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Metabolic engineering of Escherichia coli for 1-butanol

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Biochemical and Biophysical Research Communications 365 (2008) 453–458

www.elsevier.com/locate/ybbrc

Phytodegradation of organophosphorus compounds

by transgenic plants expressing a bacterial organophosphorus hydrolase

Xiaoxue Wang a , Ningfeng Wu a, *, Jun Guo a , Xiaoyu Chu a , Jian Tian a ,

Bin Yao b , Yunliu Fan a

a Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China

b Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China

Received 29 October 2007

Available online 26 November 2007

Abstract

Organophosphorus (OP) compounds are widely used as pesticides in agriculture but cause broad-area environmental pollution. In this

work, we have expressed a bacterial organophosphorus hydrolase (OPH) gene in tobacco plants. An assay of enzyme activity showed

that transgenic plants could secrete OPH into the growth medium. The transgenic plants were resistant to methyl parathion (Mep),

an OP pesticide, as evidenced by a toxicity test showing that the transgenic plants produced greater shoot and root biomass than did

the wild-type plants. Furthermore, at 0.02% (v/v) Mep, the transgenic plants degraded more than 99% of Mep after 14 days of growth.

Our work indicates that transgenic plants expressing an OPH gene may provide a new strategy for decontaminating OP pollutants.

Ó 2007 Elsevier Inc. All rights reserved.

Keywords: Phytodegradation; Organophosphorus compounds; Organophosphorus hydrolase; Transgenic plant; Bioremediation

Synthetic organophosphorus (OP) compounds are the

most widely used pesticides [1,2], and unacceptable levels

of environmental residues of these compounds have been

found in many countries worldwide [3–7]. Although most

OP compounds are not persistent, they still cause broadarea

pollution from continued use in agriculture and public

health [8]. These pesticides are frequently not used as recommended

both in dosage and in spills, leading to an accumulation

of OP compounds in the environment.

Additionally, many of these compounds are mobile in the

environment, which may lead to diffuse contamination

[8]; for example, traces of OP pesticides have been found

in hills where no previous application has occurred. Furthermore,

the accidental release or disposal of OP pesticides

has caused heavy pollution in certain areas. High

concentrations of the OP pesticide methyl parathion

(Mep) in soil are degraded to acceptable levels only after

many years [9].

* Corresponding author. Fax: +86 10 62136981.

E-mail address: wunf@caas.net.cn (N. Wu).

OP pesticides are acetylcholinesterase (AChE) inhibitors.

They are mostly liposoluble and pose a hazard to

humans through accumulation in the food chain from

the wide range of aquatic and terrestrial ecosystems contaminated

with OP pesticides. The loss of AChE results

in acetylcholine accumulation, which interferes with muscular

responses and produces serious symptoms in vital

organs, eventually leading to death. Therefore, it is

essential to decontaminate OP compounds in the environment.

However, although previous studies have

focused on persistent organic pollutants, little attention

has been paid to the remediation of OP pesticide

contamination.

Some progress has been made on screening for OP

pesticides detoxifying bacterial strains and developing

organophosphorus hydrolases (OPHs; EC 3.1.8.1) [10–

16]. However, given the large scope of the untreated contamination

worldwide, chemical and enzyme preparation

approaches do not seem economical or effective. An

alternative approach, phytoremediation, has been

considered as a potentially low cost, very effective, and

0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2007.10.193


454 X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458

an environmentally friendly method; it may provide a

better option for dealing with such diffuse contamination.

Plants have a natural ability to metabolize OP compounds,

as reported in a previous phytoremediation

study of the use of the hydrophyte Typha latifolia to

remove Mep pollutants from water [17]. Plants also prevent

contamination from spreading to other areas via

wind, rain or groundwater—an advantage of using phytoremediation

to address diffuse OP pollution at scattered

sites.

A substantial amount of work has been conducted to

increase the ability of plants to decontaminate areas that

have high concentrations of heavy-metal ions [18], organomercury

[19], and explosives (e.g. TNT and RDX) [20,21]

or chlorinated pesticides (e.g. atrazine) [22]. The introduction

of a detoxification gene improves the tolerance of the

engineered plants to such toxic pollutants. To date, however,

few studies have addressed the production of transgenic

plants designed to degrade and detoxify OP

compounds.

