Recombinant Protein Expression and Its Biotechnological Applications in Chlorella spp.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review: Genetic Elements for Recombinant Protein Expression in Chlorella spp.

Summary

The manuscript entitled "Genetic elements for recombinant protein expression in Chlorella spp" is an updated review of the basic genetic elements involved in the expression of recombinant proteins using the microalgae of biotechnological interest Chlorella spp. in accordance with the existing bioengineering and synthetic biology techniques. This review collects in a clear and concise way the studies, to date, in which the different Chlorella species are used for the expression of recombinant proteins, describing the most importatnt genetics elements involved on the integration of heterologous genes  such as promoters, terminators, enhancers, marker genes and tags as well as the different genetic transformation methods for this microalgae and the main applications of the recombinat proteins expresed by Chlorella spp.

General remarks

The most important milestone in the production of recombinant proteins is the achievement of platforms capable of producing this type of protein safely and high yields for a successful industrial production and subsequent biotechnological application. In this sense, the use of photosynthetic microorganisms, in particular cyanobacteria and, in this case, microalgae, represents a challenge for numerous future perspectives, since these microorganisms are easy to cultivate and have few requirements for their growth, thus showing both an economic and environmental advantage. These points are well defined in the present review, which provides relevant information to the scientific community dedicated to the investigation of these genetic factors and the production of this type of recombinant proteins. Furthermore, it is useful that this review focuses on microalgae such as those belonging to the genus Chlorella, since these species have a relevant biotechnological and industrial importance. At the same time, from a genetic point of view, the main genetic elements involved in a efficient protein expression have been conveniently summarised. However, in the present manuscript, the origins of replication are not reviewed, genetic elements that in recent years have been shown to be key for heterologous expression. 

The present review also focuses on the production of recombinant proteins with applications mainly as vaccines, therapeutic proteins or enzymes with an industrial potential.

The topic covered by the review is of an important relevance to the scientific community working in the field of genetic engineering and synthetic biology of microalgae since genetic elements are key to the efficient and suitable production of recombinant proteins and for the integration of heterologous genes. In this sense, the use of different promoters in microalgae is a widely studied window, since a strong and universal promoter covering the heterologous expression in different microalgae has not yet been obtained. In addition, transformation methods are also the subject of in-depth studies and of  relevance in the microalgae community, since there are only few species that can be easily manipulated with a simple, efficient and stable methodology. In the specific case of the different species of Chlorella, being one of the most robust microalgae, its genetic manipulation is not trivial, especially at the level of stability during the time. In the specific case of this review, it would be useful for a scientific solidity to include more detailed or complete information about the efficiency of the transformation methods in each of the Chlorella species mentioned, as well as the stability during the time of the genetic elements and heterologous genes integrated in the transformants generated as expression platforms for recombinant proteins.

Regarding references, since this is a review, it is important that they are as recent as possible and do the manuscript fluently reading. In this respect, the present review includes them in a correct and updated manner, although some are found to be from more than twenty-five years old. These references have already been updated in most recent works which would be more convenient. In terms of quantity, there is a suitable number of bibliography cited. 

Concerning another point, the fact that the authors have summarised the data using tables, mainly in relation to the genetic elements and recombinant proteins, provides flexibility and agility for consulting the information collected in the review.

To sum up, the review presented collects in a concise, clear and complete manner, updated information that would scientifically provide knowledge to the scientific community in the advancement of studies on the expression and production of recombinant proteins in microalgae. 

The main points and revisions to take in account would be:

- The missing of the replication origins as key genetic elements.

- The yields of the main recombinant proteins expressed in the different species of Chlorella.

- The transformation efficiencies and the stability during the time of the transformants obtained as platforms for the expression of the most important recombinant proteins.

Author Response

Thank you for your positive comments. We appreciate your time spent improving our manuscript.

1. The missing of the replication origins as key genetic elements.

Most microalgae cannot replicate exochromosomal DNA such as plasmid DNA. The genes have to be integrated into the nuclear or chloroplast genome and therefore do not require an origin for microalgae specifically. All cloning is performed in E. coli prior to transformation and uses typical origins like the pBR322 or pUC19 origins. For this reason this information was not included. 

2. The yields of the main recombinant proteins expressed in the different species of Chlorella.

The yields are summarized in Table 4 for all studies that have reported the data.

3. The transformation efficiencies and the stability during the time of the transformants obtained as platforms for the expression of the most important recombinant proteins.

Most studies do not report the transformation efficiency because the transformation efficiency in Chlorella spp. (or microalgae in general) is known to be very low and it has not been studied thoroughly to date. When stability information was available it was summarized in Table 4 for the studies that have reported the data. 

