Characterizing and Tailoring the Substrate Profile of a γ-Glutamyltransferase Variant.

Xenobiology is an emerging field that focuses on the extension and redesign of biological systems through the use of laboratory-derived xenomolecules, which are molecules that are new to the metabolism of the cell. Despite the enormous potential of using xenomolecules in living organisms, most noncanonical building blocks still need to be supplied externally, and often poor uptake into cells limits wider applicability. To improve the cytosolic availability of noncanonical molecules, a synthetic transport system based on portage transport was developed, in which molecules of interest "cargo" are linked to a synthetic transport vector that enables piggyback transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo conjugate is enzymatically cleaved by GGTxe, leading to the release of the cargo molecule. To deepen our understanding of the synthetic transport system, we focused on the characterization and further development of the enzymatic cargo release step. Hence, the substrate scope of GGTxe was characterized using a library of structurally diverse vector-cargo conjugates and MS/MS-based quantification of hydrolysis products in a kinetic manner. The resulting substrate tolerance characterization revealed that vector-amino acid conjugates were significantly unfavored. To overcome this shortcoming, a selection system based on metabolic auxotrophy complementation and directed evolution of GGTxe was established. In a directed evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance of residue D386 in the cargo unloading step.

Rational design of a bacterial import system for new-to-nature molecules.

Integration of novel compounds into biological processes holds significant potential for modifying or expanding existing cellular functions. However, the cellular uptake of these compounds is often hindered by selectively permeable membranes. We present a novel bacterial transport system that has been rationally designed to address this challenge. Our approach utilizes a highly promiscuous sulfonate membrane transporter, which allows the passage of cargo molecules attached as amides to a sulfobutanoate transport vector molecule into the cytoplasm of the cell. These cargoes can then be unloaded from the sulfobutanoyl amides using an engineered variant of the enzyme γ-glutamyl transferase, which hydrolyzes the amide bond and releases the cargo molecule within the cell. Here, we provide evidence for the broad substrate specificity of both components of the system by evaluating a panel of structurally diverse sulfobutanoyl amides. Furthermore, we successfully implement the synthetic uptake system in vivo and showcase its functionality by importing an impermeant non-canonical amino acid.

Engineering a synthetic energy-efficient formaldehyde assimilation cycle in Escherichia coli.

One-carbon (C1) substrates, such as methanol or formate, are attractive feedstocks for circular bioeconomy. These substrates are typically converted into formaldehyde, serving as the entry point into metabolism. Here, we design an erythrulose monophosphate (EuMP) cycle for formaldehyde assimilation, leveraging a promiscuous dihydroxyacetone phosphate dependent aldolase as key enzyme. In silico modeling reveals that the cycle is highly energy-efficient, holding the potential for high bioproduct yields. Dissecting the EuMP into four modules, we use a stepwise strategy to demonstrate in vivo feasibility of the modules in E. coli sensor strains with sarcosine as formaldehyde source. From adaptive laboratory evolution for module integration, we identify key mutations enabling the accommodation of the EuMP reactions with endogenous metabolism. Overall, our study demonstrates the proof-of-concept for a highly efficient, new-to-nature formaldehyde assimilation pathway, opening a way for the development of a methylotrophic platform for a C1-fueled bioeconomy in the future.

A microbiological system for screening the interference of XNA monomers with DNA and RNA metabolism.

We explored the toxicity and mutagenicity of a wide range of xenobiotic nucleoside triphosphates to an Escherichia coli strain equipped with a nucleoside triphosphate transporter. This bacterial test provides a tool to evaluate and guide the synthesis of nucleotides for applications such as the propagation of non-natural genetic information or the selection of potential drugs.

Synthetic Biology Pathway to Nucleoside Triphosphates for Expanded Genetic Alphabets.

