Issues Magazine

Yeast 2.0: How to Build a Genome

By Ian T. Paulsen and Isak S. Pretorius

Reprogramming the software of life takes genomics and bioengineering into a new dimension, and it incites both fascination and concern.

Synthetic biology entails the design and engineering of biologically based parts, novel devices and systems as well as the redesign of existing natural biological systems (see box). Researchers in this emerging science aim to predictably bioengineer organisms for beneficial applications – from the production of new antibiotics, renewable energy and biodegradable pesticides to purifying contaminated water. This so-called genome engineering will pose not only scientific challenges to lab researchers but also ethical and policy challenges to society at large.

The World’s First Synthetic Virus and Bacterium

As a first step towards genome engineering, the poliovirus genome was synthesised in 2002. Viruses are tiny particles (as opposed to living cells) with short and relatively uncomplicated genomes. This synthesis laid the foundation for a much more ambitious project: the design of a complete synthetic genome and its transplantation into a living organism.

In 2010, the world witnessed a breakthrough with the reprogramming of the natural “software” of bacteria. For the first time, researchers at the J. Craig Venter Institute (JCVI) in the USA successfully transplanted a chemically synthesised genome of Mycoplasma mycoides – all 1.1 million base pairs of its DNA – into a closely related bacterial cell, Mycoplasma capricolum. The process consisted of many steps during which more than 1000 separate pieces of DNA were chemically synthesised, assembled and then used to replace the natural genome of the recipient cell.

To make the synthetic bacterium recognisably different and detectable, the JCVI team deleted 14 genes they thought unnecessary from M. mycoides, and added some “watermarking signature DNA” designed from scratch. The watermark branded the synthetic M. capricolum with a unique serial number (JCVI-syn1.0) and a cipher that contains the URL of a website and three quotations (if you can work out how to decode it).

This marked a world-first: a synthetic genome of a living cell, designed on a computer and synthesised in a lab, giving life to another living creature with no ancestor. A rubicon was crossed. Suddenly it has become possible to conceive of a future world in which new bacteria and, eventually, new complex microorganisms, plants and animals are designed and built to predetermined specifications. The global synthetic biology enterprise is expected to grow to $15 billion by the end of this decade, potentially revolutionising biotechnological applications in areas such as the pharmaceuticals/diagnostics, chemical, bioenergy and biomanufacturing industries.

However, the scientific successes achieved with poliovirus and mycoplasma bacteria are yet to be extrapolated to more complicated eukaryotic cells and multicellular organisms. It is for this reason that the scientific world has turned its attention to yeast.

Yeast Set to Become World’s First Synthetic Eukaryote

Of the more than 1500 yeast species, Saccharomyces cerevisiae is the most prominent in both laboratory and industrial settings. In the laboratory, this single-celled “sugar fungus” is amenable to almost any modification that modern science can throw at a eukaryotic cell. It is one of the most intensively studied biological model systems, allowing us to probe basic cellular and metabolic processes, and to shed new light on some of life’s biggest scientific challenges, such as the biology of cancer and ageing. In industry, this food-grade workhorse drives multi-billion-dollar fermentations – pumping out a diverse range of fermented foods and beverages on one hand, and biofuel and pharmaceutical products on the other.

Of all the microorganisms, this was the first to be “domesticated” for the production of food; to be observed microscopically; to be described as a living biochemical agent of transformation; to be used as a host for the production of the first recombinant vaccine (against hepatitis B) and the first recombinant food enzyme (the milk coagulation enzyme, chymosin, for cheese-making); and to be used to reveal the entire DNA sequence of a eukaryotic genome.

Following the development of the synthetic poliovirus and mycoplasma, this intriguing and tractable yeast has just become the latest organism to be equipped with a fully synthetic chromosome. A 272,871 base-pair synthetic version (SynIII) of the natural 316,617 base-pair chromosome 3 was synthesised and used to replace the authentic chromosome III of a laboratory strain of S. cerevisiae. This breakthrough by the leader of the global “Sc2.0” project, Jef Boeke, marks the first fully functional designer eukaryotic chromosome.

