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Directed evolution of artificial metalloenzymes for in vivo metathesis



The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions1. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions2,3. 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 exist3: repurposing natural metalloenzymes for abiotic transformations2,4; in silico metalloenzyme (re-)design5,6,7; and incorporation of abiotic cofactors into proteins8,9,10,11. 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 protein12,13,14,15. 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 reaction16,17,18,19,20,21,22 without equivalent in nature. Building on previous work6 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 inhibitor15. 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.

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Figure 1: Artificial metalloenzymes for in vivo metathesis.
Figure 2: Directed evolution of an artificial metathase.
Figure 3: Characterization of artificial metathases.
Figure 4: Substrate-specific evolution of an artificial metathase.

Accession codes

Data deposits

The X-ray structures of the artificial metathases have been deposited in the Protein Data Bank (PDB) under accession numbers 5F2B and 5IRA.


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We thank P. Marlière for discussions. This work was supported by funding from the European Commission Seventh Framework Programme [289572-METACODE] and the Swiss National Science Foundation as part of the NCCR Molecular Systems Engineering. We thank M. Dessing and the single-cell facility (D-BSSE, ETH Zurich) for assistance with flow cytometry.

Author information

Authors and Affiliations



T.R.W. and S.P. conceived the project. M.J. developed the periplasmic screening platform and performed in vivo and directed evolution experiments. R.R. synthesized the cofactor and substrates. M.J. and R.R. performed the in vitro experiments. T.H. conducted the crystallography studies. C.T. performed ICP-OES and J.K. expressed and purified protein variants. T.R.W. and S.P. supervised the project. M.J., T.R.W. and S.P. wrote the manuscript.

Corresponding authors

Correspondence to Sven Panke or Thomas R. Ward.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Batch-to-batch reproducibility of different protein preparations.

The activity of the evolved artificial metathase biot-Ru–SAVmut was evaluated for three independently produced and purified protein batches and compared to two independent batches of non-mutated SAV (biot-Ru–SAV). Reactions were performed at 4 mM of substrate 1, 50 μM of biot-Ru and 100 μM of of SAV-variant binding sites, and the product 2 was quantified by fluorescence. The displayed activities represent the initial slopes of the fluorescence signal in the linear reaction phase relative to the activity of free biot-Ru, which was defined as 100%. Bar heights represent the average relative activity of four replicate in vitro experiments (n = 4), with 1 s.d. indicated as error bars.

Extended Data Figure 2 Influence of cofactor loading on the activity of the artificial metathase.

The activity of the evolved artificial metathase biot-Ru–SAVmut was evaluated for different ratios between the cofactor biot-Ru and SAVmut binding sites (biot-Ru:SAVmutb.s.). Reactions were performed at 4 mM of substrate 1, 5 μM of biot-Ru and varying concentrations of SAVmut binding sites (5–50 μM corresponding to ratios ranging from 1:1 to 1:10). The product 2 was quantified by fluorescence. The displayed activities represent the initial slopes of the fluorescence signal in the linear reaction phase. Bar heights represent the average relative activity of four replicate samples (n = 4), with 1 s.d. indicated as error bars.

Extended Data Figure 3 Conformers of the artificial metathases.

af, Close-up views of the crystal structure of the complexes biot-Ru–SAVmut (a, c, e) and biot-Ru–SAV (b, d, f) displaying pairs of cis-symmetry-related biot-Ru cofactors (view perpendicular to a two-fold crystal symmetry axis, displayed in red): conformers I–I (severe steric clash; a, b), conformers II–II (c, d) and conformers I–II (e, f). The protein was removed for displaying purposes. The cofactors are contoured with 2Fo − Fc electron density in blue at 1σ and anomalous dispersion density in red at 3.5σ.

Extended Data Figure 4 Binding of the cofactor biot-Ru in crystal structures.

ad, Binding of biot-Ru in complex with SAV (a, b) and SAVmut (c, d). For clarity, only one biot-Ru cofactor (ruthenium represented as green spheres) per opposing SAV or SAVmut monomer pair (grey and pink ribbon traces indicate two monomers facing each other) is depicted in conformations I (a, c) and II (b, d). Key amino acid residues including mutations V47A, N49K, T114Q, A119G and K121R for SAVmut (c, d) are represented in stick models. Hydrogen-bonds are depicted as dashed lines. Water is shown as red spheres.

Extended Data Figure 5 Flexibility of residues within loop-7,8.

a, Comparison of mobility in loop-7,8 before (biot-Ru–SAV) and after (biot-Ru–SAVmut) directed evolution as highlighted by Cα atom B factors (colour scale). Mutations are indicated in two opposing monomers by asterisks: *1/*1′, V47A; *2/*2′, N49K; *3/*3′, T114Q; *4/*4′, A119G; and *5/*5′, K121R. b, Normalized (B/Baverage) Cα atom B factors of residues within loop-7,8 from different SAV structures that crystalize in the space group I4122 with similar unit cell dimensions. biot-Ru–SAVmut (dark blue) and biot-Ru–SAV (light blue) are compared to PDB reference structures (shades of grey): r1, 2QCB; r2, 3PK2; r3, 2WPU; r4, 4GJV; r5, 4OKA; r6, 2IZJ; and r7, 2IZB.

Extended Data Figure 6 Structures of commercial metathesis catalysts used here.

a, b, The second-generation Hoveyda–Grubbs (HGII) catalyst (a) was obtained from Sigma Aldrich (Buchs) and the Aquamet (AQM) catalyst (b) was purchased from Apeiron Synthesis S.A.

Extended Data Figure 7 Summary of the results obtained by UPLC-MS for the 96-well plate in vivo screen of the dallyl-sulfonamide 5.

Saturation mutagenesis at position 121 (K121X) was performed on SAVmut(47A/49K/114Q/119G/121K). Heights of the navy and blue bars represent the average of three replicates (n = 3), with 1 s.d. indicated as error bars; purple bars represent the value of a single measurement (n = 1).

Extended Data Table 1 Influence of glutathione on the activity of biot-Ru–SAV
Extended Data Table 2 Turnover number of artificial metathases for the conversion of the umbelliferone precursor 1

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Jeschek, M., Reuter, R., Heinisch, T. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016).

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