We have previously isolated an OP-degrading bacterial

strain (C2-1, Pseudomonas pseudoalcaligenes) from OP-polluted

soil [16]. The gene ophc2, which encodes organophosphorus

hydrolase OPHC2, has been cloned (GenBank

Accession No. AJ605330) [23] and the enzyme has been

highly expressed in the yeast Pichia pastoris [24]. In this

study, we introduced ophc2 into tobacco plants and investigated

whether these OPHC2-expressing transgenic plants

could degrade Mep in vitro and in vivo.

Fig. 1. Western blot analysis showing expression of OPHC2 in transgenic

tobacco plants. Molecular weight is given in kDa. a Positive control using

purified OPHC2 protein expressed in Pichia pastoris. b Total protein from

wild-type tobacco. c Total protein from transgenic tobacco lines. * Transgenic

lines expressed OPHC2.

Materials and methods

Construction of the OPHC2 expression vector. The OPHC2 expression

vector was constructed according to standard protocols [25]. The gene

encoding OPHC2, ophc2, was amplified by PCR from the genomic DNA

of P. pseudoalcaligenes C2-1 using primer_F (TACGTA ACC ATG GCC

GCA CCG GCA CAA CAG AAG) and primer_R (GAGCTC TTT TTT

TTT TTT TTT TTT TTT TTT TTT TTT ATC AGC GGT CGC TAC

GGA TCG G). The underlined sequences correspond to SnaBI and SacI

sites, respectively. The PCR product was purified using the MinElute Ò

PCR purification kit (QIAGEN, USA), digested with SnaBI and SacI, and

directionally cloned into the SnaBI and SacI restriction sites of the vector

Teasy-ex, which contained a signal peptide sequence from the carrot

extensin gene [26,27]. The recombinant vector was designated pTeasyophc2.

The extensin signal sequence facilitated the extracellular expression

of OPHC2. The ophc2 with the extensin sequence was excised using SacI

and Xbal from pTeasy-ophc2 and ligated into pBI121. The final expression

cassette, which contained the cauliflower mosaic virus (CaMV) 35S

promoter, the extensin sequence, ophc2, and the nos termination sequence,

was designated pBI-E-ophc2.

Transformation of tobacco. The vector pBI-E-ophc2 was introduced

into Agrobacterium LBA4404 using electroporation (MicroPulser, Bio-

Rad, USA). The leaf disks of tobacco (Nicotiana tabacum cv. Xanthi) were

transformed according to the method of Horsch et al. [28]. The selection

medium comprised Murashige–Skoog (MS) basic medium, 3% sucrose,

0.6% agar, 1 mg/l of 6-benzylaminopurine (6-BA), 0.1 mg/l of a-naphthaleneacetic

acid (NAA), 100 mg/l kanamycin (Km), and 500 mg/l of

carbenicillin (Cb).

Plant DNA isolation and PCR analysis. Plant genomic DNA was

prepared from leaves using the Phytopure kit (TIANGEN BIOTECH, Co.

LTD. Beijing, China). Primer 1 (GCC GCA CCG GCA CAA CAG

AAG) and primer 2 (TCA GCG GTC GCT ACG GAT CGG) were used

in this assay.

Western blot analysis. Transformed and untransformed leaves

(200 mg) from plants grown in a phytotron were ground in liquid nitrogen

and resuspended in 200 ll of protein extraction buffer (50 mM/l Tris–HCl,

pH 8.0, 0.02% NaN 3 , 100 lg/ml of phenylmethylsulfonylfluoride). Total

protein was extracted and quantified in the extracts for each sample.

Purified OPHC2 expressed in P. pastoris was used as a positive control

[24]. Proteins of leaf extracts were subjected to SDS–PAGE through a 5%

polyacrylamide stacking gel above a 12% separating gel. After electrophoresis,

separated proteins on the gel were transferred electrophoretically

to polyvinylidene fluoride membranes (Millipore, USA) for Western blot

analysis. Immunodetection was achieved using a rabbit polyclonal antiserum

directed against OPHC2 as the primary antibody and peroxidaseconjugated

goat anti-rabbit IgG (Jackson ImmunoResearch, USA) as the

secondary antibody. Color development was performed in 100 ml TB

buffer (50-mM/l Tris–HCl, pH 7.6) containing 50 mg of 3,3 0 -diaminobenzidine

and 40 ll of 30% H 2 O 2.

