Virtually all living organisms construct their proteins from combinations of 20 different amino acids. To add new amino acids to the mix, scientists have re-engineered genes and other bits of protein-building machinery, resulting in proteins with unique chemical properties useful in making drugs. But the work is laborious and can typically only add one new amino acid at a time.
Now, researchers have opened the floodgates to doing much more. They report today that a broad rewrite of a bacterium’s genome lets them add numerous novel amino acids to one protein. The work could open new ways to synthesize antibiotics and antitumor drugs.
“I am very impressed by this paper,” says Chang Liu, a synthetic biologist at the University of California, Irvine. “It’s a significant milestone in the arc of genetic code re-engineering.”
The new effort has been underway for decades. One early approach to create designer proteins has been to commandeer proteinmaking cellular components and get them to insert unnatural amino acids. When cells make proteins, the DNA code of A, C, G, and T is first copied into RNA (in which U replaces T). The RNA is read as a series of three-letter words, known as codons, most of which code for one of the 20 natural amino acids to be inserted in the protein. But because there are 64 three-letter codons, there are duplicates: Six codons, for example, code for the amino acid serine. Three codons don’t encode an amino acid; instead, they instruct cells to stop building proteins.
Initially, researchers inserted unnatural amino acids by having the cellular machinery add one whenever it saw a particular stop codon. Though this approach has grown more sophisticated, it can still generally only insert one amino acid per protein, says Jason Chin, a synthetic biologist at the Medical Research Council Laboratory of Molecular Biology.
In hopes of adding more, Chin and his colleagues sought to repurpose two of the six codons that normally code for serine. In a 2019 study, the researchers used the CRISPR-Cas9 gene-editing tool to create an Escherichia coli strain known as Syn61. To make it, they replaced more than 18,000 serine codons in the bacterium’s 4-million-base-long genome. The researchers replaced UCG and UCA—and the stop codon UAG—with their “synonyms,” AGC, AGU, and UAA, respectively. That meant serine would still be incorporated into the correct spots of Syn61’s growing proteins. But the UCG, UCA, and UAG codons were now effectively “blanks” that no longer coded for anything in the protein, and were thus ready to be repurposed.
That repurposing is what Chin and his colleagues have now accomplished. Working with Syn61, the scientists deleted genes for molecules called transfer RNAs (tRNAs) that recognize UGC and UCA and insert serine into a growing protein. They also removed the chemical compound that turns off protein synthesis in response to the UAG stop codon. The researchers then added back genes with novel tRNAs that would insert unnatural amino acids whenever they encountered UGC, UCA, or UAG. Finally, they wrote those codons back into the genome where they wanted unnatural amino acids to appear. That allowed them to add three unnatural amino acids at once into individual proteins, the researchers report today in Science. They could also write multiple copies of each.
“It really made an impact,” says Abhishek Chatterjee, a synthetic biologist at Boston College. The changes allowed Chin and his colleagues to string the new amino acids together in a series of cyclic structures that closely resemble existing antibiotics and antitumor drugs. And because there are dozens of different unnatural amino acids to choose from, myriad combinations could be inserted in this fashion. That opens the door to creating vast libraries of potential new drugs, Chatterjee says. Chin adds that researchers can also extend the strategy to repurpose other redundant codons to add even more new amino acids—and more chemical variety—into the mix.
Perhaps just as interesting, Liu says, was what the wholesale genome changes meant for viruses that normally infect E. coli. In 2013, researchers reported that re-engineering E. coli’s stop codons could disrupt the ability of the viruses to replicate. That occurred because the viruses rely on E. coli’s natural codons to make functional proteins. The strategy wasn’t foolproof in stopping viral infections, because stop codons don’t occur all that often, and some viruses were able to evolve their way around the changes.
But viruses typically require many more serines in each protein. Because the modified Syn61 no longer inserted serine when its protein-building machinery read UCG or UCA codons, viruses couldn’t get Syn61 to build working viral proteins, thereby preventing them from reproducing in the bacterial cells.
“This looks much more robust” than the earlier approach, Liu says. That, he adds, could be a boon for biotech companies looking to safeguard engineered organisms that churn out medicines or other valuable chemicals.