Selecting Better Biocatalysts by Complementing Recoded Bacteria**

Abstract

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.

A robust life-or-death selection platform for enzyme evolution

“…Sequentially randomizing positions Leu217 and Phe329 enabled us to identify SmLcar_GY (named after its Leu217Gly and Phe329Tyr substitutions), which displayed shorter lag-times under selective conditions and proved >1,000-times more proficient than the wildtype carbamoylase. 16 Lastly, we also demonstrated that serial passaging could faithfully elicit SmLcar_GY from a small, controlled population containing 20 enzyme variants. 16…”

“…16 Lastly, we also demonstrated that serial passaging could faithfully elicit SmLcar_GY from a small, controlled population containing 20 enzyme variants. 16…”

“…2A ). 16 As proof-of-principle, we applied our platform to engineer a L -N-carbamoylase from Sinorhizobium meliloti strain CECT 4114, SmLcar, 31 to promote the hydrolysis of the N -carbamoylated ncAA, cam- L -3-nitro-tyrosine (cam-3nY, Fig. 2B ).…”

“…To facilitate a more widespread use of life-or-death selections for the directed evolution of biocatalysts, we have recently showcased a chemical complementation strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs, vide infra ). 16 Here, we demonstrate the scalability of our approach and its ability to autonomously elicit a diverse panel of efficient biocatalysts from highly diverse populations. Excitingly, serial passaging proved sufficient to amplify inherently ‘scarce’ variants within these populations, including enzyme variants whose improved performance results from unintended, random mutations that were introduced during PCR cycles or cell division.…”

“…1D ). 13-16 Lastly, previously employed selections for biocatalysts that directly confer a growth advantage to a host organism (e.g. chorismate mutase or β-lactamase) could be employed to identify enzyme variants with a wide range of activities.…”