Genetic Code Expansion Research Articles

Genetic Code Expansion History and Modern Innovations.

The genetic code is the foundation for all life. With few exceptions, the translation of nucleic acid messages into proteins follows conserved rules, which are defined by codons that specify each of the 20 proteinogenic amino acids. For decades, leading research groups have developed a catalogue of innovative approaches to extend nature's amino acid repertoire to include one or more noncanonical building blocks in a single protein. In this review, we summarize advances in the history of in vitro and in vivo genetic code expansion, and highlight recent innovations that increase the scope of biochemically accessible monomers and codons. We further summarize state-of-the-art knowledge in engineered cellular translation, as well as alterations to regulatory mechanisms that improve overall genetic code expansion. Finally, we distill existing limitations of these technologies into must-have improvements for the next generation of technologies, and speculate on future strategies that may be capable of overcoming current gaps in knowledge.

Fluorescent non-canonical amino acid provides insight into the human serotonin transporter

The serotonin transporter (SERT), responsible for the reuptake of released serotonin, serves as a major target for antidepressants and psychostimulants. Nevertheless, refining the mechanistic models for SERT remains challenging. Here, we expand the molecular understanding of the binding of ions, substrates, and inhibitors to SERT by incorporating the fluorescent non-canonical amino acid Anap through genetic code expansion. We elucidate steady-state changes in conformational dynamics of purified SERT with Anap inserted at intracellular- or extracellular sites. This uncovers the competitive mechanisms underlying cation binding and assigns distinct binding- and allosteric coupling patterns for several inhibitors and substrates. Finally, we track in real-time conformational transitions in response to the interaction with Na+ or serotonin. In this work, we present a methodological platform reporting on SERT conformational dynamics, which together with other approaches will deepen our insights into the molecular mechanisms of SERT.

Halogenase-Assisted Alkyne/Aryl Bromide Sonogashira Coupling for Ribosomally Synthesized Peptides.

We describe the enzymatic bromination of ribosomally synthesized peptides and develop protocols for Sonogashira coupling of peptidic aryl bromides with a panel of alkynes. Using this workflow, entirely new chemical handles are introduced onto ribosomal peptides, including but not limited to terminal alkynes, which enable further diversification via alkyne-azide click chemistry. Regiospecific enzymatic installation of the aryl bromide circumvents genetic code expansion and passivation of other reactive handles on the peptide chain, representing the applicability of biocatalysts in peptide modification chemistry.

The Biosynthesis and Applications of Protein Lipidation.

Protein lipidation dramatically affects protein structure, localization, and trafficking via remodeling protein-membrane and protein-protein interactions through hydrophobic lipid moieties. Understanding the biosynthesis of lipidated proteins, whether natural ones or mimetics, is crucial for reconstructing, validating, and studying the molecular mechanisms and biological functions of protein lipidation. In this Perspective, we first provide an overview of the natural enzymatic biosynthetic pathways of protein lipidation in mammalian cells, focusing on the enzymatic machineries and their chemical linkages. We then discuss strategies to biosynthesize protein lipidation in mammalian cells by engineering modification machineries and substrates. Additionally, we explore site-specific protein lipidation biosynthesis in vitro via enzyme-mediated ligations and in vivo primarily through genetic code expansion strategies. We also discuss the use of small molecule tools to modulate the process of protein lipidation biosynthesis. Finally, we provide concluding remarks and discuss future directions for the biosynthesis and applications of protein lipidation.

Reaching New Heights in Genetic Code Manipulation with High Throughput Screening.

The chemical and physical properties of proteins are limited by the 20 canonical amino acids. Genetic code manipulation allows for the incorporation of noncanonical amino acids (ncAAs) that enhance or alter protein functionality. This review explores advances in the three main strategies for introducing ncAAs into biosynthesized proteins, focusing on the role of high throughput screening in these advancements. The first section discusses engineering aminoacyl-tRNA synthetases (aaRSs) and tRNAs, emphasizing how novel selection methods improve characteristics including ncAA incorporation efficiency and selectivity. The second section examines high-throughput techniques for improving protein translation machinery, enabling accommodation of alternative genetic codes. This includes opportunities to enhance ncAA incorporation through engineering cellular components unrelated to translation. The final section highlights various discovery platforms for high-throughput screening of ncAA-containing proteins, showcasing innovative binding ligands and enzymes that are challenging to create with only canonical amino acids. These advances have led to promising drug leads and biocatalysts. Overall, the ability to discover unexpected functionalities through high-throughput methods significantly influences ncAA incorporation and its applications. Future innovations in experimental techniques, along with advancements in computational protein design and machine learning, are poised to further elevate this field.

