Polyamine-Mediated Growth Regulation in Microalgae: Integrating Redox Balance and Amino Acids Pathway into Metabolic Engineering

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reviewed the polyamines regulating the growth and metabolic pathway and possible mechanisms in microalgae. Actually, I don’t think the review has high innovation. The author wrote the manuscript with free play without strict logical thinking, and also there are some obvious scientific errors, so I suggest it should be rejected by your journal.

1. The Introduction part is too long with 28 references (in total 60 references), and should introduce the topic more quickly.
2. In line 120-121”Moreover, a common by-product of the metabolism of polyamines into spermidine, or its back transformation, is hydrogen peroxide (H2O2).” The expression is not right, please check it. Spermidine is a kind of polyamines, H2O2 is byproduct of metabolism of polyamines. But not into spermidine.
3. In line 125-128, “In microalgae, these polyamines, especially putrescine and spermidine can accumulate in high concentrations, leading to processes associated with cell proliferation and growth, and resistance to environmental stress, by regulating the cell cycle and antioxidant responses”, please give references.
4. In line 144-145. The balance between the number of conjugated-unconjugated
   forms controls main events related to metabolic regulation, since the unconjugated forms can regulate the phenotype at the metabolic flux or substrate-level enzyme regulation, and the conjugated form may act at a supramolecular level. The expression is not right, please give reference.
5. in line 160-166. The logical reasoning is not rigorous, and there are too many assumptions.
6.  in line 205-206，”which is the main precursor for putrescine, are heavily reduced, depreciating the ODC activity by the lack of available substrate and likely by the participation of antizyme protein as well”. This sentence is not right.
7. In line 217, what means for “Polyamine saturation”?
8. The manuscript had only two figures, which are made by the author. But it is something like a hypothesis without experimental proof. By the way, these kinds of results did not show in the abstract.

Comments on the Quality of English Language

The English is good.

Author Response

Comments and Suggestions for Authors

This manuscript reviewed the polyamines regulating the growth and metabolic pathway and possible mechanisms in microalgae. Actually, I don’t think the review has high innovation. The author wrote the manuscript with free play without strict logical thinking, and also there are some obvious scientific errors, so I suggest it should be rejected by your journal.

1. The Introduction part is too long with 28 references (in total 60 references), and should introduce the topic more quickly.

R: The introduction was reformulated accordingly. Part of the original text was transferred to other sections of the manuscript  for clarity and better coherence.

1. In line 120-121”Moreover, a common by-product of the metabolism of polyamines into spermidine, or its back transformation, is hydrogen peroxide (H2O2).” The expression is not right, please check it. Spermidine is a kind of polyamines, H2O2 is byproduct of metabolism of polyamines. But not into spermidine.

R: The sentence was modified and relocated in the session 2. Polyamines and their main role in microalgae.

“In plants, the catabolism of polyamines generates hydrogen peroxide (H₂O₂), which has been related with the induction of signaling events triggering mild antioxidative defense responses related to abiotic stress [36,37]. The oxidative de-amination of polyamines in plants by copper-containing amine oxidases (CuAO) and the flavin-containing polyamine oxidases (PAO) has been associated to modifications on the cell wall structure, wound closure, and resistance to biotic and abiotic stress (DOI: 10.1016/j.tplants.2005.12.009). In Chlamydomonas reinhardtii, at least five genes coding for polyamine oxidases (CuAOs and PAOs) have been annotated and predicted to be involved in the metabolism of spermine, spermidine and putrescine, likely generating H2O2 in the process (doi: 10.1111/pbi.13879).”

1. In line 125-128, “In microalgae, these polyamines, especially putrescine and spermidine can accumulate in high concentrations, leading to processes associated with cell proliferation and growth, and resistance to environmental stress, by regulating the cell cycle and antioxidant responses”, please give references.

R: The reference from where this information was collected now is properly cited.

1. In line 144-145. The balance between the number of conjugated-unconjugated
   forms controls main events related to metabolic regulation, since the unconjugated forms can regulate the phenotype at the metabolic flux or substrate-level enzyme regulation, and the conjugated form may act at a supramolecular level. The expression is not right, please give reference.

