Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy

doi:10.13523/j.cb.2005006

China Biotechnology

2020, Vol. 40

Issue (8): 24-32 DOI: 10.13523/j.cb.2005006

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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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

Received: 06 May 2020 Published: 10 September 2020

ZTFLH:

Q819

Corresponding Authors: Li-rong YANG E-mail: lryang@zju.edu.cn

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Cite this article:

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.

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

Table 1 Information of molecular chaperone plasmid

Fig.1 Expression region structure of the co-expression plasmids

Table 2 Information of constituent promoters

Fig.2 Schematic diagram of CRISPR genome editing modified promoter

Fig.3 SDS-PAGE analysis of molecular chaperones co-expression strains Lane M:molecular weight marker;Lane W:whole cell protein; Lane S:supernatant;Lane P:precipitation

Table 3 CbADH activity of different strategies

Fig.4 SDS-PAGE analysis of pET single plasmid co-expression strains Lane M: molecular weight marker;Lane W:whole cell protein; Lane S: supernatant;LaneP:precipitation

Fig.5 SDS-PAGE analysis of GroES-GroEL genomic over-expression strains Lane M: molecular weight marker; Lane W: whole cell protein; Lane S: supernatant; Lane P: precipitation

Fig.6 SDS-PAGE analysis of constitutive pGro7/GroES-GroEL and pET-28a(+)/CbADH dual plasmid co-expressed strains Lane M: molecular weight marker;Lane W:whole cell protein;Lane S:supernatant;Lane P:precipitation

Fig.7 Relationship between GroES-GroEL expression and CbADH activity


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