Biotechnology Notes 5 (2024) A1–A3

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The future of cell-free synthetic biology

A R T I C L E I N F O A B S T R A C T

Keywords: Cell-free synthetic biology aims at the targeted replication, design, and modification of life processes in open
Cell-free synthetic biology systems by breaking free of constraints such as cell membrane barriers and living cell growth. The beginnings of
In vitro synthetic biology this systematized technology, which took place in the last century, were used to explore the secrets of life.
Cell-free protein synthesis
Currently, with its easy integration with other technologies or disciplines, cell-free synthetic biology is devel­
Cell-free gene expression
Cell-free transcription-translation
oping into a powerful and effective means of understanding, exploiting, and extending the structure and function
Future perspectives of natural living systems. Cell-free synthesis technology has been used in basic and applied research such as
metabolic prototyping, artificial cell construction, nucleic acid engineering, protein engineering, etc. Worldwide
experts shared their views and opinions on the future development of cell-free synthetic biology, as illustrated in
this paper. With the integration of current popular technologies such as artificial intelligence and electronics,
cell-free synthetic biology will play an increasingly important role in fields such as life exploration, intelligent
manufacturing, human health, new energy, and sustainable environmental development.

1. Introduction At present, cell-free synthetic biology has entered a period of rapid

development and application. Currently, the cell-free biosynthesis
Cell-free synthetic biology, also known as in vitro synthetic biology, platform includes three major systems: multi-enzyme catalyzed syn­
solution biochemistry, etc. The core concept is to achieve life processes thesis system, component-specific transcription/translation system, and
such as transcription, translation, catalysis, metabolism, and signal crude extract-based transcription/translation system. The development
transduction in an in vitro cell-free system. The key technologies are of its chassis has originated from diverse model or non-model prokary­
called cell-free protein synthesis, cell-free transcription-translation, cell- otic or eukaryotic cells. At the basic research level, cell-free systems are
free gene expression, etc. Compared with traditional cellular systems, used to explore dark matter in living systems, design system prototypes,
cell-free systems have outstanding characteristics such as openness and construct artificial cells, and perform other fascinating research. At the
controllability, which can quickly and flexibly design and modify life application research level, cell-free systems are used for functional
processes from bottom to top, achieving a deeper understanding and molecule screening, nucleic acid synthesis, complex protein synthesis,
wider application of natural life systems with great engineering and non-natural biological molecule synthesis. Furthermore, by inte­
freedom. In addition, due to the absence of cell membranes and con­ grating with the evolving novel technologies, cell-free synthetic biology
straints on cell growth, cell-free systems can be freely fused with non- continues to radiate new vitality for technology development.
living molecules, materials, equipment, etc., greatly expanding the The future development of cell-free synthetic biology is a common
functionality and application scope of natural living systems. concern for synthetic biologists. Experts worldwide in this field were
This cell-free synthetic biology research field has gone through invited to express their opinions and perspectives. Based on their own
roughly three golden periods. The first period was in the 1960s, focusing research content and understanding of the field, experts shared their
on basic research, such as the exploration of the transcriptional trans­ views, ideas, challenges, and prospects. The specific information is
lation mechanism of the biological central dogma, which was the em­ shown below.
bryonic form of cell-free biosynthesis systems. The second period was at
the beginning of this century. Cell-free biosynthesis systems were 2. Opinions from worldwide experts
expanded to engineering and industrial applications, mainly focusing on
synthesizing difficult-to-express proteins. The third period is the present Allen Liu.
2020s. With the development of new technologies such as biology, University of Michigan
chemistry, materials, and computers, as well as the new demands of allenliu@[Link].
society for health, energy, resources, environment, and other aspects, The ability to construct programmable synthetic cell systems from
cell-free biosynthesis systems are increasingly being used in challenging the bottom up — with precise input-output relationships — promises
basic and applied research. transformative impacts on human health, biotechnology, and the

