Identification of host factors limiting the overexpression of recombinant Cu, Zn superoxide dismutase in Escherichia col
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ORIGINAL RESEARCH PAPER
Identification of host factors limiting the overexpression of recombinant Cu, Zn superoxide dismutase in Escherichia coli Shweta Guleria . Robin Joshi . Dharam Singh . Sanjay Kumar
Received: 15 February 2020 / Accepted: 3 July 2020 Ó Springer Nature B.V. 2020
Abstract Objective Superoxide dismutase (SOD) enzyme has implications in modulating the cell’s redox state. The study aims to explore the host genetic factors that limit the heterologous expression of a thermostable SOD from Potentilla atrosanguinea (Pa-SOD) in E. coli. Results It was observed that the heterologous expression of Pa-SOD in E. coli did not exhibit any enhancement after 1 h of induction. This led to the alteration in cell morphology and an increase in the doubling time of E. coli cells expressing Pa-SOD. Label-free quantification and MALDI-TOF/TOF-MS/
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-02962-6) contains supplementary material, which is available to authorized users. S. Guleria R. Joshi D. Singh S. Kumar (&) Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India e-mail: [email protected] S. Guleria e-mail: [email protected] R. Joshi e-mail: [email protected] D. Singh e-mail: [email protected]
MS analysis suggested differential expression of 81 proteins, of which 77 proteins were found to be downregulated and 4 were found to be upregulated in Pa-SOD expressing cells as compared to uninduced E. coli cells. Functional analysis of downregulated proteins shows involvement in molecular function, biological process, and were the part of a cellular component. The STRING database revealed interaction of an essential autoregulatory protein, RNase E with other proteins involved in biosynthetic processes, protein biosynthesis and folding, and cell division. Further, validation of RNase E protein revealed upregulation of rne at transcript level and downregulation of RNase E at protein level as compared to uninduced cells. Conclusions The observations suggested the operation of multifaceted mechanisms with a key role of RNase E that regulated the expression of Pa-SOD at the physiological and molecular level. Since Pa-SOD has commercial applications, identification and manipulation of these networked genetic factors could lead to improvement of host strain for large-scale production of biologically active Pa-SOD and other heterologous proteins. Keywords Heterologous expression E. coli morphology Doubling time Differential expression RNase E
S. Guleria Department of Biotechnology, Guru Nanak Dev University, Amritsar 143005, Punjab, India
123
Biotechnol Lett
Introduction Superoxide dismutase (SOD) catalyses dismutation of destructive superoxide radical into hydrogen peroxide and molecular oxygen (McCord and Fridovich 1969; Rabinowitch and Fridovich 1983). If not removed, superoxide radical reacts with biomolecules i
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