One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson-Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution in vitro. To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that "polyphosphate kinases" can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway in vitro makes AEGIS triphosphates, including third-generation triphosphates having improved ability to survive in living bacterial cells. In α-32P-labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third-generation AEGIS triphosphates perform better with natural enzymes than second-generation AEGIS triphosphates.

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization.

Chemical cell surface modification is a fast-growing field of research, due to its enormous potential in tissue engineering, cell-based immunotherapy, and regenerative medicine. However, engineering of bacterial tissues by chemical cell surface modification has been vastly underexplored and the identification of suitable molecular handles is in dire need. We present here, an orthogonal nucleic acid-protein conjugation strategy to promote artificial bacterial aggregation. This system gathers the high selectivity and stability of linkage to a protein Tag expressed at the cell surface and the modularity and reversibility of aggregation due to oligonucleotide hybridization. For the first time, XNA (xeno nucleic acids in the form of 1,5-anhydrohexitol nucleic acids) were immobilized via covalent, SNAP-tag-mediated interactions on cell surfaces to induce bacterial aggregation.

A third purine biosynthetic pathway encoded by aminoadenine-based viral DNA genomes.

Cells have two purine pathways that synthesize adenine and guanine ribonucleotides from phosphoribose via inosylate. A chemical hybrid between adenine and guanine, 2-aminoadenine (Z), replaces adenine in the DNA of the cyanobacterial virus S-2L. We show that S-2L and Vibrio phage PhiVC8 encode a third purine pathway catalyzed by PurZ, a distant paralog of succinoadenylate synthase (PurA), the enzyme condensing aspartate and inosylate in the adenine pathway. PurZ condenses aspartate with deoxyguanylate into dSMP (N6-succino-2-amino-2'-deoxyadenylate), which undergoes defumarylation and phosphorylation to give dZTP (2-amino-2'-deoxyadenosine-5'-triphosphate), a substrate for the phage DNA polymerase. Crystallography and phylogenetics analyses indicate a close relationship between phage PurZ and archaeal PurA enzymes. Our work elucidates the biocatalytic innovation that remodeled a DNA building block beyond canonical molecular biology.

Noncanonical DNA polymerization by aminoadenine-based siphoviruses.

Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3'-exonuclease but lacks 5'-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution.

A Path toward SARS-CoV-2 Attenuation: Metabolic Pressure on CTP Synthesis Rules the Virus Evolution.

In the context of the COVID-19 pandemic, we describe here the singular metabolic background that constrains enveloped RNA viruses to evolve toward likely attenuation in the long term, possibly after a step of increased pathogenicity. Cytidine triphosphate (CTP) is at the crossroad of the processes allowing SARS-CoV-2 to multiply, because CTP is in demand for four essential metabolic steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope, it is a critical building block of the host transfer RNAs synthesis and it is required for synthesis of dolichol-phosphate, a precursor of viral protein glycosylation. The CCA 3'-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. This unique metabolic setup allowed us to highlight and provide a raison d'être to viperin, an enzyme of innate antiviral immunity, which synthesizes 3'-deoxy-3',4'-didehydro-CTP as an extremely efficient antiviral nucleotide.

Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate.

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.

An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli.

Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform.

Vitamin-guanosine monophosphate conjugates for in vitro transcription priming.

Expanding the catalytic repertoire of ribozymes to include vitamin synthesis requires efficient labelling of RNA with the substrate of interest, prior to in vitro selection. For this purpose, we rationally designed and synthesized six GMP-conjugates carrying a synthetic pre-thiamine or biotin precursor and investigated their transcription incorporation properties by T7 RNA polymerase.

Use of ß3-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase.

Polypeptides containing ß-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of ß-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of ß3-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds ß3-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in ß3-Met commercial samples. The 1.45 Å crystal structure of the MetRS: ß3-Met complex shows that ß3-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved ß3-Met aminoacylation capabilities.

Correction: E. coli surface display of streptavidin for directed evolution of an allylic deallylase.

[This corrects the article DOI: 10.1039/C8SC00484F.].

