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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  2020, Vol. 40 Issue (8): 24-32    DOI: 10.13523/j.cb.2005006
技术与方法     
基于分子伴侣策略提高NADPH依赖型醇脱氢酶的异源可溶性表达 *
邓通,周海胜,吴坚平,杨立荣()
浙江大学化学工程与生物工程学院生物工程研究所 杭州 310027
Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy
DENG Tong,ZHOU Hai-sheng,WU Jian-ping,YANG Li-rong()
College of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China
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摘要：

目的:生物催化的氧化还原反应广泛应用于手性化合物的制备,其中很多反应涉及辅酶NADPH的原位再生。以异丙醇为辅助底物,利用醇脱氢酶再生NADPH,具有比酶活高、副产物丙酮易于分离等优势,受到越来越多的关注。选择极具应用潜力的来源于Clostridium beijerinckii的醇脱氢酶CbADH作为研究对象,针对其在大肠杆菌中的可溶性表达差、酶活低的瓶颈问题开展研究。方法: 首先通过引入诱导型质粒pGro7表达分子伴侣GroES-GroEL,将pET-28a(+)质粒表达CbADH的可溶性提高了3.57倍,酶活达到出发菌株的4.83倍。其次,考察了另外三种不同的分子伴侣表达策略:pET-28a(+)单质粒共表达、基因组强化表达GroES-GroEL和组成型改造pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达。结果: 组成型改造pGro7和pET-28a(+)双质粒共表达策略的效果最优,其CbADH的可溶性表达提高了8.07倍,酶活达到了21.79U/mg DCW,是出发菌株的9.43倍。结论: 为CbADH的工业应用奠定了良好的基础,也为外源蛋白的可溶性表达提供了参考。
关键词： 醇脱氢酶;  异源可溶性表达;  分子伴侣;  NADPH    
Abstract:

Objectives: Many of biocatalytic redox reactions which are widely used in the production of chiral chemicals involve the regeneration of the coenzyme NADPH in situ. Alcohol dehydrogenases that regenerate NADPH with isopropanol as substrate have the advantages of high specific activity and easy separation of byproduct acetone, attracting more and more attention. Therefore, an alcohol dehydrogenase from Clostridium beijerinckii, namely CbADH, was chosen as the research object for its more considerable specific activity and the most applicable potentiality within present literatures. To solve the problem of poor soluble expression of CbADH in E. coli genetically engineered strains and the consequent enzyme activity as low as 2.31 U / mg DCW, the following studies were carried out. Methods: Firstly, different chaperone proteins were expressed by inducible plasmids to increase the soluble expression level of CbADH, and the results showed that molecular chaperone GroES-GroEL significantly improved the soluble expression of CbADH by 3.57 times more than the original strain, with enzyme activity of 11.18 U/mg DCW which is 4.83 times more than the original strain. Secondly, three other different GroES-GroEL expression strategies were examined: pET-28a(+) single plasmid co-expression, genomic enhancing expression of chaperone, and constitutive-pGro7/pET-28a(+) dual plasmid co-expression. Results: The results indicated that the constitutive-pGro7/pET-28a(+) dual plasmid co-expression strategy had the best effect which improved the soluble expression of CbADH by 8.07 times more than the oringinal strain, with a CbADH activity of 21.79 U/mg DCW, which was 9.43 times higher than the oringinal strain. Conclusions: This study not only lays the foundation for the industrial application of CbADH but also provides a reference for heterologous soluble protein expression.
Key words: Alcohol dehydrogenase    Soluble heteroexpression    Chaperone    NADPH
收稿日期: 2020-05-06 出版日期: 2020-09-10
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(21476199)
通讯作者: 杨立荣     E-mail: lryang@zju.edu.cn
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引用本文:

邓通,周海胜,吴坚平,杨立荣. 基于分子伴侣策略提高NADPH依赖型醇脱氢酶的异源可溶性表达 *[J]. 中国生物工程杂志, 2020, 40(8): 24-32.

DENG Tong,ZHOU Hai-sheng,WU Jian-ping,YANG Li-rong. Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy. China Biotechnology, 2020, 40(8): 24-32.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2005006    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I8/24

分子伴侣质粒 伴侣蛋白 启动子 诱导剂 抗性
pG-KJE8 DnaK-DnaJ-GrpE-GroES-GroEL araB Pzt-1 0.5g/ml L-Arabinose+5ng/ml Tetracyclin Cm
pGro7 GroES-GroEL araB 0.5g/ml L-Arabinose Cm
pKJE7 DnaK-DnaJ-GrpE araB 0.5g/ml L-Arabinose Cm
pG-Tf2 GroES-GroEL-Tf Pzt-1 5ng/ml Tetracyclin Cm
pTf16 Tf araB 0.5g/ml L-Arabinose Cm
表1  分子伴侣质粒信息
图1  共表达质粒的表达区域结构示意图
启动子名称 序列 相对强度
J23119 ttgacagctagctcagtcctaggtataatgctagc n/a
J23100 ttgacggctagctcagtcctaggtacagtgctagc 1
J23102 ttgacagctagctcagtcctaggtactgtgctagc 0.86
J23104 ttgacagctagctcagtcctaggtattgtgctagc 0.72
J23108 ctgacagctagctcagtcctaggtataatgctagc 0.51
J23110 tttacggctagctcagtcctaggtacaatgctagc 0.33
J23114 tttatggctagctcagtcctaggtacaatgctagc 0.10
表2  组成型启动子信息①(① 组成型启动子具体信息来源于http://parts.igem.org/Promoters/Catalog/Anderson)
图2  CRISPR基因组编辑示意图
图3  分子伴侣共表达菌株的SDS-PAGE分析
采用策略 菌株名称 OD600 酶活(U/mg DCW)
出发菌株 CbADH-WT 5.91±0.34 2.31±0.3
分子伴侣共表达 A 6.24±0.38 11.18±0.96
B 5.27±0.2 3.61±0.21
C 5.17±0.31 3.98±0.14
D 7.19±0.24 4.17±0.35
E 5.97±0.23 2.88±0.09
单质粒共表达 F 5.4±0.24 10±0.35
G 5.23±0.32 7.89±0.46
H 4.88±0.28 8.02±0.43
I 5.09±0.19 12.99±0.89
基因组强化表达 J 5.23±0.35 9.13±0.31
K 5.31±0.37 7.26±0.18
L 5.28±0.25 2.3±0.24
组成型双质粒共表达 M 5.08±0.41 21.79±1.1
N 5.44±0.24 17.74±0.92
O 5.43±0.32 16.8±0.42
P 5.56±0.21 15.57±0.52
Q 5.79±0.13 15.08±0.46
R 5.55±0.32 14.82±0.32
S 5.32±0.26 12.35±0.67
表3  不同策略的CbADH发酵液酶活测定结果
图4  pET单质粒共表达菌株的SDS-PAGE分析
图5  基因组强化表达GroES-GroEL的SDS-PAGE分析
图6  组成型改造pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达菌株SDS-PAGE分析
图7  GroEL与CbADH表达量比例同CbADH酶活的关系
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