R: The expression was reformulated and the references were properly addressed: ˜The unconjugated forms have been associated to regulatory events occurring at the level of metabolic flux or substrate-level regulation, and serve as precursors of conjugated forms by interacting with  decarboxylated S-adenosylmethionine molecules, and serve also as substrates to aminopropyltransferase enzymes in polyamines elongation [44,45].˜

1. in line 160-166. The logical reasoning is not rigorous, and there are too many assumptions.

R: The idea of this paragraph was better developed into a concise statement, and replaced in session 2, closest to the description of basic participation of polyamines in plant cell wall structure.

˜In diatoms, a major group of eukaryotic microalgae, the regulation of the deposition of cell wall structure is performed unique siliceous cell walls [47] by an organic template consisting of proteins and aliphatic long chain polyamines (LCPAs) [48]. LCPAs are crucial in controlling the shape and morphology of the frustules, acting as both structural and catalytic agents in silica condensation.˜

 

1.  in line 205-206，”which is the main precursor for putrescine, are heavily reduced, depreciating the ODC activity by the lack of available substrate and likely by the participation of antizyme protein as well”. This sentence is not right.

R: The sentence was modified to empathize that multiple simultaneous alterations in ODC pathway may occur during the activation of cell quiescence that directly and indirectly affect the polyamines pools.

˜In this condition the de novo production of ornithine, which is the main precursor for putrescine in Chlamydomonas reinhardtii, is heavily reduced, as well the expression of S-adenosylmethionine synthetase (SAS1).  Therefore, the reduced production of putrescine through the ODC pathway due to the lack of available substrate and possible inhibitory effect of antizyme over ODC activity would be accompanied by the reduced generation of conjugated forms of polyamines.˜

 

1. In line 217, what means for “Polyamine saturation”?

R: We have modified the text to better explain the idea of saturation mentioned by Bachrach. In fact, what he defines as a saturation status, could be interpreted as a

“Bachrach et al. (2001), suggested that under homeostasis, cells accumulate  putrescine up to a maximum concentration, after which a metabolic homeostasis status is disrupted achieved by putrescine “saturation” moment, after which the dynamics of the interconversion of the several polyamines, specially modulating the synthesis rate of spermidine and spermine, is affected. If the internal concentration of putrescine decreases, as it was reported for microalgae cells under nitrogen starvation and mammalians cells under serum starvation, this conversion rate remains minimal [42], while microalgae cells remained with unmodified growth yields in AMX1 and AMX2 knockout mutants where putrescine was not degraded and its levels where similar to a reference type strain. This analysis indicates that a minimum concentration of putrescine is necessary to provide growth yield in microalgae, while the overexpression of ODC reduces the cell concentration and increases the cellular putrescine during cell growth”.

1. The manuscript had only two figures, which are made by the author. But it is something like a hypothesis without experimental proof. By the way, these kinds of results did not show in the abstract.

R: The Figure 3 is a consolidation of several pieces of evidence retrieved from the literature cited in the main text. It is an important illustrative scheme summarizing conclusions and results not yet combined in a general theory for explaining the C. reinhardii growth phenotype in terms of polyamines dynamics. Most importantly, it illustrates the very likely importance of putrescine as a molecule that preludes the peak or enhancement of cellular growth performance, not defying the exact growth productivity, but preparing the cells to an optimal growth which in a specific metabolic moment will be halted by several regulatory events. This is also relevant to the fact that the whole functional role of the polyamines in signaling and transcriptional regulation is still poorly understood in microalgae species. It is relevant, therefore, that the dynamic characteristic of the variation of polyamines concentration over the cell's life, not emphasized in one particular molecule, such as putrescine.  We modified the Figure legend to make it clear that it does not represent specific results from C. reinhardii experiments, but it illustrates a compendium of several conclusions drawn by previous experimentations and observations mentioned in the text. The Figure 4, is an exercise of recapitulation from the basic knowledge of the polyamines association with physiological responses and broadly to main biological processes and molecular functions that might be modulated by these molecules.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer’s comments

