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Introduction
The origin of the Last Universal Common Ancestor (LUCA) as the root of life has long been sought by science. Ever since their discovery, the submarine hydrothermal vents have been regarded as attractive sites to house prebiotic development1,2. These vents emitted hydrogen, carbon dioxide and little oxygen, thereby favoring chemical reduction over oxidation. At the vent temperatures (~350oC), energetics favored the formation of organic compounds from inorganic ones3, which would later become intermediates of the reductive tricarboxylate, tricarboxylate and glyoxylate cycles4,5. Given the dissimilar pHs of vent fluid and the surrounding sea water, bioenergy may be generated as well through chemiosmosis in the vents6.

In terms of genomics, the average divergences between alloacceptor tRNA sequences that serve as carriers of amino acids in translation differ between organisms, with a display of minimum divergence by the anaerobic hyperthermophilic archaeon Methanopyrus kandleri (Mka) isolated from the vents7, indicating that Mka is phylogenetically closest to LUCA8. This conclusion has received widely based support from the usage of the simplest wobble rules for translation by Mka, its closely related tRNA sequences for reading the two separate codon domains of Ser9, and the display of the smallest difference between the paralogous protein sequences of Val-tRNA synthetase (VARS) and Ile-tRNA synthetase (IARS) among 5000-plus species of organisms10. The Mka node was also the oldest among archaea, dating back to 2.8 Gya11.The objective of the present study is to examine some of the major landmarks in the development of the genetic code from Methanopyrus to the present.

Formation of RNA Genes
A living cell has to fulfill two basic functions: to store and replicate its organization blueprint so that it can be passed on to the next generation, and to produce gene products to perform the catalytic and structural functions required by the act of living. Proteins cannot store information, whereas nucleic acids excel in information storage and also serve as catalysts, thus leading to the concept of an RNA World in the prebiotic age12,13. Cyclic and acyclic analogues of ribose and nucleobases were obtainable from thermosynthesis and meorites14,15, suggesting that RNA-like structures preceded the formation of present day RNA through the evolution of RNA polymerase ribozymes16. The polymerization of nucleotides to yield a product RNA strand would be guided by a complementary template RNA strand via A-T and G-C basepairings, so that the product strand was bound to the template strand in a double helix17. Since only one out of 1024 random 40-mer RNA templates weighing one kilogram would include a functional 40-mer RNA with aptamer or ribozyme activity, it would need random RNA templates equal to the mass of the Earth to generate two functional RNA molecules18. Furthermore, because the vents did not undergo the temperature cycles of a polymerase-chain-reaction (PCR) incubator, the double helices formed by the old-new RNA strands would remain mostly as dead-end duplexes that can no longer replicate themselves.

Although primer extension could bring about limited strand displacement, the problem posed by the rarity of functional RNA strands amidst a flood of random non-functional ones was an intractable obstacle to the development of a functional RNAome. To resolve this problem, it became essential to single out the minute fraction of duplexes that included an RNA strand with aptamer or ribozyme function, and induce their melting through the binding of a ligand of the aptamer or ribozyme to the functional strand, thus freeing up the other strand for continual replication. This replicator induction by metabolites (REIM) mechanism brought about the selective replication of duplexes containing functional RNAs while the mass of non-functional random duplexes remained unmelted and underwent decay in time, allowing the recycling of their constituents to suport the proliferation of the functional duplexes into clones of RNA genes19,9.
Coevolution Theory
The ribozymes at the vents consisted of the four nucleotidyl letters U, C, A and G, and relied on their sidechains to catalyze different types of biochemical reactions, which were barely adequate for the task20. Accordingly, they had to acquire extra catalysis-competent sidechains post- transcriptionally, as in the case of modern tRNAs and rRNAs. Where the extra sidechains included amino acids and peptides from the prebiotic surroundings, the latter’s outstanding catalytic properties soon became evident, and they were positioned at the catalytic centers of ribozymes, while the RNA moieties themselves functioned as mRNAs for the ribozyme sequences21.
Many organisms contained amino acid biosynthetic pathways that converted precursor amino acids into product amino acids. The early cells selected some of these products for encoding by the code as additional letters of the protein alphabet. Comparison of the codon locations for the 20 finalized amino acids in the code with the precursor-product biosynthetic relationships between them led to the coevolution theory which proposed that biosynthesis played an important role in determining codon locations: the Phase 1 amino acids Gly, Ala, Ser, Asp, Glu, Val, Leu, Ile, Pro and Thr in the code were selected from the prebiotic environment, whereas the Phase 2 amino acids Arg, His, Trp, Lys, Cys, Met, thermal unstable Gln and Asn, and UV-labile Phe and Tyr were selected subsequently from the products of biosynthetic pathways that coevolved with the code22,23. That the GRA 95229 Antarctic meteorite24 and bombardment of high-energy particles25 yielded only Phase 1 but no Phase 2 amino acids, together with the extremely low maximum concentrations of Gln and Asn, respectively 3.7 pM Gln and 24 nM Asn, in the prebiotic soup on account of their thermoinstability26, strongly confirmed the coevolution view and the two-stage development of the 20-amino acid code.
Mutability of the Code
Despite the invariance of the 20-amino acid code among living species from archaea to bacteria and eukaryotes, there is no evidence for true invariance. On the contrary, that the Class 2 amino acids had to be evolved to complement the Class 1 amino acids could imply that code evolution entered into a prolonged pause due to the existence of oligogenic barriers consisting of conservative amino acid residues in the proteome firmly opposed to the loss of particular canonical amino acids (cAAs), in which case the frozen code may resume its evolution if the strength of these opposing residues were suppressed by mutations.

