Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins

Ma, Ying; Biava, Hernán; Contestabile, Roberto; Budiša, Nediljko; Salvo, Martino L. di

doi:10.3390/molecules19011004

Cited by 55 publications

(50 citation statements)

References 39 publications

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“…This solid had a melting point of 186°C. The 1 H NMR spectrum (500 MHz, D 2 O) was δ 4.23 ( t , J = 5.0 Hz, 2H), 3.83 ( t , J = 5.0 Hz, 1H), 2.29 – 2.15 (m, 2H), 2.09 (s, 3H), as described previously (Ma et al ., ). The 13 C NMR spectrum (125 MHz, D 2 O) was δ 174.2, 173.9, 61.6, 52.8, 29.3 and 20.4.…”

Section: Methodsmentioning

confidence: 99%

Primordial‐like enzymes from bacteria with reduced genomes

Ferla

Brewster

Hall

et al. 2017

Molecular Microbiology

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The first cells probably possessed rudimentary metabolic networks, built using a handful of multifunctional enzymes. The promiscuous activities of modern enzymes are often assumed to be relics of this primordial era; however, by definition these activities are no longer physiological. There are many fewer examples of enzymes using a single active site to catalyze multiple physiologically-relevant reactions. Previously, we characterized the promiscuous alanine racemase (ALR) activity of Escherichia coli cystathionine β-lyase (CBL). Now we have discovered that several bacteria with reduced genomes lack alr, but contain metC (encoding CBL). We characterized the CBL enzymes from three of these: Pelagibacter ubique, the Wolbachia endosymbiont of Drosophila melanogaster (wMel) and Thermotoga maritima. Each is a multifunctional CBL/ALR. However, we also show that CBL activity is no longer required in these bacteria. Instead, the wMel and T. maritima enzymes are physiologically bi-functional alanine/glutamate racemases. They are not highly active, but they are clearly sufficient. Given the abundance of the microorganisms using them, we suggest that much of the planet's biochemistry is carried out by enzymes that are quite different from the highly-active exemplars usually found in textbooks. Instead, primordial-like enzymes may be an essential part of the adaptive strategy associated with streamlining.

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Section: Methodsmentioning

confidence: 99%

Primordial‐like enzymes from bacteria with reduced genomes

Ferla

Brewster

Hall

et al. 2017

Molecular Microbiology

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“…Although this is perfectly acceptable for small‐scale proof‐of‐principle experiments, it is not feasible for general synthetic applications because of the high cost and low availability of most ncAAs . Large‐scale production of proteins containing ncAAs has to rely on either readily available natural ncAAs or on the design of host cells with in situ metabolic synthesis of the respective amino acid …”

Section: Figurementioning

confidence: 99%

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

Exner

Kuenzl

et al. 2016

ChemBioChem

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The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits ar ange of biological activities. It is also as mall bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report as imple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploitedt he exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyltRNA synthetase (SacRS) capable of specifically acceptingt he small, polar amino acid instead of its long andb ulky aliphatic natural substrate. We succeeded in generating an ovel and inexpensive strategy for the incorporation of af unctionally versatile amino acid. This will help in the conversiono fo rthogonal translation from as tandard technique in academic research to industrial biotechnology.

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“…Orthogonal ribosome/ mRNAp airs [72] have also been used to introduce multiple, different unnatural amino acids into as ingle protein. [8c] Cells can also be engineered to produce orthogonal amino acids by introducing the corresponding biosynthetic pathways.I mportant representative examples include bacterial production and the incorporation of azidohomoalanine, [75] p-aminophenylalanine, [76] and pyrrolysine derivatives [77] into proteins. [74] Thus,c urrent systems are based on four orthogonal (O) parts,the O-ribosome,O-mRNA, O-tRNA, and O-aaRS, which operate in parallel to the endogenous protein synthesis machinery.D epending on the chemical modification, the introduced unnatural amino acid (e.g.…”

Section: Expansion Of the Genetic Codementioning

confidence: 99%

Synthetic Biology—The Synthesis of Biology

2017

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Synthetic biology concerns the engineering of man-made living biomachines from standardized components that can perform predefined functions in a (self-)controlled manner. Different research strategies and interdisciplinary efforts are pursued to implement engineering principles to biology. The "top-down" strategy exploits nature's incredible diversity of existing, natural parts to construct synthetic compositions of genetic, metabolic, or signaling networks with predictable and controllable properties. This mainly application-driven approach results in living factories that produce drugs, biofuels, biomaterials, and fine chemicals, and results in living pills that are based on engineered cells with the capacity to autonomously detect and treat disease states in vivo. In contrast, the "bottom-up" strategy seeks to be independent of existing living systems by designing biological systems from scratch and synthesizing artificial biological entities not found in nature. This more knowledge-driven approach investigates the reconstruction of minimal biological systems that are capable of performing basic biological phenomena, such as self-organization, self-replication, and self-sustainability. Moreover, the syntheses of artificial biological units, such as synthetic nucleotides or amino acids, and their implementation into polymers inside living cells currently set the boundaries between natural and artificial biological systems. In particular, the in vitro design, synthesis, and transfer of complete genomes into host cells point to the future of synthetic biology: the creation of designer cells with tailored desirable properties for biomedicine and biotechnology.

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Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins

Cited by 55 publications

References 39 publications

Primordial‐like enzymes from bacteria with reduced genomes

Primordial‐like enzymes from bacteria with reduced genomes

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

Synthetic Biology—The Synthesis of Biology

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