Under Boeke’s leadership, a global consortium of research laboratories from six countries has now embarked on building the ultimate yeast genome by 2017. Each participating lab takes responsibility for the design and construction of one or more of the 16 individual yeast chromosomes. In Australia we will be leading the synthesis of chromosomes 14 and 16.

With a genome size of ∼12 Mb – distributed along 16 chromosomes that encompass ∼6000 genes (of which ∼5000 are individually non-essential) – S. cerevisiae is marching on to become the world’s first eukaryote with a chemically synthesised genome. This will be a long and testing journey as SynIII accounts for less than 3% of the yeast genome, and it is yet to be proven that the same design rules will be successfully applied across the other 15 chromosomes.

In many ways, however, Boeke’s yeast group has taken a much more ambitious approach than the JCVI Mycoplasma team: rather than making a simple copy of S. cerevisiae’s chromosome III with a few added changes, they have greatly streamlined the chromosome by removing almost 20% of the original sequence, while also adding DNA sequences that will make it possible to reshuffle the genome at will. These DNA shuffling sequences can potentially be used to rapidly evolve new yeast strains with desirable properties and to help figure out the function of yeast genes. The team also added unique sequence tags to each gene in the chromosome and used these to check that all of the genes in their final chromosome were synthetic.

The team designing and constructing SynIII included undergraduate and secondary school students. Each student assembled short, overlapping 75 base-pair pieces of DNA into larger 750 base-pair “building blocks”. These were then assembled into mini-chunks of 3000–4000 base pairs, and groups of adjacent mini-chunks were pooled together and then transferred as mega-chunks into a yeast cell to replace part of the original chromosome III. This process was repeated across the entire length of the chromosome until the original, native chromosome III had been replaced with the new synthetic version, SynIII. Yeast cells with SynIII were able to grow just as well as yeast cells with the original chromosome III under virtually all conditions tested.

Ethics and Governance

Almost all scientific discoveries hold the potential for both benefit and harm, and synthetic biology is no exception. Concerns focus on risks to human and environmental health due to intentional or unintentional release of novel organisms, and the possibility that synthetic biology products designed to benefit society may also be capable of causing harm if placed in the wrong hands. The discussion around synthetic biology also includes concerns often associated with new technologies: the appropriateness of the research itself, the role of intellectual property, and the just distribution of risks and benefits issuing from the research.

A number of guidelines have been proposed in response to some of these issues, most of which focus on biosafety. For example, in the USA, the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules apply to NIH-funded synthetic biology research. These guidelines focus on DNA synthesis companies as the primary point of intervention. Other guidelines focus on the researchers themselves.

A Statement of Principles developed for all Sc2.0 participants seeks to strike a balance between what individual researchers can do and the responsibilities others must assume. The intent is to commit participating researchers, providers of synthetic DNA and collaborators to relevant legal and regulatory frameworks, to conducting their research in an open and transparent way for public good, and to promoting the work on Sc2.0 for the benefit of humans.

Understanding that the science is moving very quickly and that local and national policies may also shift, we will regularly review the statement to ensure that the Sc2.0 project’s policies are appropriately matched to risks and the regulatory status of the project. If risks increase, so will oversight and our accountability.

Our approach will prioritise social and ethical responsibility. Our approach to community engagement will be as ground-breaking as our science. Our objective is to conduct research that puts synthetic biology at the service of society.

Designing and Building a Better Future

If successfully constructed, Sc2.0 will provide a chassis for engineering other capabilities into yeast. This work will pave the way for the design of neo-chromosomes – purpose-built for specific applications in, for example, agriculture, medicine and energy. For now, advances with the Sc2.0 project, including constructive input from other disciplines, will be followed with interest by synthetic biologists.

In many ways the synthetic Mycoplasma and Sc2.0 projects are really just the starting point of synthetic biology. They provide a proof-in-principle that this approach works and that entire genomes or chromosomes of an organism can be replaced with a synthetic version. Synthetic biology and genome engineering will accelerate the understanding of the fundamentals of biological systems.

The statement found on the blackboard of Nobel Laureate and theoretical physicist Richard Feynman after his death – “What I cannot create, I do not understand” – rings true in the context of synthetic biology. We generally believe that research inspired by the quest for understanding the fundamentals, and the promise of future use, are the most powerful dynamo of technological and societal progress.