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. 2019 Apr;103(7):2947-2958.
doi: 10.1007/s00253-019-09690-6. Epub 2019 Feb 21.

Incorporation of Non-Standard Amino Acids Into Proteins: Challenges, Recent Achievements, and Emerging Applications

Free PMC article

Incorporation of Non-Standard Amino Acids Into Proteins: Challenges, Recent Achievements, and Emerging Applications

Xing Jin et al. Appl Microbiol Biotechnol. .
Free PMC article


The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation.

Keywords: Genetic code expansion; Non-standard amino acids; Orthogonal translational system; Synthetic biology.

Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.


Figure 1.
Figure 1.. Scheme of non-standard amino acid incorporation using an orthogonal translation system via amber suppression.
Orthogonal aminoacyl tRNA synthetase, o-aaRS; non-standard amino acid, NSAA; orthogonal tRNA, o-tRNA; elongation factor Tu, EF-Tu. The anti-amber codon sequence on o-tRNA is CUA.
Figure 2.
Figure 2.. A) NSAA incorporation using a cell-free protein synthesis reaction.
Cell extracts containing transcription/translation machinery, co-factors, energy sources and other components are added to the reaction to mimic the bacterial environment. The addition of the o-aaRS/o-tRNA pair, NSAA and plasmid DNA template containing amber sites enables NSAA incorporation. B) Genomically recoded organism evolution via multiplex automated genome engineering (MAGE). Single-strand DNA oligos are added to the MAGE cycle to mutate multiple genomic locations simultaneously. C) Redundant sense codon reassignment. Codons for the standard amino acid (SAA) are artificially reprogramed for NSAA. N denotes A, U, G or C. D) Orthogonal ribosome and quadruplet codon. The orthogonal ribosome is developed by recognizing the orthogonal ribosome binding site (o-RBS) and tethering the 50S subunit and orthogonal 30S subunit. The orthogonal ribosome translates orthogonal mRNA containing o-RBS and incorporates NSAAs via quadruplet codon and amber codon suppression.

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