Bacteria Metabolically Engineered for Enhanced Phytochelatin Production and Cadmium Accumulation

Phytochelatins (PCs) with good binding affinities for a widerange of heavy metals were exploited to develop microbial sorbentsfor cadmium removal. PC synthase from Schizosaccharomyces pombe(SpPCS) was overexpressed in Escherichia coli, resulting inPC synthesis and 7.5-times-higher Cd accumulation. The coexpressionof a variant ${gamma}$ -glutamylcysteine synthetase desensitized to feedbackinhibition (GshI*) increased the supply of the PC precursorglutathione, resulting in further increases of 10- and 2-foldin 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 µmol/g [dry weight] of cells) was more than 25-foldhigher than that in the control strain.

INTRODUCTION

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INTRODUCTION

Widespread pollution by heavy metals generated from variousindustrial and agricultural activities has serious adverse effectson human health and ecosystems (12). The increased public concernover these heavy metals has caused more-stringent control ofthe allowable limits in drinking water and soil. Although conventionaltechnologies are adequate to remove the bulk of heavy-metalcontamination, they are often inadequate to reduce heavy-metalconcentrations to acceptable regulatory standards (8). Bioremediationbased on genetically engineered bacteria is an emerging technologythat is receiving more attention as an inexpensive and efficientway of cleaning up toxic-metal contamination (10). In particular,the production of metal-binding peptides such as metallothioneinsand phytochelatins (PCs) has been shown to confer enhanced heavy-metal-bindingcapabilities (1, 2, 18).

PCs are naturally occurring peptides consisting of the repeating ${gamma}$ -Glu-Cys dipeptide unit terminated by a Gly residue (6, 21).The presence of a ${gamma}$ bond between glutamic acid and cysteine indicatesthat the synthesis of PCs cannot occur via the ribosomes. PCbiosynthesis, indeed, proceeds through the transfer of ${gamma}$ -Glu-Cysfrom glutathione (GSH) to another GSH or other PCs (21) by theenzyme PC synthase (PCS) when this enzyme is activated by heavymetals such as Cd, Cu, Hg, and Pb. PCs are known to bind heavymetals such as Cd, Hg, As, and Pb, especially cadmium, withhigh affinity through thiolate complexes (7, 16). Recently,the genes coding for PCS have been cloned from plants and fungiand functionally expressed in Escherichia coli (15). The intracellularcadmium content of the E. coli strain expressing PCS increasedmore substantially than that of the control strain. A directcorrelation 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) demonstratedthe possibility of heavy-metal remediation using PC-producingcells, a severe drop in the GSH content suggests that the GSHsupply may be the limiting step in PC synthesis. In E. coli,the biosynthesis of GSH proceeds via ${gamma}$ -glutamylcysteine synthetase(GshI) with the conversion of glutamate and cysteine into ${gamma}$ -glutamylcysteine( ${gamma}$ -EC), which is then converted into GSH by GSH synthetase (9).Since the synthesis of ${gamma}$ -EC is reported to be rate limiting (5),one potential strategy to increase the supply of GSH is to overexpressGshI.

In this work, a variant of GshI, GshI*, which is desensitizedto feedback inhibition by GSH (11), was overexpressed in E.coli, resulting in a significant increase in PC production anda corresponding increase in heavy-metal accumulation. Furtherimprovement in Cd accumulation was achieved by the coexpressionof a Cd transporter, MntA, suggesting that PC-mediated Cd accumulationcan be optimized by manipulating the GSH and PC synthesis pathwaysin a rational manner.

Expression of different PCSs and effects on cadmium accumulation.

