Ethylene synthetase and use thereof

Abstract

The application discloses ethylene synthetase and application thereof. The application provides a high-activity ethylene synthetase gene (Sbefe), and constructs the gene into a corresponding expression vector, expresses protein in Escherichia coli, and purifies and determines the enzyme activity. The activity of the ethylene synthetase (SbEFE) is about 4 times higher than that of the highest-activity Pseudomonas syringae ethylene synthetase (PsEFE) known at present, and the thermal stability of the enzyme is obviously higher than that of the ethylene synthetase known at present, which has important significance for saving cost and improving efficiency in industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an ethylene synthase and its applications. Background Technology

[0002] Ethylene is a key focus of current research in bioenergy conversion. As one of the most important industrial chemicals, ethylene has a wide range of applications. It is a basic chemical raw material for the synthesis of fibers, rubber, plastics, and ethanol, and can be relatively easily converted into C5-C10 hydrocarbons in gasoline. It also has important uses in medicine and agriculture. Currently, most ethylene is produced through petrochemical processes, which, besides being constrained by the large-scale consumption of fossil fuels and resource scarcity, also causes serious environmental pollution problems. Bio-ethylene is another industrial biotechnology topic that has received widespread attention in recent years. It mainly involves producing bioethanol from biomass, followed by industrial-scale dehydration processes to produce ethylene. Discovering and establishing a simpler, lower-consumption, and lower-cost bio-ethylene production method would be a significant breakthrough in biomass utilization and bioenergy regeneration.

[0003] The metabolic systems for ethylene production in some microorganisms have attracted considerable attention. Among them, the ethylene production pathways derived from *Pseudomonas syringae* and *Penicillium digitatum* have been the most studied. In this pathway, ethylene is produced using α-ketoglutarate (α-KG, also known as 2-oxoglutarate, 2-OG) and arginine as substrates, under the action of ethylene forming enzyme (EFE). α-ketoglutarate and arginine are very common biomolecules, present in virtually all organisms. The produced ethylene is non-toxic to organisms, and as a gaseous molecule, it is easily separated from the products during production. Studies have shown that expressing the *efe* gene for this ethylene synthase in *Escherichia coli*, yeast, *Trichoderma viride*, *Trichoderma reesei*, or cyanobacteria also enables these organisms to produce ethylene. Since Trichoderma can often use lignocellulose as a carbon source and cyanobacteria can use carbon dioxide as a carbon source, if the efe gene can be efficiently expressed in them, it will provide a completely new route for the industrial biosynthesis of ethylene.

[0004] Currently, the most studied enzyme is the EFE protein (PsEFE) derived from *Pseudomonas syringae*. According to existing literature, the activity of this enzyme in catalyzing ethylene production is 660 units / mg. Heterologous synthesis of ethylene has already been achieved in cyanobacteria such as *Synechocystis*, *Trichoderma*, and *Saccharomyces cerevisiae*. Finding more active ethylene synthases will provide an important foundation for increasing the yield of biosynthesized ethylene and accelerating its industrial production. Summary of the Invention

[0005] One object of the present invention is to provide the following protein.

[0006] The protein provided by this invention is as follows: 1) or 2) or 3) or 4):

[0007] 1) The protein shown in sequence 2 of the sequence listing;

[0008] 2) Proteins that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with 1);

[0009] 3) Proteins that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with 1) and have the same function and are derived from Oxidoreductase Streptomyces bottropensis;

[0010] 4) The protein obtained by adding a tag to the end of the sequence shown in 1), 2), or 3).

[0011] The nucleic acid molecules that encode the aforementioned proteins are also within the scope of protection of this invention.

[0012] The above-mentioned nucleic acid molecules are any one of the following DNA molecules: 1)-3)

[0013] 1) The coding region is the DNA molecule shown in sequence 1 of the sequence listing;

[0014] 2) DNA molecules that hybridize with the DNA sequence defined in 1) under strict conditions and encode proteins with the same function;

[0015] 3) DNA molecules that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in 1) and encode a protein having the same function.

[0016] Expression cassettes, recombinant vectors, or recombinant bacteria containing the aforementioned nucleic acid molecules are also within the scope of protection of this invention.

[0017] The application of the aforementioned protein in ethylene production is also within the scope of protection of this invention.

[0018] The application of the aforementioned protein in the catalytic production of ethylene from α-ketoglutarate and arginine is also within the scope of protection of this invention.