Accordingly, our group carried out rounds of iterative random mutations on the Trp-auxotrophic Bacillus subtilis QB928 strain with deleted Trp- biosynthesis, and looked for detectible growth on 4-fluoroTrp (4FTrp) on agar. This resulted in the isolation of an intermediate LC33 strain that grew well on Trp and 4FTrp, and subsequently the HR23 strain which grew well on 4FTrp but not on Trp. This HR23 mutant thus became the first organism in the history of life with a mutated code, where Trp has been displaced from the code by 4FTrp to become an inhibitor of growth on 4FTrp27-29.

Isolation of Mutated Codes
The isolation of the HR23 strain proved the intrinsic mutability of the code. This opened wide the floodgate for mutated codes, which led to the successful isolations of hundreds of mutant codes. The mutation technologies employed have included three general approaches30: Selective  pressURE  incorporation  (SPI).  Trp  was changed to 4FTrp in the HR23 code through two rounds  of  selection:  weakening  the  oligogenic barrier of conservative amino acids in the QB928 wildtype  against  the  removal  of  some  key  Trp residues to yield LC33, followed by the nurturing of an opposite oligogenic barrier of conservative amino  acids  against  the  removal  of  some  key 4FTrp   residues.   This   method   for   adding   or switching codon-amino acid affiliations has been employed    to    prepare    ‘fluorous’    Teflon-like proteins by means of thorough fluorination of Pro, Phe  and  Trp  residues  with  little  change  in  the activity  or  structure  of  the  proteins31.  In  another instance,  genetic  code  expansion  involved  the amino  acid  selenocysteine  (Sec)  found  in  some species.   Following  a   series   of   mutations,   Sec became   encodable   by   erstwhile   amber   stop codons,  expediting  the  replacement  of  labile cystine   bridges   in   the   proteome   by   Sec-Sec bridges  with  improved  resistance  to  reducing conditions32.
Stop   codon   SUPPRession   (SCI).