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Expression of different PCSs...
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Previously, the gene for Arabidopsis thaliana PCS (AtPCS) wasfunctionally expressed in E. coli, resulting in a significantincrease in the cellular cadmium content (15). To investigatewhether other PCSs could function similarly, the effect of PCSfrom Schizosaccharomyces pombe (SpPCS) (3) on cadmium accumulationwas compared with that of AtPCS. The expression of both PCSgenes was under the control of a T5 tac promoter, and a sequenceencoding a hexahistidine tag was added to the end of each genecorresponding to the C terminus of the product to allow easydetection with an anti-His tag antibody (see the supplementalmaterial for details). Transformed JM109 cells were grown inLuria-Bertani medium with appropriate antibiotics to an opticaldensity of 0.5 before the addition of 20 µM CdCl₂ and0.8 mM isopropyl-ß-D-1-thiogalactopyranoside (IPTG).For the evaluation of whole-cell metal contents, cells werewashed three times with 5 mM HEPES buffer containing 0.8% NaClbefore drying at 65°C for 24 h. The dried cell pellets weredigested with 100 µl of concentrated nitric acid for 2days according to a method modified from that of Sriprang etal. (19). The total cadmium contents were measured using atomicabsorption spectrometry with an AAnalyst 800 spectrometer (PerkinElmerInc., Waltham, MA). Although the expression of either PCS resultedin an increase of more than eightfold in the intracellular cadmiumcontent over that of the control strain (Fig. 1A), the levelof accumulation in cells expressing SpPCS was 20% higher thanthat in cells expressing AtPCS. This difference in cadmium accumulationcan be attributed partially to the twofold-lower level of expressionof AtPCS than of SpPCS (Fig. 1B) as determined by Western blotting(14) and densitometry. Cells expressing SpPCS exhibited a levelof Cd accumulation (14.9 µmol/g [dry weight] of cells)twofold higher than the levels achieved in a previous studyusing AtPCS (7.2 µmol/g [dry weight] of cells) (15). Owingto the higher level of Cd²⁺ accumulation and the improved PCSproduction, cells expressing SpPCS were investigated further.

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FIG. 1. Comparison of E. coli JM109 cells expressing AtPCS or SpPCS. (A) Cadmium accumulation in E. coli cells harboring either pQE60 (control), pQE-AtPCS (AtPCS), or pQE-SpPCS (SpPCS). The Cd content was measured by atomic absorption spectrometry. Data shown are the mean values (+ standard deviations) obtained from three independent experiments. (B) Western blot analysis of PCS expression in E. coli cells harboring either pQE60 (lane 2), pQE-AtPCS (lane 3), or pQE-SpPCS (lane 4). The molecular weight marker is shown in lane 1. The production of PCS was detected by an anti-His tag antibody, and the intensity was quantified using densitometry.

Effect of GshI* overexpression on the intracellular cadmium, PC, GSH, and ${gamma}$ -EC contents.

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Expression of different PCSs...

Effect of GshI* overexpression...

To investigate the effect of SpPCS expression on the intracellularthiol contents, the levels of ${gamma}$ -EC, GSH, and PCs were measured.The derivatization procedure with monobromobimane using fluorescencedetection was adopted from Sneller et al. (17). The expressionof SpPCS resulted in the synthesis of different forms of PC(such as PC2, PC3, and PC4), with PC2 being the dominant species(data not shown). Unlike control cells without SpPCS expression,cells expressing SpPCS no longer contained detectable intracellularGSH (Fig. 2B), indicating that all the available GSH had beenused for PC synthesis. These results strongly suggest that thesupply of GSH is limiting for PC synthesis. In contrast, thelevel of ${gamma}$ -EC actually increased in cells expressing SpPCS (Fig.2C). Since the enzyme responsible for ${gamma}$ -EC synthesis, GshI, isreported to be subject to feedback inhibition by GSH (4), thisincreased level of ${gamma}$ -EC may be the result of elevated GshI activityin the absence of GSH.

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FIG. 2. Intracellular Cd (A), GSH (B), ${gamma}$ -EC (C), and PC2 (D) contents of E. coli strain JM109 harboring either pQE60 (C), pQE-SpPCS (Sp), pMMB-gshI (G), or both pQE-SpPCS and pMMB-gshI (Sp/G). Data shown are the mean values (+ standard deviations) obtained from three independent experiments.

