小檗碱桥酶高效原核表达及生物合成l-SLR的研究  

Study on the Biosynthesis of l-SLR by Efficient Prokaryotic Expression of Berberine Bridge Enzyme

作　　者：梅欢 李玥 刘可蒙 刘吉华[1] MEI Huan;LI Yue;LIU Ke-meng;LIU Ji-hua(Jiangsu Provincial Key Laboratory of Traditional Chinese Medicine Evaluation and Transformation,China Pharmaceutical University,College of Traditional Chinese Medicine,China Pharmaceutical University,Nanjing 211100)

机构地区：[1]中国药科大学江苏省中药评价与转化重点实验室、中国药科大学中药学院,南京211100

出　　处：《生物技术通报》2023年第7期277-287,共11页Biotechnology Bulletin

基　　金：中国药科大学“双一流”建设项目(CPU2018GY32)。

摘　　要：左旋金黄紫堇碱(l-Scoulerine,l-SLR)是苄基异喹啉类生物碱(benzylisoquinoline alkaloids,BIAs)的关键中间体,由小檗碱桥酶(berberine bridge enzyme,BBE)催化左旋网状番荔枝碱(l-Reticuline,l-RL)合成。基于前期首次实现加州罂粟小檗碱桥酶(Eschscholzia californcia berberine bridge enzyme,EcBBE,EcBO)的原核表达,通过更换原核表达载体及共表达分子伴侣,构建高效表达EcBO的大肠杆菌工程菌株,实现l-RL向l-SLR的高效转化。结果表明,EcBO与分子伴侣pGro7共表达的工程菌株A的酶活力达到194.14 U/L,较原始酶活力提高了9.22倍。进一步对菌株A生物合成l-SLR的培养条件进行优化,在TB培养基中,当异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度为0.04 mmol/L,L-阿拉伯糖浓度为4 mg/mL,16℃下诱导18 h,0.2 mg/mL l-RL,37℃转化18 h,l-SLR产量为144.19 mg/L,较初始菌株提高4.72倍。综上,通过在原核系统中共表达分子伴侣pGro7与EcBO实现了EcBO高活性表达,显著提高了l-SLR的生物合成效率,为高效生物合成l-SLR及其他BIAs类生物碱提供了新的策略。l-Scoulerine(l-SLR)is a key intermediate of benzylisoquinoline alkaloids(BIAs),which are synthesized by berberine bridge enzyme(BBE).Based on the previous successful prokaryotic expression of Eschscholzia californcia berberine bridge enzyme(EcBBE,EcBO),an engineered E.coli strain with efficient expression of EcBO,which efficiently transformed l-Reticularine(l-RL)into l-scoulerine(l-SLR),was constructed by replacing the prokaryotic expression vector and co-expressing with chaperone protein.The results showed that the engineered strain A co-expressed by EcBO and chaperone pGro7 increased the activity of EcBO to 194.14 U/L,9.23 times compared with the original strain.Further optimization of the culture conditions for strain A biosynthesis l-SLR showed that in TB medium and at the conditions of 0.04 mmol/L isopropylthio-β-D-galactoside(IPTG),4 mg/mL L-arabinose,induced for 18 h at 16℃ then 0.2 mg/mL l-RL conversion for 18 h at 37℃,the l-SLR yield reached 144.19 mg/L,4.72 times higher than that by the initial strain.In conclusion,the highly active expression of EcBO was achieved by co-expressing the chaperone pGro7 and EcBO in the prokaryotic system,which significantly improved the biosynthesis efficiency of l-SLR,and provides a new strategy for the efficient biosynthesis of isoquinoline alkaloids such as l-SLR.

关 键 词：小檗碱桥酶 左旋金黄紫堇碱 生物转化 分子伴侣 原核表达 

分 类 号：Q78[生物学—分子生物学] R284[医药卫生—中药学]