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Identification of host factors limiting the overexpression of recombinant Cu, Zn superoxide dismutase in Escherichia coli

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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/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.

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Acknowledgements

SG is thankful to Indian council of Medical Research for Junior and Senior Research Fellowships. Authors gratefully acknowledge Dr Sue Lin-Chao, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan for providing RNase E antibodies as a gift. Financial support from CSIR network projects (Grant No. BSC0109, BSC0209) and in-house grant (MLP201) is duly acknowledged. The manuscript represents CSIR-IHBT communication number 4148.

Supporting Information

Fig. S1 Effect of Pa-SOD expression on growth of E. coli at 37 °C and 28 °C. Graphs on left represent E. coli growth curve at 37 °C while graphs on right represent E. coli growth curve at 28 °C a, b Growth curve of E. coli M15 strain harboring empty pQE30 vector (control). c, d Growth curve of E. coli M15 strain harboring recombinant pQE-Pa-SOD. e, f Growth curve of E. coli BL21 (DE3) harboring recombinant pET47b-Pa-SOD. The results are expressed as means ± SE of the means, n=3.

Fig. S2 SDS-PAGE analysis of differentially expressed proteins upon induction of Pa-SOD. M represents protein molecular weight marker (kDa). pQE-Pa-SOD/M15 indicates total protein obtained from E. coli M15 strain harboring pQE-Pa-SOD recombinant plasmid (genetically engineered mutant of Pa-SOD WT) before induction at 0 h and after induction at 3 h (lanes 2-3). The arrow indicates the differentially expressed proteins.

Fig. S3 Relative expressions of rne, ispF, ftsZ, ftsA, and Pa-SOD at 0, 1, 3 and 5 h of induction as analyzed by qRT-PCR. Endogenous hcaT of E. coli was used to normalize the data. The results are expressed as means ± SE of the means, n=3. Different alphabet above bars represent significant difference at p ≤ 0.05 according to Duncan’s multiple range test.

Table S1 List of primers used for qRT-PCR.

Table S2 Differentially expressed proteins identified by UHPLC-Q-TOF-IMS in E. coli upon Pa-SOD induction.

Table S3 Differentially expressed proteins identified by MALDI-TOF-MS/MS/MS in E. coli upon Pa-SOD induction.

Table S4 Functional classification and cluster analysis of differentially expressed protein obtained from PANTHER and DAVID, respectively.

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Authors and Affiliations

  1. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176061, Himachal Pradesh, India

    Shweta Guleria, Robin Joshi, Dharam Singh & Sanjay Kumar

  2. Department of Biotechnology, Guru Nanak Dev University, Amritsar, 143005, Punjab, India

    Shweta Guleria

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  1. Shweta Guleria
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Correspondence to Sanjay Kumar.

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Guleria, S., Joshi, R., Singh, D. et al. Identification of host factors limiting the overexpression of recombinant Cu, Zn superoxide dismutase in Escherichia coli. Biotechnol Lett 42, 2389–2401 (2020). https://doi.org/10.1007/s10529-020-02962-6

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