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.

Biocontainment is an essential feature when deploying genetically modified organisms (GMOs) in open system applications, as variants escaping their intended operating environments could negatively impact ecosystems and human health. To avoid breaches resulting from metabolic cross-feeding, horizontal gene transfer, and/or genetic mutations, synthetic auxotrophs have been engineered to become dependent on exogenously supplied xenobiotics, such as noncanonical amino acids (ncAAs). The incorporation of these abiological building blocks into essential proteins constitutes a first step toward constructing xenobiological barriers between GMOs and their environments. To transition synthetic auxotrophs further away from familiar biology, we demonstrate how bacterial growth can be confined by transition-metal complexes that catalyze the formation of an essential ncAA through new-to-nature reactions. Specifically, using a homogeneous ruthenium complex enabled us to localize bacterial growth on solid media, while heterogeneous palladium nanoparticles could be recycled and deployed up to five consecutive times to ensure the survival of synthetic auxotrophs in liquid cultures.

Interfacing biocompatible, small‐molecule catalysis with cellular metabolism promises a straightforward introduction of new function into organisms without the need for genetic manipulation. However, identifying and optimizing synthetic catalysts that perform new‐to‐nature transformations under conditions that support life is a cumbersome task. To enable the rapid discovery and fine‐tuning of biocompatible catalysts, we describe a 96‐well screening platform that couples the activity of synthetic catalysts to yield non‐canonical amino acids from appropriate precursors with the subsequent incorporation of these nonstandard building blocks into GFP (quantifiable readout). Critically, this strategy does not only provide a common readout (fluorescence) for different reaction/catalyst combinations, but also informs on the organism's fitness, as stop codon suppression relies on all steps of the central dogma of molecular biology. To showcase our approach, we have applied it to the evaluation and optimization of transition‐metal‐catalyzed deprotection reactions.

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.

NADP + -dependent formate dehydrogenases (FDHs) are biotechnologically relevant enzymes for cofactors regeneration in industrial processes employing redox biocatalysts. Their effective applicability is however hampered by the low cofactor and substrate affinities of the few enzymes described so far. After different efforts to ameliorate the previously studied GraFDH from the acidobacterium Granulicella mallensis MP5ACTX8, an enzyme having double (NAD + and NADP + ) cofactor specificity, we started over our search with the advantage of hindsight. We identified and characterized GraFDH2, a novel highly active FDH, which proved to be a good NAD + -dependent catalyst. A rational engineering approach permitted to switch its cofactor specificity, producing an enzyme variant that displays a 10-fold activity improvement over the wild-type enzyme with NADP + . Such variant resulted to be one of the best performing enzyme among the NADP + -dependent FDHs reported so far in terms of catalytic performance.[a] Dr. the following pH values at 25°C using 5 M NaOH or 5 M HCl: 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 12.0.