[0019] The application of the aforementioned nucleic acid molecules, expression cassettes, recombinant vectors, or recombinant bacteria in ethylene production is also within the scope of protection of this invention.

[0020] The application of the above-mentioned nucleic acid molecules, expression cassettes, recombinant vectors, or recombinant bacteria in the catalytic production of ethylene from α-ketoglutarate and arginine is also within the scope of protection of this invention.

[0021] Another object of the present invention is to provide a method for producing ethylene.

[0022] The method provided by the present invention includes the following steps: using the above-mentioned protein to catalyze the production of ethylene from α-ketoglutarate and arginine.

[0023] This invention discovers a highly active ethylene synthase gene (Sbefe), constructs it into a corresponding expression vector, expresses and purifies the protein in *E. coli*, and performs enzyme activity assays. Gas chromatography analysis shows that this ethylene synthase (SbEFE) has approximately four times the activity of the currently known highest-activity *Pseudomonas syringae* ethylene synthase (PsEFE), making it more suitable for industrial production. Furthermore, the enzyme's thermostability is significantly higher than that of currently known ethylene synthases, which is of great significance for saving costs and improving efficiency in industrial production. Attached Figure Description

[0024] Figure 1 The results of SbEFE nickel column purification are shown. M: Marker, S: Supernatant, P: Pellet, FT: FlowThrough, W / 20 / 50 / 100 / 200 / 500: Elution solutions containing different concentrations of imidazole.

[0025] Figure 2 Detection spectra of ethylene synthesis by ethylene synthase (SbEFE) and the previously reported ethylene synthase from Pseudomonas syringae (PsEFE).

[0026] Figure 3 The thermostability curve of ethylene synthase is shown.

[0027] Figure 4 This is the result of gas chromatography detection of ethylene standards.

[0028] Figure 5Preparation of the ethylene standard curve. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1: Preparation of ethylene synthase Sbefe

[0032] I. Cloning of the ethylene synthase Sbefe

[0033] 1. Obtaining the ethylene synthase gene Sbefe

[0034] The nucleotide sequence of the ethylene synthase gene Sbefe is sequence 1 in the sequence listing; the amino acid sequence of the enzyme encoding the ethylene synthase gene Sbefe is sequence 2 in the sequence listing. The ethylene synthase gene Sbefe is derived from Oxidoreductase Streptomyces bottropensis.

[0035] The above gene sequences can be synthesized artificially.

[0036] 2. Construction of the recombinant vector

[0037] The recombinant vector expressing the ethylene synthase gene Sbefe is obtained by replacing the NdeI and XhoI sites of the pET22b vector with the ethylene synthase gene Sbefe shown in sequence 1 of the sequence listing. The vector is named pET22b-Sbefe. The ethylene synthase gene Sbefe is fused with the XhoI restriction site and His tag on the vector for expression. The vector expresses the fusion protein, which consists of ethylene synthase SbEFE, Leu, Glu and 6 His from the N-terminus.

[0038] 3. Preparation of recombinant bacteria

[0039] The recombinant vector pET22b-Sbefe was transformed into BL21 competent cells, plated on LB agar plates containing ampicillin, and incubated upside down at 37°C for 12 hours to obtain recombinant bacteria.

[0040] The recombinant bacteria were sent for sequencing, and the results showed that the recombinant bacteria was obtained by transforming pET22b-Sbefe into BL21.

[0041] II. Expression of ethylene synthase SbEFE

[0042] 1. Induced expression

[0043] Single colonies of the recombinant bacteria obtained above were picked from LB plates and inoculated into 100 ml of LB medium (containing 100 μg / ml ampicillin) and cultured overnight at 37°C and 200 rpm. 8 ml of the culture was transferred to a flask containing 800 ml of LB medium (100 μg / ml Ampicillin) and cultured until the OD600 reached approximately 0.8. IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 16°C for 16 h. The precipitate was collected by centrifugation at 6000 rpm for 15 min, and the precipitate was the bacterial cells.

[0044] The collected bacterial cells were sonicated (200W, 3s sonication, 8s rest, 100 cycles). The buffer solution was a 20mM Tris-HCl solution containing 200mM NaCl, pH 8.0. The sonicated product was centrifuged at 18000rpm at 4 degrees for 50min, and the supernatant was collected.

[0045] The above-mentioned 20mM Tris-HCl solution containing 200mM NaCl was prepared by mixing 20mM Tris-HCl, 200mM NaCl, and water, and adjusting the pH to 8.0.

