Recent Advances in the Heterologous Biosynthesis of Natural Products from Streptomyces
, Chung Thanh Nguyen 1, Dipesh Dhakal 1, Hue Thi Nguyen 1, Tae-Su Kim 1 and Jae Kyung Sohng 1,2,*Round 1
Reviewer 1 Report
The manuscript entitled: “Recent advances in heterologous biosynthesis of natural products from Streptomyces”, reference 1064912.
General comments:
The manuscript is interesting and comprises an important, however in some sections the English has some issues which considerably reduce the clarity of the manuscript. However, this is not the main issue of this manuscript, in fact I would like to pin point 3 major issues:
- Novelty – a quick search in Scopus shows three recent articles encompassing several of the information depicted in this manuscript: DOI: 10.1016/j.biotechadv.2018.10.003, DOI: 10.1016/j.biotechadv.2018.10.003, and DOI: 10.1002/elsc.201800137. Please clearly justify the novelty of this work at least on the Abstract and Introduction sections.
- As the title of manuscript suggests, I was expecting a clear focus on heterologous production of natural products that are Streptomyces that are biosynthesized. However, in several sections there is a not mixture where Streptomyces is in fact the surrogate for heterologous production. I am not against the inclusion of this information, I would simply like to suggest that this information should be clearly separated.
- In my opinion the manuscript neglects an extensive and insightful definition of natural products, and their potential uses. This, in my understanding undermines the impact of the manuscript. Are these natural products that important? Can they be produced through chemical synthesis? What is their economical overview? Additional sections could be added to the manuscript to comprise all this information, and at least partilally should be included in the Conclusion section which is, in my point of view, very reductive and oversimplified.
Additional comments:
In my understanding the manuscript impact would be improved if the authors included additional quantitative data. In particular, when mentioning rapid or slow growth, i.e. specific growth rates should be depicted (an average value or a range).
The manuscript contains a vast number of acronyms, thus an acronym list would be recommended.
Point by point comments:
Line 24 to 28, contains several important statements that require support from adequate references.
Figure 1 also does not contain at least a reference that ascribes the production of these metabolites.
Line 31, BGCs acronym is not adequately defined in the manuscript text, please revise. In addition, please revise the definition of all acronyms throughout the manuscript text, namely: SSRTA, BAC, TAR.
Line 55 to 61, the acronyms definition should be performed prior not after, such as: Natural Product Domain Seeker (NaPDoS).
Line 99, the statement: “These resources provide new opportunities for cyanobacterial NP 99 research using the bottom-up approach” is in my opinion, unclear. Are the authors stating that Streptomyces are cyanobacteria? Are these tools solely directed to cyanobacteria natural products? I am confused. Please clarify.
Line 130, please consider replacing “and easily genetic manipulation” by “easily genetically manipulated” or “facile genetic manipulation”.
Line 131, “some Streptomyces host” please italicize Streptomyces and host should be in its plural form: some hosts.
Line 131 to 133, the first reference to a species must contain its full name, such as: Streptomyces albus. I understand that this manuscript is mainly focused on Streptomyces, nevertheless this is the correct procedure (example: “Saccharomyces cerevisiae” in line 155 and line 195. Moreover, after the its first reference, its abbreviated name should be used consistently. Please carefully and thoroughly revise for all species names.
Line 202, the authors state: “possibly produced” and afterwards describe quantifiable data. Please clarify, did S. cerevisiae produced or not these natural compounds at these concentrations?
Line 215 to 216, I could not understand this statement due to English issues, please revise.
Author Response
Review 1
The manuscript entitled: “Recent advances in heterologous biosynthesis of natural products from Streptomyces”, reference 1064912.
General comments:
The manuscript is interesting and comprises an important, however in some sections the English has some issues which considerably reduce the clarity of the manuscript. However, this is not the main issue of this manuscript, in fact I would like to pin point 3 major issues:
- Novelty – a quick search in Scopus shows three recent articles encompassing several of the information depicted in this manuscript: DOI: 10.1016/j.biotechadv.2018.10.003, DOI: 10.1016/j.biotechadv.2018.10.003, and DOI: 10.1002/elsc.201800137. Please clearly justify the novelty of this work at least on the Abstract and Introduction sections.
