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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (7): 75-82    DOI: 10.13523/j.cb.20180710
技术与方法     
利用SpyTag/SpyCatcher构建胞内自组装多酶复合体实现高效生物合成 *
刘璐,殷亮,黄飞,张勇,刘倩,冯雁()
上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200240
Construction of Intracellular Self-assembled Multienzyme Complex by SpyTag/SpyCatcher to Achieve Efficient Biosynthesis
Lu LIU,Liang YIN,Fei HUANG,Yong ZHANG,Qian LIU,Yan FENG()
State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiaotong University,Shanghai 200240,China
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摘要：

SpyTagr和SpyCatche可通过自发反应形成共价键,产生稳定的分子自组装体。酶分子自组装体因具有高效有序的催化特性在合成生物学和纳米技术领域具有重要的应用价值。为探索SpyTag/SpyCatcher在大肠杆菌胞内多酶复合体系形成有序自组装分子能力,将SpyTagr和SpyCatche分别与P450BM3m单加氧酶和葡萄糖脱氢酶GDH进行融合表达,以期产生具有辅酶再生循环系统、高效生物合成靛蓝分子的SpyTag/SpyCatcher双酶自组装复合体。首先,通过电泳及质谱对重组工程菌表达蛋白进行分析,证实SpyCatcher-P450BM3m与SpyTag-GDH在胞内成功形成了自组装多酶复合体;然后,系统分析不同培养条件下组装体合成靛蓝的能力。结果发现,经0.5mmol/L IPTG诱导后,菌体在16℃继续培养18h后,工程菌对吲哚(2mmol/L)与葡萄糖(4mmol/L)的全细胞催化能力最强,靛蓝产量最高达258mg/L,是未组装多酶系统的1.9倍,比P450BM3m单酶表达系统高约2.4倍;反应70min后达到反应平衡,转化率为52%。成功实现了SpyTag/SpyCatcher介导的多酶体系在大肠杆菌细胞中的自组装和高效转化体系,为胞内多酶复合物组装体的设计提供了新思路。
关键词： SpyTag/SpyCatcher;  自组装;  多酶复合物;  辅酶再生;  靛蓝    
Abstract:

SpyCatcher can form an irreversible covalent linkage to its partner SpyTag via a spontaneous isopeptide bond. To explore the potential application of SpyTag/SpyCatcher system in the self-assembly of multiple enzyme complex in Escherechia coli, SpyCatcher and SpyTag were fused with P450BM3m monooxygenase and glucose dehydrogenase(GDH), respectively. The fusion proteins co-expressed in vivo were expected to self-assemble into a double-enzyme complex with the ability of coenzyme regeneration and efficient biosynthesis of indigo. At first, the self-assembled multienzyme complex formed via SpyTag/SpyCatcher system in vivo was confirmed by SDS-PAGE and Nano-Liquid Chromatography. After that, the ability to synthesize indigo by multienzyme complex was systematically analyzed under different culture conditions. When induced at 16℃ with 0.5mmol/L IPTG, the engineered bacteria showed robust ability to catalyze indole (2mmol/L) and glucose (4mmol/L). The yield of indigo produced by self-assembled complex cells was up to 258mg/L, which was 1.9 and 2.4 times higher than that of cells with free enzymes and P450BM3m single enzyme system accordingly. The conversion rate was 52% when reaction was balanced after 70min. These findings suggest that the SpyTag/SpyCatcher system can successfully assemble multienzyme complex for efficient biosynthesis of chemicals, which provide a new idea for the design of intracellular multienzyme complex assembly.
Key words: SpyTag/SpyCatcher    Self-assembly    Multienzyme complex    Coenzyme regene-ration    Indigo
收稿日期: 2018-03-09 出版日期: 2018-08-13
ZTFLH:  Q819  
基金资助: 国家973计划(2012CB721000);国家自然科学基金国际合作与交流项目(31620103901)
通讯作者: 冯雁     E-mail: yfeng2009@sjtu.edu.cn
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引用本文:

刘璐,殷亮,黄飞,张勇,刘倩,冯雁. 利用SpyTag/SpyCatcher构建胞内自组装多酶复合体实现高效生物合成 *[J]. 中国生物工程杂志, 2018, 38(7): 75-82.

Lu LIU,Liang YIN,Fei HUANG,Yong ZHANG,Qian LIU,Yan FENG. Construction of Intracellular Self-assembled Multienzyme Complex by SpyTag/SpyCatcher to Achieve Efficient Biosynthesis. China Biotechnology, 2018, 38(7): 75-82.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180710    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/75

Plasmid Bacterial strain E.coli BL21(DE3)-X
1 2 mS1) 4 5 NS2) 7 8 S3)
pACYCDuet1-mSpyTag-GDH + - + - - - - - -
pET28a-mSpyCatcher-P450BM3m - + + - - - - - -
pACYCDuet1-GDH - - - + - + - - -
pET28a-P450BM3m - - - - + + - - -
pACYCDuet1-SpyTag-GDH - - - - - - + - +
pET28a-SpyCatcher-P450BM3m - - - - - - - + +
表1  表达重组质粒的E.coli BL21 (DE3)工程菌的构建
图1  SDS-PAGE分析SpyTag-GDH和SpyCatcher-P450BM3m组装图
图2  SpyTag-GDH和SpyCatcher-P450BM3的MS/MS图谱
Protein Specific activity (U/mg)
GDH 3.076±0.42
SpyTag-GDH 3.13±0.86
P450BM3m 180.22±17.5
SpyCatcher-P450BM3m 187.83±8.2
表2  重组蛋白酶活比较
图3  IPTG浓度对靛蓝产量的影响
图4  诱导温度对靛蓝产量的影响
图5  吲哚与葡萄糖比例对靛蓝产量的影响
图6  单酶细胞和辅酶再生细胞催化合成靛蓝反应的比较
图7  E.coli BL21 (DE3)-S合成靛蓝的催化进程
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