生物技术通报 ›› 2023, Vol. 39 ›› Issue (7): 277-287.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1517
收稿日期:
2022-12-16
出版日期:
2023-07-26
发布日期:
2023-08-17
通讯作者:
刘吉华,男,博士,教授,研究方向:中药生物技术与新药创制;E-mail: liujihua@cpu.edu.cn作者简介:
梅欢,女,硕士研究生,研究方向:中药生物技术;E-mail: 3220020340@stu.cpu.edu.cn
基金资助:
MEI Huan(), LI Yue, LIU Ke-meng, LIU Ji-hua()
Received:
2022-12-16
Published:
2023-07-26
Online:
2023-08-17
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。
梅欢, 李玥, 刘可蒙, 刘吉华. 小檗碱桥酶高效原核表达及生物合成l-SLR的研究[J]. 生物技术通报, 2023, 39(7): 277-287.
MEI Huan, LI Yue, LIU Ke-meng, LIU Ji-hua. Study on the Biosynthesis of l-SLR by Efficient Prokaryotic Expression of Berberine Bridge Enzyme[J]. Biotechnology Bulletin, 2023, 39(7): 277-287.
项目 Item | 菌株和质粒Strains and plasmids | 详细信息Detailed information | 来源Source |
---|---|---|---|
菌株Strains | BL21(DE3)-SMR-MEcBO | pMAL-c4x, Tac P-EcBO-Tac T, EcoR I/Sal I; pBR322 ori, Smr+ | Lab stock |
BL21(DE3)-pCold-II- ECBO | pCold-II, CSPA P-MBP-EcBO - CSPA T, Nde I/Xho I; pBR322 ori, Amp+ | This work | |
BL21(DE3)-pTrc99a-ECBO | pTrc99a, Trc P-EcBO - Trc T, EcoR I/Hind III; pBR322 ori, Amp+ | This work | |
BL21(DE3)-pBad24- ECBO | pBad24, araBAD P-EcBO - araBAD T, EcoR I/ Hind III; pBR322 ori, Amp+ | This work | |
BL21(DE3)-pET28a-sumo-EcBO | pET28a-sumo, T7 P-EcBO - T7 T, BamH I/Sac I ; pBR322 ori, Kan+ | This work | |
E. coli DH5α | F-φ80 lacZΔM15Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rk-, mk-)supE44λ- thi-1 gyrA96 relA1 phoA | General Biol | |
E. coli BL21(DE3) | F-ompT hsdS(rB-mB-)gal dcm(DE3) | General Biol | |
Strain A | BL21(DE3)with plasmid SMR-MEcBO and pGro7 | This work | |
Strain B | BL21(DE3)with plasmid SMR-MEcBO and pTf16 | This work | |
Strain C | BL21(DE3)with plasmid SMR-MEcBO and pKJE7 | This work | |
Strain D | BL21(DE3)with plasmid SMR-MEcBO and pG-KJE8 | This work | |
Strain E | BL21(DE3)with plasmid SMR-MEcBO and pG-Tf2 | This work | |
质粒Plasmids | pCold II | CSPA Promoter,Amp+ | Lab stock |
pET-28a-sumo | T7 Promoter,Kan+ | Lab stock | |
pTrc99a | Trc Promoter,Amp+ | Lab stock | |
pBad24 | araBAD Promoter,Amp+ | Lab stock |
表 1 菌株及质粒信息
Table 1 Strain and plasmid information
项目 Item | 菌株和质粒Strains and plasmids | 详细信息Detailed information | 来源Source |
---|---|---|---|
