The ability to write new chemistries into the proteins of living cells, known as genetic code expansion (GCE), represents a frontier in synthetic biology. By site-specifically incorporating non-canonical amino acids (ncAAs) with novel functionalities, researchers can create proteins with enhanced therapeutic properties, build novel biomaterials, and install molecular probes to illuminate complex biological processes. However, a persistent bottleneck has long constrained the field's potential: the inefficient delivery of ncAAs into the host cell. Most strategies rely on passive diffusion or the co-opting of native amino acid transporters, leading to low intracellular ncAA concentrations that cripple the efficiency of protein synthesis [2].
Early efforts to address this challenge focused on optimizing the intracellular machinery. This included engineering aminoacyl-tRNA synthetase/tRNA pairs for higher activity, designing orthogonal ribosomes to reduce competition with native translation, and deleting release factors to improve ncAA incorporation at stop codons [2]. While these innovations improved yields, they could not overcome the fundamental supply-side problem. The cell simply couldn't get enough of the raw material—the ncAA—to the factory floor, especially for ncAAs that are bulky, charged, or structurally foreign to native transport systems. This limitation has not only kept protein yields low but also restricted the chemical diversity of ncAAs that could be effectively utilized.
A recent study published in Nature by Kathrin Lang and her colleagues provides a powerful and elegant solution, shifting the focus from optimizing ncAA utilization to engineering ncAA supply [1]. Their work introduces a "Trojan Horse" strategy that hijacks a bacterial transport system to actively pump ncAAs into the cell, achieving unprecedented incorporation efficiencies.
The core of their approach is a modular tripeptide system. The team designed and synthesized tripeptides (Z-XisoK) where the desired ncAA (X) is linked via a stable isopeptide bond to a carrier scaffold. They demonstrated that these tripeptides are actively imported into Escherichia coli by the native oligopeptide permease (Opp), a type of ATP-binding cassette (ABC) transporter. Once inside the cell, native peptidases (PepN and PepA) cleave the scaffold, releasing the free ncAA at high concentrations.
The results are striking. Using this system, the intracellular concentration of the ncAA was boosted 5- to 10-fold compared to adding the free ncAA to the culture medium. This dramatic increase in bioavailability translated directly to protein expression. For a reporter protein, the tripeptide delivery method yielded protein levels comparable to wild-type expression, whereas direct addition of the free ncAA produced almost no protein at all [1].
The researchers further refined this platform in two critical ways:
G-XisoK toolbox" by demonstrating the efficient incorporation of 11 different ncAAs bearing diverse functional groups. These included bioorthogonal handles for chemical conjugation (e.g., alkynes), photo-activated crosslinkers for capturing protein-protein interactions, and mimics of post-translational modifications (PTMs) like lysine acetylation. This versatility transforms GCE from a niche technique into a broadly applicable tool for protein engineering and functional studies.Opp transporter's substrate-binding protein, OppA. Through a high-throughput fluorescence-activated cell sorting (FACS) screen, they isolated a variant, OppA-iso, with enhanced selectivity for their isopeptide-linked tripeptides over the linear peptides found in common lab media. The resulting engineered E. coli strain, IsoK12, enables robust and cost-effective ncAA incorporation even in nutrient-rich conditions. This successful optimization of the transporter via directed evolution underscores a powerful paradigm. High-throughput screening of vast genetic libraries, potentially accelerated by platforms using self-selecting vectors, can rapidly pinpoint optimal designs for complex biological functions.Finally, the team extended the strategy to enable the co-transport and dual incorporation of two different ncAAs from a single, cleverly designed tripeptide. This innovation opens the door to studying complex biological phenomena, such as the interplay between different PTMs on a single protein.
This study marks a paradigm shift for genetic code expansion. By proving that cellular import systems can be rationally hijacked and engineered, it provides a generalizable blueprint for the programmable uptake of a vast array of chemical building blocks. The construction of these tailored genetic systems, from reporter plasmids to evolved transporter variants, is a critical step. Integrated DNA design and synthesis services can streamline this process, accelerating the design-build-test-learn cycle.
The implications are far-reaching. This technology dramatically lowers the barrier to producing ncAA-modified proteins at scale, paving the way for their use in biotherapeutics, industrial biocatalysis, and advanced materials. Furthermore, the ability to efficiently incorporate PTM mimics provides a powerful tool to dissect the complex regulatory codes that govern protein function in health and disease.
Looking ahead, the challenge will be to adapt this strategy to other organisms, particularly mammalian cells, and to further expand the substrate scope of engineered transporters. Nonetheless, this work has effectively broken a long-standing bottleneck, opening a new and highly efficient gateway to expanding the language of life.
Ailurus Bio is a pioneering company building biological programs, genetic instructions that act as living software to orchestrate biology. We develop foundational DNAs and libraries, transforming lab-grown cells into living instruments that streamline complex research and production workflows. We empower scientists and developers worldwide with these bioprograms, accelerating discovery and diverse applications. Our mission is to make biology the truly general-purpose technology, as programmable and accessible as modern computers, by constructing a biocomputer architecture for all.
