Validation and Stabilization of a Prophage Lysin of Clostridium perfringens by Using Yeast Surface Display and Coevolutionary Models

Abstract

Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a Clostridium perfringens genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C. IMPORTANCE Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.

Directed evolution methods for overcoming trade‐offs between protein activity and stability

“…Moreover, they also found that mutations beneficial for both activity and solubility are extremely rare (~0.05–0.15%), which appears to explain previous observations of trade‐offs between these and related properties such as stability . This and other deep sequencing studies are enabling unprecedented analysis of trade‐offs between enzyme activity, stability and solubility, which is improving the systematic and reliable generation of enzymes with optimized properties.…”

“…Given the unimaginably large chemical diversity that is possible for all combinations of mutations within a single protein, an important future direction will be to further improve library design methods to focus amino acid diversity in protein libraries on the small fraction of sequence space that is likely to yield both highly active and stable proteins. This ambitious goal—which is starting to be realized—will require improvements in the prediction of both activity‐enhancing and stabilizing mutations, and assessment of the complex interplay between these two properties. Combining such novel library design approaches with emerging screening methods that enable unprecedented throughput are expected to lead to successful generation of proteins with levels and types of activities that have previously been unattainable to protein engineers.…”

Computationally Aided Discovery of LysEFm5 Variants with Improved Catalytic Activity and Stability

“…5). Halo formation did not occur when E. coli transformed with a plasmid encoding a phage lysin with specific activity against Clostridium perfringens (42) was plated in an identical manner (see Fig. S2 in the supplemental material).…”

“…The remaining seven libraries showed high rates of activity retention (84% to 100%) but no discernible trend in average statistical fitness. Assuming that the results of the halo assay are a monotonic function of the total lysin activity, the relationship between halo-forming variants as a function of the statistical fitness is expected to be sigmoidal in nature (42). The observed similar fractions of active variants for libraries 1 to 7 suggest that the WT lies to the far right of this sigmoid (corresponding to high fitness), such that meaningful differences in the fraction of halo-forming variants would only be seen at considerably lower statistical fitness values, such as the value observed for library 8.…”

“…It was determined that an IPTG concentration of 0.05 mM, 1.0% (wt/vol) agar, and an incubation time of 19 h were appropriate. Additionally, a saturated culture of E. coli expressing a phage lysin with specific activity against Clostridium perfringens (42) was plated alongside a positive control. At no time did halos form on the plate containing the C. perfringens-specific lysin (Fig.…”