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

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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 transketolase (TK), a thiamine diphosphate-dependent enzyme catalyzing a highly stereoselective C-C bond formation leading in one step to chiral ketoses. TK enzymes are highly specific for ketol donor substrates, stereospecific and enantioselective.The objective of this project will be to develop novel optimized TK enzyme variants by protein engineering in vitro for new synthetic applications in four directions:(i) modified stereoselectivity(ii) modified donor specificity(iii) modified acceptor scope(iv) development of new reaction cascades for accelerated synthesis of new chiral products.The project is based on a new thermostable TK from G. stearothermophilus, which promises major advantages for TK applications in synthetic biocatalysis as compared to current sources.Engineering TK variants for new properties requires protein modifications at various levels of complexity by using random mutagenesis at the active site. From the results, we expect to be able to contribute to the growing knowledge base for appropriate methods to future protein engineering tasks, particularly for the yet less-explored class of carboligation enzymes.Screening for improved variants will be conducted in three stages: 1) catalytic activity will be analyzed in high-throughput mode based on the pH-dependent assay format jointly developed and validated by both partners; 2) synthetic activity of clones will be assessed by analysis for specific product formation; 3) positive clones will be further investigated for their stereoselectivity.Subsequent objectives will be the development of integrated reactions catalyzed by optimized TK variants for innovative cascade enzymatic reactions, e.g. by coupling carboligation to in-situ donor substrate synthesis and/or in-situ product modification. This project is multidisciplinary and comprises aspects of asymmetric synthesis, chiral analysis, molecular biology, microbiology, enzyme technology, based on the complementary expertise of the French partner in enzymology/screening/mutagenesis and by the German partner in assay technology, mutant characterization for substrate tolerance, stereoselectivity, and synthetic applications. The efficiency of this consortium has been already demonstrated in a previous joint ANR/DFG project (deo TK).