Cell and peptide engineering: Environmental clean-up to bioprogramming.

摘要

Ubiquitously present metalloid arsenic is a known carcinogen and long term exposure to arsenic has been linked to high risks of cancer of the skin, kidneys, lungs as well as other disorders. Enforcing the new maximum contaminant level (MCL) of As of 10 ppb would affect several water supply systems across the country. Current methods prove to be too costly and ineffective. Therefore an alternative technology based on biological methods is demonstrated here to provide cost effective and environmentally benign selective technology for arsenic remediation.;Saccharomyces cerevisiae and Escherichia coli were engineered by combining their naturally existing arsenic detoxification pathways with defense mechanisms developed by other organisms for combating arsenic toxicity. Production of arsenic binding peptides phytochelatins (PCs) in the yeast Saccharomyces cerevisiae by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) resulted in six times higher As accumulation under a wide range of As concentrations. Subsequently functional expression of an arsenic specific peptide from Fucus vesiculosus; metallothionein (fMT) in E. coli cells led to 36- & 26-fold higher As(V) and As(III).. Co-expression of an As(III)-specific transporter GlpF with fMT further increased the arsenic accumulation and also offered high selectivity towards As. PC production in yeast and fMT expression in E. coli, also led to their suitability in resting cell systems.;Finally PCs were also produced in E. coli by overexpressing PC synthase from Schizosaccharomyces pombe (SpPCS), leading to 18 fold higher arsenic accumulation. PC production was significantly increased (more than 30 fold) by improving the levels of PC precursors Gluthathione (GSH) and gamma-glutamylcysteine (gamma-EC) by overexpressing feedback desensitized rate liming first enzyme glutamylcysteine synthetase (GshI*). Deletion of arsenic efflux pump ArsAB in E. coli, further added to arsenic accumulation resulting in 16.81 mumol/gDCW arsenic accumulation levels.;The specific interaction of metal binding peptides with respective metals and formation of metal-peptide complex was exploited for formation of highly fluorescent semiconductor nanocrystals. A bi-functional peptide selected for bulk semiconductor material binding was exploited for synthesis of core shell CdSe/ZnS nanoparticles. Highly monodisperse and photoluminescent nanocrystals were obtained. CdSe core with an average size of 4 nm followed by ZnS shell growth increasing the overall size to an average 5 nm was observed under TEM. Diffraction patterns verified the presence of characteristic zinc blend CdSe diffractions. We demonstrate core-shell semiconductor nanoparticle green chemistry low temperature synthesis with control over size and crystallinity using biological templates.