Reviewer 2 Report

Comments and Suggestions for Authors

The review, dedicated to the discussion of the using genetic elements for recombinant protein expression in representatives of Chlorella genus is important for biotechnology. The paper is very clear and relevant to the synthetic biology. In 2016 in “Biotechnology Journal” review “Chlorella species as hosts for genetic engineering and expression of heterologous proteins: progress, challenge and perspective (Bo Yang, Jin Liu, Yue Jiang, Feng Chen, DOI 10.1002/biot.201500617) was published. However, the content of these articles differs significantly, and the reviewed MS is of interest to the scientific community. The statements and conclusion are supported by discussed publications. The paper content supported by high quality figures, which allow bettering understanding of the reviewed topic.

The reviewer could be published in the journal SynBio after some corrections.

 

Major suggestions:

 1.         Please, give a brief description of the genus Chlorella and its biological features, as well as describe its biochemical and physiological characteristics in more detail.

 2.         It is necessary to add recent publication to reference. You have 93 reference, and only about 35 were published in 2019-2024. Please, try to add at least 25-30.

 Minor suggestions:

 Line 17: Try to use as keywords the terms, that are not mentioned in the title.

 Line 35 and further: Please add the authors of the taxa.

 Line 43: “Microalgae are unicellular microorganisms ubiquitous in aquatic environments”- I am not agreed with this sentence. 1) some microalgae consist of several or even more cells, for example, filamentous representatives of families Scenedesmaceae (Chlorophyta), Xanthophyceae (Heterokontophyta) and many others; 2) algae are widely distributed in terrestrial habitats also (see Ettl, Gärtner, 1995; Kostikov et al., 2021; Patova et al., 2023). Please, correct this sentence and add references for algae ecology.

 Line 89: If you use the term “Agrobacterium” to designate a genus, then you need to write it in italics.

 Line 90, 93 and further: Citations of figures should not be highlighted in bold.

 Line 158: Correct “ca” to “can”.

 Line 199: Correct “Chlorella spp..” to “Chlorella spp.”

 Line 283: Correct “[81] .” to “[81].”

 Table 3-6: Correct “Ref.” to “Reference” in the titles of the last columns.

 Line 364: Please, use the synonym for the word “recent”, because you have written it in the previous sentence.

Comments on the Quality of English Language

 Minor editing of English language required

Author Response

We thank the reviewer for the time spent reviewing our manuscript and making helpful comments. 

1. Please, give a brief description of the genus Chlorella and its biological features, as well as describe its biochemical and physiological characteristics in more detail.

We have edited the text to include some basic biological features of Chlorella, unicellular, non-motile green algae (pg 2. line 26).

2. It is necessary to add recent publication to reference. You have 93 reference, and only about 35 were published in 2019-2024. Please, try to add at least 25-30.

We have thoroughly searched the literatures for all instances in which Chlorella species have been transformation and a protein expressed and have reported them all in this review. Thus, regrettably, there are no further recent publications to include at this time.

Minor suggestions:

Line 17: Try to use as keywords the terms, that are not mentioned in the title.

We have made minor edits to the title, abstract and keywords, non of these terms are in the title. 

Line 35 and further: Please add the authors of the taxa.

Chlorella vulgaris was created in 1890 by Beijerinck. Since then, there has been significant reshuffling and multiple authors of the taxa. It is very unclear which should be used for some at this time. As a very well studied taxa, we felt this information was not critical to the reader.

Line 43: “Microalgae are unicellular microorganisms ubiquitous in aquatic environments”- I am not agreed with this sentence. 1) some microalgae consist of several or even more cells, for example, filamentous representatives of families Scenedesmaceae (Chlorophyta), Xanthophyceae (Heterokontophyta) and many others; 2) algae are widely distributed in terrestrial habitats also (see Ettl, Gärtner, 1995; Kostikov et al., 2021; Patova et al., 2023). Please, correct this sentence and add references for algae ecology.

Our apologies for the mistake, you are correct. There are some species that are small groups of cells and they are able found in soil and other habitats, although aquatic habitats certainly have the greatest abundance. 

We have modified the sentence to “Microalgae are microorganisms widely distributed throughout many natural environments”.

Line 89: If you use the term “Agrobacterium” to designate a genus, then you need to write it in italics.

Thank you. We have corrected this in the writing.

Line 90, 93 and further: Citations of figures should not be highlighted in bold.

Thank you. We have corrected this in the writing.

Line 158: Correct “ca” to “can”.

Thank you. We have corrected this in the writing.

Line 199: Correct “Chlorella spp..” to “Chlorella spp.”

Thank you. We have corrected this in the writing.

Line 283: Correct “[81] .” to “[81].”

Thank you. We have corrected this in the writing.

Table 3-6: Correct “Ref.” to “Reference” in the titles of the last columns.

Due to the width of the table, we could not easily accommodate the whole word. This short form is common practice in many publications. 

Line 364: Please, use the synonym for the word “recent”, because you have written it in the previous sentence.

Thank you. We have corrected this in the writing.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive summary of advancements in the bioengineering of Chlorella spp. for recombinant protein production. Overall, this manuscript was well-written. There was only minor comment that might be the reviewer’ attention.

