Evolving Escherichia coli to use a tRNA with a non-canonical fold as an adaptor of the genetic code.

All known bacterial tRNAs adopt the canonical cloverleaf 2D and L-shaped 3D structures. We aimed to explore whether alternative tRNA structures could be introduced in bacterial translation. To this end, we crafted a vitamin-based genetic system to evolve Escherichia coli toward activity of structurally non-canonical tRNAs. The system reliably couples (escape frequency <10-12) growth with the activities of a novel orthogonal histidine suppressor tRNA (HisTUAC) and of the cognate ARS (HisS) via suppression of a GTA valine codon in the mRNA of an enzyme in thiamine biosynthesis (ThiN). Suppression results in the introduction of an essential histidine and thereby confers thiamine prototrophy. We then replaced HisTUAC in the system with non-canonical suppressor tRNAs and selected for growth. A strain evolved to utilize mini HisT, a tRNA lacking the D-arm, and we identified the responsible mutation in an RNase gene (pnp) involved in tRNA degradation. This indicated that HisS, the ribosome, and EF-Tu accept mini HisT ab initio, which we confirmed genetically and through in vitro translation experiments. Our results reveal a previously unknown flexibility of the bacterial translation machinery for the accepted fold of the adaptor of the genetic code and demonstrate the power of the vitamin-based suppression system.

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.

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.

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.

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.

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.

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.

Phosphonomethyl Oligonucleotides as Backbone-Modified Artificial Genetic Polymers.

Although several synthetic or xenobiotic nucleic acids (XNAs) have been shown to be viable genetic materials in vitro, major hurdles remain for their in vivo applications, particularly orthogonality. The availability of XNAs that do not interact with natural nucleic acids and are not affected by natural DNA processing enzymes, as well as specialized XNA processing enzymes that do not interact with natural nucleic acids, is essential. Here, we report 3'-2' phosphonomethyl-threosyl nucleic acid (tPhoNA) as a novel XNA genetic material and a prime candidate for in vivo XNA applications. We established routes for the chemical synthesis of phosphonate nucleic acids and phosphorylated monomeric building blocks, and we demonstrated that DNA duplexes were destabilized upon replacement with tPhoNA. We engineered a novel tPhoNA synthetase enzyme and, with a previously reported XNA reverse transcriptase, demonstrated that tPhoNA is a viable genetic material (with an aggregate error rate of approximately 17 × 10-3 per base) compatible with the isolation of functional XNAs. In vivo experiments to test tPhoNA orthogonality showed that the E. coli cellular machinery had only very limited potential to access genetic information in tPhoNA. Our work is the first report of a synthetic genetic material modified in both sugar and phosphate backbone moieties and represents a significant advance in biorthogonality toward the introduction of XNA systems in vivo.

Modulation of BACE1 Activity by Chemically Modified Aptamers.

A modified DNA aptamer that binds BACE1, a therapeutic target involved in Alzheimer's disease has been developed. This ssXNA not only tightly binds to BACE1 but also inhibits its protease activity in vitro in the same range as a previously described unmodified aptamer. We report the in vitro selection of functional oligonucleotides incorporating two nucleobase modifications: 5-chlorouracil and 7-deazaadenine. The nucleoside analogue 5-chloro-2'-deoxyuridine has already been explored as a replacement for thymidine in a chemically modified genome of a bacterium. Thus, 5-chlorouracil modification is a good candidate to support genetic transfer in vivo as well as functional activity.

Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In†Vitro and In†Vivo.

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2'-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by Tm measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W-C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases).

Overcoming the membrane barrier: Recruitment of γ-glutamyl transferase for intracellular release of metabolic cargo from peptide vectors.

Semipermeable membranes of cells frequently pose an obstacle in metabolic engineering by limiting uptake of substrates, intermediates, or xenobiotics. Previous attempts to overcome this barrier relied on the promiscuous nature of peptide transport systems, but often suffered from low versatility or chemical instability. Here, we present an alternative strategy to transport cargo molecules across the inner membrane of Escherichia coli based on chemical synthesis of a stable cargo-peptide vector construct, transport through the peptide import system, and efficient intracellular release of the cargo by the promiscuous enzyme γ-glutamyl transferase (GGT). Retaining the otherwise periplasmic GGT in the cytoplasm was critical for the functionality of the system, as was fine-tuning its expression in order to minimize toxic effects associated to cytoplasmic GGT expression. Given the established protocols of peptide synthesis and the flexibility of peptide transport and GGT, the system is expected to be suitable for a broad range of cargoes.

Biotin-independent strains of Escherichia coli for enhanced streptavidin production.

Biotin is an archetypal vitamin used as cofactor for carboxylation reactions found in all forms of life. However, biotin biosynthesis is an elaborate multi-enzymatic process and metabolically costly. Moreover, many industrially relevant organisms are incapable of biotin synthesis resulting in the requirement to supplement defined media. Here we describe the creation of biotin-independent strains of Escherichia coli and Corynebacterium glutamicum through installation of an optimized malonyl-CoA bypass, which re-routes natural fatty acid synthesis, rendering the previously essential vitamin completely obsolete. We utilize biotin-independent E. coli for the production of the high-value protein streptavidin which was hitherto restricted because of toxic effects due to biotin depletion. The engineered strain revealed significantly improved streptavidin production resulting in the highest titers and productivities reported for this protein to date.

