摘要
重要工业微生物地衣芽孢杆菌(Bacillus lichenifrmis)十分缺乏合成生物学标准化元件,现有启动子主要来源于枯草芽孢杆菌等模式微生物,表达异源基因效果不甚理想。本研究首先基于转录组数据预测启动元件,然后以绿色荧光蛋白基因egfp为报告基因,建立了启动子在地衣芽孢杆菌中组成型表达强度的评价方法。进一步利用筛选出的高活性启动子P2,介导了麦芽三糖淀粉酶基因在地衣芽孢杆菌中的高效表达,并且研究了不同发酵条件对产酶的影响。荧光蛋白报告基因的结果显示,P2是地衣芽孢杆菌来源的强组成型启动子P_(shuttle-09)的28倍,是天然强启动子P43的52倍。基于新型启动子构建的表达质粒转化入地衣芽孢杆菌,实现了麦芽三糖淀粉酶的分泌表达。混合碳源麦芽糊精60 g/L,葡萄糖10 g/L以及42℃的发酵条件更有利于产酶,重组菌发酵的最高酶活达到578.47 U/mL。新的组成型强启动子的鉴定和应用为地衣芽孢杆菌表达系统的开发和完善奠定了基础。
Bacillus licheniformis,an important industrial microorganism,lacks the standardized elements of synthetic biology.The existing promoters are mainly derived from model microorganisms such as Bacillus subtilis,but the effect of expressing heterologous genes is not satisfactory.First,the promoter elements were predicted based on transcriptome data and then using green fluorescent protein egfp gene as the reporter gene,a method was established to evaluate the constitutive expression strength of the promoter in Bacillus licheniformis.Furthermore,the selected highly active promoter P2 was used to mediate the high-efficiency expression of maltotrioseproducing amylase gene in Bacillus licheniformis,and the effect of different fermentation conditions on enzyme production was investigated.The results showed that P2 was 28 times of the strong constitutive promoter P_(shuttle-09)and 52 times of the natural strong promoter P43.The expression plasmid harbouring the new promoter was transformed into Bacillus licheniformis to realize the secretion and expression of maltotriose-producing amylase.The fermentation conditions of mixed carbon source maltodextrin 60 g/L,glucose 10 g/L and 42℃were more conducive to enzyme production,and the highest enzyme activity of genetically modified bacteria by fermentation reached 578.47 U/mL.Identification and application of new constitutive strong promoter should lay a solid foundation for the development and improvement of the Bacillus licheniformis expression system.
作者
赵鑫馨
李由然
石贵阳
Zhao Xinxin;Li Youran;Shi Guiyang(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi,214122)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第11期3566-3575,共10页
Genomics and Applied Biology
基金
国家自然科学基金项目(31401674)
“十三五”国家重点研发计划(2018YFA0900504,2020YFA0907700)共同资助
关键词
启动子
地衣芽孢杆菌
表达系统
麦芽三糖淀粉酶
Promoter
Bacillus licheniformis
Expression system
Maltotriose-producing amylase