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa -Perturbed Nucleotides.

The formation of artificial metal base pairs is an alluring and versatile method for the functionalization of nucleic acids. Access to DNA functionalized with metal base pairs is granted mainly by solid-phase synthesis. An alternative, yet underexplored method, envisions the installation of metal base pairs through the polymerization of modified nucleoside triphosphates. Herein, we have explored the possibility of using thiolated and pKa -perturbed nucleotides for the enzymatic construction of artificial metal base pairs. The thiolated nucleotides S2C, S6G, and S4T as well as the fluorinated analogue 5FU are readily incorporated opposite a templating S4T nucleotide through the guidance of metal cations. Multiple incorporation of the modified nucleotides along with polymerase bypass of the unnatural base pairs are also possible under certain conditions. The thiolated nucleotides S4T, S4T, S2C, and S6G were also shown to be compatible with the synthesis of modified, high molecular weight single-stranded (ss)DNA products through TdT-mediated tailing reactions. Thus, sulfur-substitution and pKa perturbation represent alternative strategies for the design of modified nucleotides compatible with the enzymatic construction of metal base pairs.

Invading Escherichia coli Genetics with a Xenobiotic Nucleic Acid Carrying an Acyclic Phosphonate Backbone (ZNA).

A synthetic orthogonal polymer embracing a chiral acyclic-phosphonate backbone [(S)-ZNA] is presented that uniquely adds to the emerging family of xenobiotic nucleic acids (XNAs). (S)-ZNA consists of reiterating six-atom structural units and can be accessed in few synthetic steps from readily available phophonomethylglycerol nucleoside (PMGN) precursors. Comparative thermal stability experiments conducted on homo- and heteroduplexes made of (S)-ZNA are described that evince its high self-hybridization efficiency in contrast to poor binding of natural complements. Although preliminary and not conclusive, circular dichroism data and dynamic modeling computations provide support to a left-handed geometry of double-stranded (S)-ZNA. Nonetheless, PMGN diphosphate monomers were recognized as substrates by Escherichia coli (E. coli) polymerase I as well as being imported into E. coli cells equipped with an algal nucleotide transporter. A further investigation into the in vivo propagation of (S)-ZNA culminated with the demonstration of the first synthetic nucleic acid with an acyclic backbone that can be transliterated to DNA by the E. coli cellular machinery.

Towards the enzymatic formation of artificial metal base pairs with a carboxy-imidazole-modified nucleotide.

The identification of synthetic nucleotides that sustain the formation of orthogonal, unnatural base pairs is an important goal in synthetic biology. Such artificial synthons have been used for the generation of semi-synthetic organisms as well as functional nucleic acids with enhanced binding properties. The enzymatic formation of artificial metal-base pairs is a vastly underexplored and alluring alternative to existing systems. Here, we report the synthesis and biochemical characterization of 1­(2-deoxy­ß­d­ribofuranosyl) imidazole­4­carboxylate nucleoside triphosphate (dImCTP) which is equipped with a carboxylic acid moiety on the imidazole moiety in order to increase the coordination environment to [2 + 2] and [2 + 1]. A clear metal dependence was observed for the single incorporation of the modified nucleotide into DNA by the DNA polymerase from Thermus aquaticus (Taq). The presence of AgI in primer extension reactions conducted with combinations of 1­(2­deoxy­ß­d­ribofuranosyl) imidazole nucleoside triphosphate (dImTP) and dImCTP supported the unusual [2 + 1] coordination pattern. The efficiency of the tailing reactions mediated by the terminal deoxynucleotidyl transferase (TdT) was markedly improved when using dImCTP instead of dImTP. Even though products with multiple modified nucleotides were not observed, the appendage of additional metal binding ligands on the imidazole nucleobase appears to be a valid approach to improve the biochemical properties of modified triphosphates in the context of an expansion of the genetic alphabet with metal base pairs.