Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance

Abstract

Background: Plants synthesize phytochelatin peptides for protection against heavy metals. Results: Metabolic engineering in yeast and plants using a phytochelatin synthase variant leads to improved cadmium tolerance. Conclusion: Enhanced cadmium tolerance results from a balance between phytochelatin synthesis and redox state. Significance: Our results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools for environmental remediation.Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and ␥-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.Environmental heavy metal contamination, which is implicated in many diseases and agricultural losses (1-2), poses a challenge with a price tag for remediation estimated at upward of $200 billion in the United States alone (2). Heavy metals, such as cadmium (Cd 2ϩ ), undergo aberrant capping reactions with the thiol groups of proteins and some coenzymes, displace endogenous metal cofactors from their cellular binding sites, and promote the formation of reactive oxygen species (2). Engineering plants and microbes for the detoxification of heavy metal-contaminated soils and waters traditionally rely on the modification of existing metabolic pathways and/or metal transport and sequestration systems (3-7). Although transgenic methods can enhance heavy metal tolerance and accumulation, the inherent biochemical properties of the proteins introduced often limit the extent of the enhancement. Engineering the proteins that are used for this purpose can offset inherent limitations on tolerance to and/or accumulation of heavy metals so as to broaden the range of molecular tools available for environmental clean-up (8).Plants employ a combination of metabolic enzymes and transport systems for protection against toxic levels of heavy metals (9). Exposure of plants to heavy metals, such as cadmium, arsenic...

Nitric Oxide Mediated Transcriptome Profiling Reveals Activation of Multiple Regulatory Pathways in Arabidopsis thaliana

“…This enzyme catalyzes the synthesis of phytochelatin from glutathione at basal levels and in the presence of metal ions like Cd 2+ , Zn 2+ , Cu 2+ , and Fe 3+ . It has been recently used to engineer heavy metal tolerance in plants (Cahoon et al, 2015 ) and to engineer cadmium biosensers in yeast (Matsuura et al, 2013 ), and its over-expression in tobacco has been shown to enhance arsenic and cadmium detoxification (Zanella et al, 2015 ). However, the ubiquitous nature and constitutive expression of phytochelatin synthases suggest a rather more generalized function besides metal detoxification, as PCS1 was later shown to be involved in non-host resistance (Clay et al, 2009 ).…”

The next green movement: Plant biology for the environment and sustainability

“…In addition to knockout engineering, fine-tuning enzymatic activity can also tailor plant responses to toxic metals. Directed evolution of phytochelatin synthase (PCS), an enzyme that synthesizes heavy metal-binding peptides, revealed an unexpected way of improving heavy metal tolerance through the attenuation of enzymatic activity (43). A catalytically inferior PCS resulted in phenotypic superiority because it maintained cellular redox homeostasis while synthesizing peptides for detoxification of cadmium (43).…”

Diverse fission yeast genes required for responding to oxidative and metal stress: Comparative analysis of glutathione‐related and other defense gene deletions

“…No form of phytochelatin was detected; however, phytochelatin synthase activity is only detectable in the presence of heavy metal stress (Cahoon et al . ).…”