Our research moves the needle in synthetic biology from theory to reality. This work represents the biggest step yet in an international effort to construct the full genome of synthetic yeast. It is the most extensively altered chromosome ever built. But the milestone that really counts is integrating it into a living yeast cell. We have shown that yeast cells carrying this synthetic chromosome are remarkably normal. They behave almost identically to wild yeast cells, only they now possess new capabilities and can do things that wild yeast cannot.

—Dr. Boeke

Using computer-aided design, the researchers built a fully functioning 272,871–base pair designer eukaryotic chromosome, synIII, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III.They then successfully incorporated it into Saccharomyces cerevisiae.

Dr. Boeke and his team made more than 500 alterations to its genetic base, removing repeating sections of some 47,841 DNA base pairs, deemed unnecessary to chromosome reproduction and growth. Also removed was “junk DNA”, including base pairs known not to encode for any particular proteins, and “jumping gene” segments known to randomly move around and introduce mutations. Other sets of base pairs were added or altered to enable researchers to tag DNA as synthetic or native, and to delete or move genes on synIII.

When you change the genome you’re gambling. One wrong change can kill the cell. We have made over 50,000 changes to the DNA code in the chromosome and our yeast still live. That is remarkable. It shows that our synthetic chromosome is hardy, and it endows the yeast with new properties.

—Dr. Boeke

The massive effort was aided by some 60 undergraduate students enrolled in the “Build a Genome” project, founded by Dr. Boeke at Johns Hopkins. The students pieced together short snippets of the synthetic DNA into stretches of 750 to 1,000 base pairs or more. These pieces were then assembled into larger ones, which were swapped for native yeast DNA, an effort led by Srinivasan Chandrasegaran, PhD, a professor at Johns Hopkins. Chandrasegaran is also the senior investigator of the team’s studies on synIII.

Student participation kicked off what has become an international effort, Synthetic Yeast 2.0 (Sc2.0), in which several academic researchers have partnered to reconstruct the entire yeast genome, including collaborators at universities in China, Australia, Singapore, the United Kingdom, and elsewhere in the US.

Yeast chromosome III was selected for synthesis because it is among the smallest of the 16 yeast chromosomes and controls how yeast cells mate and undergo genetic change.

Yeast shares roughly a third of its 6,000 genes with humans. The team was able to manipulate large sections of yeast DNA without compromising chromosomal viability and function using a “scrambling” technique that allowed the scientists to shuffle genes like a deck of cards, where each gene is a card.

Boeke and colleagues earlier proposed SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) as an inducible evolution system. In a 2011 paper in Nature (Dymond et al.), they showed the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes.

We can pull together any group of cards, shuffle the order and make millions and millions of different decks, all in one small tube of yeast. Now that we can shuffle the genomic deck, it will allow us to ask, can we make a deck of cards with a better hand for making yeast survive under any of a multitude of conditions, such as tolerating higher alcohol levels.

—Dr. Boeke

Before testing the scrambling technique, researchers first assessed synIII’s reproductive fitness, comparing its growth and viability in its unscrambled form—from a single cell to a colony of many cells—with that of native yeast III. Yeast proliferation was gauged under 19 different environmental conditions, including changes in temperature, acidity, and hydrogen peroxide, a DNA-damaging chemical. Growth rates remained the same for all but one condition.

Further tests of unscrambled synIII, involving some 30 different colonies after 125 cell divisions, showed that its genetic structure remained intact as it reproduced. According to Dr. Boeke, individual chromosome loss of one in a million cell divisions is normal as cells divide. Chromosome loss rates for synIII were only marginally higher than for native yeast III.

To test the scrambling technique, researchers successfully converted a non-mating cell with synIII to a cell that could mate by eliminating the gene that prevented it from mating.

The total synthesis of the synIII chromosome represents a major step toward the design and complete synthesis of a novel eukaryotic genome structure using the model S. cerevisiae as the basis for a synthetic designer genome, Sc2.0. The many changes made to synIII, including intron deletion, tRNA gene removal, and loxPsym sites and PCRTags introduction, do not appear to significantly decrease the fitness or alter the transcriptome or the replication timing of the synIII strain, supporting the very pliable nature of the yeast genome and potentially allowing for much more aggressively redesigned future genome versions.

Sc2.0 represents just one of myriad possible arbitrary genome designs, and we anticipate that synthetic chromosome design will become a new means of posing specific evolutionary and mechanistic questions about genome structure and function. Rapid advances in synthetic biology coupled with ever decreasing costs of DNA synthesis suggest that it will soon become feasible to engineer new eukaryotic genomes, including plant and animal genomes, with synthetic chromosomes encoding desired functions and phenotypic properties based on specific design principles.

