Protein design and host engineering for whole-cell biocatalysis using pyrroloquinoline quinone (PQQ)-dependent oxidoreductases

使用吡咯喹啉醌 (PQQ) 依赖性氧化还原酶进行全细胞生物催化的蛋白质设计和宿主工程

• 批准号: 254226746
• 负责人: Dr. Janosch Klebensberger
• 金额: --
• 依托单位: Abteilung für Technische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/254226746?language=en
• 关键词: Protein; design; host; engineering; whole

The proposed research aims to establish pyrroloquinoline quinone (PQQ)-dependent oxidoreductases as a novel, versatile tool for the whole-cell biocatalytic synthesis of industrially relevant products. For this, a synthetic pathway for the production of glyoxylic acid, an important building block for the synthesis of many agrochemicals and polymers, via the metabolism of ethylene glycol using engineered PQQ-dependent oxidoreductases will be established. Pseudomonas putida KT2440 represents an ideal platform for such an approach, due to the presence of the necessary PQQ-dependent oxidation system including cofactor biosynthesis, the excellent available genetic tools, and the general robustness towards harsh environmental conditions. The latter is especially valuable for reactions, in which substrates - such as alcohols - or the corresponding products - such as aldehydes or acids - are toxic to the cells. The use of PQQ-dependent oxidoreductases, which are located in the periplasm of the cell and, thus, catalyze the oxidation of the substrate outside of the cytosol, represents an intelligent and designable way to further limit the toxic effects for a living biocatalyst. In addition, the unique cofactor of these enzymes (PQQ) which shuttles the electrons from the irreversible oxidation reaction directly into the respiratory chain, eliminates a specific regeneration cycle that usually is needed for other electron-transferring cofactors, such as NAD(P)H or FADH.For the successful establishment of a novel whole-cell biocatalyst, the fundamental knowledge of interfering enzyme reactions and genetic stability of the host, defined biochemical properties for the enzymatic catalyst of interest, and an understanding of the molecular determinants responsible for substrate selectivity, are required. Thus, this proposal will combine biochemical and physiological studies, genetics and ´Omics´ techniques, and bioinformatics-driven protein engineering, to provide new and exciting insights into the regulation and function of PQQ-dependent oxidation systems in Pseudomonads, and will expand the existing toolbox for bio-oxidations with a novel and highly promising biocatalyst.

拟议的研究旨在建立吡咯喹啉醌（PQQ）依赖性氧化还原酶作为一种新的，多功能的工具，全细胞生物催化合成的工业相关产品。为此，将建立一种用于生产乙醛酸的合成途径，乙醛酸是合成许多农用化学品和聚合物的重要构件，其通过使用工程化的PQQ依赖性氧化还原酶代谢乙二醇来进行。恶臭假单胞菌KT 2440代表了这种方法的理想平台，由于存在必要的PQQ依赖性氧化系统，包括辅因子生物合成，优秀的可用遗传工具，以及对恶劣环境条件的一般鲁棒性。后者对于反应特别有价值，其中底物-如醇-或相应的产物-如醛或酸-对细胞有毒。PQQ依赖性氧化还原酶位于细胞的周质中，因此催化细胞质外底物的氧化，其使用代表了进一步限制活生物催化剂的毒性作用的智能和可设计的方式。此外，这些酶的独特辅因子（PQQ）将电子从不可逆氧化反应直接穿梭到呼吸链中，消除了其他电子转移辅因子（如NAD（P）H或FADH）通常所需的特定再生循环。为了成功建立新型全细胞生物催化剂，干扰酶反应和宿主遗传稳定性的基础知识，需要确定感兴趣的酶催化剂的生物化学性质，并理解负责底物选择性的分子决定因素。因此，该提案将结合联合收割机生物化学和生理学研究，遗传学和“组学”技术，以及生物信息学驱动的蛋白质工程，为假单胞菌中PQQ依赖的氧化系统的调节和功能提供新的和令人兴奋的见解，并将扩展现有的生物氧化工具箱，具有新的和非常有前途的生物催化剂。