Half-Synthetic Yeast Engineered for the First Time
Scientists engineer a strain of yeast that has an over half synthetic genome.
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The Synthetic Yeast Genome Project 2.0 (Sc2.0) is a global consortium working to synthesize the entire yeast genome. In a collection of papers, the project declares a major milestone: it has created a yeast strain comprising over 50% synthetic DNA.
Generating synthetic genomes
Synthetic genomes are created by designing and assembling DNA sequences that are then inserted into an organism’s cells, effectively allowing researchers to “customize” the organism with certain characteristics. It’s an area of research within synthetic biology that utilizes recent advances in molecular biology, genetic engineering and gene synthesis.
“Writing a genome is in many ways a test of how well you understand its function and its evolved natural ‘design’," says Dr. Jef Boeke, a synthetic biologist at New York University (NYU) Langone Health and the leader of Sc2.0.
The first efforts to generate synthetic genomes largely focused on viruses or bacteria. In 2010, scientists from the J. Craig Venter Institute synthesized the first synthetic bacterial genome, Mycoplasma mycoides JCVI-syn1.0.
Boeke and colleagues have been working hard on Sc2.0 for at least 18 years, as synthesizing a yeast genome is more technologically challenging than a bacterial genome. “It’s substantially bigger. It’s more repetitive than bacterial genomes, and it’s made up of many individual chromosomes, whereas most bacteria have only one,” Boeke explains.
It’s a challenge that is worthwhile in Boeke’s mind, considering that humans are more closely related to yeast than bacteria, making yeast a better model for understanding how human cells work.
Yeast’s designer genome
The half-synthetic yeast has a “designer” genome that is based on Saccharomyces cerevisiae (S. cerevisiae), commonly referred to as baker’s or brewer’s yeast. Boeke and colleagues wanted to create a strain that could help them understand how the model organism functions and how it could be improved. It was therefore important that the synthetic yeast was highly modified.
Non-coding and repetitive elements of DNA were removed and a diversity generator, called Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution, or the “SCRaMbLE” system, was added.
Boeke and colleagues also created a new chromosome, which does not exist in nature. Genes encoding transfer RNA (tRNA) are often associated with regulatory elements that can lead to stability issues when integrated into a synthetic chromosome. The researchers decided to remove these genes and created a tRNA “neochromosome”.
“The tRNA neochromosome is not modeled on an existing natural chromosome but was literally designed piece by piece, on a computer, and the pieces came from at least 5 different species of microorganisms,” says Boeke. “The fact that it can function, by producing tRNAs like a natural genome does, is pretty amazing. It is also designed to avoid ‘head-on collisions’ between RNA polymerase and DNA polymerase which can lead to DNA breaks and other challenges for the cell.”
Strain generated that is over 50% synthetic
S. cerevisiae’s genome is organized into 16 chromosomes, with each team in the consortium responsible for producing one synthetic chromosome at a time. “Sc2.0 started small and grew to be a project involving researchers in 4 continents and 9 countries,” says Boeke. The teams independently assembled each chromosome to produce 16 strains, each of which comprised 15 native chromosomes and one synthetic chromosome. The next challenge was finding a way to pull all of the synthetic chromosomes together.
The researchers developed a new method called chromosome substitution to bring the synthetic chromosomes together in a single strain. “We combined two methods to do this: transfer of the synthetic chromosome to a recipient strain by doing a special kind of genetic cross. In this cross, a single chromosome moves from one nucleus to the other – a process called ‘chromoduction’,” explains Boeke.
“This leads to a cell with one extra synthetic chromosome. At this point, the resident natural chromosome corresponding to it can be destabilized by forcing RNA polymerase to traverse its centromere, a process that greatly destabilizes it. Et voila! A cell with a synthetic chromosome swapped in place of a native chromosome.” By integrating seven of the synthetic chromosomes, Sc2.0 has succeeded in generating a strain that is over 50% synthetic.
The synthetic yeast strain was found to possess genetic defects, or “bugs”, such as genetic interactions between genes located on the synthetic chromosomes. Such issues had been anticipated, so Boeke and colleagues were prepared to map the bugs and find a fix. “The defects can result from RNA not folding up as it should,” says Boeke. “Once mapped, bugs can be easily corrected by removing the changed bases and restoring the native sequence. This has already been done for every bug.”
A major milestone
The collection of papers marks a milestone in genomics and synthetic biology, but the real fun will begin once the rest of the synthetic chromosomes are integrated. “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,” says Boeke. Synthetic genomes designed for the production of medicines or biofuels might well become a reality in the near future – the foundations are being laid, after all.
Boeke is often asked, “which genome is next"? His team at NYU Langone Health is currently working on “The Dark Matter Project”, which seeks to understand what new information lies in the non-protein-coding portion of the mammalian genome.
Dr. Jef Boeke was speaking to Molly Campbell, Senior Science Writer for Technology Networks.
Zhao Y, Coelho C, Hughes A, et al. Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions. Cell. 2023. doi: 10.1016/j.cell.2023.09.025
Schindler D, Walker R, Jiang S, et al. Design, construction, and functional characterization of a tRNA neochromosome in yeast. Cell. 2023. doi: 10.1016/j.cell.2023.10.015
Shen Y, Gao F, Wang Y, et al. Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast. Cell Genomics. 2023. doi: 10.1016/j.xgen.2023.100364
McCulloch L, Sambasivam V, Hughes A, et al. Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains. Cell Genomics. 2023. doi: 10.1016/j.xgen.2023.100419