Engineering a thermostable transketolase by directed evolution: new stereoselectivity, new substrate tolerance, new product scope – thermoTK

In the field of polyols and carbohydrate chemistry, the inherent problems of regioselectivity, chemoselectivity and stereoselectivity are a challenge for traditional methods of organic synthesis, mandating to apply sophisticated protective group strategies in order to prevent undesired side reactions. In contrast, the application of biocatalysis renders protective groups unnecessary and in many cases allows reducing the number of steps and applying mild reaction conditions.
This project concerns the enzyme transketolase (TK), a thiamine diphosphate-dependent enzyme catalyzing a highly stereoselective C-C bond formation leading in one step to chiral polyols, D-threo ketoses (3S,4R). TK enzymes are highly specific for ketol donor substrates, stereospecific and enantioselective.
The objective of this project will be to develop novel optimized TK enzymes by protein engineering in vitro for new synthetic applications in four directions:
(i) enzyme variants with modified stereoselectivity
(ii) modified donor specificity
(iii) modified acceptor scope
(iv) development of new reaction cascades for accelerated synthesis of new chiral products.
All the project will be focused on a new thermostable TK from Geobacillus stearothermophilus (TKgst) recently discovered by the applicants. Its properties promise to offer major advantages for TK applications in synthetic biocatalysis as compared to current sources, including higher tolerance to non-conventional media and to destabilizing factors introduced into the protein by mutagenesis.
Engineering TKgst variants for new properties requires protein modifications at various levels of complexity by using appropriate techniques for random mutagenesis at the active site. From the results, we expect to be able to contribute to the growing knowledge base in choosing appropriate methods for future protein engineering tasks, particularly for the yet less-explored class of carboligation enzymes.
Screening for improved protein variants will be conducted in three stages. First, catalytic activity will be analyzed in high-throughput mode based on the pH-dependent assay format jointly developed and validated by both partners. Second, synthetic activity of clones will be assessed by low-throughput analysis for specific product formation by TLC/HPLC/GC analysis. On a third level, identified positive clones will be further investigated for their stereoselectivity by appropriate methods (NMR, chiral HPLC/GC analysis).
Subsequent main objectives of the project will be the development of integrated reactions catalyzed by optimized TKgst variants for innovative cascade enzymatic reactions, exemplified by coupling carboligation to in-situ donor substrate synthesis and/or further in-situ product modification. The isomerization of ketose-type products arising from TKgst catalysis into corresponding aldose-type isomers by using glucose isomerise is particularly promising for TKgst applications because the available commercial isomerases are typically thermostable enzymes that show very low activity only under ambient conditions.
This project is multidisciplinary and comprises aspects of organic asymmetric synthesis, chiral analysis, molecular biology, microbiology, enzyme technology. The coordination of tasks is based on the complementary expertise provided by the French partner in enzymology/screening/mutagenesis and by the German partner in liquid assay technology, mutant characterization for substrate tolerance, stereoselectivity, and development of synthetic applications for selected targets. The efficiency of this consortium has been already demonstrated in a previous joint ANR/DFG project (deo TK).
Beyond the expertise of the applicants, the utility of results obtained from this project will be further enhanced by external collaborations with European experts for immobilization and bioreaction engineering for optimized productivity using whole-cell catalysis or development of continuous flow systems.