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

通过定向进化设计热稳定性转酮醇酶：新的立体选择性、新的底物耐受性、新的产品范围

• 批准号: 242577053
• 负责人: Professor Dr. Wolf-Dieter Fessner
• 金额: --
• 依托单位: Arbeitsgruppe Organische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/242577053?language=en
• 关键词: Engineering; thermostable; transketolase; directed; evolution

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).

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以防止不期望的副反应。相比之下，生物催化的应用使得保护基团不必要，并且在许多情况下允许减少步骤的数量并应用温和的反应条件。本项目涉及转酮醇酶（TK），一种二磷酸硫胺素依赖性酶，催化高度立体选择性的C-C键形成，一步导致手性酮糖。TK酶对酮醇供体底物具有高度特异性、立体特异性和对映选择性，本项目的目标是通过体外蛋白质工程开发新型优化的TK酶变体，用于四个方向的新的合成应用：（i）改进的立体选择性（ii）改进的供体特异性（iii）改进的受体范围（iv）开发新的反应级联以加速新手性产物的合成。stearothermophilus的，这承诺的主要优势TK应用在合成的生物催化相比，目前的sources.Engineering TK变体的新特性需要在各种复杂程度的蛋白质修饰，通过使用随机诱变的活性位点。从这些结果中，我们期望能够为未来蛋白质工程任务的合适方法提供不断增长的知识基础，特别是对于尚未开发的碳连接酶。筛选改进的变体将分三个阶段进行：1）基于双方共同开发和验证的pH依赖性测定格式，以高通量模式分析催化活性; 2）通过分析特定产物的形成来评估克隆的合成活性; 3）将进一步研究阳性克隆的立体选择性。随后的目标将是开发由优化的TK变体催化的集成反应，用于创新的级联酶促反应，例如通过将碳化偶联到原位供体底物合成和/或原位产物修饰。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，酶技术方面，基于法国合作伙伴在酶学/筛选/诱变方面的互补专业知识，以及德国合作伙伴在分析技术，突变体表征底物耐受性，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）中得到证明。

项目成果

Engineering a thermostable transketolase for unnatural conversion of (2S)-hydroxyaldehydes

• DOI: 10.1002/adsc.201500207
• 发表时间: 2015-05
• 期刊: Advanced Synthesis & Catalysis
• 影响因子: 5.4
• 作者: Juliane Abdoul Zabar;Marion Lorillière;D. Yi;T. Saravanan;T. Devamani;L. Nauton;F. Charmantray

Engineering a thermostable transketolase for arylated substrates

• DOI: 10.1039/c6gc02017h
• 发表时间: 2017-01-21
• 期刊: GREEN CHEMISTRY
• 影响因子: 9.8
• 作者: Saravanan, Thangavelu;Reif, Marie-Luise;Fessner, Wolf-Dieter
• 通讯作者: Fessner, Wolf-Dieter

A thermostable transketolase evolved for aliphatic aldehyde acceptors.

• DOI: 10.1039/c4cc08436e
• 发表时间: 2015-01
• 期刊: Chemical communications
• 影响因子: 4.9
• 作者: D. Yi;T. Saravanan;T. Devamani;F. Charmantray;L. Hecquet;W. Fessner

One-pot, two-step cascade synthesis of naturally rare L-erythro (3S,4S) ketoses by coupling a thermostable transaminase and transketolase

• DOI: 10.1039/c6gc02015a
• 发表时间: 2017-01-21
• 期刊: GREEN CHEMISTRY
• 影响因子: 9.8
• 作者: Lorilliere, Marion;De Sousa, Maxime;Hecquet, Laurence
• 通讯作者: Hecquet, Laurence