We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic non-canonical amino acid L-β-(thieno[3,2-b]pyrrolyl)-alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp → [3,2] Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition are capable of robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.
Classical enzyme optimization exploits the chemistry confined to the 20 canonical amino acids encoded by the standard genetic code. ‘Genetic code engineering’ allows the global substitution of particular residues with synthetic analogues, endowing proteins with chemical diversity not found in nature. These proteins are congeners of the parent protein because they originate from the same gene sequence, but contain a fraction of noncanonical amino acids. Global substitutions of methionine, proline, phenylalanine, and tyrosine have been carried out with related analogues in Thermoanaerobacter thermohydrosulfuricus lipase. This study represents the first extensive report of an important biocatalyst substituted with a high number of noncanonical amino acids. The generated lipase congeners displayed special features such as enhanced activation, elevated enzyme activity (by up to 25 %) and substrate tolerance (by up to 40 %), and changes in optimal temperature (by up to 20 °C) and pH (by up to 3). These emergent features achieved by genetic code engineering might be important not only for academic research, but also for numerous economical applications in the food, detergent, chemical, pharmaceutical, leather, textile, cosmetic, and paper industries.
Our long-term goal is the in vivo expression of intrinsically colored proteins without the need for further posttranslational modification or chemical functionalization by externally added reagents. Biocompatible (Aza)Indoles (Inds)/(Aza)Tryptophans (Trp) as optical probes represent almost ideal isosteric substitutes for natural Trp in cellular proteins. To overcome the limits of the traditionally used (7-Aza)Ind/(7-Aza)Trp, we substituted the single Trp residue in human annexin A5 (anxA5) by (4-Aza)Trp and (5-Aza)Trp in Trp-auxotrophic Escherichia coli cells. Both cells and proteins with these fluorophores possess intrinsic blue fluorescence detectable on routine UV irradiations. We identified (4-Aza)Ind as a superior optical probe due to its pronounced Stokes shift of Ϸ130 nm, its significantly higher quantum yield (QY) in aqueous buffers and its enhanced quenching resistance. Intracellular metabolic transformation of (4-Aza)Ind into (4-Aza)Trp coupled with high yield incorporation into proteins is the most straightforward method for the conversion of naturally colorless proteins and cells into their blue counterparts from amino acid precursors.expanded genetic code ͉ imaging ͉ optical probes ͉ protein engineering and design ͉ red-shift A n ideal optical probe for the analysis of single proteins or whole proteomes would have the following properties: it is biocompatible, well incorporated into the target protein(s) by the endogenous translational apparatus and does not require posttranslational modifications or extensive host-engineering. Also, this chromophore should be noninvasive, i.e., it introduces minimal structural and functional perturbations into the target(s). The most promising candidates for such a chromophore are Ind analogs. They are basic structures of numerous highly important biomolecules. For example, Ind as part of the side chain of the amino acid Trp is the main source of intrinsic protein fluorescence, and purine bases of nucleic acids are Ind derivatives as well (1).The canonical amino acid Trp is one of the most suitable targets for protein engineering and design owing to its low abundance in proteins and high relevance for protein stability and function (2). However, due to its complicated photophysics and the unfavorable overlap between nucleic acid and protein fluorescence emission spectra, Trp is not always qualified as a suitable optical probe (3), and alternatives are needed. Trp analogs with their Ind side chains containing a single atom exchange (''atomic mutation'') (4) would be highly desirable. These analogs do not perturb the local environment of the substituted target protein(s) but induce considerable spectral changes relative to Trp.(Aza)Trps meet the above described criteria. In these Trp isosteres, one of the endocyclic, CH, groups of Ind is substituted with nitrogen (Fig. 1A). This substitution comprises not only the smallest possible structural alteration of all known Trp analogs but also leads to dramatic changes in the photophysics of the aromatic system. (Aza)Inds r...
The expansion of the genetic code is gradually becoming a core discipline in Synthetic Biology. It offers the best possible platform for the transfer of numerous chemical reactions and processes from the chemical synthetic laboratory into the biochemistry of living cells. The incorporation of biologically occurring or chemically synthesized non-canonical amino acids into recombinant proteins and even proteomes via reprogrammed protein translation is in the heart of these efforts. Orthogonal pairs consisting of aminoacyl-tRNA synthetase and its cognate tRNA proved to be a general tool for the assignment of certain codons of the genetic code with a maximum degree of chemical liberty. Here, we highlight recent developments that should provide a solid basis for the development of generalist tools enabling a controlled variation of chemical composition in proteins and even proteomes. This will take place in the frame of a greatly expanded genetic code with emancipated codons liberated from the current function or with totally new coding units.
Expansion of the standard genetic code enables the design of recombinant proteins with novel and unusual properties. Traditionally, such proteins have contained only a single type of noncanonical amino acid (NCAA) in their amino acid sequence. However, recently reported initial efforts demonstrate that it is possible with suppression-based methods to translate two chemically distinct NCAAs into a single recombinant protein by combining the suppression of different termination codons and nontriplet coding units (such as quadruplets). The possibility of expanding the code with various NCAAs simultaneously further increases the toolkit for the generation of multifunctionalized proteins.
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