多策略调控谷氨酸棒杆菌产丁二胺的研究

Recent advances in systems biology,synthetic biology and evolution engineering are opening a new era for metabolic engineering.Putrescine has many industrial applications as a precursor of production of anitumor, alkaloid,fungicide,and polymers. In particular, putrescine is currently used to synthesize nylon-4,6 by polycondensation with adipic acid. Thus, studies on efficent biotechnological production of putrescine show an important scicentic and pratical value. Based on our original works, we plan to globally regulate Corynebacterium glutamicum to produce putrescine on the levels of translation(codon optimization), transcription(promoter engineering and module assemble), protein(ornithine decarboxylase screening and optimization), cell(evolution engineering), supply of NADHP and supression of CO2 emmision (bypass pathway construction) by combination of systems biology, synthetic biology and evolution engineering. The metabolic mechanism of the efficient production of putrescine of Corynebacterium glutamicum will be also investigated by comprative analysis of proteomics, genomics and expression levels of key genes. The project has a significant novelty and is the foreland subject of metabolic engineering. The studies can not only drive the addavancies in metabolic engineering and synthetic biology, but also promote the development of putrescine, industrial upgrading of enterprises of industral biotechnology in China.

随着系统生物学、合成生物学和进化工程技术的发展，代谢工程进入一个全新的时代。丁二胺可作为合成抗肿瘤药物、生物碱、杀菌剂和聚酰胺纤维(尼龙4,6)的前体,具有广阔的市场前景。因此利用代谢工程新技术高效合成生物基丁二胺的研究具有重要的科学意义和实用价值。我们将在现有工作的基础上，联合应用系统生物学、合成生物学和进化工程技术，从基因翻译(密码子优化)、转录(启动子工程与模块装配)、蛋白(鸟氨酸脱羧酶筛选与优化)、细胞(进化工程)、提高NADPH供给和抑制CO2释放(旁路构建)等层次，对谷氨酸棒杆菌进行全局调控，构建不含抗性标记和表达质粒的高产丁二胺的人工合成谷氨酸棒杆菌；并通过比较蛋白质组学、比较基因组学和关键基因表达水平的比较研究，揭示人工合成谷氨酸棒杆菌高效合成丁二胺的代谢调控机制。项目研究不仅能推动代谢工程和合成生物学学科的发展，又将促进生物基丁二胺的研发，推动我国工业生物技术产业升级。

首先从E. coli, Enterobacter cloacae, Hafnia alvei, Salmonella Arizona, Salmonella enterica, Serratia liquefaciens中筛选到最适鸟氨酸脱羧酶基因E. cloacae speC1；然后以实验室已有产鸟氨酸谷氨酸帮杆菌为出发菌，敲除了丁二胺氧化酶、NCgl0281 and NCgl2582基因以避免降解途径和提高NADPH供给，从而构建了一株无质粒的产丁二胺的谷氨酸帮杆菌；采用适应性进化技术得到了一株高产丁二胺的代谢进化工程菌C. glutamicum PUT-ALE，摇床发酵可产111.4mM丁二胺、葡萄糖转化率27.4%(g/g)，其产量是目前文献报道最高值(58.1mM)的1.9倍；最后为了揭示丁二胺合成的代谢调控机制，进行了比较基因组学和蛋白质组学的研究，发现高产菌株中与丁二胺合成有关的一些蛋白表达水平发生了明显变化，丁二胺合成途径有关基因argCJBD的序列也发生了突变。项目研究大大推动了生物基丁二胺和生物塑料的发展，同时也促进了代谢工程和合成生物学学科的发展。