Thank you for your comment. In the paper with doi: 10.1016/j.biotechadv.2018.10.003, author and et al. focus on only heterologous in Streptomyces. In the paper doi: 10.1002/elsc.201800137 introduced different techniques such as CRISPR for genome editing, direct cloning,... to discover and enhance production in Streptomyces.
In our review, we included Streptomyces and other host as E. coli, P. putida,… for the heterologous expression of biosynthetic gene clusters from Streptomyces. Beside that, we also introduced recent advanced techniques that are phage Ï•BT1 integrase-mediated site-specific recombination, SIRA, SSRTA.
The abstract and introduction has been rewritten from line 12-24,
- As the title of manuscript suggests, I was expecting a clear focus on heterologous production of natural products that are Streptomyces that are biosynthesized. However, in several sections there is a not mixture where Streptomyces is in fact the surrogate for heterologous production. I am not against the inclusion of this information, I would simply like to suggest that this information should be clearly separated.
Thank you for your great suggestion. Based on that, we rearranged some sections as flow of work. Following that we also changed information in each part. We re-arranged the sections of our manuscript based on the logic and connection between the sections. We moved the section "2. Strategies for construction of the biosynthetic gene cluster" to section "4. Strategies for construction of the biosynthetic gene cluster". We also moved section "3.1. Promoter engineering" and section "3.2. RBS turning" in section "3. Host engineering approaches" to section "4. Strategies for construction of the biosynthetic gene cluster" because Promoters and RBSs are located in recombinant vectors. Engineering Promoters and RBSs are in part of biosynthetic gene cluster construction. In sections “4. Host engineering approaches”, we added section “4.1. Host cleaning”.
- In my opinion the manuscript neglects an extensive and insightful definition of natural products, and their potential uses. This, in my understanding undermines the impact of the manuscript. Are these natural products that important? Can they be produced through chemical synthesis? What is their economical overview? Additional sections could be added to the manuscript to comprise all this information, and at least partilally should be included in the Conclusion section which is, in my point of view, very reductive and oversimplified.
Thank you for your comment. In this revised manuscript, this information had been already added to the Introduction section (line 50-58).
Additional comments:
In my understanding the manuscript impact would be improved if the authors included additional quantitative data. In particular, when mentioning rapid or slow growth, i.e. specific growth rates should be depicted (an average value or a range).
The quantitative information was added with time range in line 189, 205, 211, and 242.
The manuscript contains a vast number of acronyms, thus an acronym list would be recommended.
An abbreviations list was added line number 27-41.
Point by point comments:
Line 24 to 28, contains several important statements that require support from adequate references.
The references were added number 1 to 6 line with number 47, 49.
Figure 1 also does not contain at least a reference that ascribes the production of these metabolites.
The information of compounds in figure 1 were added from line number 534-539
Line 31, BGCs acronym is not adequately defined in the manuscript text, please revise. In addition, please revise the definition of all acronyms throughout the manuscript text, namely: SSRTA, BAC, TAR.
Line 55 to 61, the acronyms definition should be performed prior not after, such as: Natural Product Domain Seeker (NaPDoS).
Acronyms were corrected as your comment.
Line 99, the statement: “These resources provide new opportunities for cyanobacterial NP 99 research using the bottom-up approach” is in my opinion, unclear. Are the authors stating that Streptomyces are cyanobacteria? Are these tools solely directed to cyanobacteria natural products? I am confused. Please clarify.
The sentence was rewritten at line 134-135.
Line 130, please consider replacing “and easily genetic manipulation” by “easily genetically manipulated” or “facile genetic manipulation”.
“easily genetic manipulation” was replaced by “easily genetically manipulated” at line 168.
Line 131, “some Streptomyces host” please italicize Streptomyces and host should be in its plural form: some hosts.
The sentence at line 131 contained “some Streptomyces host” had already rewritten and added its plural form in “hosts” line 169.
Line 131 to 133, the first reference to a species must contain its full name, such as: Streptomyces albus. I understand that this manuscript is mainly focused on Streptomyces, nevertheless this is the correct procedure (example: “Saccharomyces cerevisiae” in line 155 and line 195. Moreover, after the its first reference, its abbreviated name should be used consistently. Please carefully and thoroughly revise for all species names.
