CN112359046A - Hydroquinone degrading enzyme gene group expressed in escherichia coli and application thereof - Google Patents

Abstract

A hydroquinone degrading enzyme gene group expressed in colibacillus and application thereof comprises PnPCS, PnPDS and PnPES genes which are derived from pseudomonas and obtained by optimizing according to codon preference of the colibacillus, wherein the nucleotide sequences are shown as SEQ ID NO.1-3, the PnPCS, PnPDS and PnPES genes obtained by optimizing are respectively fused with a T7 promoter and a terminator to construct a corresponding gene expression box, then the gene expression box is connected into an colibacillus expression vector and converted into the colibacillus, the expression is successful in the colibacillus, and the obtained positive strain has good degradation effect on the hydroquinone and can completely degrade the hydroquinone with the concentration of 5mM within 24 hours.

Description

Hydroquinone degrading enzyme gene group expressed in escherichia coli and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a hydroquinone degrading enzyme gene group expressed in escherichia coli and application thereof.

Background

Hydroquinone, also known as hydroquinone, is an important chemical raw material, has wide application, is an important raw material, an intermediate and an auxiliary agent for medicines, pesticides, dyes, rubber and the like, and is mainly used in the aspects of developers, anthraquinone dyes, azo pigments, rubber anti-aging agents, monomer polymerization agents, food stabilizers, coating antioxidants, petroleum anticoagulants, synthetic ammonia catalysts and the like. At present, the application field of hydroquinone is gradually expanding, and the consumption demand is also expanding year by year.

With the mass production and consumption of hydroquinone, the substance inevitably pollutes groundwater, surface water and soil through various ways, so that hydroquinone is constantly detected in wastewater, rivers, lakes, soil, air and groundwater. Hydroquinone has toxicity, has a strong corrosive effect on skin and mucosa, can inhibit the central nervous system or damage the functions of liver and skin, and even can kill, so the hydroquinone is listed as one of the priority control pollutants by the United states Environmental Protection Agency (EPA), and the hydroquinone is classified as a 6.1 type toxic product in 2011 in China.

Hydroquinone has certain water solubility, and the treatment process by using the traditional physical and chemical method for treating pollutants is complex and can not completely remove the pollutants. The microorganisms have the characteristics of extremely strong variability, adaptability and the like, and can degrade nitrophenol under the selective pressure of the environment, so that the microorganisms become main members for degrading hydroquinone in the natural environment.

At present, the degradation research of hydroquinone mainly derives from the degradation of p-nitrophenol, since the former is a degradation intermediate product of the latter. There are two main ways to degrade p-nitrophenol: the hydroquinone pathway and the trimellitic pathway. The former is more prevalent among gram-negative bacteria, while the latter is prevalent among gram-positive bacteria. In the hydroquinone pathway, the intermediate hydroquinone is subjected to ring opening under the action of hydroquinone 1, 2-dioxygenase to form gamma-hydroxy muconic semialdehyde, which is subsequently converted into maleylacetic acid and further reduced to beta-ketoadipic acid.

Disclosure of Invention

The invention aims to provide a hydroquinone degrading enzyme genome capable of being expressed in escherichia coli and application thereof, wherein different gene segments derived from Pseudomonas putida are designed and modified by utilizing a synthetic biology technology, and a complete heterologous catabolism pathway is introduced into the escherichia coli, so that the escherichia coli has the capability of degrading and tolerating hydroquinone, the degrading efficiency and the tolerating capability of bacteria on the hydroquinone are improved, and industrial application degrading bacteria resources are enriched.

In order to achieve the purpose, the invention provides the following technical scheme:

a hydroquinone degrading enzyme genome expressed in Escherichia coli, comprising PnPCS, PnPDS and PnPES genes derived from Pseudomonas and obtained after optimization according to codon bias of Escherichia coli.

Further, after being optimized according to the codon preference of escherichia coli, the nucleotide sequence of the PnPCS gene is shown as SEQ ID No. 1; the nucleotide sequence of the PnPDS gene is shown as SEQ ID No. 2; the nucleotide sequence of the PnPES gene is shown as SEQ ID No. 3.

