Fitness effects of first five mutations fixed in G3 lineage. The first five mutations fixed in the G3 lineage (marC, miaA-hfq, rph, mdh, and groL) were constructed singly and in various combinations in E. coli EcHW24 (EcNR1 ΔmutS) using ssDNA mediated recombination; the marC::IS1 mutation was approximated by knocking out marC. The constructed mutants and E. coli EcHW24 were phenotyped by measuring the maximum specific growth rate in 0%, 0.5%, and 1% (w/v) isobutanol spiked minimal glucose media; relative fitness was calculated as mutant maximum specific growth rate (h-1) divided by E. coli EcHW24 maximum specific growth rate (h-1). Mutation combinations corresponding to the order of appearance in G3 are highlighted. Epitasis is assumed to follow a simple multiplicative fitness model , where w = relative fitness of a particular mutation combination, ε = total epistatic interaction parameter, and wi = relative fitness of single mutants; log epistasis is calculated as . Inset shows log(ε) at 1% (w/v) isobutanol for selected mutation combinations with notable epistasis.