—Annaluru et al.

By applying the scrambling technique, researchers say they will be able to more quickly develop synthetic strains of yeast that could be used in the manufacture of rare medicines, such as artemisinin for malaria, or in the production of certain vaccines, including the vaccine for hepatitis B, which is derived from yeast. Synthetic yeast, they say, could also be used to bolster development of more efficient biofuels, such as alcohol, butanol, and biodiesel.

The study will also likely spur laboratory investigations into specific gene function and interactions between genes, adds Dr. Boeke, in an effort to understand how whole networks of genes specify individual biological behaviors.

Their initial success rebuilding a functioning chromosome will likely lead to the construction of other yeast chromosomes (yeast has a total of 16 chromosomes, compared to humans’ 23 pairs), and move genetic research one step closer to constructing the organism’s entire functioning genome, says Dr. Boeke.

Dr. Boeke says the international team’s next steps involve synthesizing larger yeast chromosomes, faster and cheaper. His team, with further support from Build a Genome students, is already working on assembling base pairs in chunks of more than 10,000 base pairs. They also plan studies of synIII where they scramble the chromosome, removing, duplicating, or changing gene order.

Funding support for these experiments was provided by National Science Foundation, the National Institutes of Health, and Microsoft. Corresponding federal grant numbers are MCB-0718846 and GM-077291. Additional funding support was provided by fellowships from La Fondation pour la Recherche Médicale, Pasteur-Roux, National Sciences and Engineering Research Council of Canada, U.S. Department of Energy, and grants from the Exploratory Research Grant from the Maryland Stem Cell Research Fund and the Johns Hopkins University Applied Physics Laboratory.

Besides the teams at NYU Langone and Johns Hopkins, other scientific teams involved in the global Sc2.0 research effort are based at Loyola University in Baltimore, Md; BGI in Shenzhen, China; Tianjin University in China; Tsinghua University in China; MacQuarie University in Sydney, Australia; the Australian Wine Institute in Adelaide, Australia; the National University of Singapore; Imperial College, London, England; and the University of Edinburgh in Scotland.

Resources

• Narayana Annaluru, Héloïse Muller, Leslie A. Mitchell, Sivaprakash Ramalingam, Giovanni Stracquadanio, Sarah M. Richardson, Jessica S. Dymond, Zheng Kuang, Lisa Z. Scheifele, Eric M. Cooper, Yizhi Cai, Karen Zeller, Neta Agmon, Jeffrey S. Han, Michalis Hadjithomas, Jennifer Tullman, Katrina Caravelli, Kimberly Cirelli, Zheyuan Guo, Viktoriya London, Apurva Yeluru, Sindurathy Murugan, Karthikeyan Kandavelou, Nicolas Agier, Gilles Fischer, Kun Yang, J. Andrew Martin, Murat Bilgel, Pavlo Bohutski, Kristin M. Boulier, Brian J. Capaldo, Joy Chang, Kristie Charoen, Woo Jin Choi, Peter Deng, James E. DiCarlo, Judy Doong, Jessilyn Dunn, Jason I. Feinberg, Christopher Fernandez, Charlotte E. Floria, David Gladowski, Pasha Hadidi, Isabel Ishizuka, Javaneh Jabbari, Calvin Y. L. Lau, Pablo A. Lee, Sean Li, Denise Lin, Matthias E. Linder, Jonathan Ling, Jaime Liu, Jonathan Liu, Mariya London, Henry Ma, Jessica Mao, Jessica E. McDade, Alexandra McMillan, Aaron M. Moore, Won Chan Oh, Yu Ouyang, Ruchi Patel, Marina Paul, Laura C. Paulsen, Judy Qiu, Alex Rhee, Matthew G. Rubashkin, Ina Y. Soh, Nathaniel E. Sotuyo, Venkatesh Srinivas, Allison Suarez, Andy Wong, Remus Wong, Wei Rose Xie, Yijie Xu, Allen T. Yu, Romain Koszul, Joel S. Bader, Jef D. Boeke, and Srinivasan Chandrasegaran (2014) “Total Synthesis of a Functional Designer Eukaryotic Chromosome,” Science doi: 10.1126/science.1249252

• Jessica S. Dymond, Sarah M. Richardson, Candice E. Coombes, Timothy Babatz, Héloïse Muller, Narayana Annaluru, William J. Blake, Joy W. Schwerzmann, Junbiao Dai, Derek L. Lindstrom, et al. (2011) “Synthetic chromosome arms function in yeast and generate phenotypic diversity by design,” Nature 477, 471-476 doi: 10.1038/nature10403