Manuscript ID: synbio-3531322

Type of manuscript: Review

Title: Polyamine-mediated growth regulation in microalgae: prospecting metabolic pathways for metabolic engineering and biotechnological advancements

 

The review work entitled “Polyamine-mediated growth regulation in microalgae: prospecting metabolic pathways for metabolic engineering and biotechnological advancements” presents on the roles of polyamines in growth regulation in microalgae, covering their biosynthetic pathways, metabolic regulation mechanisms, and potential biotechnological applications. This work has made forward-looking progress in summarizing and explaining the theory behind the effects of polyamines on the physiology of green algae. However, this article still requires substantial revisions in its discussion and explanation of polyamine metabolic pathways and metabolic engineering concepts. I believe the revisions required are too extensive for the author to complete within a short period. Therefore, I recommend “Reject” based on the points raised before the manuscript is accepted for publication. Specific comments and general comments are given below:

 

Specific comments

1. Although section 5 covers other microalgae, the content from the introduction to section 4 is predominantly centered on green algae, particularly Chlamydomonas reinhardtii. It is advisable to modify the title accordingly and refine section 5 as necessary. If the title remains unchanged, the author should expand on the classification of microalgae, highlight their various biotechnological applications, and incorporate examples such as dinoflagellates and diatoms into sections 1–4.
2. It is suggested that the introduction be substantially revised, beginning with an overview of the contributions and significance of green algae in the biotechnology industry, followed by a discussion on the importance of developing methods to enhance their growth rate.
3. It is suggested to revise "death" in Fig. 1 to "stationary phase" for better accuracy.
4. Line 143, To enhance readability, it is recommended to provide a definition for "long-chain polyamines" when first mentioned, as their structure and molecular weight differ significantly from other polyamines.
5. It is recommended to annotate the concentrations to further clarify the relationship between polyamine levels and diatom growth. Additionally, since this study primarily focuses on the functions of polyamines in green algae, it would be helpful to specify the classification of other mentioned microalgae, such as Chaetoceros (diatom), Phaeodactylum tricornutum (diatom), and Alexandrium minutum (dinoflagellate).
6. Line 239, Based on published studies on spermidine synthase in diatoms (Phaeodactylum tricornutum), it does not seem likely that Spd supplementation would slow diatom growth. Maintaining a high intracellular Spd level appears to promote rapid diatom growth. Therefore, it is suggested that this statement be appropriately revised.
7. The reference format of this manuscript does not align with the journal's requirements. It is recommended to make the necessary corrections.
8. While the title mentions metabolic pathways in the context of metabolic engineering, the article offers minimal introduction and discussion of polyamine biosynthetic enzymes. It is highly recommended to include an additional figure illustrating the predicted polyamine metabolism pathways in green algae. Moreover, recent studies from the past five years on the regulation of the polyamine pathway in green algae have not been addressed. Incorporating the following references and providing a more detailed discussion would be beneficial.

Freudenberg, R.A.; Wittemeier, L.; Einhaus, A.; Baier, T.; Kruse, O. Advanced pathway engineering for phototrophic putrescine production. Plant Biotechnol. J. 2022, 20, 1968-1982.

Freudenberg, R.A.; Wittemeier, L.; Einhaus, A.; Baier, T.; Kruse, O. The spermidine synthase gene SPD1: A novel auxotrophic marker for Chlamydomonas reinhardtii designed by enhanced CRISPR/Cas9 gene editing. Cells 2022, 11, 837.

Freudenberg, R.A.; Wittemeier, L.; Einhaus, A.; Baier, T.; Kruse, O. High cell density cultivation enables efficient and sustainable recombinant polyamine production in the microalga Chlamydomonas reinhardtii. Bioresour. Technol. 2021, 323, 124542.