We revised all strains with full name in the first-time mention, and short form in the subsequence time.
Line 202, the authors state: “possibly produced” and afterwards describe quantifiable data. Please clarify, did S. cerevisiae produced or not these natural compounds at these concentrations?
The sentence was rewritten at line 202-204.
Line 215 to 216, I could not understand this statement due to English issues, please revise.
Those sentences at line 212-214 were rewritten.
Reviewer 2 Report
To whom it may concern:
The review submitted by Pham and coworkers is quite comprehensive and properly updated, and in general the cited literature is adequate. However, the contained information will require reorganization and extensive English language corrections. Many sections require important rephrasing to ensure the text reaches the level of quality required for a general international audience.
This general reorganization, in my opinion, should be based on facing the individual challenges and their particular solutions in the heterologous expression of BGCs from Streptomyces. Just as an example of what I mean: comment on the need of promoter refactoring for the expression of BGCs from Streptomyces into non-Streptomyces hosts (the native promoters may luckily work in Rhodococcus, as it is also an Actinobacteria, but cannot work in Pseudomonas, E.coli or a yeast). I know the authors talk about refactoring in another section, but I think it would be good to reorganize the information under a single heading, or at least link the information properly. If not, the text feels not well connected.
Please try to define every acronym the very first time it appears across the text, as it is not the case for many of them.
Maybe Figure 3 could provide some more information. Figure 4 is in my opinion not required. All figures will require a proper caption for the explanation of the elements described (RE?, P?, etc…)
Please comment on the advantages and disadvantages of different approaches. For example, obtaining a cosmid library is technically simple, but requires the further screening of a high number of clones, while TAR cloning is technically tricky, but once successful should provide the desired clone directly. Please comment about the need of carefully checking cloned BGCs with internal restriction enzymes, as aberrant recombination are a quite common issue both in cosmid libraries, TAR clones and many other techniques.
When talking about the traditional BGC cloning through the creation and screening of cosmid libraries for heterologous expression, the authors do not present any example: I suggest this one: Leipold, F. & Santos-Aberturas J et al (https://www.nature.com/articles/s41467-017-01975-6), as it is recent, relevant and also shows the amazing possibilities of mass spectrometry molecular networking. Another good example of the possibilities of metabolic network in Streptomyces is Crone at al. (https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201604304).
Next, I add a few examples of concepts and expressions that should be changed to increase the quality of the text. I want to remark that there are many others and that a native speaker MUST review the text to provide advice on required rephrasing and corrections. I am pretty sure the review could be quite useful and worthy of publishing if all the points I have referred to are taken into account.
Line 36 “or to increase”
Line 41 S. venezuelae
Line 71. Please specify the meaning of KS and C domains. And rephrase the following explanations, as its meaning is not clear to me.
Line 82: NeuRiPP and RiPP? The sentence is not clear to me.
Line 85: RiPPMiner
Line 119-120: It is not clear what the authors mean in the last sentence. Please clarify.
Line 129. Please substitute “characterizations” by “features”.
Line 184. These organisms are not “rare Streptomyces”, but rare Actinomycetes or non-Streptomyces Actinomycetes.
Line 192. At laboratory scale, I would not say that Streptomyces culture media are expensive and for sure that is not a reason strong enough to move to other heterologous systems.
Line 212. Better phrase as “its native biochemistry, physiology and metabolomics are extensively understood”.
Line 221: “specific genes must be introduced…”
Line 252: Pseudomonas luminescens, as the species has not been mentioned before across the text, I think.
Line 254. E.coli
Line 280-282. The sentence makes no real sense, maybe rephrase as “To increase the chance of successful heterologous expression, BGCs can be captured in a directed manner by employing different genetic engineering techniques as TAR, LLHR, ExoCET or CATCH. TAR clones can be obtained…” Please re-write the section
Lines 327-328. Please rephrase in a more understandable manner.
