Chemical Engineering Seminar
In the fitness landscape metaphor for molecular evolution, evolutionary pathways are presumed to follow uphill steps of increasing fitness. Accordingly, positive selection (i.e. selecting for variants with improved fitness) is the near universal strategy for improving protein function using directed evolution. Neutral drift is an alternative strategy in which proteins are first selected for maintenance of fitness before being subjected to positive selection. Neutral drift can offer paths to different evolutionary outcomes by expanding the diversity of robust molecules subjected to positive selection. However, neither strategy enables the crossing of fitness valleys in order to reach distant, potentially fitter peaks. We hypothesized that negative selection, here defined as the selection for variants with low but non-zero fitness, might be utilized to reach novel improved variants that are inaccessible by positive selection or neutral drift. Such a strategy might also be useful on rugged landscapes characterized by context-dependent mutational effects.
We experimentally tested our hypothesis on the antibiotic resistance gene TEM-15 β-lactamase by comparing four evolutionary strategies for increasing antibiotic resistance: positive selection, neutral drift, negative selection, and an oscillating combination of negative selection and neutral drift. All strategies ended with several rounds of positive selection. Interestingly, the strategy that started with several rounds of negative selection (i.e. selection for alleles providing low antibiotic resistance) produced superior alleles than the other three strategies. We comprehensively examined possible evolutionary pathways leading to one such high fitness allele and found that an initially deleterious mutation is key to the allele's evolutionary history. This mutation is an initial gateway to an otherwise relatively inaccessible area of sequence space and participates in higher-order, positive epistasis with a number of neutral to slightly beneficial mutations. To gain insight into potential evolutionary pathways to this allele, we developed selection-weighted attraction graphing (SWAG) a force-directed graphing method for landscape visualization that offers intuitive insight into landscape features and evolutionary pathways. The ability of negative selection to provide access to novel fitness peaks has important implications for directed evolution as well as natural evolutionary mechanisms.