Precision Fermentation as an Alternative to Animal Protein, a Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript provides a comprehensive review of precision fermentation as an emerging alternative to animal protein production, examining how it can reduce the environmental impact of food production and offering a detailed discussion on the production of animal-derived proteins used in the food and nutraceutical industries through precision fermentation. The manuscript also analyzes the potential of precision fermentation in sustainable food production, as well as the challenges and future prospects it faces, including scaling up production, establishing regulatory frameworks, and addressing ethical issues.

 

1.     The third section of the manuscript partially explains that precision fermentation can be used as a solution to existing environmental and energy problems. The author should focus on how precision fermentation can solve these issues and what ways and methods of solving the issues are? The authors could list them.

2.     The manuscript explains that proteins were obtained through precision fermentation as an alternative to traditional animal farming. However, the fundamental factor is the cost and scale of fermented proteins by which allows fermented proteins to replace animal proteins as compared to traditional animal farming, a detail discussion should be provided.

3.     One of the major differences between traditional farmed animal proteins and precision fermented proteins is that the former can be made directly into food, but the latter is usually used as a food processing raw material or excipient, and the authors need to list the cases in which fermented proteins have been used to make food products at the present.

4.     While the manuscript mentions the success of precision fermentation on a laboratory, the challenge is presented as to how to commercialize these processes and expand them to industrial scale production. The manuscript needs to discuss in more detail how to overcome these scale-up challenges.

5.     The details of the biotechnology and metabolic engineering techniques used in the precision fermentation process are insufficiently enough, especially the section on how to optimize the host microorganisms to increase the protein yields, and the authors should have stated what extend could be achieved for the current yields of the precision fermented proteins.

Author Response

The manuscript provides a comprehensive review of precision fermentation as an emerging alternative to animal protein production, examining how it can reduce the environmental impact of food production and offering a detailed discussion on the production of animal-derived proteins used in the food and nutraceutical industries through precision fermentation. The manuscript also analyzes the potential of precision fermentation in sustainable food production, as well as the challenges and future prospects it faces, including scaling up production, establishing regulatory frameworks, and addressing ethical issues.

A: We thank all 4 reviewers for their critical analysis of our manuscript, and their suggestions for improvement. We have addressed all points raised by the reviewers, which are answered in detail below. In the current revised version of our review, all inclusions/changes made in the text are market in yellow.

1. The third section of the manuscript partially explains that precision fermentation can be used as a solution to existing environmental and energy problems. The author should focus on how precision fermentation can solve these issues and what ways and methods of solving the issues are? The authors could list them.

A: We have included a more detailed discussion on this in the third section of our revised version of the manuscript, lines 160-163 and specially lines 175-210.

2. The manuscript explains that proteins were obtained through precision fermentation as an alternative to traditional animal farming. However, the fundamental factor is the cost and scale of fermented proteins by which allows fermented proteins to replace animal proteins as compared to traditional animal farming, a detail discussion should be provided.

A: We have included a discussion regarding costs and scale-up of fermented protein production in lines 196-203. We believe that with the inclusion of Table 3 in our revised version of the manuscript (as suggested by one of the reviewers), it is more visible that such proteins (products) are indeed been produced and commercialized by many companies. This would not be possible (for the companies) if costs and scale of production issues are been solved. See also the new section “6. Precision Fermentation in the World - The State of Industry”, lines 704-759.

3. One of the major differences between traditional farmed animal proteins and precision fermented proteins is that the former can be made directly into food, but the latter is usually used as a food processing raw material or excipient, and the authors need to list the cases in which fermented proteins have been used to make food products at the present.

A: We agree with the reviewer, and indeed many of the proteins made by precision fermentation will be used as food supplements, nutraceuticals or/and pharmaceutical agents. See Table 3 and also lines 436-441, 506-512, 665-672 and 713-725 for some examples were precision fermentation proteins are used directly as food products (cheeses, drinks, infant formulas, etc.).

4. While the manuscript mentions the success of precision fermentation on a laboratory, the challenge is presented as to how to commercialize these processes and expand them to industrial scale production. The manuscript needs to discuss in more detail how to overcome these scale-up challenges.

A: As mentioned above, we feels that the inclusion of Table 3 (companies and their products made by precision fermentation) gives examples of companies were these challenges are been overcome, obviously reaching industrial scale production. Unfortunately, in most cases how these companies overcome these scale-up challenges are not publically disclosed, are part of patents, or even “industrial secrets”. But a nice example of a laboratory scale-up effort to produce and purify albumin (reaching the highest production of a protein by precision fermentation) is shown in Table 2 and discussed in lines 694-703.