Line 352: “the most substantial part of the promoter activity relies in the -35 and -10 regions, as they determine the strength of the RNA polymerase binding.” Please include a citation about the classification of the promoters in the mentioned I, II and III types.
Line 391. B. subtilis
Line 392. These sentences make no sense, it is grammatically wrong. Please rephrase it.
Line 404. I do not think “production of BGCs” is the best way of expressing the idea, maybe “heterologous expression of BGCs” will work better.
Line 406. The first use of a theophylline riboswitch for bottromycin heterologous expression was reported in this paper: https://pubs.acs.org/doi/10.1021/acssynbio.8b00038
Author Response
Review 2
The review submitted by Pham and coworkers is quite comprehensive and properly updated, and in general the cited literature is adequate. However, the contained information will require reorganization and extensive English language corrections. Many sections require important rephrasing to ensure the text reaches the level of quality required for a general international audience.
We are thankful for your kind comments to improve the manuscript. According to your suggestion, we revised English, explained the information thoroughly, reformatted and revised the manuscript.
This general reorganization, in my opinion, should be based on facing the individual challenges and their particular solutions in the heterologous expression of BGCs from Streptomyces. Just as an example of what I mean: comment on the need of promoter refactoring for the expression of BGCs from Streptomyces into non-Streptomyces hosts (the native promoters may luckily work in Rhodococcus, as it is also an Actinobacteria, but cannot work in Pseudomonas, E.coli or a yeast). I know the authors talk about refactoring in another section, but I think it would be good to reorganize the information under a single heading, or at least link the information properly. If not, the text feels not well connected.
Thank you for your great suggestion. Based on that, we rearranged some sections as flow of work. Following that we also changed information in each part. We re-arranged the sections of our manuscript based on the logic and connection between the sections. We moved the section "2. Strategies for construction of the biosynthetic gene cluster" to section "4. Strategies for construction of the biosynthetic gene cluster". We also moved section "3.1. Promoter engineering" and section "3.2. RBS turning" in section "3. Host engineering approaches" to section "4. Strategies for construction of the biosynthetic gene cluster" because Promoters and RBSs are located in recombinant vectors. Engineering Promoters and RBSs are in part of biosynthetic gene cluster construction. In sections “4. Host engineering approaches”, we added section “4.1. Host cleaning”.
Please try to define every acronym the very first time it appears across the text, as it is not the case for many of them.
Acronyms were corrected as your comment.
Maybe Figure 3 could provide some more information. Figure 4 is in my opinion not required. All figures will require a proper caption for the explanation of the elements described (RE?, P?, etc…)
The information for Figure 3 was added at line 541-547. The figure 2 and 3 were replaced. Figure 4 was removed.
Please comment on the advantages and disadvantages of different approaches. For example, obtaining a cosmid library is technically simple, but requires the further screening of a high number of clones, while TAR cloning is technically tricky, but once successful should provide the desired clone directly. Please comment about the need of carefully checking cloned BGCs with internal restriction enzymes, as aberrant recombination are a quite common issue both in cosmid libraries, TAR clones and many other techniques.
The advantages and disadvantages of different approaches were mention at line 448-454
When talking about the traditional BGC cloning through the creation and screening of cosmid libraries for heterologous expression, the authors do not present any example: I suggest this one: Leipold, F. & Santos-Aberturas J et al (https://www.nature.com/articles/s41467-017-01975-6), as it is recent, relevant and also shows the amazing possibilities of mass spectrometry molecular networking. Another good example of the possibilities of metabolic network in Streptomyces is Crone at al. (https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201604304).
The information from suggested papers were added to the manuscript at line 157-159 and line 393-397.
Next, I add a few examples of concepts and expressions that should be changed to increase the quality of the text. I want to remark that there are many others and that a native speaker MUST review the text to provide advice on required rephrasing and corrections. I am pretty sure the review could be quite useful and worthy of publishing if all the points I have referred to are taken into account.
Thank you for your comment. The manuscript was revised.
Line 36 “or to increase”
It corrected in line 62.
Line 41 S. venezuelae
Its name was corrected at line 67.
Line 71. Please specify the meaning of KS and C domains. And rephrase the following explanations, as its meaning is not clear to me.