菌株Strains | BL21(DE3)-SMR-MEcBO | pMAL-c4x, Tac P-EcBO-Tac T, EcoR I/Sal I; pBR322 ori, Smr+ | Lab stock |
BL21(DE3)-pCold-II- ECBO | pCold-II, CSPA P-MBP-EcBO - CSPA T, Nde I/Xho I; pBR322 ori, Amp+ | This work | |
BL21(DE3)-pTrc99a-ECBO | pTrc99a, Trc P-EcBO - Trc T, EcoR I/Hind III; pBR322 ori, Amp+ | This work | |
BL21(DE3)-pBad24- ECBO | pBad24, araBAD P-EcBO - araBAD T, EcoR I/ Hind III; pBR322 ori, Amp+ | This work | |
BL21(DE3)-pET28a-sumo-EcBO | pET28a-sumo, T7 P-EcBO - T7 T, BamH I/Sac I ; pBR322 ori, Kan+ | This work | |
E. coli DH5α | F-φ80 lacZΔM15Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rk-, mk-)supE44λ- thi-1 gyrA96 relA1 phoA | General Biol | |
E. coli BL21(DE3) | F-ompT hsdS(rB-mB-)gal dcm(DE3) | General Biol | |
Strain A | BL21(DE3)with plasmid SMR-MEcBO and pGro7 | This work | |
Strain B | BL21(DE3)with plasmid SMR-MEcBO and pTf16 | This work | |
Strain C | BL21(DE3)with plasmid SMR-MEcBO and pKJE7 | This work | |
Strain D | BL21(DE3)with plasmid SMR-MEcBO and pG-KJE8 | This work | |
Strain E | BL21(DE3)with plasmid SMR-MEcBO and pG-Tf2 | This work | |
质粒Plasmids | pCold II | CSPA Promoter,Amp+ | Lab stock |
pET-28a-sumo | T7 Promoter,Kan+ | Lab stock | |
pTrc99a | Trc Promoter,Amp+ | Lab stock | |
pBad24 | araBAD Promoter,Amp+ | Lab stock |
名称Name | 引物序列Primer sequence(5'-3') |
---|---|
pCold-II-Nde I-F | catcatcatcatcatcatatgAAAATCGAAGAAGGTAAACTGGTAATC |
pCold-II-Xho I-R | cttgaattcggatccctcgagAATAACCACTTCACCACCATCACTG |
pTrc99a-EcoR I-F | aggaaacagaccatggaattcATGGGTAATGATCTGCTGAGCTGTCTG |
pTrc99a-Hind III-R | tccgccaaaacagccaagcttAATAACCACTTCACCACCATCACTG |
pBad24-EcoR I-F | ttgggctagcaggaggaattcATGGGTAATGATCTGCTGAGCTGTCTG |
pBad24-Hind III-R | TccgccaaaacagccaagcttAATAACCACTTCACCACCATCACTG |
pET28a-sumo- BamH I-F | cttgaattcggatccctcgagGGTAATGATCTGCTGAGCTGTCTG |
pET28a-sumo- Sac I -R | catcatcatcatcatcatatgAATAACCACTTCACCACCATCACTG |
T7 | TAATACGACTCACTATAGGG |
T7 ter | TGCTAGTTATTGCTCAGCGG |
pCold-II-F | ACGCCATATCGCCGAAAGG |
pCold-II-R | GGCAGGGATCTTAGATTCTG |
pBad-F | ATGCCATAGCATTTTTATCC |
pBad-R | GATTTAATCTGTATCAGG |
pTrc99a-F | GAGCGGATAACAATTTCACACAGG |
pTrc99a-R | GATTTAATCTGTATCAGG |
表 2 引物列表
Table 2 List of primers
名称Name | 引物序列Primer sequence(5'-3') |
---|---|
