Thifensulfuron hydrolase gene tsmE and uses thereof

Abstract

The present invention relates to the field of applied and environmental microorganism and agriculture. Disclosed are a thifensulfuron hydrolase gene tsmE and uses thereof. The thifensulfuron hydrolase gene tsmE has a nucleotide sequence of SEQ ID NO. 1, full length of 1194 bp, and G+C content of 51.09%, and encodes 398 amino acids with an amino acid sequence of SEQ ID NO. 2. The thifensulfuron hydrolase TsmE provided by the present invention can degrade completely 100 mg/L thifensulfuron within 1 hour into the herbicidally inactive product thiophenesulfonic acid; in addition, the TsmE also degrade completely 100 mg/L haloxyfop-R-methyl within 1 hour. Therefore, the thifensulfuron hydrolase gene tsmE is useful in construction of thifensulfuron-resistant transgenic crops and bioremediation of thifensulfuron or haloxyfop-R-methyl-contaminated environments.

Claims

1. A transgenic bacterium comprising a vector, wherein said vector comprises a nucleotide sequence encoding a thifensulfuron hydrolase gene tsmE, and wherein said nucleotide sequence comprises SEQ ID NO. 1.

2. A transgenic bacterium comprising a vector wherein said vector comprises a thifensulfuron hydrolase gene tsmE comprising the nucleotide sequence of SEQ ID NO. 1, and wherein the tsmE gene encodes a thifensulfuron hydrolase protein TsmE comprising the amino acid sequence of SEQ ID NO. 2.

3. A transgenic bacterium comprising a pET plasmid comprising a thifensulfuron hydrolase gene tsmE comprising the nucleotide sequence of SEQ ID NO. 1 which encodes a thifensulfuron hydrolase protein TsmE comprising the amino acid sequence of SEQ ID NO. 2.

4. A recombinant expression vector, wherein said vector comprises: (i) a heterologous regulatory element; and (ii) a thifensulfuron hydrolase gene tsmE comprising the nucleotide sequence of SEQ ID NO. 1 and encoding a thifensulfuron hydrolase protein TsmE comprising the amino acid sequence of SEQ ID NO. 2.

5. A method of removing residues of thifensulfuron and haloxyfop-R-methyl from soil or water comprising the steps of: preparing a recombinant expression vector according to claim 4; transforming a soil bacterium with the vector; and introducing the transformed bacterium to the soil or water.

6. A method of degrading thifensulfuron comprising the steps: preparing the transgenic bacterium as in claim 3; purifying the produced TsmE from said bacterium; and adding the TsmE to thifensulfuron, whereby the TsmE degrades thifensulfuron to thiophenesulfonic acid.

7. A method of degrading haloxyfop-R-methyl comprising the steps: preparing the transgenic bacterium as in claim 3; purifying the produced TsmE from said bacterium; and adding the TsmE to haloxyfop-R-methyl, whereby the TsmE degrades haloxyfop-R-methyl to propanoic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a scheme for cloning of the thifensulfuron hydrolase gene tsmE.

(2) FIG. 2 is a scheme for expression of TsmE in BL21 (pET-29a (+)).

(3) FIG. 3 is an electropherogram of the SDS-PAGE analysis of the purified TsmE.

(4) Wherein the lane 1 is protein marker, and the lane 2 is the purified TsmE.

(5) FIG. 4 is a graph of the LC-MS profile of the metabolite produced during degradation of thifensulfuron by the hydrolase TsmE.

(6) A: a liquid chromatogram for degradation of thifensulfuron by TsmE;

(7) B: a first-order mass spectra for degradation of thifensulfuron by the hydrolase TsmE.

(8) C: a second-order mass spectra for degradation of thifensulfuron by TsmE.

(9) FIG. 5 is a degradation pathway of thifensulfuron by TsmE.

(10) FIG. 6 is an MS/MS profile of haloxyfop-R-methyl degradation by TsmE,

(11) A: a first-order mass spectra for degradation of haloxyfop-R-methyl by TsmE;

(12) B: a second-order mass spectra for degradation of haloxyfop-R-methyl by TsmE.

(13) FIG. 7 is a pathway of haloxyfop-R-methyl degradation by TsmE.

DETAILED DESCRIPTION OF THE INVENTION

Embodiment 1

Cloning of the Thifensulfuron Hydrolase Gene

(14) 1.1 Extraction of Total DNA from Bacterial Genome

(15) Cells of strain S113, cultured in R2A medium for 48 h, were harvested by centrifugation and then the total DNA was extracted by the CTAB method as described by F. Osbourne et al, ed. Concise Guide to molecular biology experiments. The total DNA was dissolved in TE buffer (pH8.0), and stocked at 20 C.

