代谢工程改造大肠杆菌合成莽草酸及培养基优化  

Metabolic engineering for synthesis of shikimic acid in Escherichia coli and optimization of culture medium

作　　者：宗媛 伊进行 王雪慧 王静 王晓超 陈宁[1,2] 王小楠 ZONG Yuan;YI Jinhang;WANG Xuehui;WANG Jing;WANG Xiaochao;CHEN Ning;WANG Xiaonan(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;National and Local United Engineering Lab of Metabolic Control Fermentation Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]代谢控制发酵技术国家地方联合工程实验室,天津300457

出　　处：《食品与发酵工业》2025年第6期15-24,共10页Food and Fermentation Industries

基　　金：天津市科技计划项目(22JCQNJC01310);国家重点研发计划项目(2022YFD2101401)。

摘　　要：莽草酸是一种天然的芳香族化合物,具有抗氧化、抗炎、抗菌等功效,在医药领域具有重要的应用价值。该研究旨在通过大肠杆菌的代谢工程改造和培养基优化提高莽草酸的生物合成产量。从野生型大肠杆菌W3110出发对代谢途径进行基因组层面改造,采取敲除莽草酸消耗途径、强化莽草酸合成途径、提高前体物供给等策略构建了一株能够高效生产莽草酸的菌株。在此基础上对培养基进行优化:采用单因素实验和Plackett-Burman实验对发酵培养基中影响莽草酸生产的因素进行评估,筛选出酵母粉、蛋白胨和MgSO_(4)·7H_(2)O 3种具有显著影响的因素;应用Box-Behnken进行3因素3水平的设计及响应面模型构建与分析,获得最佳的发酵培养基配方为:初始葡萄糖10 g/L,酵母粉2.06 g/L,蛋白胨1.52 g/L,K_(2)HPO_(4)3 g/L,KH_(2)PO_(4)3.5 g/L,(NH_(4))_(2)SO_(4)3 g/L,MgSO_(4)·7H_(2)O 0.20 mmol/L,磷酸甜菜碱0.22 g/L,CaCl_(2)·2H_(2)O 15 mg/L,维生素B 10.5 mg/L,微量元素混合液1 mL/L,pH 7.0~7.2。在此条件下,莽草酸在摇瓶规模的发酵产量达到2.66 g/L,是优化前的2倍。该研究为莽草酸在大肠杆菌中的生物合成相关研究和工业化发酵生产奠定基础。Shikimic acid is a natural aromatic compound with antioxidant,anti-inflammatory,and antibacterial effects,and it has important application value in the medical field.This study aimed to enhance the biosynthesis of shikimic acid through metabolic engineering and optimization of culture medium in Escherichia coli.The metabolic pathway was modified at the genomic level from wild-type Escherichia coli W3110,and a strain that could efficiently produce shikimic acid was successfully constructed by knocking out the shikimic acid consumption pathway,strengthening the shikimic acid synthesis pathway,and improving the supply of precursors.Based on this,the medium was optimized.The factors affecting the production of shikimic acid in the fermentation medium were firstly evaluated by using the single factor experiments and the Plackett-Burman test,and 3 factors including yeast extract,peptone,MgSO_(4)·7H_(2)O were screened out with significant effects.Box-Behnken was applied for the three-factor three-level design and response surface model construction and analysis to obtain the optimal fermentation medium formulation,which was initially glucose 10 g/L,yeast extract 2.06 g/L,peptone 1.52 g/L,K_(2)HPO_(4)3 g/L,KH_(2)PO_(4)3.5 g/L,(NH4)_(2)SO_(4)3 g/L,MgSO_(4)·7H_(2)O 0.20 mmol/L,betaine phosphate 0.22 g/L,CaCl_(2)·2H_(2)O 15 mg/L,vitamin B10.5 mg/L,trace element mixture 1 mL/L,and pH 7.0-7.2.Under this condition,the yield of shikimic acid reached 2.66 g/L in the shake flask,which was twice as much as that before optimization.This study lays the foundation for scientific research and industrial fermentation production on shikimic acid biosynthesis by Escherichia coli.

关 键 词：大肠杆菌 代谢工程 莽草酸 Plackett-Burman试验设计 

分 类 号：TQ921[轻工技术与工程—发酵工程]