Enhanced Phycocyanobilin Production in Escherichia coli by Fusion-Expression of Apo-Proteins with Signal Peptides
, Jing Yu 1,†, Qian Che 1, Tianjiao Zhu 1, Dehai Li 1,2 and Guojian Zhang 1,2,3,*Round 1
Reviewer 1 Report
This manuscript by Liu et al. presents a nice work using the co-expression of chromophore-binding proteins and necessary enzymes in E.coli to increase the production yield of PCB, a critical chromophore in cyanobacteria. With well-designed plasmids and careful examination, the authors demonstrate the effects of media, co-expression of PC subunits, and signaling peptides on the PCB yield. The topic is interesting, and the experiments seem carefully performed. I appreciate that the results are presented concisely and logically. However, in order to make the work worthy of publication in Fermentation, the authors need to revise the manuscript.
Specific comments:
1. There are some issues with Table 1 and Figures S3 and S4. Is there a reason why the authors decided not to fuse CpcB with N20, considering that CpcA-N20 has the best expression? Also, the SDS PAGE in Figure S3 does not look so clear to me. Specifically, the bands of alpha subunits are blurry compared to the beta subunits in Figure S4. Do the authors know why?
Some minor points: First, the title for Table 1 is not accurate. It should be modified to include CpcB, as it is also listed. Second, in lines 233-235, “…apo-phycocyanins (7 for apo-CpcA and 6 for apo-CpcB).” If I understand this correctly, the N20 peptide was not used with CpcB? If so, it’s better to leave the space blank instead of using the ‘-‘ symbol. Third, consider rearranging the rows in Table 1 to make it consistent with the bands in Figures S3 and S4.
2. Line 177: “In addition, TB medium showed the highest cell density while SOC medium showed … PCB in TB medium (Figure 2b).” I don’t agree here. Fig. 2b and 2c, in TB media, the OD 600 was not that different from other media, such as superbroth, while the PCB yield was much higher. The high cell density here cannot fully explain the difference in PCB yield. A better explanation for the high PCB yield in TB media should be given here. For example, it seems only TB contains glycerol. Could this be related to the better PCB yield?
3. Have the authors considered adding 5-Aminolevulinate to the media? It usually helps to increase PCB production in E.coli.
Minor comments:
1. Fig. 1a. What are the blue objects near the PCB chemical structure? They should be properly labeled and noted in the figure legend.
2. Figure 3-5. I believe panel b and c legends should be switched.
3. More background information on signal peptide and translocation should be given in the introduction section. For example, the content of Lines 219-230 should be more appropriate for the introduction section but not the results section.
4. Line 12: “All samples were done in three replicates.” I suggest adding error bars for the standard curve in Fig. S2 as it is used to determine the PCB yield.
5. Overall, the manuscript lacks references. Please consider adding more refs to help readers better understand the topic. For example, more references could be added in the second paragraph of the introduction part when introducing photosynthesis and phycobilisome.
The quality of the English language can and should be improved by the authors. Please carefully read through the manuscript for obvious typos/grammar issues.
Examples: Line 278 and Line 250; please rewrite these sentences.
Author Response
- Reply to reviewer #1
(1) There are some issues with Table 1 and Figures S3 and S4. Is there a reason why the authors decided not to fuse CpcB with N20, considering that CpcA-N20 has the best expression? Also, the SDS PAGE in Figure S3 does not look so clear to me. Specifically, the bands of alpha subunits are blurry compared to the beta subunits in Figure S4. Do the authors know why?
Some minor points: First, the title for Table 1 is not accurate. It should be modified to include CpcB, as it is also listed. Second, in lines 233-235, “…apo-phycocyanins (7 for apo-CpcA and 6 for apo-CpcB).” If I understand this correctly, the N20 peptide was not used with CpcB? If so, it’s better to leave the space blank instead of using the ‘-‘symbol. Third, consider rearranging the rows in Table 1 to make it consistent with the bands in Figures S3 and S4.
Reply: Thanks for your comment. The attempts to construct the plasmid pET28a-N20-cpcB was failed. Therefore, we did not provide data related to N20-cpcB. And have tried to reproduce the SDS PAGE of alpha subunits, but all the results seemed blurry. We currently do not know why the bands of alpha subunits are blurry compared to the beta subunits, this maybe resulted from the interaction of alpha subunits with other protein in the cell extraction mixture. For other points, we have r revised the title of Table 1, and replace “-” with a blank, and make the entries in consistent with the bands in Figs. S3 and S4.
(2) Line 177: “In addition, TB medium showed the highest cell density while SOC medium showed … PCB in TB medium (Figure 2b).” I don’t agree here. Fig. 2b and 2c, in TB media, the OD 600 was not that different from other media, such as superbroth, while the PCB yield was much higher. The high cell density here cannot fully explain the difference in PCB yield. A better explanation for the high PCB yield in TB media should be given here. For example, it seems only TB contains glycerol. Could this be related to the better PCB yield?
Reply: Thanks for your kindly suggestion. You are correct that the difference in PCB yield cannot be fully explained by the high cell density present. And the TB media's high PCB yield may be better explained by the fact that only TB contains glycerol. Relative to glucose, glycerol as the alternative carbon source for E. coli, which is no need of stoichiometric amounts of phosphoenolpyruvate (PEP) for its uptake into E. coli cells (doi: 10.1007/s00253-012-4101-5) and glycerol (a C3 unit), has a higher reduction potential per C3 unit which in turn could yield higher biomass and product formation (doi: 10.1186/s12934-014-0096-1). This might be connected to the improved PCB yield. We have corrected it in the revised version.
