Fig. 3: Evolved biosynthetic pathway mediates improved Pyl protein yield.

A The best evolved variant (36A_sub-pop2) and best combinatorial variant (3f2) contained a total of four and seven mutations, of which two and four were convergent mutations (red), respectively (for further mutation analysis of subpopulations, see Supplementary Tables 2 and 3). B When tested in E. coli strain C321.∆A.exp, read-through of three amber codons in sfGFP is improved 32-fold in variant 3f2 combinatorial variant and 6.5-fold in variant 36A_sub-pop2, relative to the parental variant SUMO-pylBCD. The WT pylBCD pathway (termed “Untagged Parent”) did not have detectable activity. C Read-through of one amber codon in sfGFP is improved 2.4-fold in variant 3f2, relative to variant 36A_sub-pop2. The WT pylBCD variant and the SUMO-pylBCD did not have detectable activity in this assay. In panels B and C, fluorescence intensity shown (excitation 488 nm, emission 509 nm) was normalized by optical density (A₆₀₀) (see “Methods”). Samples were tested in biological triplicate; data shown represents mean values ± s.d.