A giant leap in sequence space reveals the intracellular complexities of evolving a new function

Abstract

20Selection for a promiscuous enzyme activity provides substantial opportunity for competition 21 between endogenous and new substrates to influence the evolutionary trajectory, an aspect 22 that has generally been overlooked in laboratory directed evolution studies. We evolved 23 the Escherichia coli nitro/quinone reductase NfsA to detoxify chloramphenicol by 24 randomising eight active site residues simultaneously and interrogating ~250,000,000 25 reconfigured NfsA variants. Analysis of every possible evolutionary intermediate of the two 26 best chloramphenicol reductases revealed complex epistatic interactions that restrict each 27 hypothetical trajectory. In both cases, improved chloramphenicol detoxification was only 28 possible after one essential substitution had eliminated activity with endogenous quinone 29 substrates. Unlike the predominantly weak trade-offs seen in previous experimental studies, 30 this substrate incompatibility suggests endogenous metabolites have considerable potential to 31 shape evolutionary outcomes. Unselected prodrug-converting activities were mostly 32 unaffected, which emphasises the importance of negative selection to effect enzyme 33 specialisation, and offers an application for the evolved genes as dual-purpose 34 selectable/counter-selectable markers. 35 enzymes possess an innate 'robustness' and stability that buffers them against the potentially 58 detrimental effects of novel mutations, coupled with a 'plasticity' that can amplify 59 promiscuous functions with relatively few mutations (Aharoni et al., 2005). More recently, 60Tokuriki and co-workers demonstrated that a robust native activity is not a prerequisite for 61 weak trade-offs, and suggested that their predominance in the literature may instead be 62 artefactual; a consequence of laboratory evolution studies being highly biased toward strong 63 selection for a new function without any selection against the native activity (Kaltenbach et 64 al., 2016). They argue that it is unclear how specialisation can occur in this manner, and that transforms this antibiotic to a product that is not discernibly toxic to bacteria (Yunis, 1988; 93 Smith et al., 2007; Crofts et al., 2019). We have observed that over-expressed native NfsA 94 confers only slight chloramphenicol protection to E. coli host cells, but reasoned that we 95 could select for improved detoxification in an extremely high throughput manner by plating 96 variant libraries on chloramphenicol-amended media. Because members of the bacterial 97 nitroreductase superfamily appear to have unusually plastic active sites (Akiva et al., 2017), 98we considered that simultaneous mass-mutagenesis of up to eight active site residues should 99 be possible. In effect, we aimed to strip NfsA of its engine, and then select for a superior 100 configuration of parts assembled within the empty chassis. By leaping directly to a new 101 fitness peak, we considered that we might arrive at synergistic combinations of substitutions 102 that would be difficult to achieve by itera...