Scalable, continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

Abstract

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolved the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity (synthesizing L-tryptophan from indole and L-serine). We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of new biomolecular functions.

Integrating continuous hypermutation with high-throughput screening for optimization ofcis,cis-muconic acid production in yeast

“…Ultimately, this could expand both the robustness and catalytic activity of further evolved PCA decarboxylases, but also increase the relatively low number of mutations observed per evolved PCA decarboxylase variant. Furthermore, toggled selection regimes of neutral drifting interrupted by selection (Rix et al, 2020;Zhong et al, 2020), may also increase the hit-rate of the continuous evolution, and limit the false-discovery rate observed in this study (>9/22, >0.45) (Figure 3).…”

“…With orthogonal in vivo evolution machineries at hand, any trait that can be coupled to growth (e.g. antibiotic resistance, tolerance to cultivation conditions, and/or complementation of auxotrophies) enables facile identification of improved target genes without need for direct screening (Esvelt et al, 2011;Ravikumar et al, 2014;García-García et al, 2020;Rix et al, 2020).…”

Rapid generation of potent antibodies by autonomous hypermutation in yeast

“…This highlights the value of experimental parallelizability available to AHEAD: through evolution experiments that kept each parent clone's lineages separate, early high achievers such as RBD6 could not outcompete the initially low performing lineages that ultimately gave rise to the most potent neutralizers. This is akin to 'demes' in natural evolution, which act to reduce clonal interference and increase overall functional diversity (25,26) Indeed, AHEAD's ability to maximize diversity of high-affinity clones through independent experiments should be valuable in all antibody generation campaigns, given that antibody performance depends on secondary features beyond affinity alone.…”