枯草芽孢杆菌合成核黄素的基因组工程研究

Genome engineering is the art of constructing a genotype that gives rise to a desired phenotype, which enables us to analyze genetic basis for desired phenotype, and finally to explore or develop the principle for controlling biological design. One of the key scientific problems for riboflavin overproduction is how to optimize the expression of different genes with different pathway at the genome scale. Aiming at resolving this, the genome reduced Bacillus subtilis is selected as a chassis, whose genome-scale metabolic network is optimized for riboflavin biosynthesis by multiplex modular engineering. Genome-scale metabolic network model is reconstructed based on the information of a reduced genome, by which targets are predicted for further modification. Genome editing system are simultaneously established and optimized, which are based on the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 (CRISPR/Cas9). Promoters are employed to coordinate the expression of genes of different pathway modular and genes in the same pathway modular by the constructed CRISPR/Cas9 system. The metabolic regulated network is deregulated and optimized to enhance its capability to synthetize riboflavin in B. subtilis. The genetic regulation mechanism can be developed or explored by correlation analysis between the different genetic modification and phenotype. This project will have a much broader impact, not only enabling us to have a deeper understanding of genetic regulation mechanism for riboflavin biosynthesis in B. subtilis, but also providing a typical case for bio-based products biosynthesis by genome engineering based on a genome reduced chassis.

基因组工程是实现菌种目标性能优化，探讨优良性状遗传调控基础，开发或发展控制生物设计法则的重要技术。本项目拟针对如何在基因组尺度优化不同途径基因的协调表达以提高核黄素合成能力的关键科学问题，以基因组简化的枯草芽孢杆菌为底盘，通过多元模块工程策略，基因组工程优化核黄素合成的代谢网络。首先根据基因组简化信息重构基因组尺度代谢网络模型，模拟预测修饰的靶位点；同时，建立和优化成簇的规律间隔的短回文重复序列关联蛋白（CRISP/Cas9）介导的基因组编辑系统，为基因组工程奠定技术基础；采用多元模块工程的策略，利用CRISPR/Cas9基因组编辑技术，基因组上优化不同途径模块间和模块内基因协调表达，解调和优化细胞的代谢调控网络，提高核黄素合成能力，探讨核黄素高产的遗传调控机理。本项目的成功实施将加深对枯草芽孢杆菌产核黄素遗传调控机理的认识，为基因组简化的底盘细胞生产生物基产品的基因组工程提供理论指导。

基因组工程是通过先进的基因组编辑手段，实现菌种目标性能优化，探讨优良性状遗传调控基础，开发或发展生物的设计法则。本项目针对如何在基因组尺度优化不同途径基因的协调表达以提高核黄素合成能力的关键科学问题，以野生型枯草芽孢杆菌为底盘，构建先进的基因组编辑技术和合成生物学调控元件，通过多元模块工程策略，基因组尺度优化核黄素合成的代谢网络，取得了系列重要结果。.构建枯草芽孢杆菌基于CRISPR/Cas9n的基因组多位点编辑系统，通过胞内单链DNA切口修复系统ligD基因修饰和系统优化，基因组三位点同时编辑效率达65%，实现3天一个循环的高效基因组编辑，达到目前枯草芽孢杆菌报道的最高多位点基因编辑效率。.基于野生型枯草芽孢杆菌168转录组数据分析，首次设计构建枯草芽孢杆菌人工合成启动子文库，文库强度调控范围广，且构建的强启动子是P43启动子的2.77倍。启动子强度在转录和翻译水平具有很好的相关性，基于发现的“位点-碱基”分布规律的启动子理性重构方法，重构强启动子和启动子文库，强化了基因表达精准调控能力。.修正和改进枯草芽孢杆菌基因组尺度代谢网络模型iBsu1147质量，基于通量平衡分析（FBA）算法，获得枯草芽孢杆菌中核黄素最优合成途径及其通量分布。利用开发的通量可变性分析（FVA），成功预测了基因扰动靶点，理性指导核黄素高产菌株的构建。.利用多元模块代谢工程策略，优化核黄素合成的嘌呤代谢途径模块、核黄素操纵子模块、戊糖磷酸途径模块和呼吸链模块，使野生型菌株核黄素摇瓶产量达4.3 g/L。结合基因组重排技术，构建的核黄素高产菌株发酵罐产量达18.5 g/L，核黄素得率0.119 g/g 葡萄糖。利用转录组分析和代谢物组分析等系统生物学手段，获得不同途径基因的协调表达导致核黄素高产的遗传调控机理，加深了枯草芽孢杆菌高产核黄素遗传调控机理的认识，为枯草芽孢杆菌高效合成其他生物基产品提供理论和技术指导。