We rewrote “KS and C domains” with their full name in the text and the sentence at line 109.
Line 82: NeuRiPP and RiPP? The sentence is not clear to me.
NeuRiPP and RiPP are acronym. We rewrite the full name at line 92-94. We define NeuRiPP and RiPP in paragraph 117-130.
Line 85: RiPPMiner
The define is written at line 123-124.
Line 119-120: It is not clear what the authors mean in the last sentence. Please clarify.
The sentence was rewritten at 154-159.
Line 129. Please substitute “characterizations” by “features”.
The word was substituted at line 167.
Line 184. These organisms are not “rare Streptomyces”, but rare Actinomycetes or non-Streptomyces Actinomycetes.
This sentence contain that words was removed.
Line 192. At laboratory scale, I would not say that Streptomyces culture media are expensive and for sure that is not a reason strong enough to move to other heterologous systems.
Thank for you suggestion, we removed that sentence and rewrote at line 189-190.
Line 212. Better phrase as “its native biochemistry, physiology and metabolomics are extensively understood”.
The sentence was substituted as suggestion line 212-213.
Line 221: “specific genes must be introduced…”
This paragraph was rewritten, the sentence was removed.
Line 252: Pseudomonas luminescens, as the species has not been mentioned before across the text, I think.
The full name of species is Photorhabdus luminescens. The information added at line 230.
Line 254. E.coli
“E. Coli” was substituted “E. coli” at line 233
Line 280-282. The sentence makes no real sense, maybe rephrase as “To increase the chance of successful heterologous expression, BGCs can be captured in a directed manner by employing different genetic engineering techniques as TAR, LLHR, ExoCET or CATCH. TAR clones can be obtained…” Please re-write the section
The section was rewritten at line 389-454.
Lines 327-328. Please rephrase in a more understandable manner.
The paraphrase was removed.
Line 352: “the most substantial part of the promoter activity relies in the -35 and -10 regions, as they determine the strength of the RNA polymerase binding.” Please include a citation about the classification of the promoters in the mentioned I, II and III types.
We had rewrote the paragraph and cited the reference line 462-469.
Line 391. B. subtilis
The name of strains was corrected at line 316.
Line 392. These sentences make no sense, it is grammatically wrong. Please rephrase it.
The phrase was rewritten from 317-319.
Line 404. I do not think “production of BGCs” is the best way of expressing the idea, maybe “heterologous expression of BGCs” will work better.
We changed some information in this section line 321-329. The sentence contains this was removed.
Line 406. The first use of a theophylline riboswitch for bottromycin heterologous expression was reported in this paper: https://pubs.acs.org/doi/10.1021/acssynbio.8b00038
Edited reference
Reference was corrected in line 327.
Round 2
Reviewer 1 Report
The manuscript entitled: “Recent advances in heterologous biosynthesis of natural products from Streptomyces”, reference applsci-1064912.
I would like to sincerely congratulate the authors for their extensive and clear improvements of the manuscript quality and impact. The authors answers were concise and enlightening. Nevertheless, I have some minor comments:
The authors state: “In our review, we included Streptomyces and other host as E. coli, P. putida,…” I agree with the authors, however, why is this information absent in the manuscript Title and Abstract? Please comment.
Line 710, “PCR targeted systems...” please do not use ellipsis in scientific writing. Please revise throughout the manuscript.
Please carefully review all the acronyms in the acronyms list. As an example, generally recognised as safe (GRAS) is not present in the acronyms list. This manuscript contains a vast number of acronyms, therefore it is easy to miss some. However, it should not happen.
Author Response
The manuscript entitled: “Recent advances in heterologous biosynthesis of natural products from Streptomyces”, reference applsci-1064912.
I would like to sincerely congratulate the authors for their extensive and clear improvements of the manuscript quality and impact. The authors answers were concise and enlightening. Nevertheless, I have some minor comments:
The authors state: “In our review, we included Streptomyces and other host as E. coli, P. putida,…” I agree with the authors, however, why is this information absent in the manuscript Title and Abstract? Please comment.
We are thankful for your kind comments to improve the manuscript. According to your suggestion, we added the information in Abstract and Introduction section at line 25 and line 91-93.