Noncanonical Amino Acid Incorporation Modulates Condensate States of Intrinsically Disordered Proteins in Escherichia coli Cells.

Biomolecular condensates are distinct subcellular structures with on-demand material states and dynamics in living cells. However, strategies for modulating their material states and physicochemical properties are lacking. Here, we report a chemical strategy for modulating the condensate states of intrinsically disordered proteins in bacterial Escherichia coli cells. This is achieved by noncanonical amino acid (DOPA) incorporation into model resilin-like proteins (RLPs) to endow autonomous oxidative and coordinative cross-linking mechanisms. Biogenesis of spherical gel-like condensates is achieved upon heterologous expression of the DOPA-incorporated RLP in the cells at 30 °C. We reveal that liquid-liquid phase separation underlies the formation of liquid condensates, and this liquid-like state is metastable and its dynamics is compromised by the oxidative and coordinative cross-linkings that ultimately drive the liquid-to-gel transition. Therefore, this study has deepened our understanding of biomolecular condensation and offers a new chemical strategy to expand the landscape of condensation phenotypes of living cells.

Expanding the Genetic Code of Bioelectrocatalysis and Biomaterials.

Genetic code expansion is a promising genetic engineering technology that incorporates noncanonical amino acids into proteins alongside the natural set of 20 amino acids. This enables the precise encoding of non-natural chemical groups in proteins. This review focuses on the applications of genetic code expansion in bioelectrocatalysis and biomaterials. In bioelectrocatalysis, this technique enhances the efficiency and selectivity of bioelectrocatalysts for use in sensors, biofuel cells, and enzymatic electrodes. In biomaterials, incorporating non-natural chemical groups into protein-based polymers facilitates the modification, fine-tuning, or the engineering of new biomaterial properties. The review provides an overview of relevant technologies, discusses applications, and highlights achievements, challenges, and prospects in these fields.

Novel Vibrational Proteins.

Genetically encoded green fluorescent protein (GFP) and its brighter and redder variants have tremendously revolutionized modern molecular biology and life science by enabling direct visualization of gene regulated protein functions on microscopic and nanoscopic scales. However, the current fluorescent proteins (FPs) only emit a few colors with an emission width of about 30-50 nm. Here, we engineer novel vibrational proteins (VPs) that undergo much finer vibrational transitions and emit rather narrow vibrational spectra (0.1-0.3 nm, roughly 3-10 cm-1). In response to an amber stop codon (UAG), a terminal alkyne bearing an unnatural amino acid (UAA, pEtF) is directly incorporated in place of Tyr64 in the chromophore of pr-Kaede by genetic code expansion. Essentially, the UAA64 further conjugates into a large π system with the contiguous two editable amino acid residues (His63 and Gly65), resulting in a programmable Raman resonance shift of the embedded alkyne. In the proof-of-concept experiment, we constructed a series of novel pEtF-VP mutants and observed fine Raman shifts of the alkynyl group in different chromophores. The genetically encoded novel VPs, could potentially label tens of proteins in the future.

Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.

Genetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons ( e.g ., the amber stop codon). Many orthogonal aaRS/tRNA pairs covering a wide range of ncAAs have been generated by directed evolution, yet the evolution of new aaRS/tRNA pairs by standard strategies remains a labor-intensive process that often produces aaRS/tRNA pairs with suboptimal ncAA incorporation efficiencies. In this study, we present a strategy for evolving aaRSs that leverages OrthoRep to drive their continuous hypermutation in yeast. We demonstrate our strategy in 8 independent aaRS evolution campaigns starting from 4 different aaRS/tRNA parents targeting 7 distinct ncAAs. We observed the rapid evolution of multiple novel aaRSs capable of incorporating an overall range of 13 ncAAs tested into proteins in response to the amber codon. Some evolved systems reached efficiencies for amber codon-specified ncAA-dependent translation comparable to translation with natural amino acids specified by sense codons in yeast. Additionally, we discovered a surprising aaRS that evolved to self-regulate its own expression for greater dependency on ncAAs for translation. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms in supporting the continued growth of GCE technologies.

Noncanonical Amino Acids: Bringing New-to-Nature Functionalities to Biocatalysis.

