**3.2 Phytochelatin synthase-involving pathway engineering for enhancing heavy metal accumulation**

Pathway engineering involved in the co-expression of both GSH synthesis and PC synthesis pathways is another strategy to preserve the balance of GSH metabolism in the cells with constitutive PC synthesis [72, 74]. A kinetic model of GSH and phytochelatin synthesis in plants suggests that at least two enzymes, γ-glutamylcysteine synthetase (γ-ECS) and PCS, should be increased to enhance PC synthesis without depleting the GSH pool [89]. In fact, the effects of modified GSH/PC synthesis pathways have been tested in *Escherichia coli* and tobacco plants, respectively [93, 94]. In these experiments, the activities of SpPCS and two enzymes catalyzing the rate-limiting steps of GSH biosynthesis, including serine acetyltransferase (SAT) and γ-ECS, were enhanced (**Figure 1**) [7, 8, 93, 94]. The *E. coli* cells co-overexpressing these enzymes accumulated significantly higher concentrations of PCs and Cd2+, while the single-gene expression in the PC synthesis pathway had limited effects [93]. These findings support the "gene stacking" approaches to enhancing heavy metal metabolism. Although the same strategy cooverexpressing these three genes in tobacco increased some classes of non-protein thiol, the Cd2+ accumulation in the transgenic plants did not change compared to the wild type [94]. These findings suggest that other mechanisms, in addition to the availability of precursors for PC synthesis, limit Cd accumulation in plants [94].

Overall, the genetic engineering approaches involved in manipulating PC synthesis have shown promising prospects for improving the performance of plants in the phytoremediation of heavy metals. However, there are also setbacks pointing at the complexity of the stress response induced by heavy metals [75]. Thus, while enhanced PC synthesis can contribute to the heavy metal chelating, other factors, such as the subsequent vacuolar sequestration or the delicate balance of GSH metabolic pathways under heavy metal stress, should be considered in order to achieve heavy metal tolerance.