Line 710, “PCR targeted systems...” please do not use ellipsis in scientific writing. Please revise throughout the manuscript.
Thank you for your comment. It corrected in line 719.
Please carefully review all the acronyms in the acronyms list. As an example, generally recognised as safe (GRAS) is not present in the acronyms list. This manuscript contains a vast number of acronyms, therefore it is easy to miss some. However, it should not happen.
Thank you for your great suggestion. We were rewritten acronyms list in line 30-47.
Author Response File: 
Author Response.docx
Reviewer 2 Report
To whom it may concern,
I think the manuscript submitted by Pham and co-workers is substantially improved from its previous version, although a thorough English language revision is still required. I also suggest some minor changes (see below). With the implementation of all these changes, I will happy to support the publication of the manuscript. Please be specially careful with the references, as some of them are doubled in the reference list and this could obviously lead to problems in the text, as numbering may be wrong.
Line 14: “many new secondary metabolites have been characterized”
Line 16 “and also that many secondary metabolites are produced in very low amounts under laboratory conditions”
Line 46: “with high GC content in their genomes”
Line 63 “many secondary metabolites BGCs”
Line 76: “several cloning methods and vector systems have been developed…”
Line 102 “some RiPPs”
Line 142 “cretain RiPPs classes”
I personally would eliminate lines 152-153. So far, NeuRiPP has not proved to be able to find new RiPP classes experimentally, although it is an excellent tool. Comparisons can be complicated at this stage.
Line 205: reference 128 is missing there.
Line 230. “rare Actinomycetes” (NOT Actinomyces). Same for line 238.
Line 247: “ and their species are many times difficult to genetically engineer”.
Line 273: I think that E.coli growth rate is wrong. E.coli cultures double their OD every 20-30 minutes under optimal conditions.
Line 275: “and it is an easy target for genetic manipulation”
Line 363: The resulting engineered strains were employed as hosts for the integration of heterologous BGCs, leading to the production of the target products.
Please re-check the reference lists, as some references are repeated and probably not well reflected in the text.
Author Response
I think the manuscript submitted by Pham and co-workers is substantially improved from its previous version, although a thorough English language revision is still required. I also suggest some minor changes (see below). With the implementation of all these changes, I will happy to support the publication of the manuscript. Please be specially careful with the references, as some of them are doubled in the reference list and this could obviously lead to problems in the text, as numbering may be wrong.
We are thankful for your kind comments to improve the manuscript. According to your suggestion, we were edited references.
Line 14: “many new secondary metabolites have been characterized”
It was edited at line 14.
Line 16 “and also that many secondary metabolites are produced in very low amounts under laboratory conditions”
The sentence was rewritten at line 16-17.
Line 46: “with high GC content in their genomes”
It was edited at line 50-51.
Line 63 “many secondary metabolites BGCs”
It was edited at line 73-74.
Line 76: “several cloning methods and vector systems have been developed…”
It was edited at line 82.
Line 102 “some RiPPs”
It was edited at line 149.
Line 142 “cretain RiPPs classes”
It was edited at line 155.
I personally would eliminate lines 152-153. So far, NeuRiPP has not proved to be able to find new RiPP classes experimentally, although it is an excellent tool. Comparisons can be complicated at this stage.
That sentence was removed line 161-163.
Line 205: reference 128 is missing there.
The reference was added line 214.
Line 230. “rare Actinomycetes” (NOT Actinomyces). Same for line 238.
They were edited at line 239 and 249.
Line 247: “ and their species are many times difficult to genetically engineer”.
The sentence was rewritten at line 257-258.
Line 273: I think that E.coli growth rate is wrong. E.coli cultures double their OD every 20-30 minutes under optimal conditions.
The information rewrote at line 284, 256, 274, and 333.
Line 275: “and it is an easy target for genetic manipulation”
It was edited at line 285.
Line 363: The resulting engineered strains were employed as hosts for the integration of heterologous BGCs, leading to the production of the target products.
It was rewritten at line 374-376.
Please re-check the reference lists, as some references are repeated and probably not well reflected in the text.
Thank you for your suggestion. We revised some repeated references.
Author Response File: Author Response.docx
