Directed evolution of transketolase to broaden its substrate range for applications in chiral drug synthesis (deoTK)
Final Report Abstract
The French-German collaborative project aimed at the engineering of transketolase by directed evolution to expand its scope for synthetic applications impossible or impractical by using the wild-type enzyme. Tolerance of the enzyme to aliphatic aldehydes became the primary goal. A versatile colorimetric assay was developed for screening of TK activity based on the pH shift occurring upon TK catalyzed decarboxylation of hydroxypyruvate. This generic assay format was validated to be amenable for simple library screening in high-throughput mode and proved highly efficient, sensitive, and reproducible. A survey conducted with different TK sources demonstrated that kinetics for a broad range of substrates could be measured accurately with the novel pH assay, spanning several orders of magnitude of TK activity. By exemplary preparative-scale experiments with ω-hydroxyaldehydes, using a newly developed method for chiral GC analysis, the TK stereoselectivity could be confirmed. For the mutagenesis work, we could establish the first TK from a thermostable organism (Geobacillus stearothermophilus) as a new scaffold for directed evolution. This protein was found to be highly thermostable, retaining 100% activity after 3 days at 65 °C, and highly stereoselective for generating the (3S) configuration. Saturation libraries were constructed at positions Leu382 and Asp470, which are believed to be responsible for binding of 2-hydroxyaldehydes. Two single-site and one double-site libraries with NNS codon degeneracy were built, and altogether ca. 3500 mutant clones were screened with propanal as substrate analog using the pH-based colorimetric assay method. Top 40 mutants verified by re-screening were sequenced and evaluated for absolute kinetics. The top candidate (D470I) showed 17, 13 and 8.6 fold activity improvement towards propanal, butanal and methoxyethanal, respectively, as structurally related substrate analogs.
Publications
-
„Recombinant Transketolases from Geobacillus Strains: Study of Thermostability and Catalytic Properties“, Poster Nr. 28, BioTrans Symposium, Giardini Naxos (IT), 2.-6.10.2011
Abdoul-Zabar, J.; Charmantray, F.; Hélaine, V.; Hecquet, L.; Sorel, I.; Louis, D.; Marlière, P.; Devamani, T.; Yi, D.; Fessner, W.-D.
-
„Remote Enantioselectivity of Transketolase in the Synthesis of Non-Natural Sugars“, Poster Nr. 27, BioTrans Symposium, Giardini Naxos (IT), 2.-6.10.2011
Devamani, T.; Yi, D.; Abdoul-Zabar, J.; Hélaine, V.; Charmantray, F.; Hecquet, L.; Fessner, W.-D.
-
“A pH-Based High-Throughput Assay for Transketolase - Fingerprinting of Substrate Tolerance and Quantitative Kinetics” ChemBioChem, 2012, 13, 2290–2300
Yi, D.; Devamani, T.; Abdoul Zabar, J.; Charmantray, F.; Helaine, V.; Hecquet, L.; Fessner, W.-D.
-
„Enzymatic Properties and Directed Evolution of Transketolase and Chemoenzymatic Synthesis of neo-Sialoconjugates“, Ph.D. Thesis, Technische Universität Darmstadt, 2012
Yi, Dong
-
„Enzymes catalyzing asymmetric carboligation: Directed evolution of substrate scope towards potential industrial applications“, Poster Nr. 12, Catalyzing Bio-economy – Biocatalysts for Industrial Biotechnology, DECHEMA Meeting, Frankfurt (DE), 24.-25.4.2012
Yi, D.; Devamani, T.; Fessner, W.-D.
-
„Transketolase Catalysis for Asymmetric Carboligation: Directed Evolution of Substrate Scope and Stereoselectivity“, Poster Nr. 32, Protein Engineering for Biocatalysis Conference, Greifswald (DE), 29.-31.8.2012
Yi, D.; Devamani, T.; Sorel, I.; Hecquet, L.; Fessner, W.-D.
-
“Thermostable Transketolase from Geobacillus stearothermophilus: Characterization and Catalytic Properties” Adv. Synth. Catal., 2013, 355, 116–128
Abdoul Zabar, J.; Sorel, I.; Helaine, V.; Charmantray, F.; Devamani, T.; Yi, D.; de Bernardinis, V.; Louis, D.; Marliere, P.; Fessner, W.-D.; Hecquet, L.
