Molecular characterization of heavy metal metabolism in transgenic microalgae (Chlamydomonas reinhardtii)

On a global basis, trace-metal pollution is one of the most pervasive environmental problems because of the toxicity of their elemental form. In this work, we report the engineering of Chlamydomonas reinhardtii for uses in heavy metal bioremediation. The construction and successful application of the a universal vector (pSSCR7, 4.3 kbp) used for nuclear gene expression in C. reinhardtii is reported.; Metallothionein-II (MT) is a low molecular weight (∼61 to 62 amono acids), cysteine-rich two domain, metal-binding protein. The β-domain preferentially binds monovalent metals and the β-domain, divalent. We have constructed a fusion gene between a chicken MT-II gene and a Chlamydomonas plasma membrane protein-encoding gene for extracellular cadmium binding and protein stabilization. To increase the specificity and the binding capacity of transformants we have constructed membrane fusions of the α- and/or β-domain of MT-II as well as a series of anchored polymers (1–5) of the MT-II domains separately. We show that these transgenic algae have substantially enhanced tolerance to toxic Cd concentrations (100 μM). In addition, the Cd binding capacity increased 2–5 fold relative to wild-type cells when grown in the presence of 50 μM Cd. Sensitivity to EDTA removal of the metal ions and EXAFS studies are consistent with coordination of the metal ions by the MT-II cysteinyl sulfides.; Free proline has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. We have expressed pyrroline-5-carboxylate synthase (Vigna PSCS from mothbean), the rate limiting step to proline synthesis, in C. reinhardtii. We show that transformants expressing the Vigna PSCS gene have 80% higher free-proline levels than wild-type cells, tolerate toxic Cd concentrations (100 μM), and bind four-fold more cadmium than wild-type cells grown in the presence of 50 μM Cd. The Cd-K edge extended X-ray absorption fine structure (EXAFS) studies reveals a predominantly tetrahedral coordination of cadmium by sulfur compared to a 2 sulfur/2 oxygen tetrahedral coordination in wild-type cells. Our results show that increases in free proline reduces free radical damage, maintains a more reducing environment and results in elevated glutathione levels in the transgenic cells.