Synthetic Yeast Sc2.0

by Anja Brickwedde.

In April 2014, the first complete synthesis of a functional eukaryotic chromosome was published in Science (Annaluru et al. 2014). Jef Boeke of New York University and coworkers announced the first one of yeast’s set of 16 Chromosomes to be synthesized in context of the Synthetic Yeast project “Sc2.0” which aims at creating the first complex organism synthesized ever. The project Sc2.0 is a worldwide collaboration of researchers from amongst others New York University, Johns Hopkins University, National University of Singapore, Macquarie University, and several industrial partners (a complete list can be found at http://syntheticyeast.org).

Recent advances in synthetic biology made this discipline become center of attention after Craig Venter and his group have generated the first synthetic bacterial genome in 2010 (Gibson et al. 2010). They re-wrote the genetic code from the bacterium Mycoplasma mycoides and replaced the original bacterial DNA with the synthesized copy.

Methodologies of synthetic biology are commonly applied to design or re-design biological molecules or systems to better understand the fundaments underlying them or to engineer biological systems and platforms for industrial applications.

The project Sc2.0 aims at synthesizing a complete set of eukaryotic chromosomes including several alterations to the original DNA of the yeast Saccharomyces cerevisiae.

The researcher’s choice for the yeast S. cerevisiae was far from being a coincidence. This organism was the first eukaryote of which the complete genome has been entirely sequenced (Goffeau et al. 1996) starting with chromosome III (ChrIII) in 1992 (Oliver et al. 1992). S. cerevisiae is in addition to that a well-studied model organism which holds the GRAS (generally recognized as safe) status by the FDA (US Food and DrugAdministration), and is commercially used in brewing, baking and in the production of bio ethanol.

In the context of the project Sc2.0, Boeke and colleagues synthesized the designer chromosome SynIII which was 272,871 base pair (bp) long, based on the 316,617 bp original ChrIII of S. cerevisiae. The fitness of an organism harboring SynIII instead of the native chromosome is reported to be close to the wild type´s one although lacking several elements. On average, the chromosomes synthesized during the project Sc2.0 are 10 to 15 percent smaller than the native ones due to removal of junk-DNA.

Boeke and colleagues used a stepwise assembly process to introduce the synthesized DNA segments in vivo. Small PCR synthesized building blocks of about 750 bp were assembled into “minichunks” of about 3 kb (kilo base pair). Those were further assembled into “chunks” of about 10 kb by in vivo homologous recombination of an overlapping building block on each fragment. The assembly of the chunks included unique auxotrophic selection markers and was performed in vivo building “megachunks” of 30 – 50 kb. Each round, the marker used for the prior assembly was removed due to recombination events and another marker was introduced. The megachunks finally replaced the S. cerevisiae wild type sequence. Additionally, an inducible “SCRaMbLE” evolution system (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) was applied adding LoxPsyn sites flanking non-essential genes enabling genome scrambling. Fitness was monitored by examination of colony size on selective medium and comparison to the parental strain. The researchers also performed competition experiments to evaluate whether small alterations in fitness have occurred and transcript profiling to detect changes in gene expression. Neither fitness nor transcriptome of the synthetic version of ChrIII showed significant differences in comparison to the original version.

The project Sc2.0 is an outstanding example for the rapid developments and numerous possibilities in the field of synthetic biology. Next to applications of yeast in industrial fermentations, S. cerevisiae serving as model eukaryote for higher organism will become of increasing importance. Sakkie Pretorius, professor at Macquarie University, Australia, stated:

“Once we can synthesise the full genetic blueprint of a yeast we can then apply the same techniques to increasingly more complex organisms. The possibilities in medicine, or the environment, for example, are truly mind-blowing.”

References

Annaluru N, et al. Total synthesis of a functional designer eukaryotic chromosome. Science. 2014; 344 (6179): 55–58; DOI: 10.1126/science.1249252.

Gibson D G, et al. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science. 2010; DOI: 10.1126/science.1190719.

Goffeau A, et al. Life with 6000 genes. Science. 1996; 274: 546, 563–7.

Oliver S G, et al. The complete DNA sequence of yeast chromosome III. Nature. 1992; 357: 38–46.

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