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Abstract Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site‐specifically incorporate biologically relevant non‐standard amino acids using orthogonal translation systems (OTSs) has proven particularly useful, providing unparalleled access to cellular mechanisms modulated by post‐translational modifications, such as protein phosphorylation. However, despite significant advances in OTS design and function, the systems‐level biology of OTS development and utilization remains underexplored. In this study, we employ a phosphoserine OTS (pSerOTS) as a model to systematically investigate global interactions between OTS components and the cellular environment, aiming to improve OTS performance. Based on this analysis, we design OTS variants to enhance orthogonality by minimizing host process interactions and reducing stress response activation. Our findings advance understanding of system‐wide OTS:host interactions, enabling informed design practices that circumvent deleterious interactions with host physiology while improving OTS performance and stability. Furthermore, our study emphasizes the importance of establishing a pipeline for systematically profiling OTS:host interactions to enhance orthogonality and mitigate mechanisms underlying OTS‐mediated host toxicity.

Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs. Here we address this limitation by customizing the cell-free protein synthesis (CFPS) machinery via chassis strain selection and orthogonal translation system (OTS) reconfiguration screening. The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis. Specifically, we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine (O-pSer) or its nonhydrolyzable analog, 2-amino-4-phosphonobutyric acid (C-pSer). This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein. Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins, which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.

Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site‐specifically incorporate biologically relevant non‐standard amino acids using orthogonal translation systems (OTSs) has proven particularly useful, providing unparalleled access to cellular mechanisms modulated by post‐translational modifications, such as protein phosphorylation. However, despite significant advances in OTS design and function, the systems‐level biology of OTS development and utilization remains underexplored. In this study, we employ a phosphoserine OTS (pSerOTS) as a model to systematically investigate global interactions between OTS components and the cellular environment, aiming to improve OTS performance. Based on this analysis, we design OTS variants to enhance orthogonality by minimizing host process interactions and reducing stress response activation. Our findings advance understanding of system‐wide OTS:host interactions, enabling informed design practices that circumvent deleterious interactions with host physiology while improving OTS performance and stability. Furthermore, our study emphasizes the importance of establishing a pipeline for systematically profiling OTS:host interactions to enhance orthogonality and mitigate mechanisms underlying OTS‐mediated host toxicity. Synopsis: Analyses of global interactions between orthogonal translation systems (OTS) and the cellular environment, enable informed design practices that improve OTS performance by minimizing OTS:host interactions that reduce biological tolerance.A phosphoserine orthogonal translation system (pSerOTS) is used to investigate system‐wide interactions between OTS components and the host cell that influence OTS fidelity.Systematic profiling of OTS‐mediated sources of cellular toxicity and mitigation of deleterious OTS:host interactions enabled re‐designed host‐centric OTS variants with enhanced orthogonality, performance, and biological tolerance.The study shpws the utility of a systematic pipeline to assess OTS:host interactions during OTS development. [ABSTRACT FROM AUTHOR]

Amber suppression has been widely used to incorporate unnatural amino acids (UNAAs) with unique structures or functional side-chain groups into specific sites of the target protein, which expands the scope of protein-coding chemistry. However, this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein, which limits the development of unnatural proteins. To address this challenge, the suppression method using multiple termination codons (TAG, TAA or TGA) was proposed, and cell-free unnatural protein synthesis (CFUPS) system was employed. By the analysis of incorporating 3 different UNAAs (p-propargyloxy-l-phenylalanine, p-azyl-phenylalanine and L-4-Iodophenylalanine) and mass spectrometry, the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation. The CFUPS conditions were further optimized for better UNAA incorporation efficiency, including the orthogonal translation system (OTS) components, magnesium ions, and the redox environment. This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs, thereby synthesizing unnatural proteins with novel physicochemical functions.

