重组大肠杆菌中靛玉红合成关键酶基因的鉴定、表达及功能研究

Indirubin has therapeutic potential. However, the indirubin produced in recombinant microbes is of low yield and low purity. Discovering the new enzymes is an important strategy to solve the current problem. Here, we aimed at the recombinant Escherichia coli strain harboring the functional gene cluster from deep sea, and indentified the gene encoding the key enzyme that catalyzed the production of indirubin in the recombinant strain. Meanwhile, the biosynthetic pathway of indirubin in Escherichia coli was also confirmed. Then the gene was cloned and introduce into Escherichia coli to construct recombinant strain. Furthermore, expression of the gene was optimized, and the enzyme was extracted and purified. The activity of the purified enzyme was detected, and its enzymatic properties were also investigated. To improve the activity of the enzyme, the gene was mutated by error-prone PCR method, and random mutant library was constructed. The positive clones were screened from the library and used for sequencing. Based on the result of sequencing, site-directed saturation mutagenesis was performed at the positions with additional amino acid substitution, and saturation site-directed mutant libraries were constructed, respectively. Mutant with higher activity was obtained after screening the libraries. Results of this study will enhance the enzyme resource for the production of indirubin in microbes, and provide basis for achieving the efficient production and medicinal development of indirubin.

靛玉红有重要的医药价值，然而当前利用酶进行微生物转化合成靛玉红存在产量低、纯度差等问题。探索新型的酶资源是解决目前问题的重要策略。本项目以含深海功能基因簇的重组大肠杆菌为研究对象，鉴定该重组菌株中靛玉红合成关键酶基因，验证靛玉红的合成途径；在此基础上，构建工程菌，优化关键酶基因的表达，并分离和纯化关键酶，从而测定酶活力并研究其酶学性质；为提高关键酶的酶活力，对关键酶基因进行随机突变，并构建随机突变文库，筛选阳性克隆后测序分析；依据测序结果，对发生氨基酸替换的突变点进行定向饱和突变，并构建文库；通过筛选文库，获得高活力的突变酶。本项目将丰富靛玉红合成关键酶的资源，为实现靛玉红在大肠杆菌中的高效生产和药用开发奠定基础。

靛玉红是传统中成药“当归龙荟丸”和“板蓝根”的有效成分，对慢性粒细胞白血病有明显的抑制作用且临床疗效可靠、毒副作用小。当前利用酶进行微生物转化合成靛玉红存在产量低、纯度差等问题。探索新型的酶资源是解决目前问题的重要策略。本项目以含深海功能基因簇的重组大肠杆菌为研究对象，鉴定了该重组菌株中靛玉红合成关键酶基因moxA和moxB，验证了其靛玉红的合成途径：由色氨酸转化生成吲哚，吲哚在MoxA的催化下生成2羟基吲哚和3羟基吲哚，后两者相互聚合生成靛玉红。在此基础上，构建了工程菌，优化了moxA的表达，并分离和纯化MoxA蛋白；测定了MoxA的活性，并研究了其酶学性质。为提高MoxA的酶活，对moxA基因进行了突变研究，通过随机突变和定向饱和突变策略，酶的活性提高了36%。此外，构建重组菌株，考察了微生物转化法生产高纯度靛玉红的潜能，通过优化菌株的培养条件，最终得到靛玉红的产量198 mg/L。本项目的开展丰富了靛玉红合成关键酶的资源，为实现靛玉红在大肠杆菌中的高效生产和药用开发奠定基础。本项目共完成了6篇一作或通讯类SCI论文（均标注），和课题内容完全一致的文章两篇，正处于投稿阶段；项目衍生的SCI论文4篇，其中3篇已发表，1篇处于退修阶段；协助培养了2名硕士毕业生。