The project aims to generate a library of laboratory orthologous genes for studying evolvability. In this study, we have taken one of histidine biosynthetic genes hisA from Salmonella enterica through alternating rounds of weak selection (simulated by random mutagenesis through error-prone PCR and screens for partial loss of hisA function) followed by strong purifying selection (simulated by additional rounds of random mutagenesis and screens for restored hisA function). We have started from the wild-type hisA gene expressed from relatively weak arabinose inducible promoter ParaBAD. Twenty different single nucleotide polymorphisms (SNPs) that cause slow growth in the absence of histidine were isolated. In twelve of these deleterious mutants the activity was restored to wild-type levels by additional SNP after another round of random mutagenesis. In this study, in some of the compensated his A variants it was difficult to isolate further deleterious mutations, suggesting that these compensating mutations can mask the effects of additional deleterious mutations. By combining the compensating mutations with known deleterious mutations, complete or partial compensation was seen. Orthologs from one of the diverging lineages were also compared for their evolvability toward TrpF activity (ability to synthesize tryptophan) using fluctuation test. Two orthologs in this lineage showed higher evolvability compared to the wild type hisA.

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