The Evolution of Cell Free Biomanufacturing
Round 1
Reviewer 1 Report
This manuscript summarizes the evolution of cell-free systems, applications, and mathematical modeling. The manuscript reads very well, providing a comprehensive understanding of the advancement of cell-free platforms.
The reviewer has minor suggestions to improve the manuscript.
1) Consider adding sub-headings in each subsection of the Applications and Modeling.
e.g., cell-free protein synthesis application, cell-free systems in synthetic biology, and cell-free metabolic engineering could be subheadings in 3 Applications. and adequate sub-headings to 4 Modeling also would be good.
2) It is suggested to include reviewing scale-up and current industrial effort using cell-free systems as their manufacturing platforms.
3) L193, Butanol to butanol.
4) L204, add abbreviation of coenzyme A and the full name of NAD+ and "+" should be superscript.
5) Consider dividing the current Conclusion into two parts (Perspective and Conclusion) to improve readability.
Author Response
See attached the rebuttal document
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The evolution of cell free biomanufacturing
This manuscript summarizes the advancement in cell free technology and its applications in protein and metabolite biosynthesis, biosensors, prototyping. The manuscript covers a wide scope of cell free biomanufacturing technology and can be published after addressing the following concerns.
Major comments
- Consider change the title of the manuscript. Although the authors have touched on the origin of technology, the “evolution” or progression was not clear. Majority of the manuscript focused on the recent development in cell-free technology. It will be good to have a timeline or summary figure to indicate the progress made for the cell free technology.
- Although the paper aims to summarize the cell free technology for protein synthesis and metabolic engineering, more focus has been placed on protein synthesis. The information on cell free metabolic engineering is briefly mentioned. Authors may provide more information on cell-free metabolic engineering, such as the following publications.
(1) Dudley, Q. M.; Nash, C. J.; Jewett, M. C. Cell-Free Biosynthesis of Limonene Using Enzyme-Enriched Escherichia Coli Lysates. Synth. Biol. 2019, 4 (1). https://doi.org/10.1093/synbio/ysz003.
(2) Opgenorth, P. H.; Korman, T. P.; Bowie, J. U. A Synthetic Biochemistry Module for Production of Bio-Based Chemicals from Glucose. Nat. Chem. Biol. 2016, 12 (6), 393–395. https://doi.org/10.1038/nchembio.2062.
(3) Valliere, M. A.; Korman, T. P.; Woodall, N. B.; Khitrov, G. A.; Taylor, R. E.; Baker, D.; Bowie, J. U. A Cell-Free Platform for the Prenylation of Natural Products and Application to Cannabinoid Production. Nat. Commun. 2019, 10 (1), 1–9. https://doi.org/10.1038/s41467-019-08448-y.
(4) Chen, X.; Zhang, C.; Zou, R.; Stephanopoulos, G.; Too, H.-P. In Vitro Metabolic Engineering of Amorpha-4,11-Diene Biosynthesis at Enhanced Rate and Specific Yield of Production. ACS Synth. Biol. 2017, 6 (9), 1691–1700. https://doi.org/10.1021/acssynbio.6b00377.
- Section 4. Mathematical modeling of cell free systems. It is not clear the relevance to the cell-free technology. Please rewrite this section and make it clearer.
- It will be good if the authors could include challenges or disadvantages of cell free biomanufacturing, and the way to address them.
Author Response
See attached the rebuttal document document
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
No further comments
