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

这项拟议的研究旨在建立依赖于吡咯喹啉醌(PQQ)的氧化还原酶作为一种新的、通用的工具，用于工业相关产品的全细胞生物催化合成。为此，将建立一条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组成部分，通过使用依赖于PQQ的工程氧化还原酶代谢乙二醇来实现。恶臭假单胞菌KT2440是这种方法的理想平台，因为存在必要的PQQ依赖的氧化系统，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普遍稳健性。后者对于底物(如醇)或相应的产物(如醛或酸)对细胞有毒性的反应特别有价值。依赖于PQQ的氧化还原酶位于细胞的周质中，从而催化胞浆外底物的氧化，这是一种智能和可设计的方法，可以进一步限制对活的生物催化剂的毒性影响。此外，这些酶的独特辅因子(PQQ)将不可逆氧化反应中的电子直接送入呼吸链，消除了其他电子转移辅因子(如NAD(P)H或FADH)通常需要的特定再生周期。为了成功地建立新型全细胞生物催化剂，需要了解干扰酶反应和宿主的遗传稳定性的基础知识，确定感兴趣的酶催化剂的生化特性，并了解负责底物选择性的分子决定因素。因此，这项建议将结合生化和生理学研究、遗传学和“组学”技术以及生物信息学驱动的蛋白质工程，为假单胞菌中依赖PQQ的氧化系统的调节和功能提供新的和令人兴奋的见解，并将用一种新的和非常有前途的生物催化剂来扩展现有的生物氧化工具箱。

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.