[0046] SDS-PAGE analysis of the supernatant was performed using a protein gel with a lower layer concentration of 12% and an upper layer concentration of 5%, with a loading volume of 8 μL for both layers. Electrophoresis was conducted at a constant voltage of 130 V. The results are as follows: Figure 1 As shown, S is the supernatant after ultrasonic disruption, P is the precipitate after ultrasonic disruption, FT is the column effluent, W is the elution buffer without imidazole Tris-HCl buffer, and 20, 50, 100, 200, and 500 are the elution buffers containing 20mM, 50mM, 100mM, 200mM, and 500mM imidazole Tris-HCl buffer, respectively. It can be seen that a fusion protein with a molecular weight of 39kD was obtained.

[0047] 2. Purification

[0048] The supernatant obtained in step 1 was combined with Ni beads and eluted with 40 ml of Tris-HCl buffer containing different amounts of imidazole (0 mM, 20 mM, 50 mM, 100 mM, 200 mM, and 500 mM). The eluent was collected.

[0049] The above Tris-HCl buffer solutions containing 0mM, 20mM, 50mM, 100mM, 200mM, and 500mM imidazole are solutions obtained by mixing imidazole of different concentrations, 200mM NaCl, 20mM Tris-HCl, and water.

[0050] The results of SDS-PAGE analysis are as follows: Figure 1As shown, S is the supernatant after ultrasonic disruption, P is the precipitate after ultrasonic disruption, FT is the column effluent, W is the elution buffer without imidazole Tris-HCl, and 20, 50, 100, 200, and 500 are the elution buffers containing 20mM, 50mM, 100mM, 200mM, and 500mM imidazole Tris-HCl buffers, respectively. It can be seen that elution with Tris-HCl buffers containing 20mM, 50mM, and 100mM imidazole yields the fusion protein, a 39kD ethylene synthase SbEFE fusion protein.

[0051] The eluent was eluted with Tris-HCl buffer containing 20 mM imidazole and dialyzed into Tris-HCl buffer containing 200 mM NaCl at pH 8.0 to obtain ethylene synthase SbEFE solution.

[0052] The above-mentioned pH 8.0 Tris-HCl buffer containing 200mM NaCl is prepared by mixing 20mM Tris-HCl, 200mM NaCl and water to obtain a solution, and then adjusting the pH to 8.0.

[0053] The protein content in the solution was measured, and the concentration of ethylene synthase SbEFE was 0.4 mg / ml.

[0054] III. Assay of Ethylene Synthetic Enzyme Activity

[0055] 1. Enzymatic reaction produces ethylene

[0056] Gas chromatography is used to detect the amount of ethylene produced by an enzyme reaction. Enzyme activity units (U) refer to the amount of enzyme required to catalyze the production of 1 nmol of ethylene per minute under specific reaction conditions. Specific enzyme activity refers to the enzyme activity per milligram of enzyme protein (U / mg protein).

[0057] Table 1 shows the sources of each substance in the enzyme reaction system.

[0058]

[0059]

[0060] The enzyme reaction system (total 1 ml) consisted of: 40 mM HEPES buffer, 0.5 mM α-ketoglutarate, 0.5 mM L-arginine, 0.2 mM ferrous sulfate, 0.4 mM L-ascorbic acid, and 10 μg / mL of the EFE protein to be tested, with the remainder being water. See Table 2 for details.

[0061] Table 2 shows the reaction system for detecting ethylene synthase activity.

[0062] EFE protein 10ug / mL Ferrous sulfate 0.2mM L-Ascorbic Acid 0.4mM α-Ketoglutarate 0.5mM L-arginine 0.5mM 500mM Hepes buffer, pH 7.5 40mM H2O Add to 1 mL

[0063] The EFE proteins to be tested are the SbEFE solution and PsEFE solution of ethylene synthase prepared in the above two steps.

[0064] The PsEFE solution is obtained by dissolving the PsEFE protein (Genbank: BAA02477.1, 2008.2.13) in a Tris-HCl buffer solution containing 200 mM NaCl at pH 8.0.

[0065] The enzyme reaction system was reacted in a sealed reaction vessel at 25°C for 3 minutes. Then, 1 ml of gas was extracted from the sealed reaction vessel after the reaction and analyzed by gas chromatography to quantify the ethylene yield, as detailed below:

[0066] Add 1 ml of the enzyme reaction mixture to a 7.5 ml blood collection tube, react at 25°C for 3 minutes, and then use a gas-tight needle to draw 1 ml of gas for sample loading. Run the program at 30°C for 3 minutes, then at 150°C for 2 minutes.