5. The details of the biotechnology and metabolic engineering techniques used in the precision fermentation process are insufficiently enough, especially the section on how to optimize the host microorganisms to increase the protein yields, and the authors should have stated what extend could be achieved for the current yields of the precision fermented proteins.

A: Thank you for the suggestion. We agree with the reviewer and we include more details about metabolic engineering techniques and how microorganisms can be optimized:

-lines 238-262 shows how the transformation efficiency of a K. phaffii strain can be improved, and the development of a license-free strain and expression kits for precision fermentation production of proteins.

-lines 265-278 shows how to transform the Crabtree-positive S. cerevisiae yeast into a respiratory yeast producing more biomass, and thus more recombinant proteins.

-lines 318-329 discusses how to overcome metabolic burdens present during recombinant protein production.

-lines 338-350 discusses the use of artificial intelligence, machine learning and robotics to develop efficient production processes.

-lines 490-504 discusses how glycoproteins can be produced by yeasts, and glycoengineering strategies to produce humanized recombinant proteins.

(see also lines 299-301)

 

Reviewer 2 Report

Comments and Suggestions for Authors

First, I wanted to thank you for entrusting me with the task of reviewing this important manuscript entitled " Precision Fermentation as an Alternative to Animal Protein: A Review" for the Fermentation journal.

I have carefully examined the manuscript; I am impressed by its comprehensive overview of the advancements in precision fermentation. The authors have done an excellent job summarizing recent developments in the field and presenting a thorough list of companies producing various recombinant proteins through this innovative technique. Moreover, the manuscript is well-organized and effectively communicates complex scientific concepts clearly. I believe that this review article will be of significant interest to both academic researchers and industry professionals working in the field of fermentation technology.

Based on my assessment, I highly recommend the acceptance of this manuscript for publication in the Fermentation. I am confident that it will make a valuable contribution to the existing body of knowledge in this area and stimulate further discussion and research in the field of precision fermentation.

Author Response

Thanks for the nice comments about our review. We thank all 4 reviewers for their critical analysis of our manuscript, and their suggestions for improvement. We have addressed all points raised by the reviewers, which are answered in detail below. In the current revised version of our review, all inclusions/changes made in the text are market in yellow.

Reviewer 3 Report

Comments and Suggestions for Authors

This review manuscript by Knychala et al. reviewed the precision fermentation for the production of animal protein. The animal-derived proteins produced by precision fermentation were discussed, and the corresponding manufactories producing animal-derived proteins by precision fermentation were also reviewed. This review manuscript generally fits the scope of Fermentation journal, and I have a comment which needs revision by the authors to the manuscript. 

1. The supplementary table S1 can be moved into the main text, after line 347. 

Author Response

This review manuscript by Knychala et al. reviewed the precision fermentation for the production of animal protein. The animal-derived proteins produced by precision fermentation were discussed, and the corresponding manufactories producing animal-derived proteins by precision fermentation were also reviewed. This review manuscript generally fits the scope of Fermentation journal, and I have a comment which needs revision by the authors to the manuscript.

A: Thanks for the nice comments about our review. We thank all 4 reviewers for their critical analysis of our manuscript, and their suggestions for improvement. We have addressed all points raised by the reviewers, which are answered in detail below. In the current revised version of our review, all inclusions/changes made in the text are market in yellow.

1. The supplementary table S1 can be moved into the main text, after line 347. 

A: Thank you for the suggestion. We have included a Table 3 in our revised manuscript (what was Supplementary Table S1), including the type of products produced.

Reviewer 4 Report

Comments and Suggestions for Authors

1. The publication is a review study. In this case, the opinion of the contribution to the subject area is very subjective. In my opinion, this is not a new approach to the subject, but only a review of the current thematic literature. But, such studies are also needed and should be appreciated. The connection between the impact of traditional animal husbandry on climate change is justified. As well as the search for alternative sources of protein for this farming. However, the form and arguments in the publication causes some doubts

2. In the first chapter, the authors refer primarily to the problem related to food production. Consequently, I would expect a continuation of this topic in the next chapter (Chapter 5), which deals with the possibility of using and producing single-cell proteins. However, the authors indicate other possibilities of using biotechnologically obtained proteins.  This is interesting and necessary, but it should be mentioned in the introduction too.

