Systematic engineering of artificial metalloenzymes for new-to-nature reactions

Abstract

Artificial metalloenzymes (ArMs) catalyzing new-to-nature reactions could play an important role in transitioning toward a sustainable economy. While ArMs have been created for various transformations, attempts at their genetic optimization have been case specific and resulted mostly in modest improvements. To realize their full potential, methods to rapidly discover active ArM variants for ideally any reaction of interest are required. Here, we introduce a reaction-independent, automation-compatible platform, which relies on periplasmic compartmentalization in Escherichia coli to rapidly and reliably engineer ArMs based on the biotin-streptavidin technology. We systematically assess 400 ArM mutants for five bioorthogonal transformations involving different metals, reaction mechanisms, and reactants, which include novel ArMs for gold-catalyzed hydroamination and hydroarylation. Activity enhancements up to 15-fold highlight the potential of the systematic approach. Furthermore, we suggest smart screening strategies and build machine learning models that accurately predict ArM activity from sequence, which has crucial implications for future ArM development.

Directed Evolution of a Surface-Displayed Artificial Allylic Deallylase Relying on a GFP Reporter Protein

“…Comparison of the in vivo and in vitro ADAse activities revealed striking differences, Figure 5 . Although variable expression levels may account for marked differences in vivo enzymatic activities ( Figure S6 ), 25 past experience with Sav expression levels 28 led us to investigate an alternative hypothesis. Indeed, we surmised that fusing each Sav monomer (16.7 kDa) with a hydrophobic Lpp-OmpA insert (15.3 kDa) may affect the quaternary structure of surface-displayed Sav.…”

“… 25 , 26 The efficiency of this system was improved via iterative saturation mutagenesis of the position S112 and K121. 17 , 27 , 28 The reaction was monitored via the fluorescence of the uncaged aminocoumarin 4 , Figure 1 . We know from previous Sav-ArM evolution campaigns, that the iterative site-saturation mutagenesis at positions S112 and K121 often leads to significant improvement in catalytic performance, as these two positions lie in close proximity to the cofactor.…”

Single-Round Remodeling of the Active Site of an Artificial Metalloenzyme using an Ultrahigh-Throughput Double Emulsion Screening Assay

“…The catalytic system used in this work is the previously described ArM based on Sav compartmentalized in the periplasm and on the biotinylated ruthenium cofactor 1. [26] The E. coli bears two different plasmids: i) a pET30b vector encoding a T7-tagged Sav fused to the signal peptide of the outer membrane protein A (OmpA) with kanamycin resistance (Fig. 1b, 1) and ii) a pSC101 vector encoding mNectarine with chloramphenicol resistance (Fig.…”

“…3a and 3b). [26] The DE sample was sorted by FACS according to the top 5 % droplets with highest coumarin FI (Fig. 3c).…”

Exporting Metal‐Carbene Chemistry to Live Mammalian Cells: Copper‐Catalyzed Intracellular Synthesis of Quinoxalines Enabled by N−H Carbene Insertions

“…In recent years, there has been an impressive progress in the creation of laboratory versions of metalloenzymes that catalyze "new-to-nature" reactions. [3][4][5][6][7][8][9] However, the application of these catalytic metalloproteins has been essentially restricted to the realm of synthetic methodology. Their use for biological purposes, in the natural environments of enzymes (living cells or organisms), is more challenging, and remains to be uncovered.…”