Functional characterization of Helianthus annuus phytochelatin synthase (HaPCS): Gene expression and protein profiles of HaPCS responding to arsenic and evaluation of arsenic accumulation in engineered bacteria expressing HaPCS

Abstract

Arsenic is the most prevalent environmental toxic element and causes a variety health effects. Phytochelatins (PCs) are naturally occurring peptides with high affinity toward metal(loid)s that are present in plants and fungi. Here, the gene encoding phytochelatin synthase from Helianthus annuus (HaPCS) was isolated and cloned in expression vectors pET28a and pET41a. The resulting constructs were transformed into Escherichia coli strains Rosetta (DE3) and Rosetta gami 2. Following the induction with Isopropyl β-D-1-thiogalactopyranoside, the enzyme GST-HaPCS was expressed in the strains Rg-41a-HaPCS and R-41a-HaPCS and the enzyme His-HaPCS was expressed in R-28a-HaPCS. The comparison of soluble and insoluble extracted fractions revealed that the expression of GST-HaPCS in the strain Rg-41a-HaPCS is the optimum method for expression of this enzyme. The recombinant and pure GST-HaPCS catalysed PCs in vitro only in the presence of Cd2+, As3+ and As5+. The presence of Cd2+ activated the enzyme GST-HaPCS more than As3+/As5+. Therefore the enzyme GST-HaPCS seems to be differentially activated by different metal(loid)s. The strain Rg-41a-PCS was able to synthesise PCs when the medium was supplemented by As3+, As5+ and Cd2+. In these conditions the new strains were able to accumulate 10.67 μmol/g DCW As5+, 7.58 μmol/g DCW As3+ and 25.97 and 25.97 μmol/g DCW in medium containing 0.5 mM of each of these metal(loid)s. Furthermore in the present work we found that there was no difference between the expressions of HaPCS in H. annuus in response to different metal(loid)s. Therefore the higher accumulation of PCs and subsequently higher accumulation of Cd2+ could be due to the higher activity of HaPCS in the presence of this metal ion. This study introduces a good candidate gene from plant source with high ability for synthesis of PCs and accumulation of arsenic and cadmium.