The   translation apparati of living organisms consist of amino acids, tRNAs, ARSs, mRNAs, and ribosomes. Their mutual interactions   are   highly   specific. Consequently, when an exogenous ARS/tRNA pair is placed into a  host  cell,  they  would  form  independent,  i.e. orthogonal,   components   for   incorporating   a noncanonical  amino  acid  (ncAA)  into  proteins  in response    to    codons    designated    for    them. Furthermore, we found that archaeal tRNAs shared with  yeast,  rat  liver  and  wheat  germ  tRNAs  a distinct    preference    for    aminoacylation    by nonbacterial ARS over bacterial ARS from E. coli and Rhodopseudomonas spheroides33. On this basis, a yeast suppressor tRNA(Phe)/ARS(Phe) pair was employed  to  incorporate  the  ncAA  p-fluoroPhe into  the  DHFR  marker  protein  in  response  to  an erstwhile amber stop codon with two-thirds of the expression level of wildtype DHFR34. This usage of orthogonal (“O”) components to encode ncAAs was soon adopted for single-site and multi-site ncAA incorporations  in  E.  coli35,30,36,37,  and  at  a  Pro residue  in  Xenopus  oocytes38.  The  search  for appropriate O-ARSs is challenging, but it can be facilitated  by  the  application  of  phage-assisted continuous   evolution   (PACE)   of   ARS39.   Spare codons  can  be  derived  by  subdividing  codon boxes that have been allocated to a single cAA40. O-tRNA/O-mRNA pairs are valuable for making quadruplet codons useful41, and UAG stop codons can be converted to sense codons by the removal of release factor F1 to restrict competition against ncAA   incorporation   by   the   chain   termination process42. 
UNNATUral Basepairs (UBP). DNA contains the four nucleobases T, C, A and G, whereas RNA contains the four nucleobases U, C, A and G. However, it was  found  that  thymine  in  E.  coli  DNA  could  be replaced by 5-chlorouracil43. This led to the usage of   unnatural   basepairs   (UBP)   to   expand   the genetic  code.  In  the  case  of  clonal  semisynthetic organisms (SSOs), O-DNA-carrying plasmids were used  to  transform  E.  coli  host  cells  harboring  an accessory   plasmid   that   encoded   a   chimeric pyrrolysyl-tRNA synthetase variant. The cells were grown with a dNaM-d5SICS UBP pair, followed by addition of the corresponding mRNA UBP pair and  the  ncAA  AzK  to  ensure  the  permanent presence of AzK at the targeted protein sites44. DNA- and RNA-like systems were also built from eight    nucleotide    letters    that    formed    four orthogonal  pairs,  and  the  synthetic  biopolymer obtained   met   the   structural   requirements   of Darwinism,  including  a  polyelectrolyte  backbone and     predictable     thermodynamic     stability. Transcription of the nc-DNA into nc-RNA gave rise to aptamers with eight-letters45.
Medical Applications
In recent years, much of the effort directed to genetic code mutation and expansion has focused on technology development, but a series of findings have demonstrated the far-reaching results that may be accomplished as illustrated by the following examples.

a.    Genetic codes have been constructed that consist of 67 codons44 or 68 codons46, which provided more spare codons for ncAAs.
b.    Enhancements of immunotherapeutics for treating disorders including tumors were exemplified by the synthesis of site-specific antibody-drug conjugates47, use of a bispecific antibody against acute myeloid leukemia48 and cancer treatment with IL-249,
c.    Pegylations of proteins at designated sites turned them into more effective therapeutic agents, as exemplified by Fgf21 for overcoming insulin resistance in diabetes50, conjugation of peptides to albumin to prolong their half-lives51, diversification of antimicrobial peptides52, control of high- density lipoprotein biogenesis53, generation of a low-immunogenic and stable form of human growth hormone54, and site-specific immobilization of Bone Morphogenetic Protein 2 to solid surfaces55.
d.    Extension of ncAA usages to whole genomes of organisms or organs. Examples included E. coli56, yeast57, animals58, mouse brain59, and compartmentalized genetic parts and circuits60.
e.    Application of ncAAs to whole organisms. Examples included using sense codon reassignments to induce viral resistance61,62, and control of cell signaling in zebra fish embros63.
f.    After an ncAA has been introduced into the proteome of an organism, a supply of the ncAA has to be secured by the addition of the ncAA as a nutrient in order to maintain the presence of the ncAA. However, external ncAA supply would become unnecessary when the host organism can biosynthesize the ncAA. Examples included the ncAAs p- aminophenylalanine64,    selenocyteine32, phosphothreonine65. 5-hydroxytryptophan66 and Met-derivatives67.
Besides such attained applications of ncAAs to medicine, it would be useful to consider a couple of more exploratory objectives over the longer term:

Aging-related tissUE degeneration. All human tissues undergo aging, but often not at the same rates. As the  world  populations  grow  older,  the  disorders associated with instances of premature degeneration such as Alzheimers disease, Parkinsons disease, type 2 diabetes    are increasingly important but remain difficult to treat. For instance, osteoarthritis of the knee often incurs collagen degradation. In a recent study, subjects were treated with acetaminophen and native type 2 collagen each day. While the walking scores of the test subjects showed about 50% improvement, surpassing   the   effects   of   glucosamine   and chondroitin, the collagen degradation markers in urine were undiminished68, suggesting that replacement of degraded collagen might require reinforcement  from  ‘fluorous’  proteins  containing fluorinated Pro and other cAA or ncAA residues31.

For other types of degeneration, frequently no underlying defective proteins are clearly identified. Nonetheless, one may postulate that fragile cAA residues could be a contributing factor, and chemically unstable cAAs such as Cys and cystine in a model animal can be replaced by stable ncAA analogues such as Sec and diselenide32 in order to determine if that would relieve some of the symptoms of the degeneration.

