A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Disrupted protein interaction dynamics in a genetic neurodevelopmental disorder revealed by structural bioinformatics and genetic code expansion.

Deciphering the structural effects of gene variants is essential for understanding the pathophysiological mechanisms of genetic diseases. Using a neurodevelopmental disorder called Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS) as a genetic disease model, we applied structural bioinformatics and Genetic Code Expansion (GCE) strategies to assess the pathogenic impact of human NR2F1 variants and their binding with known and novel partners. While the computational analyses of the NR2F1 structure delineated the molecular basis of the impact of several variants on the isolated and complexed structures, the GCE enabled covalent and site-specific capture of transient supramolecular interactions in living cells. This revealed the variable quaternary conformations of NR2F1 variants and highlighted the disrupted interplay with dimeric partners and the newly identified co-factor, CRABP2. The disclosed consequence of the pathogenic mutations on the conformation, supramolecular interplay, and alterations in the cell cycle, viability, and sub-cellular localization of the different variants reflect the heterogeneous disease spectrum of BBSOAS and set up novel foundation for unveiling the complexity of neurodevelopmental diseases.

A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides.

Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 µM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications.

Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment.

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.

Expanding the genetic code beyond the 20 canonical amino acids enables access to a wide range of chemical functionality that is inaccessible within conventionally biosynthesized proteins. The vast majority of efforts to expand the genetic code have focused on the orthogonal translation systems required to achieve the genetically encoded addition of noncanonical amino acids (ncAAs) into proteins. There remain tremendous opportunities for identifying genetic and genomic factors that enhance ncAA incorporation. Here we describe genome-wide screening strategies to identify factors that enable more efficient addition of ncAAs to biosynthesized proteins. These unbiased screens can reveal previously unknown genes or mutations that can enhance ncAA incorporation and deepen our understanding of the translation apparatus.

Genetic Code Expansion in Pseudomonas putida KT2440.

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

Practical Approaches to Genetic Code Expansion with Aminoacyl-tRNA Synthetase/tRNA Pairs.

Genetic code expansion (GCE) can enable the site-selective incorporation of non-canonical amino acids (ncAAs) into proteins. GCE has advanced tremendously in the last decade and can be used to create biorthogonal handles, monitor and control proteins inside cells, study post-translational modifications, and engineer new protein functions. Since establishing our laboratory, our research has focused on applications of GCE in protein and enzyme engineering using aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. This topic has been reviewed extensively, leaving little doubt that GCE is a powerful tool for engineering proteins and enzymes. Therefore, for this young faculty issue, we wanted to provide a more technical look into the methods we use and the challenges we think about in our laboratory. Since starting the laboratory, we have successfully engineered over a dozen novel aaRS/tRNA pairs tailored for various GCE applications. However, we acknowledge that the field can pose challenges even for experts. Thus, herein, we provide a review of methodologies in ncAA incorporation with some practical commentary and a focus on challenges, emerging solutions, and exciting developments.

Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

Information, Coding, and Biological Function: The Dynamics of Life.

In the mid-20th century, two new scientific disciplines emerged forcefully: molecular biology and information-communication theory. At the beginning, cross-fertilization was so deep that the term genetic code was universally accepted for describing the meaning of triplets of mRNA (codons) as amino acids. However, today, such synergy has not taken advantage of the vertiginous advances in the two disciplines and presents more challenges than answers. These challenges not only are of great theoretical relevance but also represent unavoidable milestones for next-generation biology: from personalized genetic therapy and diagnosis to Artificial Life to the production of biologically active proteins. Moreover, the matter is intimately connected to a paradigm shift needed in theoretical biology, pioneered a long time ago, that requires combined contributions from disciplines well beyond the biological realm. The use of information as a conceptual metaphor needs to be turned into quantitative and predictive models that can be tested empirically and integrated in a unified view. Successfully achieving these tasks requires a wide multidisciplinary approach, including Artificial Life researchers, to address such an endeavour.

In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code.

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

The time of appearance of the genetic code.

I support the hypothesis that the origin of the genetic code occurred simultaneously with the evolution of cellularity. That is to say, I favour the hypothesis that the origin of the genetic code is a very, very late event in the history of life on Earth. I corroborate this hypothesis with observations favouring the progenote's stage for the Last Universal Common Ancestor (LUCA), for the ancestor of bacteria and that of archaea. Indeed, these progenotic stages would imply that - at that time - the origin of the genetic code was still ongoing simply because this origin would fall within the very definition of progenote. Therefore, if the evolution of cellularity had truly been coeval with the origin of the genetic code - at least in its terminal part - then this would favour theories such as the coevolution theory of the origin of the genetic code because this theory would postulate that this origin must have occurred in extremely complex protocellular conditions and not concerning stereochemical or physicochemical interactions having to do with other stages of the origin of life. In this sense, the coevolution theory would be corroborated while the stereochemical and physicochemical theories would be damaged. Therefore, the origin of the genetic code would be linked to the origin of the cell and not to the origin of life as sometimes asserted. Therefore, I will discuss the late hypothesis of the origin of the genetic code in the context of the theories proposed to explain this origin and more generally of its implications for the early evolution of life.