Kim, J.-H.; Woo, A.-J.; Park, E.-J.; Choi, J.-I. Overexpression of S-Adenosylmethionine synthetase in recombinant Chlamydomonas for enhanced lipid production. J. Microbiol. Biotechnol. 2023, 33, 310-318

 

General comments

1. Line 75-77, cite Ref.
2. Line 184/ 186, The gene name of spermidine synthase enzyme must be consistent (sps1 or spd1)
3. Line 376-402, please cite Refs

Author Response

Thank you for the valuable comments. Our manuscript was extensively reviewed hoping to address most if not all the concerns raised by you.

 

Comments and Suggestions for Authors

Reviewer’s comments

Manuscript ID: synbio-3531322

Type of manuscript: Review

Title: Polyamine-mediated growth regulation in microalgae: prospecting metabolic pathways for metabolic engineering and biotechnological advancements

 

The review work entitled “Polyamine-mediated growth regulation in microalgae: prospecting metabolic pathways for metabolic engineering and biotechnological advancements” presents on the roles of polyamines in growth regulation in microalgae, covering their biosynthetic pathways, metabolic regulation mechanisms, and potential biotechnological applications. This work has made forward-looking progress in summarizing and explaining the theory behind the effects of polyamines on the physiology of green algae. However, this article still requires substantial revisions in its discussion and explanation of polyamine metabolic pathways and metabolic engineering concepts. I believe the revisions required are too extensive for the author to complete within a short period. Therefore, I recommend “Reject” based on the points raised before the manuscript is accepted for publication. Specific comments and general comments are given below:

 Thank you for the valuable comments. Our manuscript was extensively reviewed hoping to address most if not all the concerns raised by you.

Specific comments

1. Although section 5 covers other microalgae, the content from the introduction to section 4 is predominantly centered on green algae, particularly Chlamydomonas reinhardtii. It is advisable to modify the title accordingly and refine section 5 as necessary. If the title remains unchanged, the author should expand on the classification of microalgae, highlight their various biotechnological applications, and incorporate examples such as dinoflagellates and diatoms into sections 1–4.

R: The title was altered, addressing the metabolic engineering aspects directly to “green algae”. Even though some topics related to different classes of microalgae are addressed, the metabolic engineering aspect is more restricted to Chlamydomonas.

1. It is suggested that the introduction be substantially revised, beginning with an overview of the contributions and significance of green algae in the biotechnology industry, followed by a discussion on the importance of developing methods to enhance their growth rate.

R: The introduction was revised and the topic of microalgae relevance and life cycle assessment of industrial applications were included, as well some citations of good examples of applications of the microalgae biomass.

“Microalgae biomass has received global attention as a possible feedstock for several industries, from bioenergy, human food, pharmaceutical and now a days agronomic industry (https://doi.org/10.1016/j.copbio.2022.102705; DOI: 10.1007/s11274-021-03192-2). As an organism that can be cultivated associated to bioremediation processes, either in consortium or isolated, these unicellular organisms have demonstrated several positive characteristics towards a sustainable and renewable biomass feedstock (doi: 10.3389/fbioe.2023.1193424.). Besides that, microalgae area also capable of growth in exclusive photoautotrophic conditions, which implies that can be combined to decarbonizing strategies which can be economically competitive in future scenarios (DOI: 10.1016/j.jprot.2013.09.016). However, its essential to increase the industrial application of microalgae and their definitive establishment as a biomass feedstock. Unfortunately, besides their enormous potential to generate molecules of broad interest to humankind, there is a still important limitations of their application at very large scales, including their slow growth phenotype which increases cultivation costs (https://doi.org/10.1016/j.biortech.2021.126155). These characteristic limits the capacity of industrial generation of products from microalgae cells at an economically competitive cost (https://doi.org/10.1016/j.biortech.2020.122822). Therefore, the deep investigation on how cells regulate their carbon accumulation, biomass production and how they limit their cell division and growth is essential to increase the chances of a real change in the use of microalgae as a global and sustainable biomass feedstock.”

 

1. It is suggested to revise "death" in Fig. 1 to "stationary phase" for better accuracy.

R: The Figure was modified accordingly as well as its legend.

