Fitness landscapes are an increasingly prevalent tool to analyze the functional effects of mutations in a given genetic context. Fitness landscapes to date have focused on the effects of mutations in one set genetic background, providing a small slice of the adaptive steps available to a given protein. However the non-additive effects of mutations (i.e. epistasis) give rise to rugged landscapes, making predictions of the effect of multiple mutations difficult from the effects of individual mutations. To begin to capture the more relevant adaptive dynamics of evolving protein function, “stacks” of fitness landscapes in differing genetic contexts must be considered concurrently. In this study, we use high-throughput synthetic biology and deep sequencing techniques to generate and combine data from multiple fitness landscapes as a tool to characterize the effects of mutations in the model protein β-lactamase. Specifically, we generated ampicillin fitness landscapes (ampicillin being the originally optimized substrate of β-lactamase) along an adaptive path in the evolution of cefotaxime resistance in β-lactamase. In this path, mutations E104K and/or G238S are acquired, both of which decrease activity on ampicillin but aid in cefotaxime hydrolysis. We use these landscapes to quantify the effects of genetic drift and promiscuity of activity as measured by fitness and epistasis, including calculation of a third-order epistasis value on a scale beyond existing studies. The ruggedness of the fitness landscape originates primarily from G238S, a destabilizing mutation. A high resolution mapping of the effects of drift due to adaptation provides the first highly quantitative account of the secondary, unselected results of evolving functionality, as well as the largest measure of fitness and intragenic epistasis among four genetic backgrounds. Our work contributes to understanding the tradeoffs inherent in the engineering of protein function as well as the spread of antibiotic resistance in nature due to the wide array of substrates of β-lactamase, which has direct relevance to global health.

Fitness landscapes as comprehensive measures of adaptive tradeoffs in the engineering of protein function

249th National Meeting (2015)

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