[Link]
Received 4 November 2024; Accepted 5 November 2024
Available online 9 November 2024
2665-9069/© 2024 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY
license ([Link]
Y. Lu Biotechnology Notes 5 (2024) A1–A3

environment. These systems will leverage the significant progress made cultivation to meet the growing demand for individualized therapeutic
over the past decades in cell-free protein expression, DNA/RNA-based solutions. This flexibility is essential for producing tailored biologics on
gene circuits, and protein biochemistry. As the field advances toward demand, aligning with the shift toward patient-specific treatments. In
creating use-inspired synthetic cell systems to address real-world prob­ diagnostics, the simplicity and modularity of cell-free synthesis allow for
lems, it must overcome three critical challenges. First, the ability to the generation of reporter proteins in response to specific biomarkers,
reliably and functionally reconstitute membrane proteins will be critical such as proteins and nucleic acids, enhancing multi-target detection
for endowing synthetic cells with environmental sensing capabilities. capabilities. Its portability and simplicity also make it ideal for point-of-
Second, ensuring a sustainable metabolism to provide energy to fuel care applications, especially in resource-limited settings. As related
protein synthesis will be a major hurdle. Third, integrating different technology advances, cell-free synthesis is poised to redefine bio­
functional modules will significantly expand the versatility of synthetic manufacturing and diagnostics, supporting next-generation healthcare
cells. From a fundamental science perspective, there are a lot of op­ with greater efficiency and adaptability.
portunities to investigate spatial and temporal control of cytoskeleton Fernando López Gallego.
assemblies of enzyme activities. This could help recreate complex fea­ CIC biomaGUNE
tures of cellular shape changes and compartmentalized signaling, at flopez@[Link].
both the single-cell level and in multi-cellular assemblies. Assembling isolated enzymes, either in their pure or crude formu­
Arnold Driessen. lation, into biosynthetic pathways of a certain complexity (including
University of Groningen more than 4 enzymes) has given rise to a new discipline: cell-free syn­
[Link]@[Link]. thetic biology, also coined as synthetic biochemistry or systems bio­
One of the main challenges in biology is to construct life bottom-up catalysis. Most existing examples are limited to free enzymes, leading to
from individual components ideally programmed by a minimized systems with low robustness that are difficult to transfer across different
genome. As an approximation, synthetic cell research depends on in vitro reactor configurations for further intensification. The rational co-
transcription/translation systems to synthesize the protein components immobilization of multi-enzyme systems on solid porous materials is
of the cell. These can be either crude cell lysates or built from purified opening a new avenue to intensify and scale up both natural and arti­
components such as the PURE system that includes, amongst others, ficial biosynthetic pathways. Yet, their implementation in flow reactors
essential components such as ribosomes that so far resisted in vitro for their continuous operation is scarce. Hence, new advances in enzyme
reconstitution. Despite the general versatility of these systems, some immobilization technology and in the analytics for characterizing multi-
major breakthroughs will be required for cell-free systems to support the functional heterogeneous biocatalysts are demanded to increase the
autonomous replicating and dividing of synthetic cells. While cell-free complexity of biosynthetic pathways in the solid phase. Heterogeneous
synthesis of biosynthetic pathways is possible, the sheer number of cell-free synthetic biology should aim to match the complexity achieved