1.      The tittle can’t cover the entire contents of this manuscript. Please rewrite.

2.      As a review manuscript, some of the author's views should be added when summary the previous work.

3.      The conclusion section should provide some development tendency and outlook.

4.      The reference format should be uniform.

Author Response

Thank you for your time spent reviewing our manuscript. 

This manuscript provides a comprehensive summary of advancements in the bioengineering of Chlorella spp. for recombinant protein production. Overall, this manuscript was well-written. There was only minor comments that might be the reviewer’ attention.

1. The title can’t cover the entire contents of this manuscript. Please rewrite.

We have changed the title from “Genetic Elements for Recombinant Protein Expression in Chlorella spp.” to “Recombinant Protein Expression and their Biotechnological Applications in Chlorella spp.” and slightly modified the abstract and keywords.

2. As a review manuscript, some of the author's views should be added when summary the previous work.

The authors' views are discussed in most sections of this review, especially in the conclusion. For example, we have highlighted the need to understand the effects of illumination on antibiotic selection. 

3. The conclusion section should provide some development tendency and outlook.

We have edited the conclusion to include development tendency and outlook.

4. The reference format should be uniform.

The referencing was done using referencing software. We are happy to modify specific instances or leave this aspect for the copy editor, but our review did not find anything amiss. 

Reviewer 4 Report

Comments and Suggestions for Authors

This review is interesting and relevant. The Figures and Tables are informative. The reviewer makes several comments to help the authors make the text more understandable for readers.
Since this is a review of scientific literature, it is important to provide all necessary references to the literature where they belong.

1) Line 47. Please add reference(s) after " Chlorella spp. (vulgaris,
ellipsoidea, and sorokiniana)".
2) Figure 1, Line 86 Please indicate how the calculation was made. What resources did the authors use?
3) Line 84. Please provide at least a few links to relevant studies.
4) Line 192. Table 3. Table 3 should be table 2. Is that right? It is useful to provide links to the relevant literature in this table as the authors made in Table 3 (line 291), Tables 4,5,6.
5) Line 199. Please provide a few links to relevant works.
6) Line 207. Reference is missing.
7) Line 208. "Have" has to be "have".
8) Line 221.The text is unclear. Please clarify your thought.
9) Line 237. Please provide at least a few links to  relevant works.
10) Line 238. Please provide links to relevant works.
11)  Please add all abbreviations used in the text in the list of abbreviations.

Author Response

Thank you for the time you took to thoroughly review our manuscript. 

This review is interesting and relevant. The Figures and Tables are informative. The reviewer makes several comments to help the authors make the text more understandable for readers.
Since this is a review of scientific literature, it is important to provide all necessary references to the literature where they belong.

1) Line 47. Please add reference(s) after " Chlorella spp. (vulgaris, ellipsoidea, and sorokiniana)".

This sentence was only to summarize the most commonly used species from the Chlorella genus. We did not include references as the remainder of the manuscript cites many examples and this would make this sentence difficult to read. The most common species are easily found by consulting the tables presented in the following sections. 

2) Figure 1, Line 86 Please indicate how the calculation was made. What resources did the authors use?

The y-axis of figure 1 is the cumulative number of studies, which means that the calculation was made by counting the cumulative number of studies that have been published each until the corresponding year. Since this paper reviews all the recombinant protein studies within Chlorella spp., it’s based on all the studies that have been discussed in this paper. We have edited the caption to indicate that the results are from the studies reviewed in the following sections of this paper.

3) Line 84. Please provide at least a few links to relevant studies.

We have edited the writing to indicate that all the studies are reviewed in the following sections of this paper.

4) Line 192. Table 3. Table 3 should be table 2. Is that right? It is useful to provide links to the relevant literature in this table as the authors made in Table 3 (line 291), Tables 4,5,6.

Yes, thank you. Line 192 should be Table 2. We have corrected this. References were not included in this table because the antibiotics uses for each species are already reported in the other tables. Rather this table is a summary of all of the concentrations reportedly used thus far for each species intended to be a quick reference for readers.

5) Line 199. Please provide a few links to relevant works.

All the promoters used are summarized in Table 3,4,5,6 with references. If a reader is looking for more information on a specific promoter they can check the tables to quickly find the relevant papers with much more details on what was produced. This figure is intended to be a quick summary of the presented work in this review.

6) Line 207. Reference is missing.

Same response as above. All references are in the tables for easy reference to this information. There are too many to cite. 

7) Line 208. "Have" has to be "have".

Thank you, we have corrected this in the writing.

8) Line 221.The text is unclear. Please clarify your thought.

Thank you, allow should be although. We have corrected this mistake. 

9) Line 237. Please provide at least a few links to relevant works.

Same response as above. All references are in the tables for easy reference to this information. This figure is a summary. 

10) Line 238. Please provide links to relevant works.

Same response as above. All references are in the tables for easy reference to this information. This figure is a summary. 

11)  Please add all abbreviations used in the text in the list of abbreviations.

Thank you. We have carefully checked and updated the list of abbreviations to include all abbreviations.