基于锌指蛋白DNA支架的多酶体系合成类胡萝卜素研究

Carotenoids are an important class of terpenoids which are widely used in the fields of clinical medicine, food and health care because of their relatively high antioxidant activity. But for the factors of long natural synthesis pathway, multi-enzyme catalyzing and low level of the production, the large-scale applications of carotenoids are restricted. Traditional operations of genetic engineering techniques which ignore the multi-enzyme collaboration and the effects of substrate channel are unable to achieve precise control to the multi-enzyme catalyzed reactions. This study is aiming to solve the problems for the synthesis of complex compounds with multi-enzyme catalyzing, such as Deinoxanthin. Based on the idea of building modules with synthetic biology, DNA scaffold is used to achieve self-assembly of multi-enzymes according to the catalytic order for the first time. It involves conversion of individual enzymes into DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. Green fluorescent protein is used to characterize self-assembly of multi-enzymes on DNA scaffold according to catalytic order. By adjusting DNA sequences on scaffold, the stoichiometric ratio and spatial distance of enzymes in host can be regulated precisely to achieve effect of substrate channel. So an efficient channel of carotenoid synthesis is constructed. By means of synthetic biology technology and DNA scaffold, this optimized system for Deinoxanthin production will significantly increased the efficiency of carotenoid biosynthetic pathway in E. coli. DNA scaffold system can be used as a platform technology for the construction and optimization of artificial metabolic pathways as well as for the production of many functional terpenoids and complex compounds. It is helpful to establish a new method and to illustrate regulating mechanism for multi-enzyme synthesis.

类胡萝卜素是一类具有抗氧化活性的重要萜类化合物，在临床医学、食品、保健领域应用广泛，天然合成代谢途径长、多酶参与、含量低等因素成为制约它们规模化应用的瓶颈。传统改造方法忽略了多酶协同作用及底物通道效应，无法实现对催化体系的精确调控。本课题针对多酶合成复杂化合物中普遍存在的问题，以类胡萝卜素Deinoxanthin多酶合成体系为研究对象，首次尝试将DNA支架技术引入萜类化合物合成中，以合成生物学创新理念构建类胡萝卜素合成及DNA支架模块，利用与目的蛋白融合的锌指蛋白结合域特异性结合DNA的特点，以绿色荧光蛋白直观表征多酶在支架上的顺序自组装；调整支架DNA组成精确调控代谢途径中各酶的化学计量数比和酶空间距离，形成“底物通道”。本课题旨在构建一条类胡萝卜素高效合成通道，建立多酶合成调控的新方法，在多酶协同调控作用机制方面取得理论性突破，为多酶催化复杂大分子化合物的高效快速合成奠定理论基础。

类胡萝卜素是耐辐射微生物代表——耐辐射奇异球菌代谢过程中重要的功能天然产物，是优良的自由基清除剂和抗氧化剂。本项目针对类胡萝卜素多酶合成体系中存在的代谢途径长、参与酶和基因多及难以调控等问题，主要开展了四方面的研究：（一）耐辐射微生物基因组测序数据解析和类胡萝卜素功能基因解析，确定了其基因组上类胡萝卜素合成的关键基因和代谢途径；（二）类胡萝卜素合成模块在大肠杆菌中的构建及调控，通过多片段组装的方式，构建类胡萝卜素合成的不同模块，成功在大肠杆菌中引入类胡萝卜素合成途径，从外部环境、调控元件及基因顺序等方面层层递进调控；（三）基于构建模块的多样性和类胡萝卜素的强抗氧化性性质，建立了一种快速高通量筛选类胡萝卜素产量方法，获得了能够产生类胡萝卜素较高产量的基因工程菌株；（四）含DNA支架类胡萝卜素表达体系构建，通过锌指蛋白与DNA支架的引入，构建双质粒共表达菌株，进一步提高了类胡萝卜素的产量。本项目对多酶催化中多基因表达的协同作用进行研究，为类似复杂化合物的合成提供共通的调控手段及研究思路。