Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and new substrates to influence the evolutionary trajectory, an aspect that has generally been overlooked in laboratory directed evolution studies. We evolved the Escherichia coli nitro/quinone reductase NfsA to detoxify chloramphenicol by randomising eight active site residues simultaneously and interrogating ~250,000,000 reconfigured NfsA variants. Analysis of every possible evolutionary intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions that restrict each hypothetical trajectory. In both cases, improved chloramphenicol detoxification was only possible after one essential substitution had eliminated activity with endogenous quinone substrates. Unlike the predominantly weak trade-offs seen in previous experimental studies, this substrate incompatibility suggests endogenous metabolites have considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, which emphasises the importance of negative selection to effect enzyme specialisation, and offers an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

中文翻译：

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序列空间的巨大飞跃揭示了进化新功能的细胞内复杂性

混杂酶活性的选择为内源底物和新底物之间的竞争提供了实质性机会，以影响进化轨迹，这在实验室指导的进化研究中通常被忽略。我们进化了大肠杆菌硝基/醌还原酶NfsA通过同时随机化八个活性位点残基并询问约250,000,000个重新配置的NfsA变异体来使氯霉素解毒。对两种最佳氯霉素还原酶的每种可能的进化中间体的分析表明，复杂的上位性相互作用限制了每个假设的轨迹。在这两种情况下，只有在一种必要的取代消除了内源性醌底物的活性之后，才能改善氯霉素的解毒作用。与先前的实验研究中主要的较弱的取舍不同，这种底物的不相容性表明内源性代谢物具有形成进化结果的巨大潜力。未选择的前药转化活动大部分不受影响，