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

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.

Polyamine metabolizing rhizobacteria Pseudomonas sp. GBPI_506 modulates hormone signaling to enhance lateral roots and nicotine biosynthesis in Nicotiana benthamiana.

Beneficial rhizobacteria in the soil are important drivers of plant health and growth. In this study, we provide the draft genome of a root colonizing and auxin-producing Pseudomonas sp. strain GBPI_506. The bacterium was investigated for its contribution in the growth of Nicotiana benthamiana (Nb) and biosynthesis of nicotine. The bacterium showed chemotaxis towards root exudates potentially mediated by putrescine, a polyamine compound, to colonize the roots of Nb. Application of the bacterium with the roots of Nb, increased plant biomass and total soluble sugars in the leaves, and promoted lateral root (LR) development as compared to the un-inoculated plants. Confocal analysis using transgenic (DR5:GFP) Arabidopsis showed increased auxin trafficking in the LR of inoculated plants. Upregulation of nicotine biosynthesis genes and genes involved in salicylic acid (SA) and jasmonic acid (JA) signaling in the roots of inoculated plants suggested increased nicotine biosynthesis as a result of bacterial application. An increased JA content in roots and nicotine accumulation in leaves provided evidence on JA-mediated upregulation of nicotine biosynthesis in the bacterized plants. The findings suggested that the bacterial root colonization triggered networking between auxin, SA, and JA to facilitate LR development leading to enhanced plant growth and nicotine biosynthesis in Nb.

Improving the catalytic efficiency and dimeric stability of Cu,Zn superoxide dismutase by combining structure-guided consensus approach with site-directed mutagenesis.

Superoxide dismutase (SOD) leads the front line of defense against injuries mediated by the reactive oxygen species (ROS). The SOD from a high-altitude plant Potentilla atrosanguinea is a unique thermostable enzyme. In this study, we applied a structure-guided consensus approach on Cu,Zn SOD from Potentilla atrosanguinea plant, to improve its enzymatic properties. The polar uncharged amino acid (threonine) at position 97 of wild-type (WT) SOD was selected as a target residue for substitution by aspartate (T97D) through site-directed mutagenesis. The WT and T97D were examined by a combinative approach consisting of robust computational and experimental tools. The in-silico analysis indicated improved dimeric stability in T97D as compared to the WT. The strong interactions between the monomers were related to improved dimerization and enhanced catalytic efficiency of T97D. These results were validated by in-vitro assays showing improved dimer stability and catalytic efficiency in T97D than WT. Moreover, the mutation also improved the thermostability of the enzyme. The combined structural and functional data described the basis for improved specific activity and thermostability. This study could expand the scope of interface residue to be explored as targets for designing of SODs with improved kinetics.

Synergistic effect of graphene oxide and silver nanoparticles as biostimulant improves the postharvest life of cut flower bird of paradise (Strelitzia reginae L.).

The bird of paradise (Strelitzia reginae L.) is one of the important tropical cut flowers. Generally, flowers like bird of paradise (BOP) grown for the commercial ornamental market must be of high pre and postharvest quality. Thus, to improve the postharvest longevity and increase marketability, the relative efficacy of two different biologically synthesized nanoparticles (NPs) was evaluated. The novel proprietary stimulants were graphene oxide (GO) and silver nanoparticles (SNPs). The NP treatments were applied as a vase (lower concentrations) solutions. Among all the applied treatments, the synergistic effect of GO + SNPs at 1 µL L-1 vase solution significantly (p =0.05) prolongs the post-harvest life of cut flowers of BOP. Increased vase life over the deionized water (DI) control was associated with better maintenance of relative water uptake, relative fresh weight, suppressed microbial density at stem-end and delay of stem blockage, reduced electrolyte leakage, malondialdehyde (MDA), SOD, and POD activity. In contrast to control, administration of NPs gave better results for all analyzed parameters. Application of biologically synthesized NPs in combination (GO + SNPs at 1 µL L-1) extended the vase life of cut flowers by 6 days compared with control flowers, and overall, showed better results than the control. The findings of the studies revealed that the standardized NPs could have more potential in prolonging the postharvest life of cut flowers in BOP. Thus, this technique can be used as a novel postharvest technology for commercial application in cut flowers.

A thermostable Fe/Mn SOD of Geobacillus sp. PCH100 isolated from glacial soil of Indian trans-Himalaya exhibits activity in the presence of common inhibitors.

Superoxide dismutases are the enzymes involved in dismutation of superoxide radicals into oxygen and hydrogen peroxide. The present work reports a thermostable Fe/Mn SOD of Geobacillus sp. strain PCH100 (GsSOD) isolated from glacial soil. Purified recombinant GsSOD is a dimeric protein of ~57 kDa that exhibited highest activity at a temperature of 10 °C and pH of 7.8. Maximum enzyme velocity and Michaelis constant of the GsSOD were 1098.90 units/mg and 0.62 µM, respectively. At 80 °C, thermal inactivation rate constant and half-life of GsSOD were 3.33 × 10-3 min-1 and 208 min, respectively. Interestingly, GsSOD tolerated a temperature of 100 °C and 130 °C up to 15 min and 5 min, respectively. Circular dichroism and differential scanning calorimetry confirmed thermostable nature of GsSOD. Apoenzyme of GsSOD regained enzymatic activity in the presence of Fe2+ and Mn2+ as metal ion cofactors. GsSOD was stable under varying concentrations of chemicals, namely ethylenediaminetetraacetic acid, potassium cyanide, hydrogen peroxide, chloroform-ethanol, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, Tween-20, Triton X-100, urea, and guanidine hydrochloride. The enzyme exhibited >70% activity in presence of 10 mM metal ions. Owing to its thermostable nature and resistance to chemical inhibitors, GsSOD is a potential enzyme for industrial applications.

Site-directed mutagenesis (P61G) of copper, zinc superoxide dismutase enhances its kinetic properties and tolerance to inactivation by H2O2.

Superoxide dismutases (SODs) protect the cells by catalyzing the dismutation of harmful superoxide radicals (O2•-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Here, a Cu, Zn SOD (WT) from a high altitude plant (Potentilla atrosanguinea) was engineered by substituting a conserved residue proline to glycine at position 61 (P61G). The computational analysis showed higher structural flexibility and clusters in P61G than WT. The P61G exhibited moderately higher catalytic efficiency (Km = 0.029 µM, Vmax = 1488) than WT protein (Km = 0.038 µM, Vmax = 1290.11). P61G showed higher thermostability as revealed from residual activity (72.25% for P61G than 59.31% for WT after heating at 80 °C for 60 min), differential calorimetry scanning and CD-spectroscopic analysis. Interestingly, the P61G mutation also resulted in enhanced tolerance to H2O2 inactivation than WT protein. The finding on enhancing the biophysico-chemical properties by mutating conserved residue could stand as an example to engineer other enzymes. Also, the reported mutant can be exploited in food and pharmaceutical industries.