3. Chapters 2 and 3, despite their different titles, mainly concern the justification for the change in protein production technology. The arguments used are an extensive criticism of traditional animal production. This is too long a consideration and very tendentious. In my opinion, the authors should change the concepts of these chapters. Animal husbandry cannot be reduced to just a source of protein.
The new technology should be an alternative or complement to traditional product,  but not "revolutionize the food industry and completely disrupt traditional animal-based agriculture"
I suggest greater balance in the opinions and consideration of the disadvantages and limitations of using precision fermentation. Especially in the case of products in which biotechnological production of protein is possible only for a specific fraction.

4. Very well written conclusions. With more rewriting and completion, they could be the introduction of this work

Author Response

1. The publication is a review study. In this case, the opinion of the contribution to the subject area is very subjective. In my opinion, this is not a new approach to the subject, but only a review of the current thematic literature. But, such studies are also needed and should be appreciated. The connection between the impact of traditional animal husbandry on climate change is justified. As well as the search for alternative sources of protein for this farming. However, the form and arguments in the publication causes some doubts.

A: Yes, the manuscript is a review in a growing area of fermentation, and in many cases the arguments used are those extensively discussed in the cited references. We thank all 4 reviewers for their critical analysis of our manuscript, and their suggestions for improvement. We have tried to address all points raised by the reviewers, which are answered in detail below. In the current revised version of our review, all inclusions/changes made in the text are market in yellow.

2. In the first chapter, the authors refer primarily to the problem related to food production. Consequently, I would expect a continuation of this topic in the next chapter (Chapter 5), which deals with the possibility of using and producing single-cell proteins. However, the authors indicate other possibilities of using biotechnologically obtained proteins. This is interesting and necessary, but it should be mentioned in the introduction too.

A: The first chapter (Introduction) has been improved and many topics not mentioned in our previous version were now introduced (lines 55-81). The comment regarding “the next chapter (Chapter 5)” is confusing, Chapter 2 (The Environmental Impacts of Animal-Based Production) indeed continues the topic of problems related to food production. And our review is not related to “using and producing single-cell proteins”. Single-cell proteins is the use of cells (primary microorganisms) as food, and or review is the production of specific proteins (by microorganisms), and the use of these specific proteins as an alternative to animal proteins (thus, we are not talking about “single-cell proteins”).

3. 1. Chapters 2 and 3, despite their different titles, mainly concern the justification for the change in protein production technology. The arguments used are an extensive criticism of traditional animal production. This is too long a consideration and very tendentious. In my opinion, the authors should change the concepts of these chapters. Animal husbandry cannot be reduced to just a source of protein.

A: We agree with the reviewer and we would like to apologize if we give the impression of “very tendentious”. Certainly that animal production will continue, let’s say forever, and we do not think that it should disappear (certainly there are other studies addressing farmers’ risk of losing their source of income!, but this is not the focus of our review), but as we argue, the growing population will demand more production that will reach a point of unsustainability. That is why we propose (not “we”, but the public, governments, scientists, etc.) that precision fermentation might be PART of the solution (not THE ONLY solution), contributing with other options for protein production (especially for certain populations –vegans, allergic infants, etc.-) as discussed along the manuscript.

3.2. - The new technology should be an alternative or complement to traditional product,  but not "revolutionize the food industry and completely disrupt traditional animal-based agriculture" I suggest greater balance in the opinions and consideration of the disadvantages and limitations of using precision fermentation. Especially in the case of products in which biotechnological production of protein is possible only for a specific fraction.

A: Again, we apologize and the statement "revolutionize the food industry and completely disrupt traditional animal-based agriculture" was removed (see lines 48-49 of our revised manuscript). We agree that there are several disadvantages and limitations of using precision fermentation, and we tried to discuss them (for particular proteins) in the section “Animal Proteins produced by Precision Fermentation”. Very few examples of food products are presented (cheeses, drinks, infant formulas, etc.), the other proteins are specific for specialized uses (food supplements, nutraceuticals or/and pharmaceutical agents, etc.).

4. Very well written conclusions. With more rewriting and completion, they could be the introduction of this work.

A: Thank you for the suggestion. We rewritten the Introduction based on issues pointed in conclusions (lines 55-81).

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The Authors' responses to my comments are satisfactory. The corrections made to the manuscript are a good addition and do not create additional doubts.