There might be older romances out there, but by most accounts the bond between humans and yeast has been the most prolific. (Also, try to name another fungal romance.) People have been messing with yeast for millennia, ever since ancient hominins first turned wild strains of the fungus into the civilization-supporting fermenters that still make everything from beer and bread to tempeh and fish sauce. That meddling has accelerated in the last twenty years since scientists sequenced the yeast genome, yielding microbes that can burp, fart and secrete biofuels, insulin, antibiotics, and tons of other novel micro- and macromolecules useful to human industry. And soon, the takeover will be complete. Scientists have now designed an entirely artificial yeast genome and constructed more than one-third of it. They say they’ll have a 100% synthetic yeast up and fermenting by the end of the year.
In seven papers published today in Science, representing a decade of work by hundreds of scientists across four continents, the Synthetic Yeast 2.0 project reports the first fully designed, and partially completed, made-from-scratch eukaryotic genome. Eukaryotes---organisms whose cells have a nucleus and other defined organelles---encompass all complex life: yeasts, plants, hamsters, humans. So writing a custom genome for one is a big deal by itself. But the artificial yeast will have a more stable, easily manipulable genome for scientists to work with, and for the chemical, pharmaceutical, and energy industries to use for a new generation of drugs, biofuels, and novel materials.
Joel Bader was sitting in his office in the department of biomedical engineering at Johns Hopkins University School of Medicine when he heard excited voices coming from the coffee lounge outside his door. Jef Boeke, then the director of the High Throughput Biology Center at Hopkins and biochemist Srinivasan Chandrasegaran were talking about what it would take to build all the DNA in a yeast from scratch.
It was 2006, and Bader, who taught computational medicine classes, quickly pointed out that any ambitions of synthesizing a genome of that size (~11 million base pairs) would need some serious computing and software support. So he signed on as Sc2.0’s third team member. Back then, the project was based solely at Johns Hopkins, where Boeke began offering an undergraduate class called “Build a Genome.”
During the first few years, dozens of bright-eyed molecular biology majors got used to keeping odd hours---and keys to Boeke’s lab---as they learned how to string together short snippets of nucleotides into longer, 750-base pair blocks. Other researchers then assembled these chunks into larger and larger stretches of the smallest yeast chromosome, chromosome 3. Then they began putting them strategically into live yeast, which spliced these pieces together into even larger sequences using a naturally-occurring yeast pathway called homologous recombination.
Each section took a long time to build, so as Boeke’s students and colleagues finished a sequence, they’d turn it into a plasmid (a circular, self-contained piece of DNA), and inject it into yeast or E. coli for safe keeping. The lab’s freezers were often filled with hundreds of plates in various states of suspended animation, all holding different pieces of the chromosomal puzzle. Only once they were all complete could they wake up the cells, and put them in new yeasts to finish the final assembly steps.
Boeke has since moved Sc2.0's base of operations to NYU Langone, and Bader has taken over the reins at Johns Hopkins High Throughput Biology Center. Over time, the team outgrew both labs, and came to encompass more than 500 scientists in ten labs around the world in places like China, Australia, and Scotland.
Bader’s software team at Hopkins built the programs that guide and execute the project’s workflow, setting rules for chromosome design, so the different labs can work on their own chromosomes individually, parallelizing the process and speeding things way up. In 2014, the international consortium revealed its first fully artificial chromosome. Getting those first 272,871 base pairs of it took eight years.
Today's announcement adds five more chromosomes, plus the completed design of the rest---for a total of 17. Any zymologists in the crowd might notice this is one more chromosome than wild yeasts have. The story of how that last one came about starts with the fact that yeast DNA---like all DNA---is full of mistakes and redundancies.
Sc2.0 began as a project to make yeasts better at producing chemicals useful to humans. Evolution optimized yeast for lots of things, but not for industrial production of enzymes or antibiotics. That didn't require remaking the yeast genome verboten, just removing destabilizing DNA from the genome and refactoring the whole thing so future researchers could customize their yeast for whatever compound they wanted to crank out.
One of the biggest changes the researchers introduced was to place 5000 DNA tags throughout the genome that act as landing sites for a protein called “Cre” that can be used to create on-demand mutations. When the protein comes in contact with estrogen it scrambles the synthetic chromosomal sequences---deleting, duplicating, and shuffling genes at random.
By building in these “SCRaMbLE” sites---it stands for Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution---scientists can start with a test tube filled with a million genetically-identical synthetic yeast cells, randomly reshuffle their genes, and then expose them to different stresses, like heat and pressure, or ask them to make different molecules. It's kind of like natural selection on speed, and allows scientists to easily identify new strains that can survive better in specific environments, or be better factories for things like fuels and drugs.
“We’re shortcutting evolution by millions of years,” says bioengineer Patrick Cai, who first became acquainted with the project as a post-doc in Boeke’s lab in 2010. “Our goal here is not engineering a particular kind of yeast, but the kind of yeast that is amenable to engineering.” Cai now runs his own lab at the University of Edinburgh, where he’s building that extra 17th chromosome. It's the only chromosome that's built completely from scratch.
Cai took on the project after starting his own lab once he left Johns Hopkins---and by that time all 16 extant chromosome projects had been divvied up. His task was to stash all the yeast's transfer RNAs---molecules that ferry amino acids into the right order during protein synthesis. Transfer RNAs are an essential part of the cell’s protein-making machinery, but are notoriously unstable because of how often they're transcribed.
Sc2.0’s scientists figured it would be better to harvest them from their scattered chromosomal locations and put them all together in one place. They call it, the “party” chromosome. “All the troublemakers got their own dedicated chromosome where they can do whatever they want,” says Cai. “That means they’re not causing breakage everywhere else in the genome, so it’s super stable. More stable than anything that exists in nature.”
Sc2.0's yeast DNA isn't just more stable, it's more concise. After all the editing and reworking, the artificial genome is eight percent smaller than a wild yeast’s. Its structure is less prone to unpredictable mutations (the kind that stymie chemical manufacturing), and the tRNA-laden 17th chromosome will give the organisms---once the genome is fully synthesized---near-infinite possibilities for manipulation.
Which is exactly what any good industrialist wants to hear. Jay Keasling, the chief executive officer of the Joint BioEnergy Institute and a professor at UC Berkeley, where his lab engineered yeast to produce the malaria drug, arteminisin, is looking forward to the day when yeast are designed 100% from-scratch. “That gives us a lot more control to build things into the organism so that it doesn’t grow under specific conditions, or produces more of your product.” he says. “There are all kinds of possibilities for the future to make these organisms industrially relevant.” The Sc2.0 team plans to be finished before the end of this year.
Of course, for any yeast---even a completely synthetic one---to become a blockbuster application, it must have complementary systems to efficiently separate, recover, and purify the products. Sc2.0 is leaving that up to industry to figure out. They’ve already entered into one corporate partnership and have three other companies interested (although they wouldn’t share further details.) And while they haven’t yet zipped together the final As, Ts, Cs, and Gs, they’re already thinking bigger than yeast. Later this spring the group is organizing a meeting in New York to talk about driving down the cost of genome building technologies. The end goal? Move from yeasts to plants, maybe even one day to humans. “That will be at least ten times as hard,” says Boeke. “But we plan to forge ahead.” At least ten times as hard to make, and probably way harder to sell to the ethics committee.