With increased attention to excellent biocatalysts, evolving methods based on nature or unnatural amino acid (UAAs) mutagenesis have become an important part of enzyme engineering. The emergence of powerful method through expanding the genetic code allows to incorporate UAAs with unique chemical functionalities into proteins, endowing proteins with more structural and functional features. To date, over 200 diverse UAAs have been incorporated site-specifically into proteins via this methodology and many of them have been widely exploited in the field of enzyme engineering, making this genetic code expansion approach possible to be a promising tool for modulating the properties of enzymes. In this context, we focus on how this robust method to specifically incorporate UAAs into proteins and summarize their applications in enzyme engineering for tuning and expanding the functional properties of enzymes. Meanwhile, we aim to discuss how the benefits can be achieved by using the genetically encoded UAAs. We hope that this method will become an integral part of the field of enzyme engineering in the future. [ABSTRACT FROM AUTHOR]

The genetic code is the manual that cells use to incorporate amino acids into proteins. It is possible to artificially expand this manual through cellular, molecular, and chemical manipulations to improve protein functionality. Strategies for in vivo genetic code expansion are under the same functional constraints as natural protein synthesis. Here, we review the approaches used to incorporate noncanonical amino acids (ncAAs) into designer proteins through the manipulation of the translation machinery and draw parallels between these methods and natural adaptations that improve translation in extant organisms. Following this logic, we propose new nature-inspired tactics to improve genetic code expansion (GCE) in synthetic organisms. Nature has evolved different strategies to expand the repertoire of translatable proteins using the standard genetic code. The standard genetic code can be expanded through artificial cellular, molecular, and chemical manipulations. Strategies found in nature to enhance protein synthesis and functionality have been applied to improve genetic code expansion (GCE). A key aspect for efficient GCE relies on improving tRNA functionality. Novel strategies inspired on nature can be devised to further optimize GCE. [ABSTRACT FROM AUTHOR]

Incorporation of unnatural amino acids (UNAAs) into proteins currently is an active biological research area for various fundamental and applied science. In this context, cell-free synthetic biology (CFSB) has been developed and recognized as a robust testing and biomanufacturing platform for highly efficient UNAA incorporation. It enables the orchestration of unnatural biological machinery toward an exclusive user-defined objective of unnatural protein synthesis. This review aims to overview the principles of cell-free unnatural protein synthesis (CFUPS) systems, their advantages, different UNAA incorporation approaches, and recent achievements. These have catalyzed cutting-edge research and diverse emerging applications. Especially, present challenges and future trends are focused and discussed. With the development of CFSB and the fusion with other advanced next-generation technologies, CFUPS systems would explicitly deliver their values for biopharmaceutical applications.

Incorporation of unnatural amino acids (UNAAs) into proteins currently is an active biological research area for various fundamental and applied science. In this context, cell-free synthetic biology (CFSB) has been developed and recognized as a robust testing and biomanufacturing platform for highly efficient UNAA incorporation. It enables the orchestration of unnatural biological machinery toward an exclusive user-defined objective of unnatural protein synthesis. This review aims to overview the principles of cell-free unnatural protein synthesis (CFUPS) systems, their advantages, different UNAA incorporation approaches, and recent achievements. These have catalyzed cutting-edge research and diverse emerging applications. Especially, present challenges and future trends are focused and discussed. With the development of CFSB and the fusion with other advanced next-generation technologies, CFUPS systems would explicitly deliver their values for biopharmaceutical applications. [ABSTRACT FROM AUTHOR]

The incorporation of non-canonical amino acids (ncAAs) into ribosomally synthesized and post-translationally modified peptides, e.g., nisin from the Gram-positive bacterium Lactococcus lactis, bears great potential to expand the chemical space of various antimicrobials. The ncAA Nε-Boc-L-lysine (BocK) was chosen for incorporation into nisin using the archaeal pyrrolysyl-tRNA synthetase–tRNAPyl pair to establish orthogonal translation in L. lactis for read-through of in-frame amber stop codons. In parallel, recombinant nisin production and orthogonal translation were combined in Escherichia coli cells. Both organisms synthesized bioactive nisin(BocK) variants. Screening of a nisin amber codon library revealed suitable sites for ncAA incorporation and two variants displayed high antimicrobial activity. Orthogonal translation in E. coli and L. lactis presents a promising tool to create new-to-nature nisin derivatives.

