RESUMO
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.
RESUMO
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.
Assuntos
Escherichia coli , Ácidos Nucleicos , Escherichia coli/genética , DNA/química , Ácidos Nucleicos/química , Hibridização de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
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.
Assuntos
Aminoácidos/genética , Escherichia coli/genética , Metionina tRNA Ligase/genética , Metionina/metabolismo , Aminoácidos/química , Sítios de Ligação/genética , Escherichia coli/química , Metionina/química , Metionina tRNA Ligase/química , Conformação Proteica , Especificidade por SubstratoRESUMO
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.
Assuntos
Aldeído Liases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Formaldeído/metabolismo , Engenharia Metabólica/métodos , Metanol/metabolismo , Biomassa , Genes Bacterianos/genética , Glicina Hidroximetiltransferase/metabolismo , Homosserina/metabolismo , Redes e Vias Metabólicas , Serina/metabolismoRESUMO
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.
Assuntos
Escherichia coli/genética , Ácidos Nucleicos/química , Organofosfonatos/química , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ácidos Nucleicos/genética , Oligonucleotídeos/química , Oligonucleotídeos/genéticaRESUMO
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.
Assuntos
Pareamento de Bases , Fenômenos Químicos , Metais/química , Nucleotídeos/química , Nucleotídeos/metabolismo , Compostos de Sulfidrila/química , Sequência de Bases , DNA Nucleotidilexotransferase/metabolismo , Concentração de Íons de Hidrogênio , Nucleotídeos/genéticaRESUMO
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.
Assuntos
DNA/química , Organofosfonatos/química , Polímeros/metabolismo , Xenobióticos/metabolismo , DNA/metabolismo , Ligases/química , Ligases/metabolismo , Modelos Moleculares , Estrutura Molecular , Organofosfonatos/metabolismo , Polímeros/química , Engenharia de Proteínas , Xenobióticos/químicaRESUMO
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.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Adenina/análogos & derivados , Adenina/química , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Humanos , Ligação Proteica , Técnica de Seleção de Aptâmeros , Uracila/análogos & derivados , Uracila/químicaRESUMO
The synthesis, base pairing properties and inâ vitro (polymerase) and inâ vivo (E.â 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. Inâ 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).
RESUMO
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.
Assuntos
Biotina/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Melhoramento Genético/métodos , Engenharia Metabólica/métodos , Estreptavidina/biossíntese , Vias Biossintéticas/fisiologia , Biotina/metabolismo , Proteínas de Escherichia coli/genética , Redes e Vias Metabólicas/fisiologia , Estreptavidina/genética , Estreptavidina/isolamento & purificaçãoRESUMO
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.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Engenharia Metabólica/métodos , Peptídeos/metabolismo , gama-Glutamiltransferase/metabolismo , Transporte Biológico Ativo/fisiologia , Vias Biossintéticas/fisiologia , Escherichia coli/genética , Melhoramento Genético/métodos , Líquido Intracelular/metabolismo , Proteínas de Membrana Transportadoras/genética , Redes e Vias Metabólicas/fisiologia , Peptídeos/genética , gama-Glutamiltransferase/genéticaRESUMO
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.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA/química , Imidazóis/química , Nucleotídeos/química , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Imidazóis/metabolismo , Nucleotídeos/metabolismoRESUMO
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 (IleâThr, LeuâVal) 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.
Assuntos
Aminoácidos , Evolução Molecular Direcionada , Código Genético , Proteínas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Corismato Mutase/química , Corismato Mutase/genética , Methanococcales/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-AtividadeRESUMO
The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate inâ vitro and to serve as genetic templates inâ 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 inâ vivo.
Assuntos
DNA/química , Reação em Cadeia da Polimerase , Resistência a Trimetoprima , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Nucleotídeos de Desoxiguanina/química , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Escherichia coli/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , Trimetoprima/toxicidade , Tubercidina/análogos & derivados , Tubercidina/químicaRESUMO
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.
Assuntos
5-Metilcitosina/análogos & derivados , Guanina/química , Nucleosídeos/química , 5-Metilcitosina/química , Estrutura MolecularRESUMO
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.
Assuntos
Bacillus subtilis/metabolismo , Cisteína/análogos & derivados , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas , Metionina/metabolismo , Óperon , Enxofre/metabolismoRESUMO
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.
Assuntos
Pareamento de Bases , Ácidos Nucleicos/genética , Oligonucleotídeos/química , Álcoois Açúcares/química , Transformação Genética/genética , Estrutura Molecular , Ácidos Nucleicos/química , Oligonucleotídeos/síntese química , Moldes GenéticosRESUMO
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.
RESUMO
The development of new efficient syntheses of labeled reagents is a great challenge. Avoidance of overcomplicated procedures, availability and cost of starting materials are important considerations in choosing the synthetic route. In this report, we describe a facile and rapid preparation of labeled cyanate by ozonation of cyanide, a basic precursor. The crude cyanate was used without purification for the synthesis of various [(13)C] or [(14)C]ureidocarboxylic acids (20-68% yield from potassium cyanide). According to these results, cyanide ozonation may prove to be a promising alternative to traditional preparations of labeled cyanate.