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

在多元醇和碳水化合物化学领域，区域选择性、化学选择性和立体选择性的固有问题是传统有机合成方法的挑战，要求应用复杂的保护基团策略来防止不希望发生的副反应。相反，生物催化的应用使得保护基团变得不必要，在许多情况下允许减少步骤数和应用温和的反应条件。该项目涉及转酮醇酶(TK)，这是一种依赖于硫胺素的二磷酸酶，催化高立体选择性的C-C键的形成，从而一步合成手性酮糖。TK酶对酮醇供体底物、立体选择性和对映体选择性具有高度的专一性。本项目的目标是通过体外蛋白质工程开发新的优化的TK酶变体，用于四个方向的新的合成应用：(I)改良的立体选择性(Ii)改良的供体特异性(Iii)改良的受体范围(Iv)开发新的反应级联以加速合成新的手性产物。该项目基于G.stearthermophilus的一种新的耐热TK，与现有来源相比，它在TK合成生物催化中的应用具有重大优势。设计具有新性质的TK变体需要通过在活性部位进行随机突变来进行不同程度的复杂程度的蛋白质修饰。从结果中，我们期望能够为未来蛋白质工程任务的适当方法的不断增长的知识库做出贡献，特别是对于尚未被探索的一类碳化酶。对改进的变体的筛选将分三个阶段进行：1)将基于双方共同开发和验证的pH依赖的分析格式以高通量模式分析催化活性；2)将通过分析特定产物的形成来评估克隆的合成活性；3)阳性克隆的立体选择性将被进一步研究。后续的目标将是开发由优化的TK变体催化的集成反应，用于创新的级联酶反应，例如通过将碳化与原位供体底物合成和/或原位产物修饰相结合。该项目是多学科的，包括不对称合成、手性分析、分子生物学、微生物学、酶技术，基于法国在酶学/筛选/诱变方面的合作伙伴和德国在检测技术、底物耐受性突变表征、立体选择性和合成应用方面的互补专业知识。这个联合体的效率已经在以前的ANR/DFG联合项目(DEO TK)中得到了证明。

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).