Biocatalysis has become an important component of modern organic chemistry, presenting an efficient and environmentally friendly approach to synthetic transformations. Advances in molecular biology, computational modeling, and protein engineering have unlocked the full potential of enzymes in various industrial applications. However, the inherent limitations of the natural building blocks have sparked a revolutionary shift. In vivo genetic incorporation of noncanonical amino acids exceeds the conventional 20 amino acids, opening new avenues for innovation. This review provides a comprehensive overview of applications of noncanonical amino acids in biocatalysis. We aim to examine the field from multiple perspectives, ranging from their impact on enzymatic reactions to the creation of novel active sites, and subsequent catalysis of new-to-nature reactions. Finally, we discuss the challenges, limitations, and promising opportunities within this dynamic research domain.

Strategy for Construction of Homogeneous Glycoproteins in Mammalian Cells.

A general strategy that combines genetic code expansion with bio-orthogonal ligation techniques was developed and utilized to prepare homogeneously glycosylated receptors on the surface of mammalian cells. Using this approach, conjugates of the cell-surface oxytocin receptor (OTR) with oligosaccharides were efficiently generated in the cells. Cell studies revealed that glycans linked to the OTR are not essential for agonist-induced calcium flux and its internalization into cells via an OTR-mediated endocytosis.

WDR5 Binding to Histone Serotonylation Is Driven by an Edge-Face Aromatic Interaction with Unexpected Electrostatic Effects.

Histone serotonylation has emerged as a key post-translational modification. WDR5 preferentially binds to serotonylated histone 3 (H3), and this binding event has been associated with tumorigenesis. Herein, we utilize genetic code expansion, structure-activity relationship studies, and computation to study an edge-face aromatic interaction between WDR5 Phe149 and serotonin on H3 that is key to this protein-protein interaction. We find experimentally that this edge-face aromatic interaction is unaffected by modulating the electrostatics of the face component but is weakened by electron-withdrawing substituents on the edge component. Overall, these results elucidate that this interaction is governed by van der Waals forces as well as electrostatics of the edge ring, a result that clarifies discrepancies among previous theoretical models and model system studies of this interaction type. This is the first evaluation of the driving force of an edge-face aromatic interaction at a protein-protein interface and provides a key benchmark for the nature of these understudied interactions that are abundant in the proteome.

AARS1 and AARS2 sense L-lactate to regulate cGAS as global lysine lactyltransferases.

L-lactate modifies proteins through lactylation1, but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular L-lactate sensors required for L-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to L-lactate with micromolar affinity and they directly catalyse L-lactate for ATP-dependent lactylation on the lysine acceptor end. In response to L-lactate, AARS2 associates with cyclic GMP-AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of L-lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular L-lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS.

Genetic Code Expansion Approaches to Decipher the Ubiquitin Code.

The covalent attachment of Ub (ubiquitin) to target proteins (ubiquitylation) represents one of the most versatile PTMs (post-translational modifications) in eukaryotic cells. Substrate modifications range from a single Ub moiety being attached to a target protein to complex Ub chains that can also contain Ubls (Ub-like proteins). Ubiquitylation plays pivotal roles in most aspects of eukaryotic biology, and cells dedicate an orchestrated arsenal of enzymes to install, translate, and reverse these modifications. The entirety of this complex system is coined the Ub code. Deciphering the Ub code is challenging due to the difficulty in reconstituting enzymatic machineries and generating defined Ub/Ubl-protein conjugates. This Review provides a comprehensive overview of recent advances in using GCE (genetic code expansion) techniques to study the Ub code. We highlight strategies to site-specifically ubiquitylate target proteins and discuss their advantages and disadvantages, as well as their various applications. Additionally, we review the potential of small chemical PTMs targeting Ub/Ubls and present GCE-based approaches to study this additional layer of complexity. Furthermore, we explore methods that rely on GCE to develop tools to probe interactors of the Ub system and offer insights into how future GCE-based tools could help unravel the complexity of the Ub code.

Repurposing Copper(II)/THPTA as A Bioorthogonal Catalyst for Thiazolidine Bond Cleavage.

Metal-mediated chemical transformations are promising approaches to manipulate and regulate proteins in fundamental biological research and therapeutic development. Nevertheless, unlike bond-forming reactions, the exploration of selective bond cleavage reactions catalyzed by metals that are fully compatible with proteins and living systems remains relatively limited. Here, it is reported that Copper(II)/tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), commonly used in copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, can be repurposed as a new bioorthogonal catalyst for thiazolidine (Thz) bond cleavage. This process liberates an α-oxo-aldehyde group under physiological conditions, without requiring additional additives. To showcase the utility of this method, this simple catalyst system is coupled with genetic code expansion technology to achieve on-demand activation of genetically encoded Thz-caged α-oxo-aldehydes, enabling further functionalization of proteins. For the first time, this cell-compatible Thz uncaging reaction allows for the site-specific installation of α-oxo-aldehydes at the internal positions of proteins in phage and bacterial surface display systems, expanding the chemical space of proteins. Overall, this study expands the toolkit of bioorthogonal catalysts and paves the way for metal-promoted chemical reactions in living systems, potentially benefiting various applications in the future.

