD600 = 200). After the reaction, we found that there was almost no arginine in the reaction system of experimental group B3-7. And the arginine content in other experimental groups was also very low. During whole-cell transformation, the reaction solution became very viscous and difficult to sample. We speculated that this was due to the viscosity of the reaction solution due to the high cell density, and the low initial substrate concentration that limits the production of GAA. In the experiment of Figure 3b, we varied the reaction conditions. We adjusted the initial substrate concentration to 20 g/L glycine and 20 g/L arginine. At the same time, we resuspended the cells to an OD600 of 60, and other conditions remained unchanged. It was found that the production of GAA in strain B3-7 increased from 0.062 g/L to 0.18 g/L after changing the reaction conditions. I have described the corresponding conditions in the corresponding places in the manuscript.
Reviewer 3 Report
The authors are searching for an environmentally friendly and efficient way to produce GAA by using the whole-cell catalysis method. This time, they challenged to produce B. subtilis strains suitable for the whole-cell catalysis by using molecular biological methods. I think this challenge is very interesting. However, this manuscript lacks the most important and essential information to evaluate their work. Therefore, we request to revise as shown in following comments.
- One of the most important pieces of information for the whole-cell catalysis is the culture conditions of the microorganism. The authors do not describe the medium composition, incubation time or temperature, and use the expression "certain time" and “fermentation broth” in the manuscript (L91). They need to describe the culture conditions for each result in the Materials and Methods, the Results, or the Supplementary materials.
- Substrate concentration is very important as a basic condition for enzymatic reactions. Also, how efficiently arginine and glycine could be converted to GAA is an important point in this study. However, the authors only describe the substrate concentrations used in the whole cell catalysis as “certain concentrations” (L95 - 96). They need to provide the substrate concentrations used for each experiment. In addition, even though the authors measured arginine and glycine concentrations in L103, there is no information on these concentrations in the manuscript. To show the superiority of this study, data on conversion efficiency should be included. If any, a comparison with other similar studies should be discussed.
- There is a 3-fold difference in the titer by the strain B3-7 in Figure 3a and 3b. Ignoring this difference may make the results in Figure 3a meaningless (e.g. about 3-fold between B3-2 and B3-7). If it is a matter of reaction or culture conditions, the authors need to describe the condition accordingly, as described in Major Comment 1 and 2. If there is any other reason, an explanation should be describe in the manuscript.
- I checked the paper listed as the Reference 20 and could not find the condition for PCR (L159). The author need to change the reference to an appropriate citation or describe the condition in the manuscript.
- The author need to describe the method about the overexpression via Pveg (L231) in the manuscript
- L35：Remove “%” from “60ï¼…”.
- L56：The Reference 12 is not suitable for the explanation that B. subtilis is regarded as GRAS.
- L94: Which is correct, 7.4? or 7.5?
- L99: Describe the manufacturer of LC-MS.
- References: Italicize the bacterial names.
Author Response
The authors are searching for an environmentally friendly and efficient way to produce GAA by using the whole-cell catalysis method. This time, they challenged to produce B. subtilis strains suitable for the whole-cell catalysis by using molecular biological methods. I think this challenge is very interesting. However, this manuscript lacks the most important and essential information to evaluate their work. Therefore, we request to revise as shown in following comments.
Q1: One of the most important pieces of information for the whole-cell catalysis is the culture conditions of the microorganism. The authors do not describe the medium composition, incubation time or temperature, and use the expression "certain time" and “fermentation broth” in the manuscript (L91). They need to describe the culture conditions for each result in the Materials and Methods, the Results, or the Supplementary materials.
Response: Thanks for your constructive comments. We have added the experimental description in the manuscript.
Q2: Substrate concentration is very important as a basic condition for enzymatic reactions. Also, how efficiently arginine and glycine could be converted to GAA is an important point in this study. However, the authors only describe the substrate concentrations used in the whole cell catalysis as “certain concentrations” (L95 - 96). They need to provide the substrate concentrations used for each experiment. In addition, even though the authors measured arginine and glycine concentrations in L103, there is no information on these concentrations in the manuscript. To show the superiority of this study, data on conversion efficiency should be included. If any, a comparison with other similar studies should be discussed.
Response: Thanks for your constructive comment. To ensure sufficient arginine and glycine to produce GAA, we increased the substrate concentration from 0.871 g/L arginine and 0.504 g/L glycine to 20 g/L glycine and 20 g/L arginine, which has been described in the “Materials and Methods” and “Results” in the manuscript. We provided excess substrate and obtained the highest intensity of GAA production. We detected GAA, arginine and glycine simultaneously using LC-MS, but arginine and glycine is not detectable.
Q3: There is a 3-fold difference in the titer by the strain B3-7 in Figure 3a and 3b. Ignoring this difference may make the results in Figure 3a meaningless (e.g. about 3-fold between B3-2 and B3-7). If it is a matter of reaction or culture conditions, the authors need to describe the condition accordingly, as described in Major Comment 1 and 2. If there is any other reason, an explanation should be described in the manuscript.
Response: In Figures 3a and 3b, the reaction conditions were different. In Figure 3a, we added substrates with an initial concentration of 5 mM arginine (0.871 g/L arginine) and 60 mM glycine (0.504 g/L glycine). And during the whole-cell transformation, the cells were resuspended in a 5 ml reaction system to form a cell suspension (OD600 = 200). After the reaction, we found that there was almost no arginine in the reaction system of experimental group B3-7. And the arginine content in other experimental groups was also very low. During whole-cell transformation, the reaction solution became very viscous and difficult to sample. We speculated that this was due to the viscosity of the reaction solution due to the high cell density, and the low initial substrate concentration that limits the production of GAA. In the experiment of Figure 3b, we varied the reaction conditions. We adjusted the initial substrate concentration to 20 g/L glycine and 20 g/L arginine. At the same time, we resuspended the cells to an OD600 of 60, and other conditions remained unchanged. We have described the corresponding conditions in the corresponding sections in the manuscript.
Q4: I checked the paper listed as the Reference 20 and could not find the condition for PCR (L159). The authors need to change the reference to an appropriate citation or describe the condition in the manuscript.
Response: We referred to the method in Reference 20, and the PCR conditions have been added in the line 133-134 of the manuscript.
Q5: The authors need to describe the method about the overexpression via Pveg (L231) in the manuscript
Response: The method about the overexpression via Pveg promoter has been described (line 88-91).
Q1: L35：Remove “%” from “60ï¼…”.
Response: It has been modified.
Q2: L56：The Reference 12 is not suitable for the explanation that B. subtilis is regarded as GRAS.
Response: It has been modified.
Q3: L94: Which is correct, 7.4? or 7.5?
Response: The correct number is 7.4, which has been modified in the revised manuscript.
Q4: L99: Describe the manufacturer of LC-MS.
Response: The manufacturer of LC-MS has been added.
Q5: References: Italicize the bacterial names.
Response: The format of bacterial names has been modified.
Round 2
Reviewer 2 Report
The revision you sent is a good answer to the question and the center of the discussion.
And it is properly organized and reflected in the revision file.
Compared to the previous paper, the high paper quality has been improved, so this reviewer is also pleased.
Author Response
The revision you sent is a good answer to the question and the center of the discussion.
And it is properly organized and reflected in the revision file.
Compared to the previous paper, the high paper quality has been improved, so this reviewer is also pleased.
Response: Thank you for your comments.
