生物技术通报2025，Vol.41Issue(12)：74-81,8.DOI:10.13560/j.cnki.biotech.bull.1985.2025-0718
代谢工程改造益生菌E.coli Nissle 1917合成靛玉红
Modifying the Probiotic Escherichia coli Nissle 1917 for the Biosynthesis of Indirubin via Metabolic Engineering
摘要
Abstract
[Objective]Indirubin,a bi-indolic alkaloid derivative isolated from the traditional Chinese herbal medicine Indigo Naturalis,represents a potent therapeutic compound having broad-spectrum antibacterial,anti-inflammatory,and anti-tumor properties.Currently,it is clinically utilized primarily in combination therapy regimens for the treatment of chronic myeloid leukemia(CML),psoriasis,and related pathologies.This study investigates the potential of the safe Escherichia coli Nissle 1917(EcN)as a chassis for constructing a green supply system for the active components of the traditional Chinese medicine indirubin.[Method]Based on the engineering modification of the natural endogenous plasmid of EcN,the product output efficiency of the indirubin biosynthesis pathway constructed using the flavin monooxygenase fmo and its mutants(fmoK223R and fmoK223R/D317S))from Methylophaga sp.SK1 was systematically evaluated.Combined with metabolic engineering,key genes in the competitive branch of the tryptophan biosynthesis pathway were knocked out,including pheA,a key gene for phenylalanine biosynthesis,and tyrA,a key gene for tyrosine biosynthesis.Further optimization was achieved by inactivating the upstream genes of the tricarboxylic acid cycle,including pykA(pyruvate kinase)and ppc(phosphoenolpyruvate carboxylase).Finally,the product synthesis level was further improved by deleting the repressor protein gene trpR in the tryptophan pathway and strengthening the anti-feedback inhibition gene trpES40F in the tryptophan pathway.[Result]After 48 h of shaking culture in a 250 mL flask(50 mL culture),the indirubin titer reached(176.9±4.5)mg/L,representing a 3.5-fold increase compared to the parent strain harboring only the wild-type fmo gene.The production in the 5 L bioreactor was(379.3±12.3)mg/L.[Conclusion]Employing the endogenous cryptic plasmid of the probiotic EcN to express the heterologous flavin monooxygenase mutant gene fmoK223R,and integrating this with systematic metabolic engineering of endogenous tryptophan biosynthesis pathways,the production of indirubin reached over 300 mg/L,demonstrating the excellent potential of EcN as a cell factory for biosynthesis of active ingredients in traditional Chinese medicines.关键词
大肠杆菌Nissle 1917/黄素单加氧酶bFMO/中药青黛/靛玉红/生物合成Key words
Escherichia coli Nissle 1917/flavin monooxygenase bFMO/traditional Chinese medicine Indigo Naturalis/indirubin/biosynthesis引用本文复制引用
MAO Li-jing,JIN Xiao-xuan,SHI Wan-ting,HU Fei-yang,ZHANG Yuan-rong,XIONG Liang-bin,REN Lu..代谢工程改造益生菌E.coli Nissle 1917合成靛玉红[J].生物技术通报,2025,41(12):74-81,8.基金项目
国家自然科学基金项目(32100067) （32100067）