pCold-II-Nde I-F | catcatcatcatcatcatatgAAAATCGAAGAAGGTAAACTGGTAATC |
pCold-II-Xho I-R | cttgaattcggatccctcgagAATAACCACTTCACCACCATCACTG |
pTrc99a-EcoR I-F | aggaaacagaccatggaattcATGGGTAATGATCTGCTGAGCTGTCTG |
pTrc99a-Hind III-R | tccgccaaaacagccaagcttAATAACCACTTCACCACCATCACTG |
pBad24-EcoR I-F | ttgggctagcaggaggaattcATGGGTAATGATCTGCTGAGCTGTCTG |
pBad24-Hind III-R | TccgccaaaacagccaagcttAATAACCACTTCACCACCATCACTG |
pET28a-sumo- BamH I-F | cttgaattcggatccctcgagGGTAATGATCTGCTGAGCTGTCTG |
pET28a-sumo- Sac I -R | catcatcatcatcatcatatgAATAACCACTTCACCACCATCACTG |
T7 | TAATACGACTCACTATAGGG |
T7 ter | TGCTAGTTATTGCTCAGCGG |
pCold-II-F | ACGCCATATCGCCGAAAGG |
pCold-II-R | GGCAGGGATCTTAGATTCTG |
pBad-F | ATGCCATAGCATTTTTATCC |
pBad-R | GATTTAATCTGTATCAGG |
pTrc99a-F | GAGCGGATAACAATTTCACACAGG |
pTrc99a-R | GATTTAATCTGTATCAGG |
质粒Plasmid | 分子伴侣组合Chaperone | 启动子Promoter | 抗性标签Resistant marker | 诱导物Inducer |
---|---|---|---|---|
pG-KJE8 | dnaK-dnaJ-grpE groES-groEL | araB Pzt-1 | Chl+ | L-阿拉伯糖 四环素 |
pGro7 | groES-groEL | araB | Chl+ | L-阿拉伯糖 |
pKJE7 | dnaK-dnaJ-grpE | araB | Chl+ | L-阿拉伯糖 |
pTf16 | tig | araB | Chl+ | L-阿拉伯糖 |
pG-Tf2 | groES-groEL-tig | Pzt-1 | Chl+ | 四环素 |
表 3 伴侣蛋白质粒信息
Table 3 Chaperone protein information
质粒Plasmid | 分子伴侣组合Chaperone | 启动子Promoter | 抗性标签Resistant marker | 诱导物Inducer |
---|---|---|---|---|
pG-KJE8 | dnaK-dnaJ-grpE groES-groEL | araB Pzt-1 | Chl+ | L-阿拉伯糖 四环素 |
pGro7 | groES-groEL | araB | Chl+ | L-阿拉伯糖 |
pKJE7 | dnaK-dnaJ-grpE | araB | Chl+ | L-阿拉伯糖 |
pTf16 | tig | araB | Chl+ | L-阿拉伯糖 |
pG-Tf2 | groES-groEL-tig | Pzt-1 | Chl+ | 四环素 |
图 1 重组菌株基因结构图及菌落PCR验证 A:重组菌株基因结构图;B:重组菌株菌落PCR验证
Fig. 1 Gene structure mapping of recombinant strains and colony PCR validation A: Genetic structure of colonies of recombinant strains; B: PCR validation of recombinant strains
图 2 重组菌株诱导表达的SDS-PAGE分析 M:蛋白质分子质量标准;泳道1-2:BL21(DE3)-SMR-MEcBO上清、沉淀;泳道3-4:BL21(DE3)-pCold II-MBP-EcBO上清、沉淀;泳道5-6:BL21(DE3)-pTrc99a-EcBO上清、沉淀;泳道7-8:BL21(DE3)-pBad24-EcBO上清、沉淀;泳道9-10:BL21(DE3)-pET28a-sumo-EcBO上清、沉淀
Fig. 2 SDS-PAGE analysis of induced expressions of recombinant strains M: Protein molecular weight marker. Lanes 1-2: BL21(DE3)-SMR-MEcBO supernatant, precipitate. Lane 3-4: BL21(DE3)-pCold II-MBP-EcBO supernatant, precipitate. Lanes 5-6: BL21(DE3)-pTrc99a-EcBO supernatant, precipitation. Lanes 7-8: BL21(DE3)-pBad24-EcBO supernatant, precipitate. Lanes 9-10: BL21(DE3)-pET28a-sumo-EcBO supernatant, precipitate
