KRED-Origami: Synthesis and Characterization of Ketoreductase Cascades Assembled on DNA Origami Nanostructures

KRED-Origami：DNA 折纸纳米结构上组装的酮还原酶级联的合成和表征

• 批准号: 335742134
• 负责人: Professor Dr. Christof M. Niemeyer
• 金额: --
• 依托单位: Institut für biologische Grenzflächen (IBG)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/335742134?language=en
• 关键词: KRED; Origami; Synthesis; Characterization; Ketoreductase

This project aims to harness concepts of DNA-nanotechnology for the production, characterization and understanding of functional, spatially organized biomolecular networks. We will synthesize and characterize supramolecular DNA origami constructs bearing distinctive arrangements of recombinant ketoreductases (KRED) and NAD(P)H regenerating enzymes (NRE). A chromatographic assay based on the stereoselective conversion of a CS-symmetric nitrodiketone will be used as read-out to generate quantitative data on the overall stereoselectivity of the supramolecular constructs as well as on the activity of the individual KREDs built into these constructs. Specific tasks include the molecular cloning of bioconjugatable KREDs and NREs, the establishment of site-selective DNA-ligation methods, the assembly and analysis of origami-based NRE-KRED cascades, and the study of cascades bearing both (S)- and (R)-selective KREDs to control the overall stereoselectivity of the constructs. Supported by theoretical work based on reaction-diffusion models, we plan to identify construction rules for effective systems which aid in the understanding of the fundamental mechanisms of spatially organized, diffusion-based, multi-center biocatalysts and open the door to future applications.

该项目旨在利用DNA纳米技术的概念来生产、表征和理解功能的、空间上有组织的生物分子网络。我们将合成并表征具有独特排列的重组酮还原酶(KRED)和NAD(P)H再生酶(NRE)的超分子DNA折纸结构。基于CS对称的硝基二酮的立体选择性转化的层析分析将被用作读出，以产生关于超分子构造的总体立体选择性以及构建在这些构造中的单个KRED的活性的定量数据。具体任务包括生物可结合KRED和NRES的分子克隆，建立位点选择性连接方法，基于折纸的NRE-KRED级联的组装和分析，以及同时具有(S)和(R)选择性KRED的级联研究，以控制构建物的整体立体选择性。在基于反应-扩散模型的理论工作的支持下，我们计划确定有效系统的构建规则，这些规则有助于理解空间组织的、基于扩散的、多中心生物催化剂的基本机制，并为未来的应用打开大门。

项目成果

Valency engineering of monomeric enzymes for self-assembling biocatalytic hydrogels

• DOI: 10.1039/c9sc04074a
• 发表时间: 2019-11-14
• 期刊: CHEMICAL SCIENCE
• 影响因子: 8.4
• 作者: Bitterwolf, Patrick;Gallus, Sabrina;Niemeyer, Christof M.
• 通讯作者: Niemeyer, Christof M.

Self-Immobilizing Fusion Enzymes for Compartmentalized Biocatalysis

• DOI: 10.1021/acscatal.7b02230
• 发表时间: 2017-11-01
• 期刊: ACS CATALYSIS
• 影响因子: 12.9
• 作者: Peschke, Theo;Skoupi, Marc;Niemeyer, Christof M.
• 通讯作者: Niemeyer, Christof M.

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

• DOI: 10.1021/acscatal.1c02076
• 发表时间: 2021-05
• 期刊: ACS Catalysis
• 影响因子: 12.9
• 作者: F. Ott;Kersten S. Rabe;C. Niemeyer;G. Gygli

Evaluation of a Microreactor for Flow Biocatalysis by Combined Theory and Experiment

• DOI: 10.1002/cctc.202000145
• 发表时间: 2020-05-07
• 期刊: CHEMCATCHEM
• 影响因子: 4.5
• 作者: Burgahn, Teresa;Pietrek, Philip;Niemeyer, Christof M.
• 通讯作者: Niemeyer, Christof M.