Analysis of OPHC2 enzyme activity. Liquid MS growth medium

(2.5 ml) from plants grown for 10 days was concentrated to 100 ll using

10K Nanosep Ò Centrifugal Devices (PALL, USA). The 100 ll of concentrated

growth medium was added to 900 ll of assay buffer (50 mM/l

Tris–HCl, pH 8.0) containing 5 ll of 10 mg/ml Mep (99.9%) as substrate

and incubated at 37 °C for 20 min. The reaction was terminated by the

addition of 1 ml of 10% CCl 3 COOH (trichloroacetic acid), and 1 ml of

10% Na 2 CO 3 was added to display the color. Absorbance was measured at

Fig. 2. Hydrolysis of Mep by OPH.


X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458 455

410 nm. The enzyme activity could be calculated based on the amount of

the hydrolysis product, p-nitrophenol. One unit of OPH activity is defined

as the amount of enzyme that liberated 1 lM ofp-nitrophenol per minute

at 37 °C.

Mep degradation assay. Five microliters of Mep (commercial product,

50% Mep emulsifiable concentrates) was added to 30 ml of MS medium

with 0.5% agar in the flasks, under aseptic conditions, shaking to uniformity.

Wild-type and transgenic tobacco seedlings were transferred into

the flasks, grown for 14 days in 12-h day/12-h night light cycles at 25 °Cin

the growth chamber. Three flasks that did not contain plants were used as

the control. After 14 days, Mep from the medium and plants was

extracted. The extraction procedure was modified according to the

methods described in internal standard and by Steinwandter [29]. A 2-ml

mixture of each extraction was evaporated to dryness using nitrogen gas

and suspended in 1 ml of dichloromethane for quantification. Mep residue

was quantified by gas chromatography (Agilent Technologies, 6890N;

detector: flame photometric detector, detector temperature 250 °C; HP-5

5% phenyl methyl siloxane column; capillary columns: 30.0 m · 320 lm

· 0.25 lm nominal; inlets heater: 250 °C; initial oven temperature: 70 °C

for 1 min, then increased at a rate of 20 °C/min to 180 °C, held for 2 min

and ramped again at 10 °C/min to 250 °C, then held for 5 min; N 2 flow:

1.2 ml/min; split ratio: 5:1). The standard Mep solution was 100 lg/ml

(China Standards GSBG23010-92).

Results

Construction and selection of ophc2-transgenic plant lines

The transformation produced 101 independent T0-generation

kanamycin-resistant plants that were selected for

further investigation.

PCR analysis was used to determine which lines contained

the ophc2 transgene. Of the 101 lines, 80 were shown

to have the transgene, with DNA from wild-type tobacco

showing no PCR product (data not shown).

Western blot analysis was undertaken on the transgenic

plants to determine whether OPHC2 was expressed.

Twenty-four of the 60 randomly chosen transgenic plants

expressed OPHC2. Part of Western blot analysis result

was showed on Fig. 1.

OPHC2 enzymatic activity study in transgenic tobacco

plants

OPH activity was assayed in vitro to assess the secretion

and enzymatic activity of the transgene product. Under

aseptic conditions, transgenic and wild-type seedlings

(seedlings with identical biomass, root or shoot size, and

leaf amounts were chosen) were allowed to grow in liquid

MS medium for 10 days, after which the medium was collected

and concentrated 25-fold. The OPH activity of the

concentrated medium was then determined, using Mep as

the substrate. OPH can hydrolyze Mep at its phosphoester

bond, releasing p-nitrophenol (Fig. 2) and generating a yellow

product that can be quantified with a spectrophotometer

at 410 nm. Table 1 shows the enzymatic activity of

OPHC2 detected in the growth medium of different transgenic

plants. The transgenic line 4–91 showed the highest

enzymatic activity and was used in the subsequent experiments.

Furthermore, T1- and T2-generation transgenic

Table 1

In vitro OPHC2 enzyme activity a

Wild-type · 10 3 U/mL Transgenic tobacco lines · 10 3 U/mL

4–14 4–23 4–32 4–38 4–43 4–45 4–56 4–67 4–80 4–90 4–91 4–97 4–98 4–99 4–100

0.37 ± 0.51 6.28 ± 0.62 24.49 ± 1.30 14.77 ± 0.35 15.11 ± 0.62 14.9 ± 3.16 0.08 ± 0.45 17.39 ± 3.21 14.70 ± 1.51 17.90 ± 3.60 8.20 ± 0.78 25.82 ± 1.47 18.17 ± 0.40 18.84 ± 1.52 1.88 ± 1.38 18.01 ± 3.64

Transgenic and wild-type seedlings were grown aseptically in MS liquid medium for 10 days. Hundred microliter of concentrated growth medium was used as the enzyme solution for activity determination. Results shown are means and standard deviation of

triplicate measurements.

a


456 X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458

tobacco plants grew normally and still showed OPHC2

enzymatic activity (data not shown).

