Curated Papers > High-impact authoritative literature on "synthetic biology"

Cell-free synthetic biology: Engineering in an open world.

Literature Information

DOI	10.1016/j.synbio.2017.02.003
PMID	29062958
Journal	Synthetic and systems biotechnology
Impact Factor	4.4
JCR Quartile	Q1
Publication Year	2017
Times Cited	66
Keywords	Artificial cell, Cell-free protein synthesis, Cell-free synthetic biology, Metabolic engineering, Protein engineering
Literature Type	Journal Article, Review
ISSN	2405-805X
Pages	23-27
Issue	2(1)
Authors	Yuan Lu

TL;DR

This review highlights the advancements in cell-free synthetic biology, which enables the engineering of biological systems without living cells, thus allowing for more straightforward, rapid, and flexible design processes. Key findings include enhanced protein synthesis capabilities, the design of metabolic pathways for desired product production, and the development of artificial cells, all of which have significant implications for life sciences and potential applications in environmental and biomedical fields.

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Artificial cell · Cell-free protein synthesis · Cell-free synthetic biology · Metabolic engineering · Protein engineering

Abstract

Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells. It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system. This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels, including protein engineering, metabolic engineering, and artificial cell engineering. In cell-free protein engineering, the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins, toxic proteins, membrane proteins, and novel proteins with unnatural amino acids. Cell-free systems offer the ability to design metabolic pathways towards the production of desired products. Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.

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Primary Questions Addressed

1. How does cell-free synthetic biology compare to traditional cell-based approaches in terms of scalability and flexibility?
2. What are the potential ethical implications of engineering artificial cells using cell-free systems?
3. In what ways can cell-free protein engineering be applied to address challenges in drug development and production?
4. How do the recent advancements in metabolic engineering through cell-free systems impact the field of sustainable biomanufacturing?
5. What are the limitations of current cell-free synthetic biology techniques, and how might future research address these challenges?

Key Findings

Research Background and Objectives

Cell-free synthetic biology has emerged as a significant technological advancement that allows for the engineering of biological systems without the use of living cells. This approach addresses the inherent complexities and limitations of traditional cell-based systems, offering enhanced flexibility and control in biological engineering. The primary objective of this review is to explore recent developments in cell-free synthetic biology, particularly in the fields of protein engineering, metabolic engineering, and artificial cell construction.

Main Methods/Materials/Experimental Design

The review discusses three main types of cell-free systems:

1. Extract-based systems: Utilize crude extracts from organisms such as E. coli, yeast, and insect cells to perform transcription and translation.
2. Purified systems: Include systems like the PURE system that use purified components for protein synthesis.
3. Synthetic enzymatic pathway systems: Comprise multiple enzymes designed for specific bioreactions.

The following flowchart illustrates the overall technical approach:

Key Results and Findings

• Protein Engineering: Cell-free systems enable the synthesis of complex, toxic, and membrane proteins with higher yields and less complexity compared to in vivo systems. The ability to incorporate unnatural amino acids (uAAs) expands the potential for novel protein designs.
• Metabolic Engineering: These systems allow for the design of metabolic pathways independent of cellular growth, achieving higher conversion efficiencies and tolerance to toxic substrates. For example, biohydrogen production reached 97% of theoretical yields using cell-free systems.
• Artificial Cell Engineering: The review discusses the potential for constructing artificial cells using cell-free systems, which could mimic living cells' functions, facilitating advancements in drug delivery and environmental sensing.

Main Conclusions/Significance/Innovation

Cell-free synthetic biology presents a promising platform for overcoming the limitations of traditional living cell systems. Its flexibility and controllability make it suitable for various applications in biopharmaceuticals, diagnostics, and sustainable bioenergy production. The integration of uAAs and the ability to construct artificial cells highlight the innovative potential of this technology.

Research Limitations and Future Directions

Despite its advantages, challenges remain in cell-free synthetic biology, including:

• Post-translational modifications: Critical for biological functions but difficult to achieve in cell-free systems.
• Expanding the genetic code: Incorporating multiple uAAs into proteins needs further exploration.
• System reuse: Optimizing cell-free systems for repeated use remains a challenge.

Future research should focus on optimizing these systems for better regulatory control, integrating them with advanced technologies such as artificial intelligence and 3D printing, and exploring new applications in biomedicine and environmental sciences.

References

1. Practical cell-free protein synthesis system using purified wheat embryos. - Kazuyuki Takai;Tatsuya Sawasaki;Yaeta Endo - Nature protocols (2010)
2. A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. - Yutetsu Kuruma;Pasquale Stano;Takuya Ueda;Pier Luigi Luisi - Biochimica et biophysica acta (2009)
3. Cell-free expression of G-protein-coupled receptors. - Erika Orbán;Davide Proverbio;Stefan Haberstock;Volker Dötsch;Frank Bernhard - Methods in molecular biology (Clifton, N.J.) (2015)
4. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. - A Krogh;B Larsson;G von Heijne;E L Sonnhammer - Journal of molecular biology (2001)
5. Five hard truths for synthetic biology. - Roberta Kwok - Nature (2010)
6. Rapid translation system (RTS): a promising alternative for recombinant protein production. - J-M Betton - Current protein & peptide science (2003)
7. Celgene wagers on Sutro's cell-free platform to ramp up ADCs. - Mark Ratner - Nature biotechnology (2014)
8. A cell-free protein synthesis system from insect cells. - Toru Ezure;Takashi Suzuki;Eiji Ando - Methods in molecular biology (Clifton, N.J.) (2014)
9. Development of an artificial cell, from self-organization to computation and self-reproduction. - Vincent Noireaux;Yusuke T Maeda;Albert Libchaber - Proceedings of the National Academy of Sciences of the United States of America (2011)
10. Cell Break: How Cell-Free Biology Is Finally Putting the Engineering Back in Bioengineering. - Shannon Fischer - IEEE pulse (2016)

Literatures Citing This Work

1. Progress in biopharmaceutical development. - Malgorzata Kesik-Brodacka - Biotechnology and applied biochemistry (2018)
2. Bacterial cell-free expression technology to in vitro systems engineering and optimization. - Filippo Caschera - Synthetic and systems biotechnology (2017)
3. Bottom-up synthetic biology: modular design for making artificial platelets. - Sagardip Majumder;Allen P Liu - Physical biology (2017)
4. Cell-Free Approaches in Synthetic Biology Utilizing Microfluidics. - Samar Damiati;Rami Mhanna;Rimantas Kodzius;Eva-Kathrin Ehmoser - Genes (2018)
5. Cell-free synthetic biology for in vitro biosynthesis of pharmaceutical natural products. - Jian Li;Lingkai Zhang;Wanqiu Liu - Synthetic and systems biotechnology (2018)
6. Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology. - Lihong Jiang;Jiarun Zhao;Jiazhang Lian;Zhinan Xu - Synthetic and systems biotechnology (2018)
7. "The Smartphone's Guide to the Galaxy": In Situ Analysis in Space. - Joost Nelis;Christopher Elliott;Katrina Campbell - Biosensors (2018)
8. Microfluidics for Artificial Life: Techniques for Bottom-Up Synthetic Biology. - Pashiini Supramaniam;Oscar Ces;Ali Salehi-Reyhani - Micromachines (2019)
9. Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System. - Wei Gao;Ning Bu;Yuan Lu - Methods and protocols (2019)
10. Accelerating the Production of Druggable Targets: Eukaryotic Cell-Free Systems Come into Focus. - Lena Thoring;Anne Zemella;Doreen Wüstenhagen;Stefan Kubick - Methods and protocols (2019)

... (56 more literatures)

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