During the last century, Escherichia coli has become one of the most important organisms used in industry (1). It was discovered by Theodor Escherich back in 1885 in Germany and since then, it has been used in molecular biology laboratories worldwide, becoming one of the best characterised organisms on Earth among other prokaryotes. But the hunger for new functions and possibilities within synthetic biology has driven some labs to turn to eukaryotic cells instead.
There are plenty of features that have led prokaryotes to be a universal model for molecular cloning techniques and genetic tools developed using these microorganisms. One of these qualities is the rapid strain development which can reduce costs significantly for industrial development (2).
E.coli’s growth conditions have put it in the limelight as one of the best host organisms for metabolic engineering and synthetic biology. It grows rapidly, its metabolism is plastic, there is a large amount of information available concerning its biochemistry and physiology and its culture conditions are easy to handle. An extra point is that the most commonly used E. coli strains are generally considered harmless.
But the microorganisms comes, of course, with some limitations as a host. It is not suitable for culturing conditions at high (3) and low pH and high temperatures while other bacteria can thrive in harsh environmental settings. The fact that E. coli can’t get through extreme conditions presents advantages for contamination resistance, reduced titration requirements, and consolidated bioprocesses (4). There are also other disadvantages as its incapability of producing glycosylated biopharmaceutical products or proteins that require complex assembly, for example. (5).
Although different bacterial species can complement these limitations —and indeed there are other popular prokaryotic hosts like Bacillus subtilis or Pseudomonas putida, to name a couple—eukaryotic cells have a promising role to play in synthetic biology.
The new era of nuclei
A team of synthetic biologists from John Hopkins University School of Medicine tried something different in 2011. They engineered a Saccharomyces cerevisiae yeast with chromosome arms assembled from chemically synthesised DNA (6). This project represented the start of something new.
Yeasts have a long and noble history in biotechnology — besides their timeless applications in brewing and baking, they have been well studied in the laboratory, too. They are simple and count with many properties which make them appealing for synthetic biology: quick to culture, non-toxic cells and robust enough to freeze-dry them and sell them in packages (7). The most used strain by far is S. cerevisiae for its innate features.
One of key differences with bacteria, of course, is the separation of the genetic material from the rest of the cell. This protects genetic material but it’s a double-edged sword: the DNA targeting proteins need effective nuclear localisations tags, and modifications that could appear in the mRNA sequence can affect when exporting to ribosomes (7).
When it comes to the DNA recombination, there is an effective and targeted integration into host chromosomes, and it has already been proved thorugh the assembly of very large DNA fragments. But also, the repetition of sequences can result in unstable constructs because of improved recombination rates.
An interesting feature from S. cerevisiae is that it has several selectable markers. This allows for multiple constructs to be introduced into one cell, in addition to the availability of positive and negative selectable indicators and the use of auxotrophy for a non-antibiotic selection (7).
With regards to research and industry, S. cerevisiae has been studied for years so it has a well characterised genome, proteome and metabalome (7). There are already engineered libraries available and there are available databases which contain organised and in-depth information. This strain has also been used for industrial purposes since it grows in both conditions: aerobiosis and anaerobiosis. It can use a wide range of carbon sources and substrates and as a great feature: it cannot be contaminated by bacteriophages.
The road ahead to standardisation in yeasts
Nowadays, the synthetic biology community is focusing on developing standardised DNA libraries and fully characterised synthetic biology toolkits. These toolkits have gone beyond S. cerevisiae since they have been successfully modified for other industrially relevant yeasts species such as Komagatella phaffi (8).
Established toolkits, like the BioBricks, whose collection is mainly focused on bacteria, have growing tools for S. cerevisiae available. SEVA, of course, is becoming increasingly compatible as well. Among other assembly methodologies with characterised toolkits, there are also Easyclone Vectors (9) and the EasyClone-MarkerFree system (10).
There is still a world of applications to be developed in bacterial chassis, and eukaryotic microbes just expand these possibilities. As we propel synthetic biology forward with imagination, fostering crossed learning and interoperability, we will start to find solutions for industrial and environmental problems where engineered cells have not succeeded yet.
- Pontrelli, S., Chiu, T. Y., Lan, E. I., Chen, F. Y. H., Chang, P., & Liao, J. C. (2018). Escherichia coli as a host for metabolic engineering. Metabolic engineering, 50, 16-46. https://doi.org/10.1016/j.ymben.2018.04.008
- Meyer, H., & Schmidhalter, D. R. (2012). Microbial Expression Systems and Manufacturing from a Market and Economic Perspective. In (Ed.), Innovations in Biotechnology. IntechOpen. https://doi.org/10.5772/29417
- Tao, F., Miao, J. Y., Shi, G. Y., & Zhang, K. C. (2005). Ethanol fermentation by an acid-tolerant Zymomonas mobilis under non-sterilized condition. Process Biochemistry, 40(1), 183-187. https://doi.org/10.1016/j.procbio.2003.11.054
- Hasunuma, T., & Kondo, A. (2012). Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains. Process Biochemistry, 47(9), 1287-1294 https://doi.org/10.1016/j.procbio.2012.05.004
- Meyer, H. P., & Schmidhalter, D. R. (2012). Microbial expression systems and manufacturing from a market and economic-perspective. Innovations in biotechnology, 211-250. https://books.google.es/books?id=gdCcDwAAQBAJ&lpg=PA211&ots=aF5yB-eaii&lr&hl=es&pg=PA211#v=onepage&q&f=false
- Dymond, J. S., Richardson, S. M., Coombes, C. E., Babatz, T., Muller, H., Annaluru, N., … & Boeke, J. D. (2011). Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature, 477(7365), 471-476. https://doi.org/10.1038/nature10403
- Blount, B. A., Weenink, T., & Ellis, T. (2012). Construction of synthetic regulatory networks in yeast. FEBS letters, 586(15), 2112-2121. https://doi.org/10.1016/j.febslet.2012.01.053
- Ordozgoiti E. et al. (2021). Standardisation in synthetic biology: a white book.
- Stovicek, V., Borja, G. M., Forster, J., & Borodina, I. (2015). EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains. Journal of Industrial Microbiology and Biotechnology, 42(11), 1519-1531. https://doi.org/10.1007/s10295-015-1684-8
- Jessop‐Fabre, M. M., Jakočiūnas, T., Stovicek, V., Dai, Z., Jensen, M. K., Keasling, J. D., & Borodina, I. (2016). EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9. Biotechnology journal, 11(8), 1110-1117. https://doi.org/10.1002/biot.201600147