The incorporation of non-canonical amino acids (ncAAs) into ribosomally synthesized and post-translationally modified peptides, e.g., nisin from the Gram-positive bacterium Lactococcus lactis, bears great potential to expand the chemical space of various antimicrobials. The ncAA Nε-Boc-L-lysine (BocK) was chosen for incorporation into nisin using the archaeal pyrrolysyl-tRNA synthetase--tRNAPyl pair to establish orthogonal translation in L. lactis for read-through of in-frame amber stop codons. In parallel, recombinant nisin production and orthogonal translation were combined in Escherichia coli cells. Both organisms synthesized bioactive nisin(BocK) variants. Screening of a nisin amber codon library revealed suitable sites for ncAA incorporation and two variants displayed high antimicrobial activity. Orthogonal translation in E. coli and L. lactis presents a promising tool to create new-to-nature nisin derivatives. [ABSTRACT FROM AUTHOR]

ABSTRACT The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs. Here we address this limitation by customizing the cell-free protein synthesis (CFPS) machinery via chassis strain selection and orthogonal translation system (OTS) reconfiguration screening. The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis. Specifically, we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine (O-pSer) or its nonhydrolyzable analog, 2-amino-4-phosphonobutyric acid (C-pSer). This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein. Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins, which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

This protocol describes the recombinant expression of proteins in E. coli containing phosphoserine (pSer) installed at positions guided by TAG codons. The E. coli strains that can be used here are engineered with a ∆ serB genomic knockout to produce pSer internally at high levels, so no exogenously added pSer is required, and the addition of pSer to the media will not affect expression yields. For "truncation-free" expression and improved yields with high flexibility of construct design, it is preferred to use the Release Factor-1 (RF1) deficient strain B95(DE3) ∆ A ∆ fabR ∆ serB , though use of the standard RF1-containing BL21(DE3) ∆ serB is also described. Both of these strains are serine auxotrophs and will not grow in standard minimal media. This protocol uses rich auto-induction media for streamlined and maximal production of homogeneously modified protein, yielding ~100-200 mg of single pSer-containing sfGFP per liter of culture. Using this genetic code expansion (GCE) approach, in which pSer is installed into proteins during translation, allows researchers to produce milligram quantities of specific phospho-proteins without requiring kinases, which can be purified for downstream in vitro studies relating to phosphorylation-dependent signaling systems, protein regulation by phosphorylation, and protein-protein interactions. Graphical abstract., Competing Interests: Competing interests The authors declare no conflicts of interest or competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in Escherichia coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remain limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNA CUA MaPyl support the incorporation of ncAAs into proteins produced in Saccharomyces cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the activity of previously reported Methanosarcina mazei , Methanosarcina barkeri , and chimeric M. mazei and M. barkeri PylRSs. Using S. cerevisiae RJY100 and pairing these PylRSs with the M. mazei tRNA CUA , we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS/tRNA CUA MaPyl to the orthogonal translation machinery toolkit in S. cerevisiae potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

