Cheng, F., Kardashliev, T., Pitzler, C., Shehzad, A., Lue, H., Bernhagen, J., Zhu, L., Schwaneberg, U. 2015. A competitive flow cytometry screening system for directed evolution of therapeutic enzyme. ACS Synth. Biol., 4, 768-775.
A ligand-mediated eGFP-expression system LiMEx was developed as a novel flow cytometry for the detection of subtle differences in intracellular concentrations of metabolites. The screening system is based on competitive relationship between metabolite’s enzymatic depletion and its role in the regulation of reporter expression governs the magnitude of the output signal. The described LiMEx screening platform relies on a competitive conversion/binding of arginine between arginine deiminase and arginine repressor. The principle of LiMEx was validated by evolving arginine deiminase for stronger inhibition of tumor growth. After screening of ∼8.2 × 106 clones in three iterative rounds of epPCR libraries, PpADI variant M31 with reduced S 0.5 value - 0.17 mM compared to 1.23 mM WT, and enhanced activity at physiological arginine concentration - M31: 6.14 s−1 with WT: not detectable was identified. Moreover, M31 showed a significant inhibitory effect against SK-MEL-28 and G361 melanoma cell lines - IC50 = 0.02 μg/mL for SK-MEL-28 and G361.
Figure 1. The Arg-LiMEx high-throughput screening system. Detection principle of the Arg-LiMEx system. eGFP fluorescence depends on intracellular arginine concentration which is determined by PpADI’s performance in converting arginine. Left panel: eGFP expression is inhibited when the argG promoter is blocked in presence of arginine by the ArgR-arginine-ArgR complex; Right panel: eGFP is produced when PpADI can efficiently deplete the intracellular arginine.
Sauer, D. F., Bocola, M., Broglia, C., Arlt, M., Zhu, L., Brocker, M., Schwaneberg, U., Okuda, J. 2015. Hybrid ruthenium ROMP catalysts based on an engineered variant of β-barrel protein FhuA ΔCVFtev: effect of spacer length. Chem. Asian J., 10, 177-182.
Philippart, F., Arlt, M., Gotzen, S., Tenne, J., Bocola, M., Chen, H.H., Zhu, L., Schwaneberg, U.*, Okuda, J.* 2013. A hybrid ring-opening metathesis polymerization catalyst based on engineered ß-barrel protein FhuA. Chemistry, 19, 13865-13871.Bio VI
A FhuA based biohybrid catalyst was developed by covalently anchoring a Grubbs–Hoveyda type olefin metathesis catalyst at a single accessible cysteine amino acid in the barrel interior of FhuAΔCVFtev. Activity of this hybrid catalyst type was successfully demonstrated by ring-opening metathesis polymerization ROMP of a 7-oxanorbornene derivative in aqueous solution. Shortening of the spacer between the N-heterocyclic carbene ligand and the cysteine site increased the ROMP activity toward a water-soluble 7-oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the biohybrid catalyst.
Frauenkron-Machedjou, V. J., Fulton, A., Zhu, L., Bocola, M., Jaeger, K.-E. and Schwaneberg, U. 2015. Towards Understanding Directed Evolution: More than Half of All Amino Acid Positions Contribute to Ionic Liquid Resistance of Bacillus subtilis Lipase A. ChemBioChem, 16, 937-945.
Ionic liquids, IL, are attractive cosolvents for biocatalysis. However, in high concentration of IL solution >10%, enzymes usually show decreased activity. In order to elucidate general principles for guiding protein engineering to improve IL resistance of enzymes, we performed a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete bsla. Screening in presence of four [BMIM]-based ILs revealed two unexpected lessons on directed evolution: a) resistance improvements were obtainable at 50-69% of all amino acid positions, explaining the success rate of small sized random mutant libraries; b) in total 6-13% of substitutions led to improved resistance. Among the beneficial substitutions 66-95% were substituted by chemically different amino acids, e.g. aromatic to polar/aliphatic/charged amino acids, indicating that mutagenesis methods introducing transversions should at least for lipases like BSLA be favored to improve IL resistance.Bio VI
Figure 3. 3D model of BSLA (Protein Databank code: 1I6W) displaying regions which contribute to BSLA resistance in presence of [BMIM]Cl in a color scale. Positions at which no substitution led to improvements are colored grey. Positions with one substitution leading to improvements are colored blue. Positions with two to seven substitutions leading to improvements are colored magnenta. Red represents positions with eight or more beneficial substitutions.