To test whether increasing the GSH supply could improve PC synthesis,a variant form of the rate-limiting enzyme involved in the biosynthesisof GSH, GshI*, which was desensitized to feedback inhibition,was expressed with SpPCS. Plasmid pMMB-gshI (13), which containedthe GshI* gene under the control of a tac promoter, was usedfor GshI* overexpression alone. The intracellular GSH and ${gamma}$ -ECcontents of cells with and without GshI* overexpression werecompared. The introduction of pMMB-gshI into E. coli strainJM109 resulted in two- and sixfold increases in the intracellularGSH and ${gamma}$ -EC contents, respectively (Fig. 2B and C). The expressionof GshI* with SpPCS resulted in a 10-fold increase in the PC2content (Fig. 2D). This result confirms that increasing thesupply of GSH can effectively alleviate the bottleneck in PCsynthesis. Interestingly, the intracellular ${gamma}$ -EC content alsoincreased by 200-fold compared to that of the control, whileonly a small amount of GSH was detected (Fig. 2B and C). Thisresult suggests that at the elevated level of GshI* and PCSexpression, the conversion of ${gamma}$ -EC into GSH by GSH synthetasebecomes rate limiting. Unlike the intracellular PC2 contentthat increased by 10-fold, the level of cadmium accumulationincreased by only a further twofold over that of cells expressingonly SpPCS, suggesting that other factors such as cadmium uptakemay be limiting (Fig. 2A). It has been reported previously thata recombinant E. coli strain harboring the manganese transportgene (mntA) could significantly increase specific cadmium accumulationdue to the enhanced uptake (6); a similar strategy was usedto further improve the bioaccumulation of cadmium.

Coexpression of MntA, GshI*, and SpPCS further enhances the intracellular cadmium content.

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Coexpression of MntA, GshI*,...

To investigate whether the overall Cd accumulation could befurther enhanced by increasing Cd transport, the Cd transporterMntA was overexpressed along with GshI* and SpPCS. Two separatevectors, one (pQE-SpPCS-GshI*) harboring the genes coding forGshI* and SpPCS as an operon and a second carrying the mntAgene on pZH3-5 (6), were used. Interestingly, the PC2 levelsin cells carrying the synthetic operon were fourfold lower thanthose in the previously tested cells in which GshI* and SpPCSwere expressed separately (see Fig. S1 in the supplemental material)due to the lower level of SpPCS expression. However, the intracellularCd contents of cells harboring the synthetic operon remainedthe same, and the co-overexpression of MntA further increasedthe intracellular Cd accumulation by 1.5-fold. The resultinglevel of Cd accumulation (31.6 µmol/g [dry weight] ofcells) was more than 25-fold higher than that in the controlstrain. This result indicates that the uptake of Cd is indeeda key rate-limiting step in the overall Cd accumulation.

It should be noted that Wawrzynska et al. (20) recently reportedthe coexpression of GshI, SpPCS, and serine acetyltransferasein E. coli. However, Cd accumulation was increased by only fivefold,and no quantitative analysis of the nonprotein thiols was reported.This lower level of Cd enhancement was likely a result of usingthe wild-type GshI, which is subjected to feedback inhibitionby GSH. In the present study, we thoroughly analyzed differentbottlenecks in PC synthesis and Cd uptake to achieve high levelsof whole-cell Cd accumulation. These levels of accumulationcould potentially be further improved by additional manipulationsof Cd uptake and PC synthesis.

In conclusion, the overexpression of PCS and GshI* in E. coliimproves not only the intracellular PC content but also thesubsequent Cd accumulation. Furthermore, the coexpression ofMntA, SpPCS, and GshI* leads to an additional increase in Cdaccumulation. Because of the high level of heavy-metal accumulationafforded by this strategy, the engineered strains can be easilyadapted in immobilized bioreactors for wastewater cleanup. Thispotential is particularly attractive if resting cells that producea high level of PCs and are optimized for Cd uptake can be employed.It has been demonstrated previously that resting cells expressingMntA and metallothionein can be used for selective Cd removal(6). The ability of resting cells expressing MntA and SpPCSfor effective Cd removal will be similarly investigated.

ACKNOWLEDGMENTS

This work was supported by grants from the NSF (BES0422791 andBES0329482).

We thank Thomas K. Wood, Texas A&M University, and DavidWilson, Cornell University, for plasmids pMMB-gshI and pZH3-5,respectively.

FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521. Phone: (951) 827-2473. Fax: (951) 827-5696. E-mail: wilfred{at}engr.ucr.edu

Published ahead of print on 3 August 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

These authors contributed equally to this paper.

Present address: Department of Chemical and Biomolecular Engineering,Cornell University, 120 Olin Hall, Ithaca, NY 14853-5201.

^¶ Present address: Department of Molecular Science and Technology,Ajou University, Suwon, Korea 442-7491.

REFERENCES

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