Orthogonal Translation for Site-Specific Installation of Post-translational Modifications.

Post-translational modifications (PTMs) endow proteins with new properties to respond to environmental changes or growth needs. With the development of advanced proteomics techniques, hundreds of distinct types of PTMs have been observed in a wide range of proteins from bacteria, archaea, and eukarya. To identify the roles of these PTMs, scientists have applied various approaches. However, high dynamics, low stoichiometry, and crosstalk between PTMs make it almost impossible to obtain homogeneously modified proteins for characterization of the site-specific effect of individual PTM on target proteins. To solve this problem, the genetic code expansion (GCE) strategy has been introduced into the field of PTM studies. Instead of modifying proteins after translation, GCE incorporates modified amino acids into proteins during translation, thus generating site-specifically modified proteins at target positions. In this review, we summarize the development of GCE systems for orthogonal translation for site-specific installation of PTMs.

Characterizing lysine acetylation of glucokinase.

Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose-6-phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site-specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl-phosphate without enzymes and deacetylated by CobB deacetylase.

"Not-so-popular" orthogonal pairs in genetic code expansion.

During the past decade, genetic code expansion has been proved to be a powerful tool for protein studies and engineering. As the key part, a series of orthogonal pairs have been developed to site-specifically incorporate hundreds of noncanonical amino acids (ncAAs) into proteins by using bacteria, yeast, mammalian cells, animals, or plants as hosts. Among them, the pair of tyrosyl-tRNA synthetase/tRNATyr from Methanococcus jannaschii and the pair of pyrrolysyl-tRNA synthetase/tRNAPyl from Methanosarcina species are the most popular ones. Recently, other "not-so-popular" orthogonal pairs have started to attract attentions, because they can provide more choices of ncAA candidates and are necessary for simultaneous incorporation of multiple ncAAs into a single protein. Here, we summarize the development and applications of those "not-so-popular" orthogonal pairs, providing guidance for studying and engineering proteins.

Studying Acetylation of Aconitase Isozymes by Genetic Code Expansion.

Aconitase catalyzes the second reaction of the tricarboxylic acid cycle, the reversible conversion of citrate and isocitrate. Escherichia coli has two isoforms of aconitase, AcnA and AcnB. Acetylomic studies have identified acetylation at multiple lysine sites of both E. coli aconitase isozymes, but the impacts of acetylation on aconitases are unknown. In this study, we applied the genetic code expansion approach to produce 14 site-specifically acetylated aconitase variants. Enzyme assays and kinetic analyses showed that acetylation of AcnA K684 decreased the enzyme activity, while acetylation of AcnB K567 increased the enzyme activity. Further in vitro acetylation and deacetylation assays were performed, which indicated that both aconitase isozymes could be acetylated by acetyl-phosphate chemically, and be deacetylated by the CobB deacetylase at most lysine sites. Through this study, we have demonstrated practical applications of genetic code expansion in acetylation studies.

Genome-Wide Screening of Oxidizing Agent Resistance Genes in Escherichia coli.

The use of oxidizing agents is one of the most favorable approaches to kill bacteria in daily life. However, bacteria have been evolving to survive in the presence of different oxidizing agents. In this study, we aimed to obtain a comprehensive list of genes whose expression can make Escherichiacoli cells resistant to different oxidizing agents. For this purpose, we utilized the ASKA library and performed a genome-wide screening of ~4200 E. coli genes. Hydrogen peroxide (H2O2) and hypochlorite (HOCl) were tested as representative oxidizing agents in this study. To further validate our screening results, we used different E. coli strains as host cells to express or inactivate selected resistance genes individually. More than 100 genes obtained in this screening were not known to associate with oxidative stress responses before. Thus, this study is expected to facilitate both basic studies on oxidative stress and the development of antibacterial agents.

Introducing noncanonical amino acids for studying and engineering bacterial microcompartments.

Bacterial microcompartments (BMCs) with selectively permeable shells and encapsulated enzyme cores are well-suited candidates for nano-bioreactors because of their advantages of enhancing pathway flux and protection against toxic products. To better study and engineer protein-based BMCs, a series of protein chemistry approaches are adopted. As one of the most advanced techniques, genetic code expansion can introduce various noncanonical amino acids (ncAAs) with diverse functional groups into target proteins, thus providing powerful tools for protein studies and engineering. This review summarizes and proposes useful tools based on current development of the genetic code expansion technique towards challenges in BMC studies and engineering.