[0067] Parameters: The gas chromatograph used was an Agilent 7890B, the column was an Agilent 19095P-Q04 HP-PLOTQ, the injection port temperature was 230℃, the detector used was an FID detector with a temperature of 250℃, the carrier gas was nitrogen, and the column oven temperature was 30℃.

[0068] The standard used is nitrogen-based ethylene standard (ethylene content 1 / 1000), provided by the National Institute of Metrology, China.

[0069] The retention time of the standard was 2.14 min. Figure 4 );

[0070] The results are as follows Figure 2 As shown, both SbEFE and PsEFE solutions catalyze the ethylene synthase, yielding the target product ethylene with a retention time of 2.14 min, proving that the SbEFE of this invention is an ethylene synthase.

[0071] 2. Calculate the specific activity of the enzyme

[0072] 1) Preparation of standard curve

[0073] A standard curve was plotted using ethylene at a concentration of 10-100 ppm as a standard (vertical axis) and the corresponding peak areas obtained by gas chromatography as described in section 1 above (horizontal axis). Figure 5 As shown in Figure A.

[0074] A standard curve was plotted using ethylene at concentrations of 100 ppm-1000 ppm as a standard (vertical axis) and the corresponding peak areas obtained by gas chromatography as described in section 1 above (horizontal axis). Figure 5 As shown in B.

[0075] 2) Calculation of enzymes

[0076] By definition, specific enzyme activity = amount of ethylene / reaction time / amount of enzyme in the reaction system.

[0077] Molecular weight of ethylene: Substitute the peak area values ​​catalyzed by ethylene synthase SbEFE solution and PsEFE solution into the corresponding... Figure 5 A or Figure 5 In the ethylene standard curve, the calculated amount of ethylene gas in 1 ml of manually sampled gas (e.g., 49.5475) is given. Since the total gas volume in the reaction vessel is 6.5 ml, the amount of ethylene gas produced in the entire 1 ml reaction system over 3 minutes is:

[0078] 49.5475 * 6.5 = 322.37725nl

[0079] 322.37725(nl) / 22.4(L / mol)=14.3918nmol

[0080] Reaction time: 3 minutes

[0081] The amount of enzyme in the reaction system is 10 μg / mL.

[0082] The results are as follows: the specific activities of ethylene synthases SbEFE and PsEFE were 2109.3±210.7 units / mg and 550.1±76.2 units / mg, respectively. The activity of ethylene synthase (SbEFE) was about 4 times higher than that of Pseudomonas syringae ethylene synthase (PsEFE), which has the highest known activity.

[0083] IV. Determination of the thermal stability of ethylene synthase SbEFE

[0084] The SbEFE solution of ethylene synthase obtained in step two above and the existing PsEFE solution of ethylene synthase were placed in water baths at 30℃, 35℃, 40℃ and 45℃ respectively, and incubated for 30 min to obtain ethylene synthases after heat treatment. After heat treatment, they were placed on ice in preparation for activity measurement.

[0085] The ethylene synthases after the above heat treatment were subjected to enzyme reactions according to the method described in section three above, with normal activity (placed at 0℃ for 30 min) as the control.

[0086] The results are shown in Table 3 and Figure 3 As shown in the figure, the thermostability of ethylene synthase SbEFE is significantly higher than that of the currently known ethylene synthase PsEFE.

[0087] Table 3 shows the thermal stability results.

