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In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non‐canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild‐type enzyme for ncAA‐precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts. [ABSTRACT FROM AUTHOR]

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4‐oxalocrotonate tautomerase (4‐OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem‐fused 4‐OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4‐OT was then subjected to eleven rounds of directed evolution to give variant 4‐OT(F11), which showed an up to 320‐fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4‐OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins. [ABSTRACT FROM AUTHOR]

Rücktitelbild: Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme (Angew. Keywords: Biocatalysis; Directed Evolution; Enzyme Engineering; Gene Fusion; Michael Addition EN Biocatalysis Directed Evolution Enzyme Engineering Gene Fusion Michael Addition 1 1 1 02/09/22 20220214 NES 220214 B Die Genfusion b ist ein wichtiger natürlicher Prozess zur Erzeugung neuer Enzyme aus einfachen Vorläufern. Biocatalysis, Directed Evolution, Enzyme Engineering, Gene Fusion, Michael Addition. [Extracted from the article]

Keywords: Biocatalysis; Directed Evolution; Enzyme Engineering; Gene Fusion; Michael Addition EN Biocatalysis Directed Evolution Enzyme Engineering Gene Fusion Michael Addition 1 1 1 02/09/22 20220214 NES 220214 B Gene fusion b is an important natural process to create new enzymes from simpler ancestors. Biocatalysis, Michael Addition, Directed Evolution, Enzyme Engineering, Gene Fusion Back Cover: Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme (Angew. [Extracted from the article]