Directed evolution of artificial metalloenzymes for in vivo metathesis

Abstract

The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist: repurposing natural metalloenzymes for abiotic transformations; in silico metalloenzyme (re-)design; and incorporation of abiotic cofactors into proteins. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature. Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the 'metathase' because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.

CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9

“…Directed evolution is a powerful approach and has been extensively used for functional analysis and optimization of DNA sequences, gene functions and protein structures (Buller et al, 2015;Jeschek et al, 2016). In order to employ directed evolution, both the strategy for creating mutant libraries, as well as the assays for screening and selection of improved variants in high throughput, need to be considered (DeLoache et al, 2015;Körfer et al, 2016;Wong et al, 2004).…”

Expanding the boundary of biocatalysis: design and optimization of in vitro tandem catalytic reactions for biochemical production

“…Using cellular membrane as a barrier to protect metal catalysts from cellular inhibitors is an attractive strategy. Recently, Ward and coworkers created an E. coli strain for periplasmic expression of Sav with a biotinylated Hoveyda-Grubbs catalyst for olefin metathesis in vivo (Jeschek et al, 2016). The periplasm provided an environment with low concentrations of inhibitors for metathesis and thus enabled the high-throughput directed evolution of artificial metalloenzymes in vivo .…”

Metal Ions Introduced to Proteins by Supramolecular Ligands

“…In practice, however, this is a very challenging task. An interesting solution is proposed in [19], where a metal ion was attached to streptavidin by connecting the Ru complex with biotin via a carboxyl group. This resulted in catalytic activity even though the complexed ion was located nearly on the surface (the biotin binding cavity has a depth of approximately 15Ǻ, which coincides with the length of the complex) [20].…”