Bacteria Metabolically Engineered for Enhanced Phytochelatin Production and Cadmium Accumulation

Abstract

Phytochelatins (PCs) with good binding affinities for a wide range of heavy metals were exploited to develop microbial sorbents for cadmium removal. PC synthase from Schizosaccharomyces pombe (SpPCS) was overexpressed in Escherichia coli, resulting in PC synthesis and 7.5-times-higher Cd accumulation. The coexpression of a variant ␥-glutamylcysteine synthetase desensitized to feedback inhibition (GshI*) increased the supply of the PC precursor glutathione, resulting in further increases of 10-and 2-fold in PC production and Cd accumulation, respectively. A Cd transporter, MntA, was expressed with SpPCS and GshI* to improve Cd uptake, resulting in a further 1.5-fold increase in Cd accumulation. The level of Cd accumulation in this recombinant E. coli strain (31.6 mol/g [dry weight] of cells) was more than 25-fold higher than that in the control strain.Widespread pollution by heavy metals generated from various industrial and agricultural activities has serious adverse effects on human health and ecosystems (12). The increased public concern over these heavy metals has caused more-stringent control of the allowable limits in drinking water and soil. Although conventional technologies are adequate to remove the bulk of heavy-metal contamination, they are often inadequate to reduce heavy-metal concentrations to acceptable regulatory standards (8). Bioremediation based on genetically engineered bacteria is an emerging technology that is receiving more attention as an inexpensive and efficient way of cleaning up toxic-metal contamination (10). In particular, the production of metal-binding peptides such as metallothioneins and phytochelatins (PCs) has been shown to confer enhanced heavy-metal-binding capabilities (1,2,18).PCs are naturally occurring peptides consisting of the repeating ␥-Glu-Cys dipeptide unit terminated by a Gly residue (6,21). The presence of a ␥ bond between glutamic acid and cysteine indicates that the synthesis of PCs cannot occur via the ribosomes. PC biosynthesis, indeed, proceeds through the transfer of ␥-Glu-Cys from glutathione (GSH) to another GSH or other PCs (21) by the enzyme PC synthase (PCS) when this enzyme is activated by heavy metals such as Cd, Cu, Hg, and Pb. PCs are known to bind heavy metals such as Cd, Hg, As, and Pb, especially cadmium, with high affinity through thiolate complexes (7, 16). Recently, the genes coding for PCS have been cloned from plants and fungi and functionally expressed in Escherichia coli (15). The intracellular cadmium content of the E. coli strain expressing PCS increased more substantially than that of the control strain. A direct correlation between PC content and metal accumulation was observed, indicating that PC is primarily responsible for the metal sequestration.Although the initial results of the previous study (15) demonstrated the possibility of heavy-metal remediation using PCproducing cells, a severe drop in the GSH content suggests that the GSH supply may be the limiting step in PC synthesis. In E. coli, the biosynthesis of GSH proceeds via ...

Bioremediation of Heavy Metals from Soil and Aquatic Environment: An Overview of Principles and Criteria of Fundamental Processes

“…Expression of different proteins and peptides by the Escherichia coli regulates the range of accumulation of cadmium [83]. Co-expression of precursor glutathione (GSH) along with phytochelatins (PC) resulted in the 10 fold increase in PC that finally increased cadmium accumulation twofold [84]. Natural resistant pathways for heavy metals (Hg and Ar) in microorganisms have been regulated by metalloregulatory protein [85].…”

Glutathione and Transition-Metal Homeostasis inEscherichia coli

“…The sequestration of heavy metal cations by phytochelatin and the consecutive transport of the bis-glutathionato complexes into vacuoles seem to be the main route of heavy-metal detoxification in these organisms. Phytochelatins can be produced successfully in transgenic bacteria, leading to enhanced metal accumulation in the bacterial cytoplasm (3,11,28,69,72) without the compartmentation ability of a vacuole.Why do bacteria detoxify heavy metals mainly by efflux (49, 50) even though they should be able to sequester metals to the phytochelatin educt GSH? In GSH, two cysteine residues and four carboxyl groups should be able to form octahedral bisglutathionato complexes that are very stable (76).…”

Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli

“…Construction of plasmids pQE-SpPCS & pQE-SpPCS-GshI Ã has been reported previously (Kang et al, 2007). For constructing pQE-SpPCS-GshI Ã -GlpF, the GlpF gene was amplified from pMT-GlpF (Singh et al, 2008b) with the forward primer GCCGGGATCC AAGGAGATATACAT-CAGAAGGAGATATACATATGAGTCA and the reverse primer TAGGATCC GTCGACTCTAGAGCTCTTCAAG-TTAATGGTGATGGT GATGGT.…”

“…PCs are synthesized (Fig. 1) by phytochelatin synthase (PCS) via the transfer of g-Glu-Cys from glutathione (GSH) to another GSH or other PCs (Mehra and Winge, 1991;Vatamaniuk et al, 1999;Zenk, 1996) and can be easily fine-tuned for high-level production (Kang et al, 2007). …”