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

Gordon Rix,Chang C Liu,Patrick J Almhjell,Frances H Arnold,Christina E Boville,Ella J Watkins-Dulaney

doi:10.1038/s41467-020-19539-6

Gordon Rix, Chang C Liu + Show 4 more

Open Access

https://doi.org/10.1038/s41467-020-19539-6

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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 evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of 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 biomolecular functions.

Highlights

Enzyme orthologs sharing identical primary functions can have different promiscuous activities
Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent coupling of L-serine and indole to generate L-tryptophan (Trp) in the presence of the tryptophan synthase α-subunit, TmTrpA19
The selection on TmTrpB’s primary activity would be multidimensional—standalone function, temperature, and neutral drift implemented when desired— and could result in complex evolutionary pathways that serve our goal of maximizing functional variant diversity across replicate evolution experiments

Summary

Introduction

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. Such variation may be attributed to the deep and distinct evolutionary histories shaping each ortholog, including long periods of neutral drift, recalibration of primary activity, and adaptation to new host environments such as temperature These rich histories act to produce extensive genetic diversity, which underpins different promiscuity profiles[2]. Inspired by the remarkable ability of enzyme orthologs to encompass promiscuous activities, we asked whether we could extend the substrate scope of useful enzymes by evolving multiple versions of an enzyme in the laboratory, selecting only for its primary function This idea has been explored before using classical directed evolution approaches, most notably through the generation of cryptic genetic variation with neutral drift libraries[11,12,13,14], we recognized that our recently developed continuous evolution system, OrthoRep, may be considerably better poised for this challenge[15,16]. Systems that better mimic the depth and scale of natural enzyme evolution, but on laboratory timescales, are needed for the effective generation of enzyme variants that begin to approach the genetic and promiscuity profile diversity of orthologs

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