orthogonal suppressor tRNA holds the key to the incorporation efficiency. Recently, improved incorporation is achieved in E. coli strain C321.ΔA, in which all amber codons, accounting for 8% of stop codons in E. coli, [1] are replaced and prfA is removed away from the genome. [2] However, it will be extremely lethal for a cell to simultaneously interfere with all three stop codons. Saturation mutagenesis for molecular evolution is another application restricted by termination mechanisms. Stop codon involved in NNN randomized codon causes undesired premature termination. One stop codon is still generated even with NNK or NNS, moreover, the reduced codon set excludes codons with high usage frequency (Figure S1, Supporting Information). We anticipate that complete codon-dependent termination defect protein translation could efficiently liberate all codons for sense function and improve all the genetic code engineering applications (Figure 1).In the cell, termination of translation includes both the essential mechanism of class-I release factors and alternative mechanisms, [3] such as tmRNA (ssrA) mediated transtranslation [4] and translation stalling rescue. [5] As alternative termination ArfT in F. tularensis, [6] ResQ in Bacillus subtilis [7] are reported. In contrast to RF1, there is still no feasible approach for deletion of RF2, major class-I release factor in charge of the essential termination and plays a critical role in alternative terminations as well, i.e., post-peptidyl transfer quality control and alternative ribosome rescue. [8] However, cell viability becomes a barrier for in vivo assessment of global termination function. Therefore, there is strong curiosity about what if losing all essential termination machineries.Essential genes cannot be genetically deleted in a living cell. Pdt peptide containing 27 amino acids in length evolved from Mesoplasma florum Lon protease (mf-Lon) is demonstrated as being able to specifically target protein to efficient degradation in E. coli. [9] Pdt-tag is fused to nascent chain release factor RF1 and RRF respectively in E. coli MG1655pro, but failed on prfB encoding release factor RF2. Thus far, complete removal of all termination functions in either living cell or cell lysate was not yet achieved.Here, we present one efficient in vitro protein synthesis with 64 sense codons (iPSSC). The mf-Lon directed protein degradation is demonstrated as one efficient approach for the Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs...
Manipulating transfer RNAs (tRNAs) for emancipating sense codons to simplify genetic codons in a cell-free protein synthesis (CFPS) system can offer more flexibility and controllability. Here, we provide an overview of the tRNA complement protein synthesis system construction in the tRNA-depleted Protein synthesis Using purified Recombinant Elements (PURE) system or S30 extract. These designed polypeptide coding sequences reduce the genetic codon and contain only a single tRNA corresponding to a single amino acid in this presented system. Strategies for removing tRNAs from cell lysates and synthesizing tRNAs in vivo/vitro are summarized and discussed in detail. Furthermore, we point out the trend toward a minimized genetic codon for reducing codon redundancy by manipulating tRNAs in the different proteins. It is hoped that the tRNA complement protein synthesis system can facilitate the construction of minimal cells and expand the biomedical application scope of synthetic biology.
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