Shweta Guleria1,2, Robin Joshi1, Dharam Singh1, Sanjay Kumar3. 1. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176061, Himachal Pradesh, India. 2. Department of Biotechnology, Guru Nanak Dev University, Amritsar, 143005, Punjab, India. 3. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176061, Himachal Pradesh, India. sanjaykumar@ihbt.res.in.
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.
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.
Entities:
Keywords:
Differential expression; Doubling time; E. coli morphology; Heterologous expression; RNase E
Authors: Cecília M Arraiano; José M Andrade; Susana Domingues; Inês B Guinote; Michal Malecki; Rute G Matos; Ricardo N Moreira; Vânia Pobre; Filipa P Reis; Margarida Saramago; Inês J Silva; Sandra C Viegas Journal: FEMS Microbiol Rev Date: 2010-06-24 Impact factor: 16.408
Authors: Kathrin Baumgardt; Pornsri Charoenpanich; Matthew McIntosh; Adam Schikora; Elke Stein; Sebastian Thalmann; Karl-Heinz Kogel; Gabriele Klug; Anke Becker; Elena Evguenieva-Hackenberg Journal: J Bacteriol Date: 2014-01-31 Impact factor: 3.490