On the enzymatic incorporation of an imidazole nucleotide into DNA.

The expansion of the genetic alphabet with an additional, artificial base pair is of high relevance for numerous applications in synthetic biology. The enzymatic construction of metal base pairs is an alluring strategy that would ensure orthogonality to canonical nucleic acids. So far, very little is known on the enzymatic fabrication of metal base pairs. Here, we report on the synthesis and the enzymatic incorporation of an imidazole nucleotide into DNA. The imidazole nucleotide dIm is known to form highly stable dIm-Ag+-dIm artificial base pairs that cause minimal structural perturbation of DNA duplexes and was considered to be an ideal candidate for the enzymatic construction of metal base pairs. We demonstrate that dImTP is incorporated with high efficiency and selectivity opposite a templating dIm nucleotide by the Kf exo-. The presence of Mn2+, and to a smaller extent Ag+, enhances the efficiency of this polymerization reaction, however, without being strictly required. In addition, multiple incorporation events could be observed, albeit with modest efficiency. We demonstrate that the dIm-Mn+-dIm cannot be constructed by DNA polymerases and suggest that parameters other than stability of a metal base pair and its impact on the structure of DNA duplexes govern the enzymatic formation of artificial metal base pairs.

Directed evolution of a model primordial enzyme provides insights into the development of the genetic code.

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.

Chemical Morphing of DNA Containing Four Noncanonical Bases.

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.

Isoguanine and 5-methyl-isocytosine bases, in vitro and in vivo.

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoC(Me) ) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)-DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoC(Me) bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoC(Me) misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoC(Me) /isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.

Paralogous metabolism: S-alkyl-cysteine degradation in Bacillus subtilis.

Metabolism is prone to produce analogs of essential building blocks in the cell (here named paralogous metabolism). The variants result from lack of absolute accuracy in enzyme-templated reactions as well as from molecular aging. If variants were left to accumulate, the earth would be covered by chemical waste. The way bacteria cope with this situation is essentially unexplored. To gain a comprehensive understanding of Bacillus subtilis sulphur paralogous metabolism, we used expression profiling with DNA arrays to investigate the changes in gene expression in the presence of S-methyl-cysteine (SMeC) and its close analog, methionine, as sole sulphur source. Altogether, more than 200 genes whose relative strength of induction was significantly different depending on the sulphur source used were identified. This allowed us to pinpoint operon ytmItcyJKLMNytmO_ytnIJ_rbfK_ytnLM as controlling the pathway cycling SMeC directly to cysteine, without requiring sulphur oxygenation. Combining genetic and physiological experiments, we deciphered the corresponding pathway that begins with protection of the metabolite by acetylation. Oxygenation of the methyl group then follows, and after deprotection (deacetylation), N-formyl cysteine is produced. This molecule is deformylated by the second deformylase present in B. subtilis DefB, yielding cysteine. This pathway appears to be present in plant-associated microbes.

Probing ambiguous base-pairs by genetic transformation with XNA templates.

The templating potential of anhydrohexitol oligonucleotides bearing ambiguous bases was studied in vivo, by using a selection screen for mosaic heteroduplex plasmids in Escherichia coli. 1,5-Anhydro-2,3-dideoxy-2-(5-nitroindazol-1-yl)-D-arabino-hexitol showed the greatest ambiguity among the three nucleosides tested. At most two successive ambiguous bases could be tolerated on hexitol templates read in bacterial cells. Hexitol nucleosides bearing simplified heterocycles thus stand as promising monomers for generating random DNA sequences in vivo from defined synthetic oligonucleotides.

Systematic analysis of tRNA transcription unit deletions in E. coli reveals insights into tRNA gene essentiality and cellular adaptation.

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis, decoding mRNA sequences into amino acids. In E. coli K-12 MG1655, 86 tRNA genes are organized in 43 transcription units (TUs) and the essentiality of individual tRNA TUs in bacterial physiology remains unclear. To address this, we systematically generated 43 E. coli tRNA deletion strains in which each tRNA TU was replaced by a kanamycin resistance gene. We found that 33 TUs are not essential for survival, while 10 are essential and require the corresponding TU to be provided on plasmid. The analysis revealed E. coli's tolerance to alterations in tRNA gene copy number and the loss of non-essential tRNAs, as most strains exhibited minimal to no growth differences under various conditions compared to the parental strain. However, deletions metZWV, alaWX and valVW led to significant growth defects under specific conditions. RNA-seq analysis of ∆alaWX and ∆valVW revealed upregulation of genes involved in translation and pilus assembly. Our results provide valuable insights into tRNA dynamics and the cellular response to tRNA TU deletions, paving the way for deeper understanding of tRNA pool complexity.