Assessment of Mep toxicity in an ophc2-transgenic tobacco

line

A toxicity study was conducted to determine the effect of

Mep on the growth of both wild-type and transgenic line

4–91 seedlings. Identically sized cuttings of wild-type and

transgenic plantlet were transferred into sterile flasks containing

solid MS medium with 0, 0.01%, 0.02%, 0.03% or

0.04% (v/v) Mep (commercial product, 50% Mep emulsifiable

concentrates). After 14 days of culture, the transgenic

4–91 plants could tolerate 0.02% Mep and showed no signs

of phytotoxicity. At 0.03% Mep, line 4–91 plants kept

growing, with a slightly stunted root and fewer root hairs.

At 0.04% Mep, the radicles of line 4–91 plants were swollen,

with no secondary roots and no visible root hairs. By

contrast, the wild-type seedlings displayed symptoms at

0.02% Mep that were similar to those observed in the

0.04% Mep-treated transgene plants (Fig. 3). Growth after

14 days in the presence of various concentrations of Mep

was also quantified as wet plant weight for both the wildtype

and the transgenic line 4–91 (Table 2).

There was a negligible difference in the root growth of

4–91 seedlings on 0%, 0.01%, and 0.02% (v/v) Mep medium,

the amount of roots was approximately 100 (Fig. 3A), and

the wet weight gain was 6.5-fold for each concentration

(Table 2). At a concentration of 0.01% Mep, wild-type

tobacco plants exhibited phytotoxicity in the form of

fewer root branches (50–60), reduced root length, and

darkened leaves compared with plants not exposed to Mep

(Fig. 3B and D), and the wet weight gain was nearly 3-fold

(Table 2). At 0.02% Mep, growth of the wild-type tobacco

was very stunted—less than 20 small, bare root branches

were produced, the shoot and root length (about 2.3 cm

and 1.5 cm respectively) were both markedly reduced compared

with 4–91 (about 3.2 cm and 2.8 cm, respectively)

(Fig. 3E), and the wet weight gain was only 2-fold (Table

2). No difference was observed between line 4–91 and wildtype

plants grown in medium without Mep, which indicates

that the ophc2 insertion had no deleterious effect on the

plants (Fig. 3C). Wild-type plants grown in medium without

Mep appeared healthy, indicating that the toxic effects

observed were caused solely by Mep.

Mep degradation in seedlings and growth medium

To assess whether transgenic plants expressing

OPHC2 can degrade environmental OP toxins, we tested

the ability of the transgenic line 4–91 plants to degrade

Mep. After 14 days of growth on solid MS medium

Fig. 3. Growth of transgenic line 4–91 and wild-type tobacco plants on Mep-containing medium 14 days after transplantation. Each sample was tested in

triplicate. (A, B) Root morphology in the presence of 0%, 0.01% or 0.02% (v/v) Mep. (C–E) Plant morphology difference between 4–91 and wild-type: (C)

medium without Mep, (D) medium with 0.01% (v/v) Mep, (E) medium with 0.02% (v/v) Mep.

Table 2

Seeding weight gain following Mep exposure a

Mep % (v/v) Wild-type Line 4–91

Weight before Mep (g) Weight after Mep (g) Weight gain (g) Weight before Mep (g) Weight after Mep (g) Weight gain (g)

0 0.60 ± 0.01 4.47 ± 0.02 3.87 ± 0.03 0.56 ± 0.03 4.24 ± 0.54 3.67 ± 0.55

0.01 0.63 ± 0.04 2.48 ± 0.44 1.85 ± 0.40 0.62 ± 0.04 4.53 ± 0.31 3.59 ± 0.40

0.02 0.63 ± 0.08 1.78 ± 0.20 1.15 ± 0.26 0.64 ± 0.05 4.00 ± 0.60 3.36 ± 0.56

a The identically sized cuttings of wild-type and transgenic line 4–91 plantlet were transferred into sterile flasks containing solid MS medium with 0,

0.01%, 0.02% (v/v) Mep (commercial product, containing 50% Mep). After 14 days of growth, wet weights were measured to establish plant biomass gain,

which was used as an indicator of phytotoxicity. Results shown are means and standard deviation of three replicate flasks.


