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De novo synthesis of a recoded genome with expanded decoding capacity

Based on a better understanding of synonymous codon choices, we would like to combine the most viable synonymous codon substitutions together and test their feasibility to simultaneously recode all 11 target codons. When implemented in the whole genome, this combined recoding will remove 11 triplet codons from the decoding box, and free-up 17% (11 out of 64) of all triplet decoding capacity for reassignment to encode unnatural amino acids. 

A synthetic genome with all 11 target codons reassigned is indeed ambitious and potentially risky, and can only proceed following the identification of viable recoding schemes for all target codons. One possible compromise with significantly reduced technological risk is to solely remove and reassign a subset of the 11 proposed target codons (Figure 3). This also allows us to individually test the feasibility of removing and reassigning an isolated subset genome-wide before combining them with the remaining target codons.

We further plan to expand the number of reassigned codons by a factor of four using quadruplet decoding (Figure 3, step vi). When fully utilized, this can create up to 44 distinct quadruplet codons based on the initial 11 freed-up triplet codons to dramatically expand the number of unnatural amino acids that can be incorporated simultaneously. 

Kai De novo synthesis Figure 3. Expanding decoding capacity through removing and reassigning decoding redundancy. 

Step i -identifies target codons for removal (e.g. serine codon TCG and TCA, and stop codon TAG; in grey). 

Step ii-identifies the best synonymous replacements by testing defined recoding schemes. For example, TAA is identified as a viable replacement to TAG. 
Step iii- systematically recodes the target codons to the identified synonymous replacements in the de novo synthesized genome across all genomic positions. 
Step iv- removes the redundant tRNAs (Ser tRNACGA and Ser tRNAUGA, in grey) and release factor 1 (RF1, in grey) to free-up the decoding capacity of removed codons TCG, TCA, and TAG. 
Step v- introduces evolved synthetase/tRNA pairs (in blue) to encode unnatural amino acids (u.a.a., blue star) quantitatively in response to reassigned triplet codons TCG, TCA, and TAG. Through the five defined steps, the previous serine codon TCG and TCA, and stop codon TAG in the wildtype genome have been reassigned to solely encode unnatural amino acids in the synthetic genome. 
Step vi expands the reassigned decoding capacities by a factor of four by introducing 12 quadruplet codons and corresponding quadruplet decoding evolved synthetase/tRNA pairs (in orange).