Pull out all the stops (Image: Pgiam/Getty Images)

A bacterium has had its genome recoded so that the standard language of life no longer applies. Instead, one of its words has been freed up to impart a different meaning, allowing the addition of genetic elements that don’t exist in nature.

The work has been described as the first step towards a new biology because the techniques used should open the door to reinventing the meaning of several genetic words simultaneously, potentially creating new types of biomaterials and drugs.

The engineered bacteria, dubbed genetically recoded organisms (GROs), have the added advantage of being resistant to many existing viruses. They are also less likely to escape the lab and survive than conventional genetically modified organisms, which should make them more palatable for commercial use.


Alien architecture

The four letters of the genetic code are usually read by a cell’s protein-production machinery, the ribosome, in sets of three letters called codons. Each codon “word” provides instructions about which amino acid to add next to a growing peptide chain.

Although there are 64 ways of combining four letters, only 61 codons are used to encode the 20 amino acids found in nature. This means that some of the codons encode the same amino acid – a phenomenon called redundancy. The three combinations left over, UAG, UAA and UGA, act like a full stop or period – telling the ribosome to terminate the process at that point. When this happens, rather than an amino acid getting added to the chain, a release factor binds and triggers the release of the peptide, so that it can be folded and processed into a protein.

A team of synthetic biologists led by Farren Isaacs at Yale University have now fundamentally rewritten these rules. They took Escherichia coli cells and replaced all of their UAG stop codons with UAAs. They also deleted the instructions for making the release factor that usually binds to UAG, rendering UAG meaningless.

Next they set about assigning UAG a new meaning, by designing molecules called tRNAs and accompanying enzymes that would attach an unnatural amino acid – fed to the cell – whenever they spotted this codon. Many such amino acids have been designed, but they can’t usually be processed by living organisms and incorporated into their proteins. By reintroducing UAGs at specific locations within genes, the team were able to add unnatural amino acids into proteins at will.

“We now have an organism that has a new code, and we can reliably and efficiently open up the chemical diversity of proteins by introducing a whole new array of amino acids using UAG as the codon,” says Isaacs.

For example, amino acids could be added that bestow proteins with new properties, like the capability to bind to metals – resulting in new adhesives. Or enzymes could be developed that are resistant to digestion in the gut or which only become activated in the presence of another molecule – resulting in new types of drug.

A new field

“This is a big step forward,” says Steve Benner of the Foundation for Applied Molecular Evolution in Gainesville, Florida. “Once you have a cell that’s able to handle your concept of DNA and your concept of amino acids, then you are opening up an entirely new field of science.”

“The genetic code is conserved for all of life, so this is a fundamental step forward,” says Philipp Holliger from the MRC Laboratory of Molecular Biology in Cambridge, UK. He adds that because the code is largely redundant, there might be quite a few more codons that could potentially be removed and then reassigned to expand the chemistry of living organisms.

“It has great potential for the future to not just replace one here and there, but to replace loads of them and have completely new types of biopolymers made in cells. It’s a first step down the road to a new biology,” he says.

Although other groups have previously modified bacteria – and even fruit flies – to produce proteins that contained unnatural amino acids, this required the addition of a synthetic ribosome into the cell that was in competition with the cell’s existing machinery.

It also offers an alternative to Craig Venter’s approach of building a genome from scratch in order to impart new properties to cells – difficult and laborious because even the smallest error kills the cell.

“This is a major result in that it counters the Venter synthesis of genomes,” says Paul Freemont of Imperial College London. “[Isaac’s team] argue that they can recode living existing genomes quickly and efficiently which can increase biological diversity.”

Resistant to viruses

The feat was achieved using a technique called multiplex automated genome engineering, or MAGE, which involves designing fragments of single-stranded DNA that, with the assistance of viral enzymes, would replace existing UAG codons in RNA with UAA when E. coli cells were zapped with electricity.

Not every stop codon was replaced on the first hit, so a second technique called CAGE was then used to merge strains with different changes, until a hybrid containing a completely recoded genome was produced.

The genetically recoded E. coli has the added advantage of being resistant to many viruses. Because viruses incorporate their genes into a host cell’s DNA to produce viral proteins, any viral genes containing the UAG stop codon can no longer be read.

This is useful, because industrial production of proteins using bacteria, to make biofuels or penicillin, for example, often gets contaminated, meaning entire vats of bacteria have to be thrown away.

What’s more, there’s little threat of recoded E. coli swapping genes with its natural counterpart, were GROs ever to escape into the wild. Reassigned stop codons would be read as normal stop codons in natural cells, resulting in the premature termination of protein production. Preliminary studies also suggest that GROs cannot grow in the absence of the unnatural amino acid they’ve been designed to incorporate – adding another level of security.

Journal reference: Science, DOI: 10.1126/science.1241459