E. coli surface display of streptavidin for directed evolution of an allylic deallylase

Abstract

An artificial deallylase is constituted on the E. coli surface and genetically optimized for the deprotection of caged aminocoumarin.

Systematic Engineering of Artificial Metalloenzymes for New-to-Nature Reactions

“…These residues are in close proximity to the biotinylated cofactor 32 (Fig. 1b) and have repeatedly been found to have a substantial impact on the activity of ArMs for diverse cofactors and reactions [33][34][35] . More specifically, we created a combinatorial Sav 112X 121X library (X representing all 20 canonical amino acids) consisting of all 400 possible amino acid combinations for these two positions.…”

“…In order to establish a generalizable first engineering step for ArMs, we sought to generate a library of Sav variants that offers a high likelihood of identifying active variants at a minimized screening effort. Based on previous experience with Sav expression and whole-cell ArM catalysis 34,35 , we selected a periplasmic compartmentalization strategy for this library. Secretion to the periplasm and subsequent ArM assembly represents a good trade-off between accessibility to the cofactor, expression levels and compatibility of the reaction conditions 39 .…”

“…While in some cases wild-type Sav imparts some selectivity or rate acceleration on the reaction, protein engineering is usually required in order to obtain proficient biocatalysts [27][28][29][30][31] . Initial studies in this direction relied on screening (semi-)purified Sav mutants 32,33 , but more recently a trend towards whole-cell screening with Escherichia coli has emerged for Sav-based ArMs 34,35 as well as other artificial enzymes 8,36,37 . This allows significantly increased throughput and is the method of choice if variants that are functional under in vivo conditions are desired, which is an important prerequisite for synthetic biology applications.…”

“…However, a number of challenges arise for cell-based ArM assays, most notably insufficient cofactor uptake into the cell as well as cofactor poisoning by cellular components such as reduced glutathione 38 . In order to circumvent these challenges, Sav has been exported to the periplasmic space 34 or the cell surface 35 . While these studies have established the possibility of engineering ArMs using whole-cell screenings, they did so using case-specific engineering strategies and with a focus on reactions affording fluorescent products.…”

Enabling protein-hosted organocatalytic transformations

“…In addition, the protein hosts can still be engineered via rational design or laboratory evolution. 46,48,[127][128][129] To this end, the supramolecular approach is an important technique for creating articial enzymes. As a rule of thumb, the supramolecular catalytic complexes are built based on protein-ligand interactions that have dissociation constants (K D ) ranging from low mM to pM.…”