A team of Yale synthetic biologists report that they were able to re-write the genetic code of E. coli—a novel genomically recoded organism (GRO) with one stop codon—using a cellular platform that they developed enabling the production of new classes of synthetic proteins. These synthetic proteins, the researchers say, offer the promise of many medical and industrial applications that can benefit society and human health.
The creation of the GRO, known as “Ochre,” which fully compresses redundant, or “degenerate” codons, into a single codon, is described in a new study “Engineering a genomically recoded organism with one stop codon” in Nature.
“This research allows us to ask fundamental questions about the malleability of genetic codes,” said Farren Isaacs, PhD, professor of molecular, cellular and developmental biology at the Yale School of Medicine and of biomedical engineering at the Yale School of Engineering & Applied Science, who is co-senior author of the paper. “It also demonstrates the ability to engineer the genetic code to endow multi-functionality into proteins and usher in a new era of programmable biotherapeutics and biomaterials.”
Synthetic approaches
“The genetic code is conserved across all domains of life, yet exceptions have revealed variations in codon assignments and associated translation factors. Inspired by this natural malleability, synthetic approaches have demonstrated whole-genome replacement of synonymous codons to construct genomically recoded organisms (GROs) with alternative genetic codes,” wrote the investigators.
“However, no efforts have fully leveraged translation factor plasticity and codon degeneracy to compress translation function to a single codon and assess the possibility of a non-degenerate code. Here we describe construction and characterization of Ochre, a GRO that fully compresses a translational function into a single codon. We replaced 1,195 TGA stop codons with the synonymous TAA in ∆TAG Escherichia coli C321.∆A. We then engineered release factor 2 (RF2) and tRNATrp to mitigate native UGA recognition, translationally isolating four codons for non-degenerate functions.
“Ochre thus utilizes UAA as the sole stop codon, with UGG encoding tryptophan and UAG and UGA reassigned for multi-site incorporation of two distinct non-standard amino acids into single proteins with more than 99% accuracy. Ochre fully compresses degenerate stop codons into a single codon and represents an important step toward a 64-codon non-degenerate code that will enable precise production of multi-functional synthetic proteins with unnatural encoded chemistries and broad utility in biotechnology and biotherapeutics.”
The advance builds on a 2013 study by the team, published in Science, which described the construction of the first GRO. In that study, the researchers demonstrated new solutions for safeguarding genetically engineered organisms and for producing new classes of synthetic proteins and biomaterials with “unnatural,” or human-created, chemistries.
Ochre is a major step toward creating a non-redundant genetic code in E. coli, specifically, which is ideally suited to produce synthetic proteins containing multiple, different synthetic amino acids, according to Jesse Rinehart, PhD, an associate professor of cellular and molecular physiology at the Yale School of Medicine and co-senior author on the study. He called the breakthrough a “profound piece of whole genome engineering based on over 1,000 precise edits at a scale an order of magnitude greater than any engineering feat we have previously done.”
Specifically, the researchers eliminated two of the three stop codons that terminate protein production. The recoded genome reassigned four codons to non-degenerate functions, including the two recoded stop codons dedicated to encoding nonstandard, or unnatural, amino acids into protein. In addition to introducing thousands of precise edits across the genome, the work required AI-guided design and re-engineering of essential protein and RNA translation factors to create a strain capable of adding two nonstandard amino acids into its recipe book.
These nonstandard amino acids imbue proteins with multiple new properties, such as programmable biologics with reduced immunogenicity or biomaterials with enhanced conductivity.
Isaacs is excited about what he describes as the potentially “killer” applications for programmable protein biologics that the new platform will make possible. One such application involves engineering protein drugs with synthetic chemistries to decrease the frequency of dosing or undesirable immune responses. The team reported such an application using their first-generation GRO in a 2022 study. In that study they encoded non-standard amino acids into protein, demonstrating a safer, controllable approach to precisely tune the half-life of protein biologics.
The new Ochre cell expands these capabilities for use in the construction of multi-functional biologics. Isaacs and Rinehart are currently acting as advisors to Pearl Bio, a Yale biotechnology spin-off that has licensed the technology for commercializing programmable biologics.
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