For the first time, researchers have fused snippets of synthetic DNA to build a eukaryotic chromosome in yeast, which like plants and animals, bundles its genetic material into a nucleus. The technical feat is reported in the 28 March issue of Science.
Critically, the researchers designed the chromosome with switches flanking certain genes so these genes could be turned on or off at will. The effort is an important step on the road to constructing a eukaryotic genome that is completely synthetic, equipped with a full set of chromosomes that can be engineered to give new properties to an organism.
"You can put it as a kind of milestone," co-author and New York University geneticist Jef Boeke told Science's Elizabeth Pennisi, who reported on the breakthrough, "like [sequencing] the ﬁrst human genome was a milestone for genomics."
Jef Boeke discusses the process for building the synthetic chromosome. | Video courtesy of NYU Langone Medical Center, 2014
The chromosome Boeke and colleagues synthesized was from one of the best-studied organisms on the planet: baker's yeast, or Saccharomyces cerevisiae. Yeast is already used to make beer, biofuel and medicine. But once yeast is equipped with a full set of changeable chromosomes like the one designed in the new study, this single-celled organism could produce even better versions of these commodities, including new antibiotics or cleaner biofuels.
In addition to serving as a highly versatile industrial chemical factory, a fully synthetic eukaryotic yeast genome also could help researchers learn more about how genomes are built and organized. It may even assist them in answering some longstanding evolutionary questions, like determining the maximum number of nonessential genes that can be deleted in an organism without a catastrophic loss of fitness.
"To better understand the universal principles of life," co-author and Johns Hopkins research scientist Narayana Annaluru said, "it is important to know the minimal set of genes required for metabolism and replication."
In recent years, DNA synthesis techniques have been rapidly improving. Leveraging these techniques, scientists have been able to assemble the single prokaryotic chromosome of the bacterium Mycoplasma genitalium, which has no cell nucleus.
The baker's yeast genome comprises 12 million nucleotides, or genetic letters, strung together in a particular order. A team led by Boeke and Annaluru homed in on yeast chromosome III, comprising more than 2.5 percent of these nucleotides. "Chromosome III is a sentimental favorite of yeast geneticists," Boeke explained. "It was the first yeast chromosome to be sequenced because it contains the genes controlling sexual behavior."
Illustrated representation of the yeast designer chromosome SYNIII. Full Size | Lucy Reading-Ikkanda
Boeke and his team made small changes to a computer model of yeast chromosome III. The chromosome's genome encodes about 6,000 genes, of which roughly 5,000 are nonessential. The scientists couldn't eliminate bunches of nonessential genes, however, because they didn't know what effect that would have on the whole organism. So they created a chromosome in which nonessential genes were bordered by short sequences of DNA called loxPsym sites. These sites could be chemically activated to delete or alter nonessential genes, giving researchers a way to manipulate the yeast genome at will and observe these effects.
They also used the software to make a few more changes, most notably to remove some of the repetitive regions of DNA between genes that can lead to genomic instability. The researchers then set about building the re-engineered chromosome in real life by stringing together individual nucleotides. To expedite their work, Boeke set up a summer class at Johns Hopkins University. Called the Build-A-Genome class, it was a major hit. The process took almost 50 students a year and a half. "Every student in the class contributed directly to the success of the project," Boeke said.
Once the artificial chromosome was built, the researchers put it in living yeast cells and tested the ability of the altered cells to grow on different nutrients and in different conditions. In each case, the version equipped with a synthetic chromosome functioned indistinguishably from native yeast. Boeke and colleagues were delighted. "Not only can we make designer changes to a genome on a computer, but we can make them throughout an entire chromosome, put that chromosome in yeast, and get a product that still looks, smells and behaves like regular yeast."
The researchers further manipulated yeast cells by activating various loxPsym sites to alter or delete genes. They found that some cells grew more slowly, once altered. Others, with different recombination of genes, grew very quickly.
The scientists and their collaborators aim to make synthetic versions of all of the organism's 16 chromosomes in the near future. Boeke is particularly enthusiastic about one application for the synthesized genome. "We are excited about engineering yeast as models for human disease. Right now we are rebuilding a network of human genes inside of yeast," he said. "We hope to use this system to learn more about human metabolic diseases underlying certain neurologic diseases, and forms of mental retardation, autism and cancer."
Annaluru is also looking ahead. "Once we complete the synthesis of whole yeast genome, I am most excited about scrambling the genome towards developing a yeast strain that can tolerate higher ethanol levels" and thereby lead to greater ethanol yields, he said.