Scientists have developed a synthetic strain of Escherichia coli that can construct artificial polymers from building blocks that are not found in nature, by following instructions that the researchers encoded in their genes. The scientists, led by a team at the Medical Research Council (MRC) Laboratory of Molecular Biology, engineered the genetic code of the E. coli strain to include several nonstandard amino acids, and found that this synthetic genome made the bacteria entirely resistant to infection by viruses.

The work is some of the first to design proteins that incorporate multiple non-canonical amino acids. The team suggests that their work, and achievement could lead to the development of new polymers such as proteins and plastics, and drugs including antibiotics, as well as making it easier to manufacture drugs reliably using bacteria.

The newly reported achievement builds on previous ground-breaking work by researchers who, in 2019, developed a new techniques to create the biggest ever synthetic genome—constructing the entire E. coli genome from scratch. Commenting on the latest  developments, study lead, Jason Chin, PhD, from the MRC Laboratory of Molecular Biology, said, “These bacteria may be turned into renewable and programmable factories that produce a wide range of new molecules with novel properties, which could have benefits for biotechnology and medicine, including making new drugs, such as new antibiotics.”

The investigators report on their latest development in Science, in a paper titled, “Sense codon reassignment enables viral resistance and encoded polymer synthesis.”

The genetic code instructs a cell how to make proteins, which are constructed by joining together strings of natural (canonical) amino acid building blocks. The genetic code in DNA is made up of four bases, represented by the letters: A, T, C and G. When a peptide or protein is being constructed, the four letters in DNA are read in groups of three letters, or codons—for example “TCG.” Each codon tells the cell to add a specific amino acid to the peptide chain, which it does via molecules called transfer RNA (tRNA). Each codon is recognized by a specific tRNA, which then adds the corresponding amino acid. For example, the tRNA that recognizes the codon “TCG,” brings the amino acid serine.

With four letters in groups of three, there are 64 possible combinations of letters, but there are only 20 different canonical amino acids that cells commonly use. So, several different codons can be synonymous—they all code for the same amino acid —for example, TCG, TCA, AGC and AGT all code for serine. There are also codons which tell a cell when to stop making a protein, such as TAG and TAA.

It’s thought that removing certain codons and the transfer RNAs that read them from the genome and replacing them with noncanonical amino acids (ncAAs) may enable the creation of synthetic cells with properties not found in natural biology, including powerful viral resistances and enhanced biosynthesis of novel proteins. “However, these hypotheses have not been experimentally tested,” the authors wrote. And to date, the approach has been largely restricted to the incorporation of a single ncAA into a polypeptide chain. As the team noted, “… limitations preclude the synthesis of noncanonical heteropolymer sequences composed entirely of noncanonical monomers.”

In 2019, the team at the MRC Laboratory of Molecular Biology created the first entire genome synthesized from scratch for the commonly studied bacteria, E. coli. They also took the opportunity to simplify its genome. In this engineered strain the scientists replaced some of the codons with their synonyms. So, they removed every instance of TCG and TCA and replaced them with the synonyms AGC and AGT. They also removed every instance of the “stop” codon TAG and replaced it with its synonym TAA. This meant that the modified bacteria no longer had the codons TCG, TCA and TAG in their genome, but they could still make normal proteins and live and grow.

The MRC scientists’ goal was to utilize their new technology to create the first cell that can assemble polymers entirely from building blocks that are not found in nature. For the newly reported studies, the scientists further modified the bacteria to remove the tRNA molecules that recognize the codons TCG and TCA. This means that, even if there are TCG or TCA codons in the genetic code, the cell no longer has the molecule that can read those codons.

This is fatal for any virus that tries to infect the cell, because viruses replicate by injecting their genome into a cell and hijacking the cell’s machinery. Virus genomes still contain lots of the TCG, TCA and TAG codons, but the modified bacteria are missing the tRNAs to read these codons. So when the machinery in the modified bacteria tries to read the virus genome, it fails every time it reaches a TCG, TCA or TAG codon.

When the researchers infected their bacteria with a cocktail of viruses, they confirmed that while unmodified, control bacteria were killed by these pathogens, the modified bacteria were resistant to infection and survived.

Many drugs—for example, protein drugs, such as insulin, and polysaccharide and protein subunit vaccines—are manufactured by growing bacteria that contain instructions to produce the drug. So making bacteria that are resistant to viruses could make manufacturing certain types of drugs more reliable and cheaper. Chin explained, “If a virus gets into the vats of bacteria used to manufacture certain drugs then it can destroy the whole batch. Our modified bacterial cells could overcome this problem by being completely resistant to viruses. Because viruses use the full genetic code, the modified bacteria won’t be able to read the viral genes.” The team further wrote, “We have synthetically uncoupled our strain from the ability to read the canonical code, and this advance provides a potential basis for bioproduction without the catastrophic risks associated with viral contamination and lysis.”

By creating bacteria with synthetic genomes that do not use certain codons, the researchers also effectively freed up those codons to be used for other purposes, such as coding for synthetic building blocks (monomers). “We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids,” the authors explained. For the studies detailed in Science, the team engineered the bacteria to produce tRNAs coupled with artificial monomers, which recognized the newly available codons (TCG and TAG).

They inserted genetic sequences with strings of TCG and TAG codons into the bacteria’s DNA. These were read by the altered tRNAs, which assembled chains of synthetic monomers in the sequence defined by the sequence of codons in the DNA. The cells were programmed to string together monomers in different orders by changing the order of TCG and TAG codons in the genetic sequence. Polymers composed of different monomers were also made by changing which monomers were coupled to the tRNAs. “We incorporated three distinct ncAAs into ubiquitin, in response to TCG, TCA, and TAG,” the team explained. “We demonstrated the generality of our approach by synthesizing seven distinct versions of ubiquitin, each of which incorporated three distinct ncAAs.”

Using their approach the scientists were able to create polymers made of up to eight monomers strung together. They joined the ends of these polymers together to make macrocycles—a type of molecule that forms the basis of some drugs, such as certain antibiotics and cancer drugs.

Chin said, “This system allows us to write a gene that encodes the instructions to make polymers out of monomers that don’t occur in nature. We’d like to use these bacteria to discover and build long synthetic polymers that fold up into structures and may form new classes of materials and medicines. We will also investigate applications of this technology to develop novel polymers, such as biodegradable plastics, which could contribute to a circular bioeconomy.”

The synthetic monomers were linked together by the same chemical bonds that join together amino acids in proteins, but the researchers are in addition investigating how to expand the range of linkages that can be used in the new polymers. In their paper, they concluded, “Future work will expand the principles we have exemplified herein to further compress and reassign the genetic code. We anticipate that, in combination with ongoing advances in engineering the translational machinery of cells, this work will enable the programmable and encoded cellular synthesis of an expanded set of noncanonical heteropolymers with emergent, and potentially useful, properties.”

Commenting in an accompanying Perspective in the same issue of Science, D. Jewel, and A. Chatterjee, from Boston College in Chestnut Hill, acknowledged, “The ability to generate designer proteins using multiple non-natural building blocks will unlock countless applications, from the development of new classes of biotherapeutics to biomaterials with innovative properties.”

Megan Dowie, PhD, head of molecular and cellular medicine at the MRC, which funded the study, said, “Dr. Chin’s pioneering work into genetic code expansion is a really exciting example of the value of our long-term commitment to discovery science. Research like this, in synthetic and engineering biology, clearly has huge potential for major impact in biopharma and other industrial settings.”