质粒 Plasmid | 启动子强度 Promoter strength | 启动子 Promoter | 特点 Peculiarity | 可溶性蛋白比例Proportion of soluble protein/% | l-SLR产量 l-SLR production/(mg· L-1) | 参考文献References |
---|---|---|---|---|---|---|
pMal-c4x | 强 | Tac | 由Trp和Lac合成的人工杂交启动子,效率高于Lac启动子 | 67.9 | 20.89 | [ |
pET-28a-sumo | 最强 | T7 | 效率高,受T7RNA聚合酶调节,不被大肠杆菌RNA 聚合酶调节 | 47.24 | 0 | [ |
pCold II | 中等 | CSPA | 低温高效表达 | 52.78 | 0 | [ |
pTrc99a | 弱 | Trc | 由Trp和Lac合成的人工杂交启动子,效率高于Lac启动子 | 0 | 0 | [ |
pBad24 | 中等 | araBAD | 表达强度由阿拉伯糖的浓度决定 | 0 | 0 | [ |
表 4 涉及启动子及对应的重组菌株蛋白表达情况
Table 4 Protein expressions of promoters and corresponding recombinant strains
质粒 Plasmid | 启动子强度 Promoter strength | 启动子 Promoter | 特点 Peculiarity | 可溶性蛋白比例Proportion of soluble protein/% | l-SLR产量 l-SLR production/(mg· L-1) | 参考文献References |
---|---|---|---|---|---|---|
pMal-c4x | 强 | Tac | 由Trp和Lac合成的人工杂交启动子,效率高于Lac启动子 | 67.9 | 20.89 | [ |
pET-28a-sumo | 最强 | T7 | 效率高,受T7RNA聚合酶调节,不被大肠杆菌RNA 聚合酶调节 | 47.24 | 0 | [ |
pCold II | 中等 | CSPA | 低温高效表达 | 52.78 | 0 | [ |
pTrc99a | 弱 | Trc | 由Trp和Lac合成的人工杂交启动子,效率高于Lac启动子 | 0 | 0 | [ |
pBad24 | 中等 | araBAD | 表达强度由阿拉伯糖的浓度决定 | 0 | 0 | [ |
图 3 分子伴侣蛋白筛选的SDS-PAGE分析及催化活性影响 A: M:蛋白质分子质量标准;泳道1-2:SMR-MEcBO上清、沉淀;泳道3-4:菌株A上清、沉淀;泳道5-6:菌株B上清、沉淀;泳道7-8:菌株C上清、沉淀;泳道9-10:菌株D上清、沉淀;泳道11-12:菌株E上清、沉淀;B:伴侣蛋白对EcBO可溶性表达的影响;C:伴侣蛋白共表达菌株生物合成l-SLR的产量
Fig. 3 SDS-PAGE analysis of molecular chaperone protein screening and catalytic activity of EcBO A: M: Protein molecular weight marker. Lanes 1-2: SMR-MEcBO supernatant, precipitate. Lanes 3-4: Strain A supernatant, precipitate. Lanes 5-6: Strain B supernatant, precipitate. Lanes 7-8: Strain C supernatant, precipitate. Lanes 9-10: Strain D supernatant, precipitate. Lanes 11-12: Strain E supernatant, precipitate. B: Effect of chaperone proteins on soluble expression of EcBO. C: Production of l-SLR biosynthesis by chaperone co-expression strains
图 5 共表达菌株A的培养条件优化 A. l-RL浓度; B. L-阿拉伯糖 浓度; C. IPTG 浓度;D.温度
Fig. 5 Optimization of cultivating conditions for co-expressed strain A A. l-RL concentration. B. L-Arabinos concentration;C. IPTG concentration ;D. temperature
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