Lessons from the Minimal Cell

In 2016, J. Craig Venter and his team (J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI)) announced the construction of a “minimal” bacterial cell that encoded only the genes necessary for cell life and growth in rich laboratory media. This organism, Mycoplasma mycoides JCVI-syn3.0—a minimized version of the bacterium M. mycoides commonly found in the guts of goats and similar animals, had the smallest genome (531,490 bp) of any known free living organism and contained 438 protein-coding genes and 35 RNA-coding genes.¹

The expectation was that the evolution of this organism was subject to strong constraints because any mutation could lethally disrupt one or more cellular functions. In addition, its streamlined genome has fewer targets upon which positive selection can act. In 2023, researchers reported that evolution of JCVI-syn3.0 for 300 days produced a version that outcompeted the minimal unevolved version.² Even more, the organism, with all its perceived constraints, effectively recovered much of the fitness it had lost due to genome streamlining. The authors state in their concluding remarks, “Despite reducing the sequence space of possible trajectories, we conclude that streamlining does not constrain fitness evolution and diversification of populations over time. Genome minimization may even create opportunities for evolutionary exploitation of essential genes, which are commonly observed to evolve more slowly.”

This is a fundamental insight, arguably impossible to achieve without a minimal cell at hand. This put closure to an argument that I have been having with myself since 2016 (which also mirrored discussions about the topic that I heard from many colleagues) – should I care about the Venter minimal cell?

We all should care greatly. The minimal cell is an important technical and conceptual advance, upon which many are starting to build new knowledge. Nonetheless, it is a case in point about three principles that our community is well-advised to keep front of mind.

Principle 1 — New ideas are almost always old ideas. Courageous execution is what makes the difference.

Ideas are almost always a continuum. That of a minimal cell is no exception. Indeed, it dates back to the 1940s when physicist Max Delbrück founded the American Phage Group. This group of physicists, chemists, and biologists was dedicated to the idea that understanding the first principles of cellular life would come through study of its simplest form.³ In a lecture in 1984, origin-of-life expert, Harold Morowitz, recognized that the mycoplasmas were the simplest cells capable of autonomous growth and proposed that these bacteria be used as models for understanding the basic principles of life.⁴ He argued that such studies might invigorate basic biology in the same way that studying the hydrogen atom sharpened questions for physics and chemistry. (Unfortunately, Morowitz died two days before the JCVI-syn3.0 work was published online).  In 2006, Forster and Church proposed an in vitro system, consisting of 151 genes (113 proteins and 38 RNAs) encoded by a 113-kb genome, that is capable of replication and evolution and fed only by small molecule nutrients.⁵ This system was never built nor successfully tested. While many wrote, discussed and theorized about the minimal cell, the implementation only came years later after many technological advances and the laser-focused and courageous work of Venter and his team.

Principle 2 – Technology is often the driver of science’s punctuated moments. 

Before M. mycoides JCVI-syn3.0, there was a M. mycoides JCVI-syn1.0, the first living cell with an entirely artificial chromosome.⁶ Before that, there were many large-scale sequencing projects where the genome of the bacterium was artificially recreated — DNA fragments were synthesized in the laboratory, and then assembled in the right order using the genetic information of the Mycoplasma genitalium bacteria as a template.⁷ It was sequencing and other genomic technologies, and the availability of whole-genome sequences for mycoplasma species (as well as other bacteria) that made this effort possible.

Several technologies again propelled the next development in 2016 – bottom-up synthesis of the minimal M. mycoides JCVI-syn3.0. These include methods for synthesis of large DNA molecules and for genome transplantation.^7–9 In addition, an ingenious rapid design-build-test cycle, as well as methods for swift troubleshooting were developed (the details of which can be gleaned from the series of papers that start in 2008 and culminate in the 2016 minimal cell publication). In a series of three such design, synthesis and test cycles, M. mycoides JCVI-syn3.0 was born!

Principle 3 – More than the icing. Computation is half of the cake.

