摘要
鲨烯是一种在食品、医药等行业有着广泛应用的三萜类化合物。大肠杆菌被认为是非常有潜力的合成鲨烯的底盘细胞。为提高大肠杆菌内鲨烯的合成水平,以三个诱导型启动子p3-lac、p6-lac、p10-lac强度为水平,以dxs、idi、ispA三个4-磷酸甲基赤藓糖醇途径(methylerythritol 4-phosphate pathway,MEP)的限速酶基因作为因素,进行正交设计,同时引入鲨烯合酶基因,构建了9株重组菌株进行鲨烯发酵实验。通过绿色荧光蛋白表达水平确定了p3-lac启动子强度相对最强,p10-lac相对最弱。通过分析正交发酵实验数据的极差,发现dxs是影响鲨烯产量最主要的因素,其次是ispA,最后是idi,且三个因素最佳水平组合所对应的菌株为1063/DE3,产量达到了450 mg/L。最终获得了一株鲨烯产量相较于出发菌株提高了681倍的产鲨烯重组大肠杆菌菌株,并为提高大肠杆菌内鲨烯产量提供了新的策略。
Squalene is a triterpenoid compound which has been widely used in the food and pharmaceutical industries.Escherichia coli is considered a highly promising chassis cell for the synthesis of squalene.In order to increase squalene production in E.coli,an orthogonal design was conducted using three genes dxs,idi and ispA in MEP pathway as factors and three inducible promoters of different strengths,p3-lac,p6-lac,p10-lac as levels to construct nine recombinant strains for fermentation by inducing squalene synthase gene.The relative strength of the three promoters was determined by the expression level of green fluorescent protein.The p3-lac promoter had the strongest relative strength,while the p10-lac promoter had the weakest relative strength.By analyzing the extreme deviation of the fermentation data,it was found that dxs was the most dominant factor affecting squalene yield,followed by ispA and idi.The strain corresponding to the optimal level combination of the three factors was 1063/DE3,which achieved a yield of 450 mg/L.Overall,an E.coli strain with a 681-fold increase in squalene production compared to the original strain has been obtained and a new strategy for increasing squalene production in E.coli has been provided in this study.
作者
厉元
韩姝
张伟
胡又佳
谢丽萍
LI Yuan;HAN Shu;ZHANG Wei;HU You-jia;XIE Li-ping(Shanghai Institute of Pharmaceutical Industry,China State Institute of Pharmaceutical Industry,Shanghai 201203,China)
出处
《天然产物研究与开发》
CAS
CSCD
北大核心
2024年第10期1721-1731,共11页
Natural Product Research and Development
关键词
鲨烯
MEP途径
限速酶
启动子强度
大肠杆菌
squalene
MEP pathway
rate-limiting enzyme
promoter strength
Escherichia coli
