One aim of synthetic biology is to generate complex synthetic organisms. Now, a stage in this process has been achieved in yeast cells — an entire yeast chromosome has been converted to a synthetic sequence in a stepwise manner.
A biological cell is much like a computer — the genome can be thought of as the software that encodes the cell's instructions, and the cellular machinery as the hardware that interprets and runs the software. Advances in DNA technology have made it possible for scientists to act as biological 'software engineers', programming new biological 'operating systems' into cells. Indeed, in 2010, the entire genome of the bacterium Mycoplasma mycoides was replaced with a rewritten synthetic genome, generating the first synthetic cell1. Now, in a paper published in Science, Annaluru et al.2 describe how they have begun rewriting the genome of a more complex organism, that of the yeast species Saccharomyces cerevisiae. The researchers report the design and generation of a functional, synthetic chromosome in this yeast, a milestone that they have had in their sights for almost ten years.
A quote by the theoretical physicist Richard Feynman, “What I cannot create, I do not understand”, has inspired synthetic biologists around the world. But this does not necessarily mean that what can be created is understood. There is an enormous gap between our ability to build DNA and our understanding of the instructions it encodes. The production of a synthetic chromosome in yeast (the first such achievement in a eukaryote — the class of organisms comprising plants, animals and fungi) represents a step towards closing that gap. Generation of synthetic versions of widely used model organisms such as S. cerevisiae will enable scientists to investigate the requirements of life in eukaryotes, because synthetic organisms can be easily manipulated.
The process began at the computer, where Annaluru and colleagues downloaded the publicly available DNA sequence of a S. cerevisiae chromosome (chromosome III). Next, they designed genetic changes, which can be thought of as software edits, with the aim of introducing specific alterations into the chromosome. These edits included the deletion of dispensable DNA sequences; the incorporation of unique sequences to enable the researchers to differentiate between natural and engineered DNA; and the replacement of one particular region of DNA that terminates gene transcription with another that performs the same task.
In addition, the authors flanked each non-essential gene with DNA sequences designed to cause deletion of the flanked genes on a given signal. This flanking allows the size of the chromosome to be reduced, a feature designed to help determine the smallest cohorts of genes required to perform a given function or necessary for survival under a particular growth condition. This information is crucial if we are to write biological software in a more predictable fashion, thus ensuring that cells can be engineered to reliably carry out the tasks they have been programmed to perform.
To generate the synthetic chromosome, Annaluru and co-workers broke down their designer DNA sequence into overlapping stretches of 70 nucleotides, which were chemically synthesized in parallel. Students in the Build-a-Genome course at Johns Hopkins University in Baltimore, Maryland, stitched together these DNA stretches into constructs approximately 3 kilobases long, using established DNA-assembly methods3,4,5,6.
Next, the original chromosome sequence was systematically replaced with the synthetic DNA in vivo, by introducing up to 12 overlapping 3-kb synthetic fragments at a time into the yeast cell, in 11 successive rounds of integration. The product of this work is a yeast strain that contains an extensively engineered chromosome III, and that grows just as well as the original strain. The 273-kb designer chromosome contains more than 50,000 sequence alterations, and is 14% shorter than the natural sequence.
This work is important because it begins to address unanswered questions about how genome design can be used to manipulate the rules of biology in a eukaryotic model. For example, can superfluous DNA between genes be removed? Can unnecessary genetic code be altered? So far, the sequence alterations made by Annaluru and colleagues have not reduced the fitness of the yeast, which bodes well for future modifications.
If two or more genes perform a similar function, can one be deleted? Which combinations of the 5,000 yeast genes that are thought to be dispensable can be simultaneously removed? The authors have already begun cataloguing genes that can be simultaneously deleted without adversely affecting the fitness of the yeast. Answering these fundamental questions is a goal that the synthetic-yeast group shares with efforts to recode the genome of the bacterium Escherichia coli7, and with current research programmes1,8 that aim to understand better how to build bacterial genomes containing only the genes necessary to sustain life.
Two bacterial genomes have previously been chemically synthesized — the 583-kb genome of Mycoplasma genitalium4 and the 1,078-kb M. mycoides genome1 — and so synthesis of a chromosome of 273 kb is not in itself unusual. The genomes of both M. genitalium and M. mycoides were assembled and propagated in yeast, acting as an extra chromosome. Like incompatible Macintosh software in a Windows computer, synthetic bacterial genomes are unable to 'boot up' in yeast-cell machinery, and so cannot produce self-replicating bacterial cells in this setting. In a process called genome transplantation9,10, the complete bacterial genome must be moved to a bacterial host with compatible hardware, converting the recipient cell into a new, synthetic species (Fig. 1a). By contrast, Annaluru et al. gradually converted the natural yeast chromosome into a fully functional designer chromosome within their target species, the yeast cell itself (Fig. 1b).
In comparison to the completely synthetic M. mycoides cell1, chromosome III accounts for less than 3% of the yeast genome. Now the question is: can these design rules be successfully applied across the entire yeast genome? The scientists attempting to generate synthetic yeast cells still have a long way to go before they make a fully reprogrammed yeast genome. However, by demonstrating the success of their design principles and assembling an international team of scientists to build the remaining 15 chromosomes, Annaluru and colleagues have laid the groundwork for making this happen in the near future.
Advances such as these are of interest to the entire field of synthetic biology. Each innovation not only enhances our general understanding of biology, but also creates a framework on which we can build and expand, allowing us to work towards the goal of basing our economy on synthetic biology — a development that will have a positive impact on all of society.
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Science and Engineering Ethics (2017)