Researchers have achieved a significant milestone in synthetic biology by successfully integrating more than seven synthetic chromosomes into a single yeast cell, yielding a strain with over 50% synthetic DNA that can survive and replicate similarly to wild yeast strains. This represents a pivotal moment in the Synthetic Yeast Genome Project (Sc2.0), an international collaboration dedicated to constructing the first synthetic eukaryote genome from scratch. The team has now synthesized and debugged all sixteen yeast chromosomes. The half-synthetic yeast is featured in the journal Cell as part of a collection of papers across Cell, Molecular Cell, and Cell Genomics that showcase the Project.
This achievement is a testament to the power of collaborative research. “Sc2.0 is not only the first synthetic eukaryotic genome to be constructed, but it is the first synthetic genome to be completed by the international community,” states co-author and senior author of two other papers in the collection Patrick Yizhi Cai, a synthetic biologist from the University of Manchester. “The team has now re-written the operating system of the budding yeast, which opens up a new era of engineering biology—moving from tinkering a handful of genes to de novo design and construction of entire genomes."
Creating a yeast strain with a significant proportion of synthetic chromosomes posed a challenge, precisely what the team expected. “Our overarching aim was to build a yeast that can teach us new biology.” says senior author and Sc2.0 leader Jef Boeke, a synthetic biologist at NYU Langone Health. Each chromosome was assembled independently, resulting in sixteen yeast strains each with a single synthetic replacement chromosome. To begin building a strain with a fully synthetic genome, the researchers employed a method akin to Mendel's pea experiments, progressively breeding different partially synthetic yeast strains until individuals harboring both synthetic chromosomes were located. Using this slow but effective method, the team eventually consolidated all previously synthesized chromosomes (six full chromosomes and one chromosome arm) into a single cell, resulting in a strain more than 31% synthetic with normal morphology and only slight growth defects compared to wild-type yeast.
But they wouldn’t stop there. To transfer specific chromosomes more efficiently between strains, the researchers developed a new method, chromosome substitution, which is discussed in another paper in the collection. As a proof of concept for this method, the team transferred chromosome IV, the largest of the synthetic chromosomes, to the part-synthetic strain, resulting in a strain that is more than 50% synthetic.
However, this breakthrough revealed several genetic defects, or ‘bugs,’ hidden in the strains carrying a single synthetic chromosome. Caused by the additive effects of many minor genetic defects being compiled in one cell and the interactions between genes on synthetic chromosomes, these issues posed a new challenge. “We knew in principle that this might happen—that we might have a huge number of things that had tiny little effects and that when you put them all together, it might result in death by a thousand cuts,” said Boeke. By mapping the defects and fixing them using a method based on CRISPR/Cas9, the researchers were able to increase the fitness of the synthetic yeast significantly. "We've now shown that we can consolidate essentially half of the genome with good fitness, which suggests that this is not going to be a big problem,” emphasizes Boeke. “And from debugging, we learn new twists on the rules of life."
The synthetic "designer" yeast genome diverges significantly from the natural Saccharomyces cerevisiae genome. “We decided that it was important to produce something that was very heavily modified from nature’s design,” stated Boeke. Researchers meticulously removed non-coding DNA and repetitive elements and introduced new genetic sequences to not only distinguish between synthetic and native genes but also create a more flexible genome. “We have built-in much more engineering designs, which allows the synthetic yeast to have novel features such as [the ability to] combinatorially re-arrange its own genome to cope with different environments while maintaining really high fitness.” Cai elaborated. The built-in gene shuffler “SCRaMbLE” allows massive chromosome rearrangements upon induction to quickly generate a wealth of genotypically diverse strains.
To increase genome stability, they also developed a "neochromosome" entirely comprised of relocated transfer RNA (tRNA) genes. “Unlike the other 16 “syn” chromosomes, the tRNA Neochromosome is not modeled on an existing natural chromosome but was literally designed piece by piece, on a computer. The pieces came from at least five different species of microorganisms!” said Cai. “There is nothing like it in nature. The fact that it can function by producing tRNAs like a natural genome does is pretty amazing.” Cai emphasized the careful design of the neochromosome, with special attention paid to avoiding collisions between RNA polymerase and DNA polymerase, which can cause a number of challenges for the cell.
With the integration of remaining synthetic chromosomes on the horizon, this research signifies the dawn of a new era in synthetic biology. "Now we're just this far from the finish line of having all 16 chromosomes in a single cell,” Boeke remarked. “I like to call this the end of the beginning, not the beginning of the end, because that's when we're really going to be able to start shuffling that deck and producing yeast that can do things that we've never seen before."
This groundbreaking research was made possible through the support of organizations, including the National Science Foundation, the National Institutes of Health, the Laura and Isaac Perlmutter Cancer Center, and Volkswagen Stiftung.
In addition to the research published in Cell, the Synthetic Yeast Genome Project has presented several papers in a collection spanning Cell, Molecular Cell, and Cell Genomics. These papers explore the intricate details of this remarkable scientific endeavor, marking a significant breakthrough that highlights the boundless possibilities within the realm of synthetic biology.