The invention provides application of the hydroquinone degrading enzyme genome in escherichia coli.

A multigene colibacillus transforming carrier includes colibacillus expressing carrier and the gene expression cassettes of PnPCS, PnPDS and PnPES genes, which are derived from pseudomonads and obtained by optimizing according to the codon preference of colibacillus, and are respectively fused with T7 promoter and terminator.

Preferably, the Escherichia coli expression vector is pET-28 a.

A method for obtaining transgenic Escherichia coli capable of completely degrading hydroquinone comprises the following steps:

1) optimizing PnPC gene, PnPD gene and PnPE gene derived from pseudomonas according to the codon preference of escherichia coli, respectively obtaining PnPCS gene, PnPDS gene and PnPES gene after optimization, respectively fusing the five optimized genes with a T7 promoter and a terminator by using an overlap extension PCR technology, and respectively constructing gene expression cassettes;

2) connecting the three gene expression cassettes constructed in the step 1) into an escherichia coli expression vector in sequence according to the sequence of PnPCS, PnPDS and PnPES to obtain a polygenic escherichia coli transformation vector of the three gene expression cassettes;

3) and (3) transferring the polygenic escherichia coli transformation vector in the step 2) into escherichia coli to obtain the escherichia coli capable of completely degrading hydroquinone.

Preferably, in the step 1), the nucleotide sequence of the T7 promoter is shown as SEQ ID NO. 4; the T7 terminator sequence is shown in SEQ ID NO. 5.

Preferably, in step 2), the E.coli expression vector is pET-28 a.

In step 3), the Escherichia coli strain is BL 21-AI.

In the present invention, the PnPC, PnPD and PnPE genes of Pseudomonas (Pseudomonas putida) are optimized according to the codon preference of Escherichia coli, and the optimization is performed according to the following principle: optimizing gene codon, and improving gene translation efficiency according to the preference of escherichia coli codon; (II) eliminating recognition sites of restriction enzymes EcoRI and HindIII in the gene, so as to facilitate the construction of an expression cassette; (III) eliminating reverse repeated sequences and stem-loop structures which are adjacent to the T7 promoter or terminator and within 100 bp; fourthly, eliminating reverse repeated sequences and stem-loop structures within 200bp adjacent between two genes; (V) eliminating transcription termination signals, balancing GC/AT in the gene, and improving the stability of RNA; (VI) making the gene coding protein accord with the N-terminal principle so as to improve the stability of the translation protein; and (seventhly) optimizing the free energy of the secondary structure of the mRNA to improve the gene expression efficiency.

Compared with the prior art, the invention has the following beneficial effects:

in the invention, the PnPC gene, the PnPD gene and the PnPE gene are combined and successfully expressed in escherichia coli after optimization, and the obtained positive strain can completely degrade 1mM of hydroquinone within 8 hours, completely degrade 5mM of hydroquinone within 24 hours and degrade 5% of 10mM of hydroquinone within 24 hours.

The gene combination of the hydroquinone degrading enzyme genome can be used for preparing microorganisms for degrading hydroquinone, and has application potential in the fields of wastewater treatment, environmental remediation and the like.

Drawings

FIG. 1 is a schematic diagram showing the structure of an E.coli transformation vector for three genes PnPCS, PnPDS, and PnPES in example 1 of the present invention.

FIG. 2 shows the results of PCR detection of foreign genes from positively cloned plasmid DNA in example 3 of the present invention.

FIG. 3 shows the result of RT-PCR detection of exogenous genes of the positive strain in example 3 of the present invention.

FIG. 4 shows the effect of removing hydroquinone by the positive strain in example 4 of the present invention.

FIG. 5 is a mass spectrum of the final product, beta-ketoadipic acid, produced in the positive strain in example 4 of the present invention.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention.