Methyl-Coenzyme M Reductase and Its Post-translational Modifications.

The methyl-coenzyme M reductase (MCR) is a central enzyme in anaerobic microbial methane metabolism, which consists of methanogenesis and anaerobic oxidation of methane (AOM). MCR catalyzes the final step of methanogenesis and the first step of AOM to achieve the production and oxidation of methane, respectively. Besides a unique nickel tetrahydrocorphinoid (coenzyme F430), MCR also features several unusual post-translational modifications (PTMs), which are assumed to play important roles in regulating MCR functions. However, only few studies have been implemented on MCR PTMs. Therefore, to recapitulate current knowledge and prospect future studies, this review summarizes and discusses studies on MCR and its PTMs.

A Synthetic Reporter for Probing Mistranslation in Living Cells.

Aminoacyl-tRNA synthetases (AARSs) play key roles in maintaining high fidelity of protein synthesis. They charge cognate tRNAs with corresponding amino acids and hydrolyze mischarged tRNAs by editing mechanisms. Impairment of AARS editing activities can reduce the accuracy of tRNA aminoacylation to produce mischarged tRNAs, which cause mistranslation and cell damages. To evaluate the mistranslation rate of threonine codons in living cells, in this study, we designed a quantitative reporter derived from the green fluorescent protein (GFP). The original GFP has multiple threonine codons which could affect the accuracy of measurement, so we generated a GFP variant containing only one threonine residue to specifically quantify mistranslation at the threonine codon. To validate, we applied this single-threonine GFP reporter to evaluate mistranslation at the threonine codon with mutations or modifications of threonine-tRNA synthetase and compared it with other methods of mistranslation evaluation, which showed that this reporter is reliable and facile to use.

Characterizing lysine acetylation of Escherichia coli type II citrate synthase.

The citrate synthase (CS) catalyzes the first reaction of the tricarboxylic acid cycle, playing an important role in central metabolism. The acetylation of lysine residues in the Escherichia coli Type II CS has been identified at multiple sites by proteomic studies, but their effects remain unknown. In this study, we applied the genetic code expansion strategy to generate 10 site-specifically acetylated CS variants which have been identified in nature. Enzyme assays and kinetic analyses showed that lysine acetylation could decrease the overall CS enzyme activity, largely due to the acetylation of K295 which impaired the binding of acetyl-coenzyme A. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to explore the acetylation and deacetylation processes of the CS, which indicated that the CS could be acetylated by acetyl-phosphate chemically, and be deacetylated by the CobB deacetylase.

The Application of Cell-Free Protein Synthesis in Genetic Code Expansion for Post-translational Modifications.

The translation system is a sophisticated machinery that synthesizes proteins from 20 canonical amino acids. Recently, the repertoire of such composition has been expanded by the introduction of non-canonical amino acids (ncAAs) with the genetic code expansion strategy, which provides proteins with designed properties and structures for protein studies and engineering. Although the genetic code expansion strategy has been mostly implemented by using living cells as the host, a number of limits such as poor cellular uptake or solubility of specific ncAA substrates and the toxicity of target proteins have hindered the production of certain ncAA-modified proteins. To overcome those challenges, cell-free protein synthesis (CFPS) has been applied as it allows the precise control of reaction components. Several approaches have been recently developed to increase the purity and efficiency of ncAA incorporation in CFPS. Here, we summarized recent development of CFPS with an emphasis on its applications in generating site-specific protein post-translational modifications by the genetic code expansion strategy.

Site-Specifically Studying Lysine Acetylation of Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (AARSs) charge their cognate tRNAs with corresponding amino acids, playing key roles in ribosomal protein synthesis. A series of proteomic studies have demonstrated that AARSs have levels of lysine acetylation much higher than those of other proteins in Escherichia coli. To study AARS acetylation, 25 site-specifically acetylated variants of four AARSs were generated by the genetic code expansion strategy. Kinetic analyses were performed to biochemically characterize the impact of site-specific acetylation on AARS functions, including amino acid activation, tRNA aminoacylation, and editing activities. The results showed that impacts of acetylation were different between class I and class II AARSs and also varied among the same class of AARSs. The results also showed that acetylation of threonyl-tRNA synthetase (ThrRS) could affect its editing function. Both in vivo and in vitro studies were further performed to explore the acetylation and deacetylation processes of ThrRS. Although nonenzymatic acetylation and CobB-dependent deacetylation were concluded, the results also indicated the existence of additional modifying enzymes or mechanisms for ThrRS acetylation and deacetylation.

