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

Abstract:

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.

Key words: berberine bridge enzyme, l-Scoulerine, biotransformation, molecular chaperone, prokaryotic expression