Genetic code expansion has allowed for extraordinary advances in enhancing protein chemical diversity and functionality, but there remains a critical need for understanding and engineering genetic code expansion systems for improved efficiency. Incorporation of noncanonical amino acids (ncAAs) at stop codons provides a site-specific method for introducing unique chemistry into proteins, though often at reduced yields compared to wild-type proteins. A powerful platform for ncAA incorporation supports both the expression and evaluation of chemically diverse proteins for a broad range of applications. In yeast, ncAAs have been used to study dynamic cellular processes such as protein-protein interactions and also allow for exploration of eukaryotic-specific biology such as epigenetics. Furthermore, yeast display is an advantageous technology for engineering and screening the properties of proteins in high throughput. The protocols presented in this chapter describe detailed methods for the yeast-based genetic encoding of ncAAs in proteins intracellularly or on the yeast surface. In addition, methods are presented for modifying proteins on the yeast surface using bioorthogonal chemical reactions and evaluating reaction efficiency. Finally, protocols are included for the preparation of libraries that involve genetic code expansion. Libraries of proteins that contain ncAAs or libraries of the cellular machinery required to encode ncAAs can be constructed and screened in high throughput for many biological and chemical applications. Efficient incorporation of ncAAs facilitates elucidation of fundamental eukaryotic biology and advances tools for enzyme and genome engineering to evolve host cells that are better able to accommodate alternative genetic codes., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Amber suppression has been widely used to incorporate unnatural amino acids (UNAAs) with unique structures or functional side-chain groups into specific sites of the target protein, which expands the scope of protein-coding chemistry. However, this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein, which limits the development of unnatural proteins. To address this challenge, the suppression method using multiple termination codons (TAG, TAA or TGA) was proposed, and cell-free unnatural protein synthesis (CFUPS) system was employed. By the analysis of incorporating 3 different UNAAs (p-propargyloxy-l-phenylalanine, p-azyl-phenylalanine and L-4-Iodophenylalanine) and mass spectrometry, the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation. The CFUPS conditions were further optimized for better UNAA incorporation efficiency, including the orthogonal translation system (OTS) components, magnesium ions, and the redox environment. This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs, thereby synthesizing unnatural proteins with novel physicochemical functions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Noncanonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, in vivo incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proofreading aa-tRNAs, which substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Here, we demonstrate an alternative strategy leveraging computational analysis to broaden EF-Tu's substrate specificity. Evolutionary analysis of EF-Tu and a naturally evolved specialized elongation factor, SelB, provide the opportunity to engineer EF-Tu by targeting amino acid residues that are associated with functional divergence between the two ancient paralogues. Employing amber codon suppression, in combination with mass spectrometry, we identified two EF-Tu variants with non-native substrate compatibility. Additionally, we present data showing these EF-Tu variants contribute to host organismal fitness, working cooperatively with components of native and engineered translation machinery. These results demonstrate the viability of our computational method and lend support to corresponding assumptions about molecular evolution. This work promotes enhanced polyspecific EF-Tu behavior as a viable strategy to expand ncAA scope and complements ongoing research emphasizing the importance of a comprehensive approach to further expand the genetic code.

Cellular signaling and regulatory cascades often rely on post-translational modification of proteins, particularly phosphorylation, to quickly and effectively relay signals from a variety of inputs. Numerous kinases, the effectors of phosphorylation, and kinase networks have been implicated in human diseases. Until recently, an inability to produce high yields of physiologically phosphorylated proteins has proven to be a substantial barrier toward our understanding of many enzymatic processes. Orthogonal translation systems provide the means to overcome many of these limitations by enabling site-specific incorporation of phosphorylated amino acids into recombinantly expressed proteins. Site-by-site, combinatorial assessment of phosphorylation site function is unique to orthogonal translation system based approaches and offers unmatched precision in the study of PTM-enzymology, extending well beyond the scope of kinase biology., (© 2019 Elsevier Inc. All rights reserved.)

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of Methanocaldococcus jannaschii ( Mj) tRNA Tyr -derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native E. coli RNA-modifying enzymes, whereas low abundance evolved heterologous tRNAs are less affected by these same enzymes. We employed mass spectrometry to map ms 2 i 6 A37 and Ψ39 in the anticodon arm of two high abundance tRNAs (Nap1 and tRNA Opt CUA ), which provides (to our knowledge) the first direct evidence that MiaA and TruA post-transcriptionally modify evolved heterologous amber suppressor tRNAs. Changes in total tRNA modification profiles were observed by mass spectrometry in cells hosting these and other evolved suppressor tRNAs, suggesting that the demonstrated interactions with host enzymes might disturb native tRNA modification networks. Together, these results suggest that heterologous tRNAs engineered for specialized amber suppression can evolve highly efficient suppression capacity within the native post-transcriptional modification landscape of host RNA processing machinery.

The addition of new and versatile chemical and biological properties to proteins pursued through incorporation of non-canonical amino acids is at present primarily achieved by stop codon suppression. However, it is critical to find new "blank" codons to increase the variety and efficiency of such insertions, thereby taking 'sense codon recoding' to center stage in the field of genetic code expansion. Current thought optimistically suggests the use of the pyrrolysine system coupled with re-synthesis of genomic information towards achieving sense codon reassignment. Upon review of the serious experimental challenges reported in recent studies, we propose that success in this area will depend on the re-synthesis of genomes, but also on 'rewiring' the mechanism of protein synthesis and of its quality control., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)