An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids.
The key prerequisites to expand the genetic code are: the non-standard amino acid to encode, an unused codon to adopt, a tRNA that recognizes this codon, and a tRNA synthetase that recognizes only that tRNA and only the non-standard amino acid.
Expanding the genetic code is an area of research of synthetic biology, an applied biological discipline whose goal is to engineer living systems for useful purposes. The genetic code expansion enriches the repertoire of useful tools available to science.
In May 2019, researchers, in a milestone effort, reported the creation of a new synthetic (possibly artificial) form of viable life, a variant of the bacteria Escherichia coli, by reducing the natural number of 64 codons in the bacterial genome to 61 codons (eliminating two out of the six codons coding for serine and one out of three stop codons) – of which 59 used to encode 20 amino acids.
Introduction
It is noteworthy that the genetic code for all organisms is basically the same, so that all living beings use the same 'genetic language'. In general, the introduction of new functional unnatural amino acids into proteins of living cells breaks the universality of the genetic language, which ideally leads to alternative life forms. Proteins are produced thanks to the translational system molecules, which decode the RNA messages into a string of amino acids. The translation of genetic information contained in messenger RNA (mRNA) into a protein is catalysed by ribosomes. Transfer RNAs (tRNA) are used as keys to decode the mRNA into its encoded polypeptide. The tRNA recognizes a specific three nucleotide codon in the mRNA with a complementary sequence called the anticodon on one of its loops. Each three-nucleotide codon is translated into one of twenty naturally occurring amino acids. There is at least one tRNA for any codon, and sometimes multiple codons code for the same amino acid. Many tRNAs are compatible with several codons. An enzyme called an aminoacyl tRNA synthetase covalently attaches the amino acid to the appropriate tRNA. Most cells have a different synthetase for each amino acid (20 or more synthetases). On the other hand, some bacteria have fewer than 20 aminoacyl tRNA synthetases, and introduce the "missing" amino acid(s) by modification of a structurally related amino acid by an aminotransferase enzyme. A feature exploited in the expansion of the genetic code is the fact that the aminoacyl tRNA synthetase often does not recognize the anticodon, but another part of the tRNA, meaning that if the anticodon were to be mutated the encoding of that amino acid would change to a new codon. In the ribosome, the information in mRNA is translated into a specific amino acid when the mRNA codon matches with the complementary anticodon of a tRNA, and the attached amino acid is added onto a growing polypeptide chain. When it is released from the ribosome, the polypeptide chain folds into a functioning protein.
In order to incorporate a novel amino acid into the genetic code several changes are required. First, for successful translation of a novel amino acid, the codon to which the novel amino acid is assigned cannot already code for one of the 20 natural amino acids. Usually a nonsense codon (stop codon) or a four-base codon are used. Second, a novel pair of tRNA and aminoacyl tRNA synthetase are required, these are called the orthogonal set. The orthogonal set must not crosstalk with the endogenous tRNA and synthetase sets, while still being functionally compatible with the ribosome and other components of the translation apparatus. The active site of the synthetase is modified to accept only the novel amino acid. Most often, a library of mutant synthetases is screened for one which charges the tRNA with the desired amino acid. The synthetase is also modified to recognize only the orthogonal tRNA. The tRNA synthetase pair is often engineered in other bacteria or eukaryotic cells.
In this area of research, the 20 encoded proteinogenic amino acids are referred to as standard amino acids, or alternatively as natural or canonical amino acids, while the added amino acids are called non-standard amino acids (NSAAs), or unnatural amino acids (UAAs; term not used in papers dealing with natural non-proteinogenic amino acids, such as phosphoserine), or non-canonical amino acids.
Non-standard amino acids
The first element of the system is the amino acid that is added to the genetic code of a certain strain of organism.
Over 71 different NSAAs have been added to different strains of E. coli, yeast or mammalian cells. Due to technical details (easier chemical synthesis of NSAAs, less crosstalk and easier evolution of the aminoacyl-tRNA synthase), the NSAAs are generally larger than standard amino acids and most often have a phenylalanine core but with a large variety of different substituents. These allow a large repertoire of new functions, such as labeling (see figure), as a fluorescent reporter (e.g. dansyl alanine) or to produce translational proteins in E. coli with Eukaryotic post-translational modifications (e.g. phosphoserine, phosphothreonine, and phosphotyrosine).
The founding work was performed by Rolf Furter, who used the yeast tRNA/PheRS pair to incorporate p-fluorophenylalanine in E. coli.
Unnatural amino acids incorporated into proteins include heavy atom-containing amino acids to facilitate certain x-ray crystallographic studies; amino acids with novel steric/packing and electronic properties; photocrosslinking amino acids which can be used to probe protein-protein interactions in vitro or in vivo; keto, acetylene, azide, and boronate-containing amino acids which can be used to selectively introduce a large number of biophysical probes, tags, and novel chemical functional groups into proteins in vitro or in vivo; redox active amino acids to probe and modulate electron transfer; photocaged and photoisomerizable amino acids to photoregulate biological processes; metal binding amino acids for catalysis and metal ion sensing; amino acids that contain fluorescent or infra-red active side chains to probe protein structure and dynamics; α-hydroxy acids and -amino acids as probes of backbone conformation and hydrogen bonding interactions; and sulfated amino acids and mimetics of phosphorylated amino acids as probes of post-translational modifications.
