A giant leap in sequence space reveals the intracellular complexities of evolving a new function
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Abstract
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.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 19 2020, follows.
Summary
This manuscript describes a laboratory evolution experiment designed to explore effects that may shape evolutionary trajectories in a native host environment. The model system is E. coli nitro/quinone reductase NfsA, a promiscuous FMN-dependent oxidoreductase that reduces toxic compounds and has the basal ability to reduce the antibiotic chloramphenicol. This function was used to select for improved detoxification by mass-mutagenizing eight active-site residues and isolating variants with up to tenfold higher tolerance against chloramphenicol. The five best variant proteins were purified and characterized, showing that their kcat/Km was only marginally improved, with worse kcat but improved Km, …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 19 2020, follows.
Summary
This manuscript describes a laboratory evolution experiment designed to explore effects that may shape evolutionary trajectories in a native host environment. The model system is E. coli nitro/quinone reductase NfsA, a promiscuous FMN-dependent oxidoreductase that reduces toxic compounds and has the basal ability to reduce the antibiotic chloramphenicol. This function was used to select for improved detoxification by mass-mutagenizing eight active-site residues and isolating variants with up to tenfold higher tolerance against chloramphenicol. The five best variant proteins were purified and characterized, showing that their kcat/Km was only marginally improved, with worse kcat but improved Km, indicating that the improvements in detoxification were driven by enhanced substrate affinity. For the top two variants, all possible evolutionary trajectories were recreated and their EC50's tested to determine the most likely possible step-wise paths from NfsA to the final variants. The authors found that iterative evolutionary strategies could have generated similar variants, but that there were only few accessible pathways, indicating epistatic effects. The analysis also showed that for both variants, elimination of arginine at position 225 in the first step enabled further improvements to take hold and played a role in the loss of wildtype 1,4-benzoquinone activity. The sensitivity to four out of five tested prodrugs was however unchanged. Turnover of the fifth prodrug, namely reduction of metronidazole, which yields a toxic product, was on the other hand increased in the evolved variants, and could be used as a counter-selectable marker. This was briefly tested showing the potential of such an application.
Essential Revisions
This study presents a wealth of data, and is well reasoned, carefully executed and clearly laid out. However, although it states that its aim was to study the evolution of a promiscuous function within the native host environment and thus under metabolic interference of the native substrate, this was not the approach taken. Instead, a fitness peak for the promiscuous function was identified through mass mutagenesis at eight positions followed by selection, and then two potential evolutionary paths leading from the wild type to this peak were inferred based on an analysis of all possible mutant combinations at the mutagenized positions. The authors need to make clear throughout the paper that the variants able to detoxify chloramphenicol were not evolved and did not arise against metabolic interference of the native substrate. This is an important point as the considerable potential of endogenous metabolites to shape evolutionary outcomes (Abstract) is purely inferred from the observation that the first mutation in both reconstructed evolutionary paths appears to have been a mutation at R225, which led to a substantial drop in the turnover rate of the endogenous substrate. From this the authors conclude (very prominently throughout the paper) that the evolution of a new activity is only possible after loss of activity against the original substrate.
From the data presented, it is however not clear to what extent this conclusion is supported.
According to the data in Figure 4 and Table S1, mutation of R225 alone is accompanied by a ~2-fold increase in kcat/Km for chloramphenicol. This seems to be sufficient to explain the ~2-fold increase in EC50 for chloramphenicol without invoking loss of quinone reductase activity. The control experiment in Figure 5, showing that substitution of R225 has no effect on most promiscuous activities of NfsA, also seems to indicate that the loss of native activity is not required for the evolution of chloramphenicol resistance. It would be important to determine the kinetic parameters of 1,4-benzoquinone reduction for NfsA and the purified R225V and R225D mutants in order to establish the loss of quinone reductase activity in the postulated first step of the evolutionary path. It would also be useful to study the effect of 1,4-benzoquinone competition on the chloramphenicol reductase activity of the mutants, at least the first ones along the proposed path, in order to show that they rapidly become insensitive to the native substrate.
After the initial screening of the transformed library of NfsA variants, 0.05% of gene variants are reported to be more effective in chloramphenicol detoxification than the wild type. In the next steps, this number is reduced to the top 30 variants, as characterized by their improvement of chloramphenicol EC50 values (Fig. 1D). However, it is not clear from the presentation whether these observations were controlled for the expression levels of the different NfsA mutants. Protein variants are often expressed at different levels in vivo, which can have a significant effect on the activity measured. Fig. 1D was used for selection of the "best" variants for the rest of the study and to support this choice and the conclusions of the manuscript, relative enzyme expression levels should be reported (and if significantly different, should be corrected for). Such expression levels are reported later on for the 36_37 and 20_39 variants, but are missing at this early stage.
While mutation of R225 appeared to be required for improved chloramphenicol detoxification in this study, the authors only considered the effects of substitutions at eight positions. This is probably the main weakness of the combinatorial mutagenesis approach used here. It seems plausible that substitutions at other positions could also increase chloramphenicol tolerance, possibly opening a path without loss of quinone reductase activity. If the authors were able to perform one round of error-prone PCR on NfsA with selection for improved chloramphenicol resistance and obtain mainly variants with substitution of R225, this would substantially strengthen their claim that evolution of increased chloramphenicol resistance can only occur through loss of quinone reductase activity.
Even with additional experimental support for the main conclusion of the article, it seems fundamentally problematic to extrapolate from two instances to a general principle of evolution. The authors should tone done the claims that improved chloramphenicol detox activity is ONLY possible after elimination the native activity and instead comment on the two characterized mutant pathways as examples of this phenomenon, within the limitations of the experimental setup.
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