In the examples, the Escherichia coli used was stored in the plant genetic engineering research institute of academy of agricultural sciences of Shanghai city, and the test methods used were all conventional molecular biology methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are commercially available reagents and materials; the HPLC analysis in the examples adopts an Agilent 1100 high performance liquid chromatography system; the GC-MS detection adopts an Agilent 7890B-7000C gas chromatograph-mass spectrometer.

Example 1

1. Optimized synthesis of three genes

The three genes are optimized by taking the PnPC, PnPD and PnPE genes (GenBank No. FJ376608.2) of pseudomonas (Pseudomonas putida) as a template according to the following principle: optimizing gene codon, and improving gene translation efficiency according to the preference of escherichia coli codon; (II) eliminating recognition sites of restriction enzymes EcoRI and HindIII in the gene, so as to facilitate the construction of an expression cassette; (III) eliminating reverse repeated sequences and stem-loop structures which are adjacent to the T7 promoter or terminator and within 100 bp; fourthly, eliminating reverse repeated sequences and stem-loop structures within 200bp adjacent between two genes; (V) eliminating transcription termination signals, balancing GC/AT in the gene, and improving the stability of RNA; (VI) making the gene coding protein accord with the N-terminal principle so as to improve the stability of the translation protein; and (seventhly) optimizing the free energy of the secondary structure of the mRNA to improve the gene expression efficiency.

After optimization, PnPC, PnPD and PnPE genes (GenBank No. FJ376608.2) of pseudomonas (Pseudomonas putida) are taken as templates to respectively synthesize DNA sequences PnPCS, PnPPDS and PnPES shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the DNA sequences are respectively cloned to a plasmid vector and sequenced to determine the sequences.

The sequence was synthesized by reference to Nucleic Acids Research,2004, 32(12) e 98.

EXAMPLE 2 construction of multigenic E.coli transformation vectors

1. Construction of three Gene expression cassette elements

1.1 synthesizing a DNA sequence T7 promoter shown in SEQ ID NO.4 by taking a T7 promoter as a template, cloning the promoter to a plasmid vector, and sequencing to determine the sequence of the promoter; the T7 terminator of the DNA sequence shown in SEQ ID NO.5 is synthesized by taking the T7 terminator as a template, cloned to a plasmid vector and sequenced to determine the sequence, and the sequence synthesis method refers to Nucleic Acids Research,2004 and 32(12) e 98.

The elements were spliced according to a modified "overlap extension PCR" technique (Appl Microbiol Biotechnol.2006, 73 (1): 234-40).

1.2 construction of PnPCS Gene expression cassette

A pair of primers P1F and P1R are designed to be connected in series according to the sequences of the chemically synthesized T7 promoter, PnPCS gene and T7 terminator, the length of the primers is 60bp, an EcoRI restriction site and the promoter are arranged on the primer P1F, and a terminator is arranged on the primer P1R, and the specific sequences are as follows:

P1F:5’-GAATTCTAATACGACTCACTATAGGATGACTGATCATTACAAGGCTGTGGAGGCACTGAT-3’；

P1R:5’-CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTACTCTGCCTCCATC-3’。

1.3 construction of PnPDS Gene expression cassette: a pair of primers P4F and P4R are designed according to the T7 promoter, PnpDS gene and T7 terminator sequence which are chemically synthesized and are connected in series, the length of the primers is 60bp, a promoter is arranged on the primer P2F, and a terminator is arranged on the primer P2R. The specific sequence is as follows:

P2F:5’-TAATACGACTCACTATAGGATGCAAAACCTTCTTTTCATCGATGGTCGTTTTGTTGAGGC-3’；

P2R:5’-CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAACGCTTGAAGTGT-3’。

1.4 construction of PnPES Gene expression cassette: a pair of primers P3F and P3R are designed according to the sequences of the chemically synthesized T7 promoter, PnPES gene and T7 terminator for tandem connection, the length of the primers is 60bp, the primer P3F is provided with the promoter, the P3R is provided with the terminator and HindIII enzyme cutting sites, and the specific sequences are as follows:

P5F:5’-TAATACGACTCACTATAGGATGAATCCATTCGTGTACCAATCACTGCCAACTCGTGTTGT-3’；

P5R:5’-CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTATGCAGGAGACCAA-3’。

2. construction of Polygenic E.coli transformation vectors

The three gene expression cassettes synthesized above are connected in sequence according to the sequence of PnPCS, PnPDS, PnPES to obtain the PnPCS-PnPDS-PnPE recombinant gene expression cassette.