(16) 1.2 Digestion of the Total DNA of Strain S113

(17) The total DNA of strain S113 was partial digested by Sau3AI

(18) 1.3 DNA Recovery

(19) The restricted total DNA was purified by electrophoresis (TAE buffer), and recovered by a recovering kit (Axygen Biosciences, china). The recovered DNA was dissolved in 10 mmol/L Tris-HCl (pH8.0) and stocked at 20 C.

(20) 1.4 Enzymatic Ligation

(21) The reaction system was as follows:

(22) TABLE-US-00001 pUC118 (BamHI) 0.1 g Total DNA fragment 0.1 g 10 ligase buffer 1 l T4DNA ligase 0.5 l Double distilled water Ad to 10 l

(23) Incubation at 16 C. for 12 hours.

(24) 1.5 Preparation of the E. coli DH10B-Competent Cells

(25) The E. coli DH10B was commercially available from the Shanghai Invitrogen Biotechnologies Co., Ltd. The AHAS II isozymes gene ilvG of the E. coli DH10B was activated using Red-mediated recombination to create E. coli DH10B(ilvG.sup.+). The competent cells of E. coli DH10B(ilvG.sup.+) was prepared according to the method described by F. Osbourne et al, ed. Concise Guide to molecular biology experiments. P22-23.

(26) 1.6 Transformation

(27) 10 l ligated product was transformed into 200 l E. coli DH10B(ilvG.sup.+)-competent cells according to the method described by F. Osbourne et al, ed. Concise Guide to molecular biology experiments. P23. The library was spread on MSM (minimum salt medium) plates containing 5 g/L glucose, 100 mg/L ampicillin, 200 mg/L valine, 200 mg/L leucine, and 10 mg/L thifensulfuron and incubated at 37 C. for 24 h. The visible colonies were picked, purified, and tested further for the ability to degrade thifensulfuron using HPLC analysis. The base salt medium was formulated from 5.0 glucose, 1.0 NH.sub.4NO.sub.3, 1.0 NaCl, 1.5 K.sub.2HPO.sub.4, 0.5 KH.sub.2PO.sub.4, and 0.2 MgSO.sub.4.7H.sub.2O pH 7.0.

(28) 1.7 Gene Nucleotide Sequencing

(29) The inserted fragment in the positive transformant capable of transforming thifensulfuron to thiophenesulfonic acid was sequenced using an automated sequencer (model 3730; Applied Biosystems) by Shanghai Invitrogen Biotechnologies Co., Ltd. The nucleotide sequence of the thifensulfuron hydrolase gene tsmE is presented in SEQ ID NO. 1, and the deduced 398 amino acid sequence was presented in SEQ ID NO. 2.

Embodiment 2

Expression of the Thifensulfuron Hydrolase Gene tsmE in E. coli BL21 (pET-29a (+))

(30) 2.1 PCR Amplification of tsmE

(31) tsmE was amplified by PCR from genomic DNA of strain S113 (CGMCC 1479) with forward primer: 5-TGCAGACATATGGAAACCGATAAAAAAAC-3 (SEQ ID NO. 3) and reverse primer: 5-TGCAGAGAATTCCCTTCCATAAGAGCGCCGAT-3 (SEQ ID NO. 4).

(32) The system for amplification was as follows:

(33) TABLE-US-00002 Primer star DNA Polymerase (5 U/l) 0.5 l 5 PCR Buffer II (Mg.sup.2+Plus) 10 l dNTP Mixture (each 2.5 mM) 5 l Template DNA 10 ng Forward primer (20 M) 1 l Reverse primer (20 M) 1 l Sterilized distilled water Ad to 50 l

(34) PCR amplification procedure was as follows:

(35) a. denaturation at 95 C. for 3 min;

(36) b. denaturation at 95 C. for 1.5 min, annealing at 53 C. for 0.5 min, Extension at 72 C. for 1.5 min, for 25 cycles;

(37) c. Terminal extension at 72 C. for 10 min.

(38) 2.2 Digestion of PCR Products with NdeI and HindIII

(39) The system for digestion was as follows:

(40) TABLE-US-00003 NdeI 1 l HindIII 1 l DNA 1 g Sterilized distilled water Ad to 20 l

(41) Reaction was carried out for about 3 hours in a water bath at 37 C. The digested product was recovered from gel after running on 2% agarose gel electrophoresis.

(42) 2.3 Digestion of pET-29a(+) with NdeI and HindIII (with Reference to 2.2).

(43) 2.4 Transformation

(44) The recovered fragment in 2.2 was linked to the NdeI and HindIII-digested pET-29a (+) in 2.3 (with reference to 1.5). The linked pET-29a (+) recombinant plasmid containing the tsmE was transformed into the host bacteria E. coli BL21 (DE3) to generate the recombinant microorganism BL21 (TsmE).