1. Line 143, To enhance readability, it is recommended to provide a definition for "long-chain polyamines" when first mentioned, as their structure and molecular weight differ significantly from other polyamines.

R: Now there’s a new section only discussing long-chain polyamines in diatoms, as an opportunity to present polyamine’s role in cell structure, and also underline the main composition of long-chain polyamines found in microalgae.

1. It is recommended to annotate the concentrations to further clarify the relationship between polyamine levels and diatom growth. Additionally, since this study primarily focuses on the functions of polyamines in green algae, it would be helpful to specify the classification of other mentioned microalgae, such as Chaetoceros (diatom), Phaeodactylum tricornutum (diatom), and Alexandrium minutum (dinoflagellate).

R: The group or class of the microalgae discussed in the text is now properly addressed.

1. Line 239, Based on published studies on spermidine synthase in diatoms (Phaeodactylum tricornutum), it does not seem likely that Spd supplementation would slow diatom growth. Maintaining a high intracellular Spd level appears to promote rapid diatom growth. Therefore, it is suggested that this statement be appropriately revised.

R: We carefully revised the statements from the mentioned citation and we could not find ground to keep this information. We could not find the original experimental data reported in the cited review manuscript. Therefore, we decided to remove the whole sentence.

1. The reference format of this manuscript does not align with the journal's requirements. It is recommended to make the necessary corrections.

R: References format was corrected accordingly.

1. While the title mentions metabolic pathways in the context of metabolic engineering, the article offers minimal introduction and discussion of polyamine biosynthetic enzymes. It is highly recommended to include an additional figure illustrating the predicted polyamine metabolism pathways in green algae. Moreover, recent studies from the past five years on the regulation of the polyamine pathway in green algae have not been addressed. Incorporating the following references and providing a more detailed discussion would be beneficial.

Freudenberg, R.A.; Wittemeier, L.; Einhaus, A.; Baier, T.; Kruse, O. Advanced pathway engineering for phototrophic putrescine production. Plant Biotechnol. J. 2022, 20, 1968-1982. doi: 10.1111/pbi.13879

Freudenberg, R.A.; Wittemeier, L.; Einhaus, A.; Baier, T.; Kruse, O. The spermidine synthase gene SPD1: A novel auxotrophic marker for Chlamydomonas reinhardtii designed by enhanced CRISPR/Cas9 gene editing. Cells 2022, 11, 837. DOI: 10.3390/cells11050837

Freudenberg, R.A.; Wittemeier, L.; Einhaus, A.; Baier, T.; Kruse, O. High cell density cultivation enables efficient and sustainable recombinant polyamine production in the microalga Chlamydomonas reinhardtii. Bioresour. Technol. 2021, 323, 124542. DOI: 10.1016/j.biortech.2020.124542

Kim, J.-H.; Woo, A.-J.; Park, E.-J.; Choi, J.-I. Overexpression of S-Adenosylmethionine synthetase in recombinant Chlamydomonas for enhanced lipid production. J. Microbiol. Biotechnol. 2023, 33, 310-318 https://doi.org/10.4014/jmb.2212.12009

R: The references mentioned were discussed in the main text, specially in the Final considerations session.

 

 

General comments

1. Line 75-77, cite Ref.

R: The reference was cited.

1. Line 184/ 186, The gene name of spermidine synthase enzyme must be consistent (sps1 or spd1)

R: The excerpt was altered to “The spermidine synthesized can be metabolized into spermine or thermospermine by spermine synthase (sps1 gene) or thermospermine synthase (acl5 gene), respectively, even though the concentration of spermine compound is undetectable experimentally in some reports, including the microalgae C. reinhardtii [28,45].” Now we properly indicated the genes related to the different enzymes in discussion as spd1: Sperimidine synthase and sps1: Spermine synthase.

 

1. Line 376-402, please cite Refs

R: The references were included and some points better explained.