The recombinant plasmid of the PnPCS-PnPDS-PnPES gene expression cassette obtained above was digested with EcoRI and HindIII and ligated to the pET-28a vector digested with the same, to obtain a multigene E.coli expression vector pET-HQ containing three genes, whose structure can be represented as pET-PnPCS-PnPDS-PnPES (shown in FIG. 1).

EXAMPLE 3 transformation of E.coli

1. Obtaining and identifying transgenic escherichia coli

1.1 preparation and transformation of E.coli

1) A single colony of Escherichia coli was inoculated into 20mL of LB medium and cultured with shaking at 37 ℃ overnight.

2) Inoculating into 20mL LB medium according to the inoculum size of 1%, and culturing at 37 deg.C 230 r/min with shaking for 3 h.

3) 1ml of the culture was taken, ice-cooled for 30min, centrifuged at 4000r/min at 4 ℃ for 3min, and the supernatant was removed.

4) Adding 500. mu.L of ice-cold 0.1mol/L CaCl₂The solution is resuspended in bacterial pellet, centrifuged at 4000r/min at 4 ℃ for 3min and the supernatant removed.

5) Add 100. mu.L of ice cold 0.1mol/L CaCl₂The solution is resuspended in bacterial pellet, centrifuged at 4000r/min at 4 ℃ for 3min and the supernatant removed.

6)50 μ L volume of ice cold 0.1mol/L CaCl₂The solution is used for resuspending the bacterial pellet, and the bacterial solution can be used immediately or stored by freezing at-70 ℃.

7) To 1.5mL of eppendorf was added 80. mu.L of the bacterial suspension and 1 to 2. mu.L of plasmid DNA (0.4pg to 0.3. mu.g), and after leaving on ice for 20 minutes, the mixture was placed in a 42 ℃ water bath and heat-shocked for 90 seconds, and then quickly placed on ice.

8) After heat shock transformation, 1.0mL of expression medium was added to the cells, incubated at 29 ℃ for 1 hour, plated on 2YT plates (kanamycin 50. mu.g/mL, X-gel 30. mu.g/mL) containing antibiotics, and incubated overnight at 37 ℃ to obtain positive clones. By utilizing the transformation program, the Escherichia coli expression vector pET-HQ is transformed into Escherichia coli BL21-AI by heat shock, and an Escherichia coli positive strain BL-HQ is obtained.

2. Identification of transgenic E.coli

2.1 DNA sequencing of plasmids in Positive clones

Positive clones obtained by transferring a polygenic Escherichia coli expression vector pET-HQ are extracted by an alkaline lysis method, and exogenous genes PnPCS, PnPDS and PnPES are respectively detected by taking the plasmids as templates and using a PCR amplification method.

The primers used were as follows:

PnpCS：F:5’-ATGACTGATCATTACAAGGCTG-3’，

R:5’-CTCCATCACGAACTCGTAGT-3’。

PnpDS：F:5’-ATGCAAAACCTTCTTTTCATCG-3’；

R:5’-ACGCTTGAAGTGTGCAGGAATAG-3’。

PnpES：F:5’-ATGAATCCATTCGTGTACCAATC-3’；

R:5’-TGCAGGAGACCAACCGTTCCATG-3’。

the amplification procedure used: 30s at 94 ℃, 30s at 54 ℃, 120s at 72 ℃ for 45 cycles, and finally a further extension at 72 ℃ for 10min, with the strain transformed with only the empty vector (BL-control) as the control strain, see FIG. 2 for the results.

As can be seen from FIG. 2, the control strain failed to amplify the foreign gene, while the positive clone amplified the above three genes, indicating that the foreign gene was completely integrated into the E.coli genome.