(3) Have the authors considered adding 5-Aminolevulinate to the media? It usually helps to increase PCB production in E. coli.
Reply: Thanks for your comment. According to some reports, up-regulating the ALA and heme biosynthetic pathway has no impact on PCB generation and simply serves to increase ALA and heme synthesis. This may be due to the accumulation of ALA and heme will cause negative feedback to the biosynthetic process (doi: 10.1016/j.procbio.2018.05.011). Therefore, instead of concentrating on adding 5-Aminolevulinate to increase BCB production, we simply focused on adding fusion-expression of apo-proteins and further introducing signal peptides to increase PCB production.
(4) Fig. 1a. What are the blue objects near the PCB chemical structure? They should be properly labeled and noted in the figure legend.
Reply: Thanks for pointing out. The blue objects near the PCB chemical structure represent the blue color of PCB, which has no special meaning. We have labeled and noted them in the figure legend.
(5) Figure 3-5. I believe panel b and c legends should be switched.
Reply: Thanks for your kindly suggestion. we have revised the Figure 3-5.
(6) More background information on signal peptide and translocation should be given in the introduction section. For example, the content of Lines 219-230 should be more appropriate for the introduction section but not the results section.
Reply: Thanks for your kindly suggestion. We have revised it in the revised one.
(7) Line 12: “All samples were done in three replicates.” I suggest adding error bars for the standard curve in Fig. S2 as it is used to determine the PCB yield.
Reply: Thanks for your kindly suggestion. We have revised the Figure S2.
(8) Overall, the manuscript lacks references. Please consider adding more refs to help readers better understand the topic. For example, more references could be added in the second paragraph of the introduction part when introducing photosynthesis and phycobilisome.
Reply: Thanks for your comment. We have added references in the revised version.
(9) The quality of the English language can and should be improved by the authors. Please carefully read through the manuscript for obvious typos/grammar issues. Examples: Line 278 and Line 250; please rewrite these sentences.
Reply: Thanks for pointing out the mistakes. We have corrected it in the revised one.
Reviewer 2 Report
Title: Enhanced phycocyanobilin production in Escherichia coli by fusion-expression of apo-proteins with signal peptides
Authors: Liu et al.,
Summary: The study described in this manuscript deals with recombinant production of phycocyanobilin (PCB) in E. coli. Given its multifarious applications, biological production of PCB is relevant. Therefore, the described study has merit and would be of interest to readers.
Specific comments:
1. The manuscript needs considerable English language editing. For instance:
A. Line 151: two gens are referred to but singular verb is used. Please correct.
B. Lines 156 – 157 are not clear. Please correct for clarity.
C. Lines 162 – 173: Please edit the figure legend for clarity.
D. Line 174: What is meant by “mediums”? Media?
2. Please edit the following for clarity – “3.2. Enhance the PCB Yield by Introducing Apo-protein” (line 186).
- “3.3. Enhance PCB production by Fusion-expression of Apo-proteins with Signal peptides” (line 218)
3. Please denote what the color changes in figures 2,3 and 4 represent in the legends.
4. The discussion section is unclear, lacks detail, and needs extensive English language editing. The entire discussion section needs to be completely re-written.
Title: Enhanced phycocyanobilin production in Escherichia coli by fusion-expression of apo-proteins with signal peptides
Authors: Liu et al.,
Summary: The study described in this manuscript deals with recombinant production of phycocyanobilin (PCB) in E. coli. Given its multifarious applications, biological production of PCB is relevant. Therefore, the described study has merit and would be of interest to readers.
Specific comments:
1. The manuscript needs considerable English language editing. For instance:
A. Line 151: two gens are referred to but singular verb is used. Please correct.
B. Lines 156 – 157 are not clear. Please correct for clarity.
C. Lines 162 – 173: Please edit the figure legend for clarity.
D. Line 174: What is meant by “mediums”? Media?
2. Please edit the following for clarity – “3.2. Enhance the PCB Yield by Introducing Apo-protein” (line 186).
- “3.3. Enhance PCB production by Fusion-expression of Apo-proteins with Signal peptides” (line 218)
3. Please denote what the color changes in figures 2,3 and 4 represent in the legends.
4. The discussion section is unclear, lacks detail, and needs extensive English language editing. The entire discussion section needs to be completely re-written.
Author Response
(1) The manuscript needs considerable English language editing. For instance:
- Line 151: two gens are referred to but singular verb is used. Please correct.
- Lines 156 – 157 are not clear. Please correct for clarity.
- Lines 162 – 173: Please edit the figure legend for clarity.
- Line 174: What is meant by “mediums”? Media?
Reply: Thanks for pointing out the mistakes. Here in the revised version, we have corrected that.
(2) Please edit the following for clarity – “3.2. Enhance the PCB Yield by Introducing Apo-protein” (line 186). - “3.3. Enhance PCB production by Fusion-expression of Apo-proteins with Signal peptides” (line 218)
Reply: Thanks for your comment. We have revised it in the revised version.
(3) Please denote what the color changes in figures 2, 3 and 4 represent in the legends.
Reply: Thanks for your comment. We have revised the legends of Figures 2, 3 and 4 in the revised version.
(4) The discussion section is unclear, lacks detail, and needs extensive English language editing. The entire discussion section needs to be completely re-written.
Reply: Thanks for your comment. We have re-written the entire discussion section in the revised version.
All the revisions have been marked red in the manuscript.