Significance of genetic code module structure in gene expression and GC content enhancement in RNA sequences.

The existent algebraic models of the genetic code contribute to the understanding of the physio-chemical characteristics of the amino acids. However, the process of translating a gene into a phenotype is highly complex. Moreover, the intricacy of gene expression gets further multiplied due to the biases in the codon usage. This paper explores an algebraic structure called module on the set of codons as well as on that of RNA sequences. We study the potential implications of these structures on gene expression and the GC content of an RNA sequence. The base order {C,U,G,A} appears to possess greater biological significance than many of the orders previously studied. We have developed a novel algorithm to generate RNA sequences with high GC content, aiming to enhance the thermostability of biomolecules. The insights gained from this investigation may have applications in biomolecular modeling and docking, protein engineering, drug development, and related fields.

The tri-flow adaptiveness of codes in major evolutionary transitions.

Life codes increase in both number and variety with biological complexity. Although our knowledge of codes is constantly expanding, the evolutionary progression of organic, neural, and cultural codes in response to selection pressure remains poorly understood. Greater clarification of the selective mechanisms is achieved by investigating how major evolutionary transitions reduce spatiotemporal and energetic constraints on transmitting heritable code to offspring. Evolution toward less constrained flows is integral to enduring flow architecture everywhere, in both engineered and natural flow systems. Beginning approximately 4 billion years ago, the most basic level for transmitting genetic material to offspring was initiated by protocell division. Evidence from ribosomes suggests that protocells transmitted comma-free or circular codes, preceding the evolution of standard genetic code. This rudimentary information flow within protocells is likely to have first emerged within the geo-energetic and geospatial constraints of hydrothermal vents. A broad-gauged hypothesis is that major evolutionary transitions overcame such constraints with tri-flow adaptations. The interconnected triple flows incorporated energy-converting, spatiotemporal, and code-based informational dynamics. Such tri-flow adaptations stacked sequence splicing code on top of protein-DNA recognition code in eukaryotes, prefiguring the transition to sexual reproduction. Sex overcame the spatiotemporal-energetic constraints of binary fission with further code stacking. Examples are tubulin code and transcription initiation code in vertebrates. In a later evolutionary transition, language reduced metabolic-spatiotemporal constraints on inheritance by stacking phonetic, phonological, and orthographic codes. In organisms that reproduce sexually, each major evolutionary transition is shown to be a tri-flow adaptation that adds new levels of code-based informational exchange. Evolving biological complexity is also shown to increase the nongenetic transmissibility of code.

Transfer RNA modifications and cellular thermotolerance.

RNA molecules are modified post-transcriptionally to acquire their diverse functions. Transfer RNA (tRNA) has the widest variety and largest numbers of RNA modifications. tRNA modifications are pivotal for decoding the genetic code and stabilizing the tertiary structure of tRNA molecules. Alternation of tRNA modifications directly modulates the structure and function of tRNAs and regulates gene expression. Notably, thermophilic organisms exhibit characteristic tRNA modifications that are dynamically regulated in response to varying growth temperatures, thereby bolstering fitness in extreme environments. Here, we review the history and latest findings regarding the functions and biogenesis of several tRNA modifications that contribute to the cellular thermotolerance of thermophiles.

Ordering events in a developing genetic code.

Preexisting partial genetic codes can fuse to evolve towards the complete Standard Genetic Code (SGC). Such code fusion provides a path of 'least selection', readily generating precursor codes that resemble the SGC. Consequently, such least selections produce the SGC via minimal, thus rapid, change. Optimal code evolution therefore requires delayed wobble. Early wobble encoding slows code evolution, very specifically diminishing the most likely SGC precursors: near-complete, accurate codes which are the products of code fusions. In contrast: given delayed wobble, the SGC can emerge from a truncation selection/evolutionary radiation based on proficient fused coding.

Decoding the language of immunity.

Optimized transfer RNA (tRNA) codon use can speed up antibody generation.

Nucleoside modification-based flexizymes with versatile activity for tRNA aminoacylation.

Extensive research has focused on genetic code reprogramming using flexizymes (Fxs), ribozymes enabling diverse tRNA acylation. Here we describe a nucleoside-modification strategy for the preparation of flexizyme variants derived from 2'-OMe, 2'-F, and 2'-MOE modifications with unique and versatile activities, enabling the charging of tRNAs with a broad range of substrates. This innovative strategy holds promise for synthetic biology applications, offering a robust pathway to expand the genetic code for diverse substrate incorporation.