Harnessing Nature-Inspired Catechol Amino Acid to Engineer Sticky Proteins and Bacteria.

3,4-Dihydroxy-L-phenylalanine (DOPA) serves as a post-translational modification amino acid present in mussel foot proteins. Mussels exploit the exceptional adhesive properties of DOPA to adhere to a wide range of surfaces. This study presents the development of sticky proteins and bacteria through the site-specific incorporation of DOPA using Genetic Code Expansion Technology. Through the optimization of the DOPA incorporation system, proteins containing DOPA demonstrate significantly improved binding abilities to various organic and metallic materials. The material-binding capabilities of DOPA to combat different types of biofoulings are harnessed by integrating it into intrinsically disordered proteins. Beyond the creation of adhesive proteins for anti-biofouling purposes, this highly efficient DOPA incorporation system is also applied to engineer adhesive bacteria, resulting in a remarkable increase in their binding capability to diverse materials including 400 folds of improvement to polyethylene terephthalate (PET). This substantial enhancement in PET binding of these bacteria has allowed to develop a unique approach for PET degradation, showcasing the innovative application of Genetic Code Expansion in cell engineering.

Efficient genetic code expansion without host genome modifications.

Supplementing translation with noncanonical amino acids (ncAAs) can yield protein sequences with new-to-nature functions but existing ncAA incorporation strategies suffer from low efficiency and context dependence. We uncover codon usage as a previously unrecognized contributor to efficient genetic code expansion using non-native codons. Relying only on conventional Escherichia coli strains with native ribosomes, we develop a plasmid-based codon compression strategy that minimizes context dependence and improves ncAA incorporation at quadruplet codons. We confirm that this strategy is compatible with all known genetic code expansion resources, which allowed us to identify 12 mutually orthogonal transfer RNA (tRNA)-synthetase pairs. Enabled by these findings, we evolved and optimized five tRNA-synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Lastly, we extend these resources to an in vivo biosynthesis platform that can readily create >100 new-to-nature peptide macrocycles bearing up to three unique ncAAs. Our approach will accelerate innovations in multiplexed genetic code expansion and the discovery of chemically diverse biomolecules.

Recoded gene circuits for multiplexed genetic code expansion.

Recoded gene circuits for multiplexed genetic code expansion.

Rapid discovery and evolution of nanosensors containing fluorogenic amino acids

Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, biosensor discovery is slow and requires tedious steps in rational design, screening, and characterization. Here we report on a platform that streamlines biosensor discovery and unlocks directed nanosensor evolution through genetically encodable fluorogenic amino acids (FgAAs). Building on the classical knowledge-based semisynthetic approach, we engineer ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. An optimized genetic code expansion chemistry with FgAAs further enables rapid (~3 h) ribosomal nanosensor discovery via the cell-free translation of hundreds of candidates in parallel and directed nanosensor evolution with improved variant-specific sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform could accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.

Biosynthesis of Halogenated Tryptophans for Protein Engineering Using Genetic Code Expansion.

Genetic Code Expansion technology offers significant potential in incorporating noncanonical amino acids into proteins at precise locations, allowing for the modulation of protein structures and functions. However, this technology is often limited by the need for costly and challenging-to-synthesize external noncanonical amino acid sources. In this study, we address this limitation by developing autonomous cells capable of biosynthesizing halogenated tryptophan derivatives and introducing them into proteins using Genetic Code Expansion technology. By utilizing inexpensive halide salts and different halogenases, we successfully achieve the selective biosynthesis of 6-chloro-tryptophan, 7-chloro-tryptophan, 6-bromo-tryptophan, and 7-bromo-tryptophan. These derivatives are introduced at specific positions with corresponding bioorthogonal aminoacyl-tRNA synthetase/tRNA pairs in response to the amber codon. Following optimization, we demonstrate the robust expression of proteins containing halogenated tryptophan residues in cells with the ability to biosynthesize these tryptophan derivatives. This study establishes a versatile platform for engineering proteins with various halogenated tryptophans.