Rewriting the Genetic Code

Abstract

The genetic code-the language used by cells to translate their genomes into proteins that perform many cellular functions-is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

Keywords: codon usage; genetic code; orthogonal; synthetic biology; translation engineering.

Figure 1

Rewriting the genetic code. (a) Three methods used to augment the genetic code with ncAAs: selective pressure incorporation, site-specific incorporation, and codon reassignment. These methods are not mutually exclusive. (b) A proposed form of an organism having a new genetic code and amino acid repertoire. The ncAA (orange star) is either supplemented in the media and taken up by the cell through a transporter or produced by the cell. Enzymes that degrade the ncAA are inactivated, and an orthogonal aaRS charges the ncAA onto its devoted tRNA. Panel a adapted from Sakamoto (129). Abbreviations: aaRS, aminoacyl-tRNA synthetase; aa-tRNA, aminoacyl-tRNA; mRNA, messenger RNA; ncAA, noncanonical amino acid; RF, release factor; tRNA, transfer RNA.

Figure 2

Deviation from the standard genetic code in nature. (a) Codon reassignment occurred in some bacteria and eukaryotes (nuclear genetic code), whereas dual or triple usage of a particular codon, including the assignment of selenocysteine (Sec) and pyrrolysine (Pyl), is found in all three domains of life. (b) Some bacteriophages change the genetic code of their host cells for late gene expression. The full map of codon reassignment (organisms and organelles) can be found in Supplemental Figure 1.

Figure 3

Engineering the orthogonal translation systems. (a) aaRS engineering with an example of Methanocaldococcus jannaschii (Mj) TyrRS. (b) EF-Tu engineering. The changed residues are shown. Ser66 was modified to alanine to improve azido-phenylalanine recognition (37). (c) tRNA engineering. Colored residues were mutated to change the indicated properties. (d) Ribosome engineering. The PTC, A site, anti-SD sequence, and mutated ribosomal RNA residues are indicated. Abbreviations: aaRS, aminoacyl-tRNA synthetase; anti-SD, anti-Shine-Dalgarno; ncAAs, noncanonical amino acids; PTC, peptidyl transfer center; rRNA, ribosomal RNA; tRNA, transfer RNA; TyrRS, tyrosyl-tRNA synthetase.

Figure 4

State-of-the-art recombination methods used to engineer the codon usage in the Escherichia coli genome. For serial genome engineering, iterative multiplex oligo-mediated recombination of multiple alleles produced E. coli genomes having more than 120 intended mutations (99, 102) (a). Alternatively, a set of recoded genome segments was prepared by iterating oligo-mediated recombination (MAGE; 51) (b) or by de novo DNA synthesis subjected to assembling in yeast (113, 149) (c). These sets of recoded genome segments were assembled by hierarchical CAGE (51, 69), CAGE 2.0 (105, 113), or iterating REXER (149) (d). The latter two methods are optimized for assembling de novo synthesized segments (50–120 kbp). Hierarchical CAGE was used for assembling 32 segments, each having 10 UAG-to-UAA stop codon changes, into one genome devoid of any UAG stop codon to produce the E. coli C321.ΔA strain (69). Abbreviations: BAC, bacterial artificial chromosome; CAGE, conjugative assembly genome engineering; CoS, coselection; dsDNA, double-stranded DNA; MAGE, multiplex automated genome engineering; REXER, replicon excision for enhanced genome engineering through recombination; ssDNA, single-stranded DNA.