大肠杆菌基于莽草酸途径后修饰合成天麻素及调控机制研究

Gastrodin is the major bioactive component of the notable Chinese herb Tianma (Gastrodia elata Bl). Gastrodin has multiple therapeutic effects including anti-convulsion and analgesic. Tianma is expensive, and the content of gastrodin is low. Extraction and purification procedures for natural products from plants are complicated. Chemical synthesis is a multi-steps procedure and leads to serious environmental pollution. In the previous study, we constructed an artificial pathway for the biosynthesis of gastrodin in Escherichia coli starting from shikimate pathway via p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-hydroxybenzyl alcohol. Thus biosynthesis of gastrodin from glucose was achieved for the first time. The post-modification procedures such as carboxylic acid reduction and glucosylation have become the limiting steps for high yield production of gastrodin. In this study, we are aiming to acquire carboxylic acid reductases and glucosyltransferases with higher activity and improved substrate specificity by gene screening and enzyme engineering. The supply of cofactor NADPH and precursor UDP-glucose will also be enhanced by metabolic engineering. In the end, systemic regulation and optimization will be applied to improve the production of gastrodin by engineered E. coli. The study will reveal mechanisms of enzymatic action and metabolic regulation in the biosynthesis of gastrodin.

天麻素是我国著名中药天麻的主要活性成分，具有镇静及抗惊厥等作用。天麻价格昂贵，天麻素含量低，提取工艺复杂。化学合成步骤多，造成严重环境污染。前期我们在大肠杆菌中创建一条起始于莽草酸途径，经由对羟基苯甲酸、对羟基苯甲醛、对羟基苯甲醇等中间体，首次打通了以葡萄糖为原料微生物合成天麻素的路线。羧酸还原、糖基化等后修饰过程是大肠杆菌高效合成天麻素的主要限速步骤。本项目拟通过基因发掘及改造，获得催化活性及专一性提高的羧酸还原酶及糖基转移酶；通过调控，增加天麻素生物合成辅因子及前体UDP-葡萄糖的供给；系统优化天麻素的代谢通路，提高天麻素产量。本研究将揭示大肠杆菌高效合成天麻素的酶学及代谢调控机制。

天麻素是我国著名中药天麻的主要活性成分，具有镇静及抗惊厥等作用。天麻价格昂贵，天麻素含量低，提取工艺复杂。化学合成步骤多，造成严重环境污染。前期我们在大肠杆菌中创建一条起始于莽草酸途径，经由对羟基苯甲酸、对羟基苯甲醛、对羟基苯甲醇等中间体，首次打通了以葡萄糖为原料微生物合成天麻素的路线，产量达到0.60克/升。本项目：（1）对羧酸还原酶和糖基转移酶进行了筛选和改造，莽草酸途径调控、强化NADPH和UDP-葡萄糖合成通路，并对发酵条件进行了优化，天麻素产量达到18克/升，并成功完成了30升中试放大，进入产业化推广阶段；（2）通过基因组分析，系统发掘了大肠杆菌内源性的醛酮还原酶与醇脱氢酶，实现了对羟基苯甲醛的积累，对工程菌株进行了系统优化，对羟基苯甲醛葡萄糖苷菌株产量达到9.3克/升；（3）首次创建合成天麻素的酵母菌细胞工厂，在摇瓶水平发酵，天麻素产量达2.1克/升。