(45) 2.5 Functional Expression and Purification of TsmE

(46) E. coli BL21 (TsmE) was cultured in the LB culture medium until OD600 nm was reached 0.6 to 0.8; 1 mM IPTG was added to the culture for induction, and the culture was incubate for another 4 hours at 30 C. 100 ml culture was centrifuged, and the cell was re-suspended in 10 ml PBS buffer (50 mM, pH 7.0), ultrasonicated (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 5 minutes, and then centrifuged. The supernatant was collected and the recombinant TsmE was purified to homogeneity using Ni-nitrilotriacetic acid affinity chromatography (FIG. 3).

(47) 2.6 Enzyme Assay.

(48) The enzymatic activities towards thifensulfuron and haloxyfop-R-methyl were assayed in 1 ml of 50 mM potassium phosphate buffer (pH 7.0) at 30 C. for 20 min. The reactions were initiated by the addition of 50 l purified TsmE to the final concentration of 0.2 mM thifensulfuron or 0.2 mM haloxyfop-R-methyl. Reactions were stopped by adding 3 ml dichloromethane and cooling in liquid nitrogen. The organic phase was dried over anhydrous sodium sulfate, and then the concentration of thifensulfuron and haloxyfop-R-methyl were determined by reverse HPLC. One unit of enzyme activity was defined as the amount of enzyme that converted 1 M of thifensulfuron or haloxyfop-R-methyl to its parent acid form. The results of degradation test showed that the purified TsmE degrade completely 100 mg/L thifensulfuron or haloxyfop-R-methyl within 1 hour, and the specific enzyme activities of TsmE to thifensulfuron and haloxyfop-R-methyl were 67 U/mg and 55 U/mg protein, respectively.

(49) 2.7 Identification of the Metabolites

(50) 2.7.1 Identification of Metabolites of Thifensulfuron

(51) The supernatants of the enzyme reaction were filtered through a 0.2 m-pore-size filter and analyzed by LC-MS with the liquid chromatography conditions: chromatographic column: Agilent Zorbax XDB-C18 column (2.150 mm, 3.5 m), mobile phase: methanol:water=80:20, flow rate: 0.25 ml/min, and UV detection wavelength: 255 nm. The first-order MS conditions: ion detection mode: multi-reaction ion detection; ion polar: negative ion; ionization mode: electro-spray ionization; capillary voltage: 4000 volts; drying gas temperature: 330 C.; flow rate of drying gas: 10.0 L/min; atomized gas pressure: 35 psi; collision voltage: 135 volts; mass scanning range (m/z): 300-500. The second-order ion MS conditions: collision voltage: 90 volts; mass scanning range (m/z): 30-400.

(52) The results of the LC-MS spectrogram (see FIG. 4A) showed that the metabolite of thifensulfuron has a retention time of 1.95 min. In the first-order mass spectrogram (see FIG. 4B), there was a negative ion peak of its molecule with m/z 372.30; and in the second-order mass spectrogram (see FIG. 4C, there were the fragments with m/z 162.10, 188.10 and 206.20, consistent with thiophenesulfonic acid. Therefore, TsmE catalyzed the de-esterification of thifensulfuron that gave rise to thiophenesulfonic acid. (see FIG. 5).

(53) 2.7.2 Identification of Metabolites from Degradation of Haloxyfop-R-Methyl

(54) The metabolite of haloxyfop-R-methyl was determined by tandem mass spectroscopy. 2 ml supernatants of the enzyme reaction was extracted and blew to dryness and dissolved in 100 L methanol. The metabolite was analysed with tandem mass spectroscopy at the following conditions: MS/MS (Finnigan TSQ Quantum Ultra AM, Thermal, U.S.A.), electro-spray ionization, detection with both positive and negative ion, and mass scanning range (m/z): 30-1200.

(55) In the first-order MS/MS mass spectrogram (see FIG. 6A), there was a negative ion peak with m/z 359.87; and in the second-order mass spectrogram (see FIG. 6B), there was a fragment with m/z 287.60, consistent with the metabolite 2-[4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy]propanoic acid. Therefore, TsmE catalyzed the hydrolysis of haloxyfop-R-methyl to 2-[4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy]propanoic acid (see FIG. 7).

(56) The microorganisms used in the embodiments were available from:

(57) TABLE-US-00004 pUC118 (BamHI) TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD. E. coli DH10B Shanghai Invitrogen Biotechnologies Co., Ltd. E. coli expression vector pET-29a(+) Novegen Co. Host E. coli BL21 (DE3) for expression Shanghai Invitrogen Biotechnologies Co., Ltd.
Deposit Information of the Strain

(58) Strain Methylophilus sp. S113 was deposited in the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences. NO. 1 Beichen West Road, Chaoyang District, Beijing 100101. China, with accession number CGMCC 1479, dated Oct. 12, 2005.