Pseudomonas putida中靛蓝色素合成代谢调控机制研究

Production of the blue pigment by microbial fermentation is an effective way to meet the needs of a large number of this scarce natural pigment in the global market. The screened bacteria strain Pseudomonas putida B3 could produce the indigo by oxygenation using the indole as the substrate. However, the key enzymes catalyzing the indigo biosynthesis and the regulatory mechanism of the gene expression are still not clear. In this program, P. putida B3 was selected as the research target. We will utilize the genetic and metabolic engineering to first identify the key enzymes, the encoding genes, the regulatory elements and the reaction nodes for the indigo biosynthesis. Then the inductive effect of the substrate and the molecular regulation mechanism of the key genes’ expression will be revealed by the electrophoretic mobility shift assay (EMSA) and the isothermal titration calorimetry (ITC) analysis. Furthermore, we will explore the regulatory effect of the coenzyme NAD(P)H on the indigo production and the coordinated regulation of the NAD(P)H regeneration system and the regulation factors of gene expression on the anabolism of the indigo. The research results will illustrate the molecular mechanisms of coordinated regulation in the anabolism of the indigo based on the coenzyme NAD(P)H and the substrate in P. putida B3. This program will provide fundamental theoretical basis and technological guidance for design of the genetically engineered bacterial cell factory for efficient natural indigo production.

利用微生物发酵生产蓝色素是解决当前天然蓝色素稀缺的有效途径。本项目组分离筛选出一株以吲哚为底物通过加氧反应生成靛蓝色素的Pseudomonas putida B3，但其靛蓝生物合成代谢的关键酶及其基因表达调控机制并不清楚。项目以P. putida B3为研究对象，综合运用基因工程、代谢工程技术识别靛蓝生物代谢合成的关键酶基因（簇）及其调控元件；利用凝胶迁移（EMSA）、等温滴定量热（ITC）等技术，研究靛蓝合成代谢中底物诱导效应、表达调控因子与关键功能基因的互作效应，探索辅酶因子NAD(P)H对靛蓝合成的调节效应以及NAD(P)H再生系统与表达调控因子对靛蓝合成代谢的协同调控效应，揭示基于辅酶因子NAD(P)H和诱导物的P. putida B3 靛蓝色素合成关键代谢协同调控分子机制。研究成果将为天然靛蓝色素高效生产细胞工厂的定向设计构建提供理论基础和技术支持，具有重要研究价值和应用前景。

靛蓝颜色鲜艳而持久，作为染料已有2500年的使用历史，天然靛蓝主要是从木蓝、菘蓝等植物中提取，来源少，利用微生物发酵生产靛蓝色素是解决当前天然蓝色素稀缺的有效途径。项目利用基因工程技术构建多株基因超表达工程菌和基因敲除突变菌，结合生物信息学方法，研究识别了Pseudomonas putida B3靛蓝合成代谢的关键酶、基因簇及其调控元件；利用凝胶迁移（EMSA）技术等，研究靛蓝色素合成代谢中底物诱导效应、表达调控因子与关键功能基因的互作效应，探索辅酶因子NAD(P)H对靛蓝合成代谢的调节效应、NAD(P)H再生系统增强与表达调控因子对靛蓝合成代谢的协同调控效应，明晰了基于辅酶因子NAD(P)H和诱导物的P. putida B3 靛蓝色素合成关键代谢协同调控分子机制；选育高产靛蓝色素菌株在3L发酵罐补料发酵48h时靛蓝产量达到2.5 g/L以上，目前正在合作企业开展液体深层发酵技术中试研究。研究成果将为天然靛蓝色素高效生产细胞工厂的定向设计构建提供理论基础和技术支持，具有重要研究价值和应用前景。