Experimental verification of the error minimization theory using non-standard genetic codes constructed in vitro

All living systems use an almost identical standard genetic code (SGC), in which 20 amino acids are assigned non-randomly. According to the error minimization theory, amino acids are arranged to minimize the mutational effect on protein function, while experimental verification remains limited. Here, we constructed 10 non-standard genetic codes (non-SGCs) in vitro by reassigning three amino acids (Ala, Ser, and Leu) in vacant codons of the minimal genetic code consisting of 21 tRNAs. Most of these non-SGCs have a higher cost of amino acid replacement than the SGC, calculated based on three amino acid properties: polar requirement (PR), molecular volume (MV), and hydropathy index (HI). The protein function of three reporter genes expressed using these non-SGCs decreased similarly when random mutations were introduced into the genes, implying that the effect of mutations was similar across all the non-SGCs tested here. This result provides direct experimental evidence that mutational robustness does not significantly change in individual reporter protein activity within the range of mutational cost tested in this study (Cost_PR: 5.29-5.77, Cost_MV: 1848-2348, and Cost_HI: 3.27-5.10), which covers approximately 18.4% (PR), 37.6% (MV), and 50.8% (HI) of the possible cost range achievable among one million randomly-generated genetic codes.