genes to be translated in sufficient numbers for a synthetic cell still by cell-free systems in solution, where up to 54 enzymes have been
provides a formidable challenge. Further, efficient methods are needed assembled, while also enhancing product titers and enzyme and cofactor
to trap cell-free systems into giant unilamellar vesicles while main­ turnover numbers.
taining the permeability barrier of the membrane. Finally, specifically Filippo Caschera.
for membrane proteins, their synthesis and membrane insertion should Consultant
be coordinated to prevent protein aggregation and misfolding and, filippocaschera@[Link].
hence, poor productivity of the synthetic cell. Cell-Free Protein Synthesis (CFPS) will supercharge drug discovery,
Atsushi Ogawa. improving human health. CFPS is an easily accessible platform for
Ehime University expressing and prototyping proteins at unprecedented speed and
[Link]@[Link]. throughput with robotic workstations and microfluidic systems. New
As the name implies, cell-free synthetic biology seeks to create living macromolecules will be designed using Generative AI or Active Learning
cells from cell-free components. While it is academically important to – Assisted Directed Evolution. The Design-Build-Test-Learn cycle will
create artificial cells that are identical or similar to living cells found in enable the discovery of antibodies and antigens conjugates to develop
nature, creating artificial cells with functions that surpass those of nat­ more efficient cancer therapies and fight infectious diseases. New par­
ural cells for the benefit of society would also be a major step forward in adigms will be advanced towards antimicrobial activity by engineering
this field. Many challenges must be overcome to achieve these goals, and new peptides and phage therapies. Moreover, CPFS is the favourable
one of the most pressing is how to assemble well-controlled and complex method to produce membrane proteins such as GPCRs, which represent
genetic circuits. Our research subject, cell-free artificial riboswitches, the major target for drug development. Consequently, Cell-Free Protein
will help to address this challenge. Synthesis will be a platform used in every pharmaceutical and
Bo Zheng. biotechnology company worldwide.
Shenzhen Bay Laboratory Jamin Koo.
bozheng@[Link]. Hongik University
The research of in vitro reconstitution of ribosomes has made sig­ jaminkoo@[Link].
nificant progress in recent years. In the near future, we expect to witness Future trends in cell-free synthetic biology will revolutionize the
a cell-free transcription-translation (TX-TL) system that is capable of synthesis of novel metalloproteins with tailored shapes and structures.
self-replication. However, the construction of self-replicating synthetic By bypassing the complexities of living cells, cell-free systems enable
cells based upon the self-replicating cell-free TX-TL system still faces precise control over protein design, including the incorporation of non-
mounting challenges, such as the complexity of membrane regulation. natural amino acids and cofactors. This will facilitate the creation of
Membraneless porous particles enriched with cell-free TX-TL system metalloproteins with enhanced or entirely new functions, expanding
could circumvent the challenges and be the stepping stones to the first their applications in catalysis, bioremediation, and therapeutic devel­
self-replicating synthetic cells. opment. Recent advances, such as the use of cell-free systems to syn­
Dong-Myung Kim. thesize complex metalloproteins, suggest a promising path toward the
Chungnam National University rapid production of custom-designed biomolecules for diverse
dmkim@[Link]. industries.
Cell-free synthetic biology will play transformative roles in person­ Jian Li.
alized medicine and diagnostics. Cell-free protein synthesis enables ShanghaiTech University
rapid, customizable protein production, bypassing traditional cell lijian@[Link].