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth.

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies.

Nowadays advanced mass spectrometry techniques make the identification of protein posttranslational modifications (PTMs) much easier than ever before. A series of proteomic studies have demonstrated that large numbers of proteins in cells are modified by phosphorylation, acetylation and many other types of PTMs. However, only limited studies have been performed to validate or characterize those identified modification targets, mostly because PTMs are very dynamic, undergoing large changes in different growth stages or conditions. To overcome this issue, the genetic code expansion strategy has been introduced into PTM studies to genetically incorporate modified amino acids directly into desired positions of target proteins. Without using modifying enzymes, the genetic code expansion strategy could generate homogeneously modified proteins, thus providing powerful tools for PTM studies. In this review, we summarized recent development of genetic code expansion in PTM studies for research groups in this field.

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli.

The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.

Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously.

Post-translational modifications (PTMs) play important roles in regulating a variety of biological processes. To facilitate PTM studies, the genetic code expansion strategy has been utilized to cotranslationally incorporate individual PTMs such as acetylation and phosphorylation into proteins at specific sites. However, recent studies have demonstrated that PTMs actually work together to regulate protein functions and structures. Thus, simultaneous incorporation of multiple distinct PTMs into one protein is highly desirable. In this study, we utilized the genetic incorporation systems of phosphoserine and acetyllysine to install both phosphorylation and acetylation into target proteins simultaneously in Escherichia coli. And we used this system to study the effect of coexisting acetylation and phosphorylation on malate dehydrogenase, demonstrating a practical application of this system in biochemical studies. Furthermore, we tested the mutual orthogonality of three widely used genetic incorporation systems, indicating the possibility of incorporating three distinct PTMs into one protein simultaneously.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

Genetically encoding thioacetyl-lysine as a non-deacetylatable analog of lysine acetylation in Escherichia coli.

Reversible lysine acetylation is one of the most widely distributed post-translational modifications; it is involved in a variety of biological processes and can be found in all three domains of life. Acetyltransferases and deacetylases work coordinately to control levels of protein acetylation. In this work, we applied the genetic code expansion strategy to site-specifically incorporate Nε-thioacetyl-l-lysine (TAcK) as an analog of Nε-acetyl-l-lysine (AcK) into green fluorescent protein and malate dehydrogenase in Escherichia coli. We showed that TAcK could serve as an ideal functional mimic for AcK. It could also resist the bacterial sirtuin-type deacetylase CobB. Thus, genetic incorporation of TAcK as a non-deacetylatable analog of AcK into proteins will facilitate in vivo studies of protein acetylation.

Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl-tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic-code-expansion strategy to site-specifically incorporate Nϵ -acetyl-l-lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E. coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5-related N-acetyltransferase family in E. coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl-CoA or acetyl-phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation.

Studying the Lysine Acetylation of Malate Dehydrogenase.

Protein acetylation plays important roles in many biological processes. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, has been identified to be acetylated in bacteria by proteomic studies, but no further characterization has been reported. One challenge for studying protein acetylation is to get purely acetylated proteins at specific positions. Here, we applied the genetic code expansion strategy to site-specifically incorporate Nε-acetyllysine into MDH. The acetylation of lysine residues in MDH could enhance its enzyme activity. The Escherichia coli deacetylase CobB could deacetylate acetylated MDH, while the E. coli acetyltransferase YfiQ cannot acetylate MDH efficiently. Our results also demonstrated that acetyl-CoA or acetyl-phosphate could acetylate MDH chemically in vitro. Furthermore, the acetylation level of MDH was shown to be affected by carbon sources in the growth medium.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

BACKGROUND: Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. METHODS: We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. RESULTS: By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNALys, tRNATyr, and tRNAGln(CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. CONCLUSIONS: Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. GENERAL SIGNIFICANCE: We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.