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, Lavandosque and Winck present a review regarding the biosynthesis, function, and metabolic impact of main polyamines (putrescine, spermidine, and spermine) in microalgae, with a focus on the model organism Chlamydomonas reinhardtii. This review is of relevance to biotechnology, since polyamines are involved in regulating processes that control growth, stress resilience, and lipid accumulation in microalgae, and integration of current knowledge can prompt metabolic engineering strategies designed to enhance growth and optimization of microalgae biomass production.  The text is very well written, integrating knowledge worth to be considered for publication in SynBio journal.

There are several issues that the authors need to address before the manuscript can be accepted for publication.

• Section 2: The authors are encouraged to include a scheme indicating the polyamine chemical structures, also pointing to the known metabolic pathways/biological processes known to be regulated by the respective polyamine in microalgae (combine with Figure 2). Alternatively, this could be presented as a table, with a column dedicated to relevant references.
• Lines 117-118: please elaborate on the structure of conjugated polyamines.
• Section 3: Please include a scheme pinpointing the polyamine biosynthesis in microalgae.
• Section 4: please include a scheme indicating the processes that are influenced by exogenous polyamines.

Author Response

 Thank you for the valuable comments. Our manuscript was extensively reviewed hoping to address most if not all the concerns raised by you.

 

Comments and Suggestions for Authors

In this manuscript, Lavandosque and Winck present a review regarding the biosynthesis, function, and metabolic impact of main polyamines (putrescine, spermidine, and spermine) in microalgae, with a focus on the model organism Chlamydomonas reinhardtii. This review is of relevance to biotechnology, since polyamines are involved in regulating processes that control growth, stress resilience, and lipid accumulation in microalgae, and integration of current knowledge can prompt metabolic engineering strategies designed to enhance growth and optimization of microalgae biomass production.  The text is very well written, integrating knowledge worth to be considered for publication in SynBio journal.

There are several issues that the authors need to address before the manuscript can be accepted for publication.

• Section 2: The authors are encouraged to include a scheme indicating the polyamine chemical structures, also pointing to the known metabolic pathways/biological processes known to be regulated by the respective polyamine in microalgae (combine with Figure 2). Alternatively, this could be presented as a table, with a column dedicated to relevant references.

R: A new Figure was included (please see Figure 1), with the chemical structures and their related biosynthesis pathways.

• Lines 117-118: please elaborate on the structure of conjugated polyamines.

R: A new session was added in the main text to describe and discuss the structure of the conjugated polyamines. Please see session 4. The role of long-chain polyamines in cell architecture.

• Section 3: Please include a scheme pinpointing the polyamine biosynthesis in microalgae.

R: A Figure (Figure 1) representing the polyamine biosynthesis was provided, also including their molecular structures.

• Section 4: please include a scheme indicating the processes that are influenced by exogenous polyamines.

R: A new Figure (Figure 2) was included in the main text providing the processes affected by the main exogenous polyamines tested in C. reinhardtii (Put, Spd, Spm and Cad).

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript had been revised and make some improvement in its present version, but there are also not well enough to be published in the journal of Synbio. The real problem is the innovation and logic of the MS.

Comments on the Quality of English Language

need to be polished.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer’s comments

Manuscript ID: synbio-3531322

Type of manuscript: Review

Title: Polyamine-mediated growth regulation in microalgae: prospecting metabolic pathways for green algae metabolic engineering and biotechnological advancements

 

 

The revised version of the manuscript entitled “Polyamine-mediated growth regulation in microalgae: prospecting metabolic pathways for green algae metabolic engineering and biotechnological advancements” demonstrates substantial improvement. The content and conceptual framework are sound, with no major concerns identified. However, there are still some minor omissions in the manuscript that need to be supplemented. I suggested this work may be accepted for publication in “SynBio” after a “minor revision”. General comments are given below:

 

 

General comments

1. Line 61, [9–14][15–19] à [9–19].
2. Line 63 [20] à delete
3. Line 75-79, Ref needs to be re-cited in the correct place
4. The reference format of this manuscript still does not align with the journal's requirements, such as on page number. It is recommended to make the necessary corrections.

Comments for author File: Comments.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed very well all reviewer's concerns.