2.2 RT-PCR detection of Positive strains

RNA in the positive strain is extracted by using an RNA extraction kit of a biological company, the extracted RNA is reversely transcribed into cDNA by using a reverse transcription kit of a whole gold organism company, the RT-PCR detection of exogenous PnPCS, PnPDS and PnPES genes is carried out by using the following primers and amplification conditions, the bacterial 16SrRNA is used as an internal reference, and the following primers are used:

PnpCS：F:5’-TGGTCGTTACCGTTCTGAC-3’；

R:5’-GTCTTCGTACTTCACTTGC-3’。

PnpDS：F:5’-TGGGTCCACTGACTTCTG-3’；

R:5’-GTCCACAGACCAGAACCCA-3’。

PnpES：F:5’-CGTGGATGACCCTGAACA-3’；

R:5’-GGGATACCAAGTTTCTCG-3’。

16S rRNA：F:5’-AGAGTTTGATCCTGGCTCAG-3’；

R:5’-TACCTTGTTACGACTT-3’。

the amplification procedure used: 30s at 94 ℃, 30s at 54 ℃, 30s at 72 ℃ for 45 cycles, and finally 10min at 72 ℃ for further extension, see fig. 3.

The results show that the positive strains can amplify the three genes (see figure 3), which shows that the exogenous genes are all correctly transcribed and expressed in the transgenic escherichia coli, and the relative expression amount is more than 1.6 times.

EXAMPLE 4 degradation of Hydroquinone by Positive strains

1. Preparation of samples

The positive strain (BL-HQ) and the control strain (strain transformed with only empty vector) in example 3 were inoculated in 100 ml of M9 liquid culture medium containing 1% glycerol and 50. mu.g/ml kanamycin, shaken at 37 ℃ for 24 hours (150rpm), centrifuged to remove the supernatant, the cells were washed once with sterilized distilled water, and then resuspended in 10 ml of M9 liquid culture medium containing 1% glycerol, 0.2% arabinose, 50. mu.g/ml kanamycin and 1mM IPTG, 1mM, 5mM and 10mM of hydroquinone were added to the positive strain, 1mM of hydroquinone was added to the control strain medium, shaken at 37 ℃ and treated for various times, and the residual hydroquinone content thereof was detected by HPLC; the content of the finally generated beta-ketoadipic acid is detected by gas mass spectrometry.

2. Hydroquinone residue detection and product analysis

2.1 HPLC analysis and content determination of Hydroquinone in Positive Strain

1ml of the bacterial solution was centrifuged, and the supernatant was collected and filtered through a 0.22 μm organic filter for further use.

The HPLC conditions for measuring hydroquinone are as follows: c18 column (

4.6X 150mm, 5 μm); the mobile phase is methanol: water 30: 70, the flow rate is 0.5 ml/min; the column temperature is 30 ℃; the detection wavelength is 270 nm; the amount of sample was 20. mu.L.

Referring to fig. 4, the positive strain can rapidly degrade hydroquinone in the culture medium within 8 hours, while the control strain cannot degrade or utilize hydroquinone. At higher hydroquinone concentrations, e.g. 5mM, the positive strains were also completely degraded within 24 hours; hydroquinone, 10mM, positive strain was also more tolerant and achieved 5% degradation in 24 hours.

2.2 GC-MS detection of the final product, beta-ketoadipic acid, in positive strains:

and putting 100 mu L of sample into a gas phase sample injection bottle, pre-freezing the sample, placing the sample into a freeze dryer for freeze drying, adding 100 mu L of pyridine and 100 mu L of BSTFA into the freeze-dried sample, placing the sample in an oven at 60 ℃ for reacting for 2 hours, filtering the reacted sample by a 0.22 mu m filter membrane, and injecting a sample for analysis.