Availability of the non-standard amino acid requires that the organism either import it from the medium or biosynthesize it. In the first case, the unnatural amino acid is first synthesized chemically in its optically pure L-form. It is then added to the growth medium of the cell. A library of compounds is usually tested for use in incorporation of the new amino acid, but this is not always necessary, for example, various transport systems can handle unnatural amino acids with apolar side-chains. In the second case, a biosynthetic pathway needs to be engineered, for example, an E. coli strain that biosynthesizes a novel amino acid (p-aminophenylalanine) from basic carbon sources and includes it in its genetic code. Another example is the production of phosphoserine, a natural metabolite, which required alteration of its pathway flux to increase its production.
Codon assignment Another element of the system is a codon to allocate to the new amino acid.
A major problem for the genetic code expansion is that there are no free codons. The genetic code has a non-random layout that shows tell-tale signs of various phases of primordial evolution, however, it has since frozen into place and is near-universally conserved. Nevertheless, some codons are rarer than others. In fact, in E. coli (and all organisms) the codon usage is not equal, but presents several rare codons (see table), the rarest being the amber stop codon (UAG).
+ Codon usage in E. coli Codon Amino acid Abundance (%) UUU Phe (F) 1.9 UUC Phe (F) 1.8 UUA Leu (L) 1.0 UUG Leu (L) 1.1 CUU Leu (L) 1.0 CUC Leu (L) 0.9 CUA Leu (L) 0.3 CUG Leu (L) 5.2 AUU Ile (I) 2.7 AUC Ile (I) 2.7 AUA Ile (I) 0.4 AUG Met (M) 2.6 GUU Val (V) 2.0 GUC Val (V) 1.4 GUA Val (V) 1.2 GUG Val (V) 2.4 UCU Ser (S) 1.1 UCC Ser (S) 1.0 UCA Ser (S) 0.7 UCG Ser (S) 0.8 CCU Pro (P) 0.7 CCC Pro (P) 0.4 CCA Pro (P) 0.8 CCG Pro (P) 2.4 ACU Thr (T) 1.2 ACC Thr (T) 2.4 ACA Thr (T) 0.1 ACG Thr (T) 1.3 GCU Ala (A) 1.8 GCC Ala (A) 2.3 GCA Ala (A) 0.1 GCG Ala (A) 3.2 UAU Tyr (Y) 1.6 UAC Tyr (Y) 1.4 UAA Stop 0.2 UAG Stop 0.03 CAU His (H) 1.2 CAC His (H) 1.1 CAA Gln (Q) 1.3 CAG Gln (Q) 2.9 AAU Asn (N) 1.6 AAC Asn (N) 2.6 AAG Lys (K) 3.8 AAA Lys (K) 1.2 GAU Asp (D) 3.3 GAC Asp (D) 2.3 GAA Glu (E) 4.4 GAG Glu (E) 1.9 UGU Cys (C) 0.4 UGC Cys (C) 0.6 UGA Stop 0.1 UGG Trp (W) 1.4 CGU Arg (R) 2.4 CGC Arg (R) 2.2 CGA Arg (R) 0.3 CGG Arg (R) 0.5 AGU Ser (S) 0.7 AGC Ser (S) 1.5 AGA Ser (S) 0.2 AGG Ser (S) 0.2 GGU Gly (G) 2.8 GGC Gly (G) 3.0 GGC Gly (G) 0.7 GGA Gly (G) 0.9
Amber codon suppression The possibility of reassigning codons was realized by Normanly et al. in 1990, when a viable mutant strain of E. coli read through the UAG ("amber") stop codon. This was possible thanks to the rarity of this codon and the fact that release factor 1 alone makes the amber codon terminate translation. Later, in the Schultz lab, the tRNATyr/tyrosyl-tRNA synthetase (TyrRS) from Methanococcus jannaschii, an archaebacterium, was used to introduce a tyrosine instead of STOP, the default value of the amber codon. This was possible because of the differences between the endogenous bacterial syntheses and the orthologous archaeal synthase, which do not recognize each other. Subsequently, the group evolved the orthologonal tRNA/synthase pair to utilize the non-standard amino acid O-methyltyrosine. This was followed by the larger naphthylalanine and the photocrosslinking benzoylphenylalanine, which proved the potential utility of the system.
The amber codon is the least used codon in Escherichia coli, but hijacking it results in a substantial loss of fitness. One study, in fact, found that there were at least 83 peptides majorly affected by the readthrough. Additionally, the labelling was incomplete. As a consequence, several strains have been made to reduce the fitness cost, including the removal of all amber codons from the genome. In most E. coli K-12 strains (viz. Escherichia coli (molecular biology) for strain pedigrees) there are 314 UAG stop codons. Consequently, a gargantuan amount of work has gone into the replacement of these. One approach pioneered by the group of Prof. George Church from Harvard, was dubbed MAGE in CAGE: this relied on a multiplex transformation and subsequent strain recombination to remove all UAG codons—the latter part presented a halting point in a first paper, but was overcome. This resulted in the E. coli strain C321.ΔA, which lacks all UAG codons and RF1. This allowed an experiment to be done with this strain to make it "addicted" to the amino acid biphenylalanine by evolving several key enzymes to require it structurally, therefore putting its expanded genetic code under positive selection.
Rare sense codon reassignment In addition to the amber codon, rare sense codons have also been considered for use. The AGG codon codes for arginine, but a strain has been successfully modified to make it code for 6-N-allyloxycarbonyl-lysine.