Microrganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over-express either gamma-GCS or GS and found that gamma-GCS over-expression resulted in inclusion body formation and impaired cell physiology, whereas GS over-expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over-expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild-type cells in size (2-5 mn) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was approximate to 2.5 times that attained in the wild-type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli-based biosynthesis offers a more energy-efficient and eco-friendly method as opposed to chemical processes requiring high temperature and toxic solvents. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1260-1266, 2009