Engineering a thermostable transketolase by directed evolution: new stereoselectivity, new substrate tolerance, new product scope
Final Report Abstract
The French-German collaborative project aimed at the enzyme re-engineering of a new thermostable transketolase from Geobacillus stearothermophilus by directed evolution to expand its scope for synthetic applications impossible or impractical by using the wild-type enzyme. In search of enzyme variants active on aliphatic aldehydes, two NNK libraries complementary to the initial L382X/D470X library were constructed for the L191X/D470X and F435X/D470X combinations and screened for activity and stereoselectivity with propanal. The most important outcome from the combined investigation of ca. 10 000 clones is that only a very small subset of variants incorporating the L382F replacement was able to guarantee a product ee >99% for aliphatic non-hydroxylated aldehyde electrophiles. This is all the more impressive as no correlated result had ever been observed by other research groups studying the TK from E. coli and yeast. Our structure-independent colorimetric assay based on the pH shift upon TK catalyzed decarboxylation of a nucleophilic substrate proved to be extremely versatile for screening different aspects of substrate variation for TK activity. This generic assay format was used in high-throughput mode for screening libraries for TK activity against arylated substrates, nonnatural (2S) configurated substrates, and non-natural non-hydroxylated nucleophilic components. The assay proved to be highly reliable also for continuous kinetic measurements, always matching preparative verification of synthetic conversions. This is an important advance to other reported assay strategies that rely on discontinuous, laborious sample preparation for single-point HPLC or GC data collection, and allowed rapid progress along the various project objectives. A new thermostable L-serine transaminase was found and applied for the cascade coupling of in situ generation of the unstable hydroxypyruavte substrate with subsequent consumption in situ for stereoselective carboligation. By this way, new preparative applications could be realized for the synthesis of unusual L-ketose sugars. This approach is a first step into rendering the TK catalysis economically viable, as this principle of substrate generation could potentially be transferred to fermentative whole-cell technology. An impressive outcome certainly is the successful re-engineering of the TK enzyme to accept non-hydroxylated 2-oxoacids as alternative nucleophilic substrates. For the first time this development allowed to generate products with flexible determination of structural variations of the backbone that hitherto were not accessible by enzymatic carboligation. The study also demonstrates convincingly the superiority of random over rational protein engineering, as the transfer of a functional environment from another protein scaffold did not yield the significant improvement as expected.
Publications
- “New Applications of Transketolase: Cascade Reactions for Assay Development” in Cascade Biocatalysis (Eds. Riva. S; Fessner, W.-D.), Wiley-VCH, Weinheim, 2014, 315-337
Hecquet, L.; Fessner, W.-D.; Helaine, V.; Charmantray, F.
- “A Thermostable Transketolase Evolved for Aliphatic Aldehyde Acceptors” Chem. Commun., 2015, 51, 480-483
Yi, D.; Saravanan, T.; Devamani, T.; Charmantray, F.; Hecquet, L.; Fessner, W.-D.
(See online at https://doi.org/10.1039/c4cc08436e) - “Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes” Adv. Synth. Catal., 2015, 357, 1715-1720
Abdoul Zabar, J.; Lorilliere, M.; Yi, D.; Saravanan, T.; Devamani, T.; Nauton, L.; Charmantray, F.; Helaine, V.; Fessner, W.-D.; Hecquet, L.
(See online at https://doi.org/10.1002/adsc.201500207) - “Donor Promiscuity of a Thermostable Transketolase Engineered by Directed Evolution – Efficient Complementation of 1-Deoxy-D-xylulose-5-phosphate Synthase Activity” Angew. Chem. 2017, 129, 5442-5447; Angew. Chem. Int. Ed. 2017, 56, 5358-5362
Saravanan, T.; Junker, S.; Kickstein, M.; Hein, S.; Link, M.-K.; Ranglack, J.; Witt, S.; Lorilliere, M.; Hecquet, L.; Fessner, W.-D.
(See online at https://doi.org/10.1002/anie.201701169) - “Engineering a thermostable transketolase for arylated substrates” Green Chem., 2017, 19, 481-489
Saravanan, T.; Reif, M.L.; Yi, D.; Lorilliere, M.; Charmantray, F.; Hecquet, L.; Fessner, W.-D.
(See online at https://doi.org/10.1039/c6gc02017h) - “One-pot, two-step cascade synthesis of naturally rare L-erythro (3S,4S) ketoses by coupling thermostable transaminase and transketolase” Green Chem., 2017, 19, 425-435
Lorilliere, M.; De Sousa, M.; Bruna, F.; Heuson, E.; Gefflaut, T.; de Bernardinis, V.; Saravanan, T.; Yi, D.; Fessner, W.-D.; Charmantray, F.; Hecquet, L.
(See online at https://doi.org/10.1039/c6gc02015a) - “Second Generation Engineering of Thermostable Transketolase (TKgst) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity” ChemBioChem, 2017, 18, 455-459
Zhou, C.; Saravanan, T.; Lorilliere, M.; Wei, D.; Charmantry, F.; Hecquet, L.; Fessner, W.-D.; Yi, D.
(See online at https://doi.org/10.1002/cbic.201600609)