While M. mycoides JCVI-syn3.0 would not have been possible without genome sequencing as a technology, computational biology and comparative genomics that developed in parallel was just as crucial. Suffice to say that as early as 1996, Mushegian and Koonin compared orthologs between the two genomes of M. genitalium and Haemophilus influenza Rd, one Gram-negative and one Gram-positive, and determined a set of 256 genes as a seed of an ensemble of genes that specify the core functions of a minimal cell.¹⁰

A computationally determined set of 206 protein-coding genes was subsequently proposed as a possible minimum gene set necessary to sustain life.¹¹ These were highly conserved in both near-minimal organisms such as M. genitalium and endosymbiotic bacteria such as Buchnera aphidicola, as well as Escherichia coli and B. subtilis. Remarkably, these genes are almost all present in both the minimal cell JCVI-Syn3.0 and in the near-minimal species M. genitalium.

So, what do you do with a minimal cell?

At a cost of 2010’s approximately $40 million and countless work hours, Venter and his team had converted a digitized DNA sequence into a living entity capable of growth and self-replication.

Writing in Nature, Ewen Callaway asked, “an explosion in powerful ‘gene-editing’ techniques, which enable relatively easy and selective tinkering with genomes, raises a niggling question: why go to the trouble of making new life forms when you can simply tweak what already exists?”¹²

The sensational answer of the time put-forth by both the scientific and popular press was that these minimal cells will serve as templates for lab-made organisms that make medicines and agricultural products at rates that are yet unheard of, as well as churn out molecules not yet imagined. This, unfortunately, has not yet materialized.

It seems that the legacy of M. mycoides JCVI-syn3.0 is more fundamental. The principles gleaned from its evolution in the recent study are a case in point. The fact that 149 of its genes (One third of this stripped-down minimalist genome!) were on unknown function in 2016 was a moment of truth about how much we still don’t know even about the basic tenets that create and maintain life. And its technological legacy (e.g. the assembly of large DNA in episomes¹³) still inspires remarkable developments.

References

1. Hutchison CA 3rd, Chuang R-Y, Noskov VN, et al. Design and synthesis of a minimal bacterial genome. Science 2016;351(6280):aad6253; doi: 10.1126/science.aad6253.
2. Moger-Reischer RZ, Glass JI, Wise KS, et al. Evolution of a minimal cell. Nature 2023; doi: 10.1038/s41586-023-06288-x.
3. Morange M. A History of Molecular Biology. Harvard University Press; 2000.
4. Morowitz HJ. The completeness of molecular biology. Isr J Med Sci 1984;20(9):750–753.
5. Forster AC, Church GM. Towards synthesis of a minimal cell. Mol Syst Biol 2006;2:45; doi: 10.1038/msb4100090.
6. Gibson DG, Glass JI, Lartigue C, et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 2010;329(5987):52–56; doi: 10.1126/science.1190719.
7. Gibson DG, Benders GA, Andrews-Pfannkoch C, et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 2008;319(5867):1215–1220; doi: 10.1126/science.1151721.
8. Lartigue C, Glass JI, Alperovich N, et al. Genome transplantation in bacteria: changing one species to another. Science 2007;317(5838):632–638; doi: 10.1126/science.1144622.
9. Benders GA, Noskov VN, Denisova EA, et al. Cloning whole bacterial genomes in yeast. Nucleic Acids Res 2010;38(8):2558–2569; doi: 10.1093/nar/gkq119.
10. Mushegian AR, Koonin E V. A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc Natl Acad Sci U S A 1996;93(19):10268–10273; doi: 10.1073/pnas.93.19.10268.
11. Gil R, Silva FJ, Peretó J, et al. Determination of the core of a minimal bacterial gene set. Microbiol Mol Biol Rev 2004;68(3):518–37, table of contents; doi: 10.1128/MMBR.68.3.518-537.2004.
12. Callaway E. ‘Minimal’ cell raises stakes in race to harness synthetic life. Nature 2016;531(7596):557–558; doi: 10.1038/531557a.
13. Zürcher JF, Kleefeldt AA, Funke LFH, et al. Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly. Nature 2023;619(7970):555–562; doi: 10.1038/s41586-023-06268-1.

Hana El-Samad ([email protected]) is Editor in Chief of GEN Biotechnology.

The GEN Biotechnology Journal, published by Mary Ann Liebert, Inc., is the new, marquee peer-reviewed journal publishing outstanding original research and perspectives across all facets of the biotech industry. The above article was first published in the August, 2023 issue of the GEN Biotechnology Journal.