Alleviation of food crises. During the past decades, extreme weather has caused widespread floods and draught, compounding the food crises due to population pressure and conflicts. Therefore it would be useful if genetic code expansion can be deployed to increase food production. Possible approaches in this regard would include the following:
i. While vascular plants are used to normal sunlight with photosynthetically active radiation (PAR) of 100-2,000 micromole photons m-2s-1, algae at 300 m below sea surface can utilize dim light of 0.02 m-2s-1 for photosynthesis69. Such dim-light photosynthetic systems can be introduced into edible macro- and micro-algae grown rapidly on optimized cAA-ncAA mixtures to yield biomass.
ii. Wind power, tidal power, fusion power and power from solar panels covering all the world’s highlands will be converted into photons for algal photosynthesis. The performance of the photosynthetic apparatus can be enhanced by ncAAs, as suggested by the kinetic alteration caused by the replacement of a single Trp residue in the Orange Carotenoid Protein of Synechocystis by benzothienyl-L-Ala70.
iii. Autotrophic centers’ can be established to culture archaeal methanogens on the hydrogen and carbon dioxide emissions from hydrothermal vents and boreholes, supported by cAA-ncAA mixtures for rapid growth.
iv. Synthesize starch chemoenzymatically from carbon dioxide, which can produce starch 8.5- fold faster than maize71, using enzymes bearing cAA-ncAA mixtures to accelerate the rate of synthesis.
v. Water-compatible artificial spherical chromatophore nanomicelles comparable to the chromatophore of Rhotobacter sphaeroides has enabled the reduction of carbon dioxide to methane with 15% solar-to-fuel efficiency72. Admixture with protein and membrane components of R. spheroides, grown on optimized cAA-ncAA mixtures, may generate even higher efficiency.
The biomass and carbohydrates derived from these approaches can serve directly as food for humans, or fed to edible yeasts so that the ncAAs and archaeal lipids may be removed prior to human consumption.
Moreover, while collisions between medium-sized meteorites and the Earth can be avoided by diverting them from their collision courses, collisions with too-big-to-divert meteorites will bring about dust clouds that cut agricultural output. It is also predicted that the sun will turn into a red giant with decreased temperatures in about 5 Gyrs, but unforeseen factors might cause an extremely unlikely but not impossible premature occurrence of red-giant formation, massively reducing agricultural output. Under these circumstances, measures such as (i) to (v) can be activated to improve food supply. On the other hand, in the likely event that red-giant formation occurs only 5
 
Gyrs from now, these measures could eliminate or at least decrease the severity of the food shortages that are as old as human societies. Continuation of the bane of these shortages for another 5 Gyrs is not a tolerable outcome. In this regard, the ncAAs offer a rare opportunity to upgrade the productivity of food production processes.

Conclusions
The genetic code, a centerpiece of the DNA-RNA- protein information system unique to life, began to evolve its familiar 20-amino acid alphabet prior to the appearance of Methanopyrus. This encoded alphabet has furnished an invaluable foundation for microbes, plants, animals and humans in their movement, flight, sensory signaling and memory. Now that the code has revealed its malleability, and begun to encode hundreds of ncAAs in addition to the cAAs, the effects of these new genetic codes and the ncAAs they can encode are not readily surmised. However, based on the new advances on a broad front of ncAA applications illustrated in the preceding section, it is evident that the chemical diversity of proteins will be steeply enhanced by the genetic encoding of an enlarged array of organic compounds. Known enzymic reactions will be accelerated, and enzymic reactions hitherto not observed in living organisms may be devised, so that medicine and health will benefit from novel approaches for the treatment of cancers, degenerative diseases and other disorders.

Furthermore, humans the space travelers are searching for habitable planets in our galaxy. If human hibernation can be achieved, the distance of travel to suitable planets will be increased, and attractive planets as goals of migration identified. Wherever the top choice might be, the local environment will not be identical to that on Earth, and the 20 amino acid alphabet and eight nucleobase building blocks may need to be revised to fit the local conditions and resources. Consequently, the ncAAs and UBPs currently investigated in genetic-code expansion research could pave the way for humans to re-optimize our molecular biology in adaptation to life on the new planet.

Acknowledgments
This research was funded by grant number 5112- 703-0110D-42002 from the University Grants Council of Hong Kong to Applied Genomics Center of Hong Kong University of Science and Technology.
 
Author contributions
Conceptualization and writing, T.F.W. and H.X.; both authors have read and agreed to the published version of the manuscript.

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