Supplementary Figure 11: Benefits of rational mutagenesis for sampling novel adaptive genotypes.

a) 500 μg/mL rifampicin b) 500 μg/mL erythromycin c) 10 g/L acetate and d) 2 g/L furfural. While naturally evolving systems or error-prone PCR are highly biased towards sampling single nucleotide polymorphisms (i.e. 1 nt mutations, red) these histograms illustrate the potential advantages for rational design approaches that can identify rare or inaccessible mutations (2 and 3 nt, green and blue respectively). For example, the highest fitness solutions appear to be biased toward these rare mutations in rifampicin, erythromycin and furfural selections to varying degrees. These results indicate that procedures such as CREATE should allow more rapid and thorough analysis of fitness improving mutations, in much the same way that computational approaches are being used to improve directed evolution for protein engineering.