X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458 457

(0.5% agar) with 0.02% Mep, the concentration of Mep

residue in the medium and plant material was then measured

using gas chromatography. The total absolute

quantity of Mep reduction was 99.6% in the flasks of

line 4–91 plants and 27% in the flasks of wild-type

plants, compared with the flasks without plants

(Fig. 4A).

The absolute quantity of Mep in the tissue of wild-type

plants was 163-fold that of the tissue of line 4–91 plants

(Fig. 4B). This indicates either higher degradation or lower

absorption of Mep by the 4–91 line. However, Mep residue

extracted from the MS medium exhibited an even higher

(173-fold) absolute quantity of Mep in the wild-type

growth flask (Fig. 4B), so we suggest that the lower level

of Mep residue in the transgenic plant flask was due to

the increased degradation of Mep. Mep residue in line

4–91 plant material extract was 0.16 ± 0.01 lg/g plant

material. However, it was 16.74 ± 2.33 lg/g plant material

in the wild-type extract. The growth medium of wild-type

plants contained 68.94 ± 4.55 lg Mep/g MS medium,

which was much higher than that of the transgenic line

4–91 (0.39 ± 0.01 lg Mep/g MS medium).

A

absolute quantity of Mep(ug)

B

absolute quantity of Mep (ug)

2400

2100

1800

1500

1200

900

600

300

0

1800

1600

1400

1200

1000

800

600

400

200

0

blank flask WT 4-91

Total extraction from the whole plant and its medium

WT plant 4-91 plant WT medium 4-91 medium

Extraction from plant and growth medium

Fig. 4. Extraction of Mep from plant tissue and growth medium after 14

days of culture in MS medium with 0.5% agar containing 0.02% (v/v)

Mep. Results shown are means and standard deviation of three replicate

flasks.

Discussion

A wide range of OP compounds present in pesticides

and chemical warfare agents can be degraded by OPHs

because many OP compounds—aside from displaced

groups—have analogous structures. We selected Mep as

a test substrate for OPHC2 degradation of OP pesticides

in this study.

Many investigations have focused on the bioremediation

of persistent organic pollutants, but few have been

conducted for OP compounds. Our approach shows that

an OPH, OPHC2, identified in bacteria isolated from a

polluted site can be redeployed in plants to increase the

ability of the plants to degrade Mep. In the analysis of

Mep degradation in both seedlings and growth media,

there was a reduction of total Mep residue in the flasks

containing wild-type tobacco compared with the flask

without any plants. This could be because of the natural

metabolism of Mep by tobacco plants. However, it is not

enough to reduce the amount of OP pesticide residue to

acceptable levels. Total Mep residue in wild-type plant

flasks still remained 73% (on average) of that of the control

flask. And the amount of Mep residue was much

higher in the medium than in the plants themselves, which

indicated that limited Mep was absorbed by the plants at

high Mep concentrations. Furthermore, because of the

low metabolism efficiency for the absorbed Mep, there

was still a high level of Mep in the tissue of wild-type

plants. The transgenic line 4–91 plants were able to

degrade Mep to a much lower level, which we expect to

be sufficient to decontaminate environmental OP pollution.

In addition, effective degradation of Mep in the

transgenic plant tissue can keep the plants healthy, which

is essential for phytoremediation.

Pollution caused by OP pesticides is wide ranging; most

OP pesticides are directly liberated by spraying, and a perceptible

amount is released through evaporation from

water surfaces, leaves and soil [30], causing air pollution

and long-range impacts via atmospheric transport. However,

there is currently no economical way to block the diffusion

of OP in the environment. The improved

degradation of OP pesticides by transgenic plants highlights

a new way to combat such diffuse contamination.

In contrast to previous studies that aimed to increase the

removal rate of pollutants, our results make possible a substantial

increase in OP degradation in the environment.

Because of its constitutive extracellular expression, OPHC2

was detected in both the transgenic plant tissue and the rhizosphere.

With the continued release of OPHC2 by the

expanded root system of plants, OP compounds diffused

in soils or water could be detoxified. Combined with an

OP pollution study, our investigation takes a step towards

determining whether OPHC2 secretion by transgenic plant

leaves can counteract the contaminating effects of OP-polluted

rain, fog or snow. Further studies should be conducted

to investigate how ophc2-transgenic plants work

in natural conditions.


458 X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458

Acknowledgments

We thank Sheng Gao for gas chromatography analysis.

This work was supported by the National Natural Science

Foundation of China (Grant No. 30470031).

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