The gas chromatography conditions were: an HP-5 capillary chromatography column (30 m.times.0.25 mm. times.0.25 μm) was used; carrier gas He (99.999%), flow rate 1 mL/min; the sample inlet temperature is 280 ℃; column temperature procedure: the initial temperature is maintained at 80 ℃, the temperature is increased to 150 ℃ at 20 ℃/min, the temperature is increased to 280 ℃ at 10 ℃/min, the sample injection amount is 1 mu L, a split-flow sample injection mode is adopted, and the split-flow ratio is 10: 1.

the GC-MS mass spectrum analysis conditions are as follows: electron impact ion source (EI), ionization energy 70 eV; the ion source temperature is 230 ℃ and the quadrupole rod temperature is 150 ℃. Collision gas N₂Ions 169 are monitored using the SIM monitoring mode.

Referring to FIG. 5, it can be seen from the mass spectrum that the presence of beta-ketoadipic acid is clearly detected in the positive strain, indicating that hydroquinone is completely degraded, and the final product, beta-ketoadipic acid, can enter the tricarboxylic acid cycle of the microorganism and participate in the synthesis of substances in the microorganism.

Therefore, the degradation rate and concentration of the hydroquinone of the Escherichia coli transferred with the hydroquinone degrading enzyme genome are greatly improved.

Sequence listing

<110> Shanghai city academy of agricultural sciences

<120> hydroquinone degrading enzyme gene group expressed in escherichia coli and application thereof

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Claims (10)

1. A hydroquinone degrading enzyme genome expressed in Escherichia coli, comprising PnPCS, PnPDS and PnPES genes derived from Pseudomonas and obtained after optimization according to codon bias of Escherichia coli.

2. The hydroquinone degrading enzyme genome of claim 1, wherein the nucleotide sequence of the PnPCS gene is represented by SEQ ID No.1 after being optimized according to the codon preference of Escherichia coli; the nucleotide sequence of the PnPDS gene is shown as SEQ ID No. 2; the nucleotide sequence of the PnPES gene is shown as SEQ ID No. 3.

3. Use of the hydroquinone degrading enzyme genome according to claim 1 or 2 in escherichia coli.

4. A multigene colibacillus transforming carrier includes colibacillus expressing carrier and the gene expression cassettes of PnPCS, PnPDS and PnPES genes, which are derived from pseudomonads and obtained by optimizing according to the codon preference of colibacillus, and are respectively fused with T7 promoter and terminator.

5. The multigenic E.coli transformation vector according to claim 4, wherein the E.coli expression vector is pET-28 a.

6. A method for obtaining transgenic Escherichia coli capable of completely degrading hydroquinone comprises the following steps:

1) optimizing PnPC gene, PnPD gene and PnPE gene derived from pseudomonas according to the codon preference of escherichia coli, respectively obtaining PnPCS gene, PnPDS gene and PnPES gene after optimization, respectively fusing the optimized three genes with a T7 promoter and a terminator by using an overlap extension PCR technology, and constructing a gene expression cassette;

2) connecting the three gene expression cassettes constructed in the step 1) into an escherichia coli expression vector in sequence according to the sequence of PnPCS, PnPDS and PnPES to obtain a polygenic escherichia coli transformation vector of the three gene expression cassettes;

3) and (3) transferring the polygenic escherichia coli transformation vector in the step 2) into escherichia coli to obtain the escherichia coli capable of completely degrading hydroquinone.

7. The method for obtaining a protein according to claim 6, characterized in that the nucleotide sequence of the PnPCS gene is shown as SEQ ID No. 1; the nucleotide sequence of the PnPDS gene is shown as SEQ ID No. 2; the nucleotide sequence of the PnPES gene is shown as SEQ ID No. 3.

8. The method for obtaining a polypeptide according to claim 6 or 7, wherein in step 1), the nucleotide sequence of the T7 promoter is shown as SEQ ID No. 4; the T7 terminator sequence is shown in SEQ ID NO. 5.

9. The method of claim 6 or 7, wherein in step 2), the E.coli expression vector is pET-28 a.

10. The method for obtaining the protein of claim 6, wherein in the step 3), the Escherichia coli is BL 21-AI.

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