Until the end of the 19th century, it had been accepted that fermentation required live yeast cells. At that time, a debate had arisen as to whether fermentation could take place without living cells or whether such a «life force» was necessary.  In 1897, the German chemist Eduard Buchner found out that yeast extract could form alcohol from a sugar solution without the living yeast fungi. He covered a group of cells with sand until they were completely destroyed, extracted the remaining liquid and added it to a sugar solution. To the surprise of many, the liquid produced fermentation.

For this discovery he was awarded the Nobel Prize in Chemistry in 1907. Years later, in 1961 Nirenberg and Matthaei found cell-free synthesis of polyphenylalanine from extracts of E. coli [1]. Following these findings, cell-free biosynthesis began to be considered as a field for further research. It was the beginning of something, we suddenly realised that biochemical processes could be produced without the need for living cells and that opened a new world full of possibilities. Since that moment huge progress has been made, but in the last decade there has been a rise of cell-free systems development [2].

The way to overcome major constraints in synbio research?

Many researchers are frustrated by the restrictions found when working with living cells, as the requirement of a cellular host entails significant constraints. There are concerns over biosafety linked to the self-replicability of the cell and the risk of contamination that could lead to an impact on human health or food security. Moreover, they are much harder and slower to work with than cell-free systems, not only because of the high level of complexity in design but also because they require laborious steps to import the previously designed vector into the cell. There is also possible cross-talk with internal mechanisms of the cell that must be taken into account since cells are focused on their own growth and division — their natural evolutionary progress has not optimised them to produce things for us.

Cell-free systems (CFS) open up the possibility of minimising some of these constraints. Their main advantages are their simplicity and portability. CFS are much faster than cell-based methods, can be made sterile by simple filtration and there is no need for selective markers or tedious cloning steps. Bearing in mind that they still have many shortcomings, mainly linked to cost, yield and reproducibility, and that much more development is needed to overcome their weaknesses, their potential in some major fields of synthetic biology is worth noting.

For starters, they could be the new revolution in drug discovery. There are many proteins in the Protein Data Bank (PDB) without characterised function, and as the potential for many of them as functional drugs remain unknown, CFS systems provide a fast method to synthetise and characterise these proteins to test them [3]. But their range of applications is wide and does not stop here; CFS are being used in other fields, too.

In the wake of the Covid-19 pandemic, we have realised that we need more effective and faster methods to deal with such a global emergency. CFS were key in characterising and identifying viral proteins during the pandemic [4]. It caught everyone off guard and since then there has been a change in mentality and awareness about being prepared for possible new zoonoses.

They are also useful for the development of vaccines. Just this January, a vaccine for bacterial pneumococcal disease using a cell-free biosynthesis strategy was developed [6]. This possibility might also be key to combating possible future health threats. Last year, a team of researchers developed an eukaryotic CFS that was able to synthesise all the proteins derived from SARS-CoV-2 using a lysate derived from Chinese hamster ovary cells. This breakthrough might ensure a quick response, providing fast and efficient synthesis and characterization of viral proteins and antibody validation [5].

Another major concern of our times is the seemingly unstoppable increase of antibiotic resistance by pathogens. These systems may speed up the process of discovering potential antibiotic-applicable genes and they are capable of producing antimicrobial compounds, including antimicrobial peptides and small molecule drugs [7].

Stepping into the near future of cell-free systems

Cell-free systems might be the forthcoming revolution in some fields of synthetic biology. After some years of development, they are becoming increasingly cost-effective, however, there is still a road ahead to improve efficiency and production quality.

Adding to the potential of these systems in the face of a new public health threat, they have a high level of functionality as diagnostic sensors. They also have the potential to produce antibodies, which could be a breakthrough in the pharmaceutical industry. But for all their merits, as of today, they still need more precision to make many of these applications a useful reality.

Besides the more glaring limitations, some key processes that occur to proteins in vivo are difficult to achieve with CFS. And even if these shortcomings can be solved, it is unlikely that CFS will ever fully replace cell-based systems, which also continue to make steady progress and have their own large scope of application. However, CFS are still a step forward in synbio research and we need to highlight the advantages they provide and optimise their strengths.

We envision a future where these two systems —both cell-based and cell-free— will each develop to their strengths, and we will be following with great interest what CFS brings us in the coming years.


  1. Nirenberg, M.W. and Matthaei, J.H. (1961) “The dependence of cell-free protein synthesis in coli upon naturally occurring or synthetic polyribonucleotides,” Proceedings of the National Academy of Sciences, 47(10), pp. 1588–1602. https://doi.org/10.1073/pnas.47.10.1588.
  2. Tinafar, A., Jaenes, K. and Pardee, K. (2019) “Synthetic Biology goes cell-free,” BMC Biology, 17(1). https://doi.org/10.1186/s12915-019-0685-x.
  3. Dondapati, S.K. et al. (2020) “Cell-free protein synthesis: A promising option for future drug development,” BioDrugs, 34(3), pp. 327–348. https://doi.org/10.1007/s40259-020-00417-y.
  4. Combating covid-19 with cell-free expression (August 19, 2020) The Scientist Magazine®. Available at: https://www.the-scientist.com/sponsored-article/combating-covid-19-with-cell-free-expression-67840 (Accessed: March 15, 2023).
  5. Ramm, F. et al. (2022) “The potential of eukaryotic cell-free systems as a rapid response to novel zoonotic pathogens: Analysis of SARS-COV-2 viral proteins,” Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.896751.
  6. Arnold, C. (2023) “How cell-free processes could speed up vaccine development,” Nature, 13 March. https://www.nature.com/articles/d41586-023-00760-4
  7. Martemyanov, K.A. et al. (2001) “Cell-free production of biologically active polypeptides: Application to the synthesis of antibacterial peptide Cecropin,” Protein Expression and Purification, 21(3), pp. 456–461. https://doi.org/10.1006/prep.2001.1400.