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Y. Lu Biotechnology Notes 5 (2024) A1–A3

Cell-free synthetic biology has evolved from a fundamental research systems will likely see increased integration with artificial intelligence
tool into a versatile platform with great potential. Historically, cell-free for protein design and optimization, enabling faster iteration and high-
systems played a pivotal role in deciphering genetic codes. Looking throughput screening. Besides, despite being in the early stages, syn­
ahead, I envision cell-free systems emerging as powerful biotechnolog­ thetic cells based on cell-free systems are emerging as powerful plat­
ical tools for biomanufacturing and the bioeconomy. My research fo­ forms for mimicking cellular functions without the complexity of living
cuses on the cell-free biosynthesis of complex pharmaceutical natural cells. By enabling precise control over reaction conditions and the
products, and I believe these systems will provide efficient pathways for components involved, synthetic cells offer a streamlined environment
natural product biosynthesis, significantly accelerating discovery and for the development of complex gene circuits, allowing rapid testing and
development processes. The future of cell-free synthetic biology is iteration of circuit designs.
promising, poised to drive innovations that will transform the field of Yoshihiro Shimizu.
biotechnology. RIKEN
Kei Fujiwara. yshimizu@[Link].
Keio University We’re strongly interested in synthetic cell projects that aim to answer
fujiwara@[Link]. fundamental questions: what is life? Recently, we have been studying
Advances in cell-free technology are bridging the gap between pu­ crucial modifications on tRNAs, beneficial for the tRNA reconstitution,
rified systems and living cells. This progress enables more detailed as well as how to assemble active ribosomes from their molecular parts,
emulation of cellular processes and will clarify the fundamental differ­ both performed in cell-free systems. We are also interested in synthe­
ences between in vitro and in vivo environments, which were often dis­ sizing beneficial products with cell-free systems to solve biological
missed as artifacts. Consequently, we are gaining insights into the questions in other research fields. We have discovered that cell-free
essential blueprint of life, which holds the potential for a deeper un­ synthetic biology can contribute to quantitative proteome analysis,
derstanding of biological systems. This achievement transforms molec­ and we are promoting research in such a direction for the development
ular biology from a field that observes and identifies components of life of medicine and analysis of the molecular status in higher organisms.
into one that literally assembles and understands biology at the molec­
ular level. 3. Conclusion
Lei Kai.
Jiangsu Normal University The perspectives shared by these fourteen experts worldwide on the
lkai@[Link]. future development of cell-free synthetic biology would inspire students,
The exciting pursuit of building a cell through bottom-up approaches teachers, researchers, government decision-makers, and business
has captured the attention of multidisciplinary communities, emerging personnel engaged in synthetic biology or biotechnology-related fields.
as a distinct research field. Over the past two decades, significant ad­ The announcement of this year’s Nobel Prize has once again pushed the
vancements have been made in constructing synthetic cellular mimics application of artificial intelligence in the field of biology to another
characterized by compartmentalization, metabolism, replication, and climax. Artificial intelligence tools can accelerate the design of bio­
emergent properties such as evolution, responsiveness, and movement. molecules, but there are two important challenges that need to be
However, challenges remain in developing key technologies to integrate addressed after design: how to synthesize them through experiments,
these components effectively. This includes refining tunable compart­ and how to quickly screen for such a large sample size. In this context,
mentalization techniques and creating long-lasting, autonomous cell- cell-free synthetic biology will leverage its unique and significant ad­
free expression systems. Continuous improvement in encapsulation ef­ vantages to address the dual issues of synthesis and screening. With the
ficiency and system homogeneity, even through microfluidic platforms, development and demand of important fields such as new energy, ma­
is essential. Additionally, enhancing ribosome efficiency within cell-free terials science, bioelectronics, neuroscience, and carbon neutrality, cell-
systems, especially toward constructing a fully synthetic ribosome, re­ free synthetic biology will also play its unparalleled advantages and
mains vital. In addition, other enabling technologies, such as DNA roles.
synthesis, imaging technologies, automation, computational modeling
as well as AI, would greatly accelerate such progress. Finally, large-scale
collaboration across laboratories, consortia, and continents is necessary Declaration of competing interest
to enable seamless communication and efficient technology transfer,
ultimately paving the way toward the creation of a minimal synthetic The author is an Editor-in-Chief for Biotechnology Notes and was not
cell. involved in the editorial review or the decision to publish this article.
Mark Styczynski.
Georgia Institute of Technology
[Link]@[Link]. Acknowledgments
Cell-free synthetic biology has tremendous potential for broad sci­
entific and societal impact. As costs come down and productivities go Many thanks to the worldwide experts for kindly contributing their
up, cell-free synthetic biology is destined for increasingly broad indus­ perspectives and sharing their thoughts on the future trends of cell-free
trial applications that could substantially impact biomanufacturing even synthetic biology. These kind experts are Allen Liu, Arnold Driessen,
if it cannot displace several well-established biomanufacturing strate­ Atsushi Ogawa, Bo Zheng, Dong-Myung Kim, Fernando López Gallego,
gies. In the intervening time, small-volume applications like diagnostics Filippo Caschera, Jamin Koo, Jian Li, Kei Fujiwara, Lei Kai, Mark Styc­
are likely to be the main vehicle for impact, from biomedical to envi­ zynski, Shaobin Guo, and Yoshihiro Shimizu. These experts are sorted by
ronmental applications. As these applications make their way through their first names in alphabetical order.
regulatory approval and adoption, they will be the first ways that we put
the benefits of cell-free approaches into the hands of everyone. Yuan Lu
Shaobin Guo. Department of Chemical Engineering, Tsinghua University, Beijing 100084,
Fuzhou University China
sguo@[Link]. Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua
As this year’s Nobel Prize in Chemistry recognizes breakthroughs in University, Beijing 100084, China
rational protein design and AI-driven structure prediction, cell-free E-mail address: yuanlu@[Link].

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