Directed evolution has revolutionized biomolecular engineering by applying cycles of mutation, amplification and selection to genes of interest (GOIs). However, classical directed evolution methods that rely on manually staged evolutionary cycles constrain the scale and depth of the evolutionary search that is possible. We describe genetic systems that achieve cycles of rapid mutation, amplification and selection fully inside living cells, enabling the continuous evolution of GOIs as cells grow. These systems advance the scale, evolutionary search depth, ease and overall power of directed evolution and access important new areas of protein evolution and engineering.
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.
The standard proteinogenic amino acids grant access to a myriad of chemistries that harmonize to create life. Outside of these twenty canonical protein building blocks are countless noncanonical amino acids (ncAAs), either found in nature or created by man. Interest in ncAAs has grown as research has unveiled their importance as precursors to natural products and pharmaceuticals, biological probes, and more. Despite their broad applications, synthesis of ncAAs remains a challenge, as poor stereoselectivity and low functional-group compatibility stymie effective preparative routes. The use of enzymes has emerged as a versatile approach to prepare ncAAs, and nature’s enzymes can be engineered to synthesize ncAAs more efficiently and expand the amino acid alphabet. In this tutorial review, we briefly outline different enzyme engineering strategies and then discuss examples where engineering has generated new ‘ncAA synthases’ for efficient, environmentally benign production of a wide and growing collection of valuable ncAAs.
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