[0088] SEQUENCE LISTING <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> An ethylene synthase and its application <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1041 <212> DNA <213> Artificial sequence <400> 1 atgactgaat tacaaacatt tgtactaccc ccggtcgtgg atggctctga tgtggacaag 60 gccttaggcc aggcgctgat tgccgcgtgg caggcggacg ggatcttcca gattcaggca 120 acgccggaac aagaagctgc gactgaaaga gcactggaag caagccgtgg tttctttggt 180 cgcccgtttg aagagaaggc tggccatgtt agcgacctga cctattctgg ttacgtagcg 240 agcggtgagg aggagacggc gggcgaaaag gacggctccg aaatttatac cgtttgcccg 300 gacatcccgg aagatgacgc gcgcgtggtg gacaagtggc cttgtcatgg tccagcaccg 360 tggccatcag aacaatatgc tgacgctatg aaaggttaca tgggtgcagt tggcgacatc 420 ggtgagcgcc tgttgcgttt ggttgcgctg ggtttaggcc tggacgacat ggatcacttt 480 accaaactga cggaggatgg atggcaccac atgcgtgtgc tgcgttttcc gcgtgcagac 540 gctaccagcg agcgtggtat tggctcccac accgattacg gtttgctggt gattgcggtc 600 caggatgatg ttggtggtct gtatatccgt ccaccggttc cgggtgagac tcgtggccgt 660 aattggctgg cggatgagag catggcaggc cgttacgaga acgaagagcc gtggaccttt 720 gttaccccgg tctccgcggt gttcaccgtg ttcccgggtg acatcatgca gtttatcacc 780 ggtggtacac tgttgtcgac cccgcataaa gttcgtctcg cggaccgtga acgctacacc 840 atcgcgtatt tccacgaacc gagcttccaa gcggtggccc gtccgctgga cggcggtggc 900 caagatgaat tcatccacta cggcacccac ttcaccaata tgtttatgcg ctgctacccg 960 gatcgtgctg ccactgcgag aattgaaaac gaggagcgct tggcggtcct tgagcgcctg 1020 cgtaaagaag ccctgaacag c 1041 <210> 2 <211> 347 <212> PRT <213>Artificial sequence <400> 2 Met Thr Glu Leu Gln Thr Phe Val Leu Pro Pro Val Val Asp Gly Ser 1 5 10 15 Asp Val Asp Lys Ala Leu Gly Gln Ala Leu Ile Ala Ala Trp Gln Ala 20 25 30 Asp Gly Ile Phe Gln Ile Gln Ala Thr Pro Glu Gln Glu Ala Ala Thr 35 40 45 Glu Arg Ala Leu Glu Ala Ser Arg Gly Phe Phe Gly Arg Pro Phe Glu 50 55 60 Glu Lys Ala Gly His Val Ser Asp Leu Thr Tyr Ser Gly Tyr Val Ala 65 70 75 80 Ser Gly Glu Glu Glu Thr Ala Gly Glu Lys Asp Gly Ser Glu Ile Tyr 85 90 95 Thr Val Cys Pro Asp Ile Pro Glu Asp Asp Ala Arg Val Val Asp Lys 100 105 110 Trp Pro Cys His Gly Pro Ala Pro Trp Pro Ser Glu Gln Tyr Ala Asp 115 120 125 Ala Met Lys Gly Tyr Met Gly Ala Val Gly Asp Ile Gly Glu Arg Leu 130 135 140 Leu Arg Leu Val Ala Leu Gly Leu Gly Leu Asp Asp Met Asp His Phe 145 150 155 160 Thr Lys Leu Thr Glu Asp Gly Trp His His Met Arg Val Leu Arg Phe 165 170 175 Pro Arg Ala Asp Ala Thr Ser Glu Arg Gly Ile Gly Ser His Thr Asp 180 185 190 Tyr Gly Leu Leu Val Ile Ala Val Gln Asp Asp Val Gly Gly Leu Tyr 195 200 205 Ile Arg Pro Pro Val Pro Gly Glu Thr Arg Gly Arg Asn Trp Leu Ala 210 215 220 Asp Glu Ser Met Ala Gly Arg Tyr Glu Asn Glu Glu Pro Trp Thr Phe 225 230 235 240 Val Thr Pro Val Ser Ala Val Phe Thr Val Phe Pro Gly Asp Ile Met 245 250 255 Gln Phe Ile Thr Gly Gly Thr Leu Leu Ser Thr Pro His Lys Val Arg 260 265 270 Leu Ala Asp Arg Glu Arg Tyr Thr Ile Ala Tyr Phe His Glu Pro Ser 275 280 285 Phe Gln Ala Val Ala Arg Pro Leu Asp Gly Gly Gly Gln Asp Glu Phe 290 295 300 Ile His Tyr Gly Thr His Phe Thr Asn Met Phe Met Arg Cys Tyr Pro 305 310 315 320 Asp Arg Ala Ala Thr Ala Arg Ile Glu Asn Glu Glu Arg Leu Ala Val 325 330 335 Leu Glu Arg Leu Arg Lys Glu Ala Leu Asn Ser 340 345

Claims

1. Use of a protein in catalyzing production of ethylene from α-ketoglutarate and arginine. The protein is the protein represented by SEQ ID NO: 2 in the sequence listing.

2. Use of a nucleic acid molecule encoding the protein as claimed in claim 1 or an expression cassette, a recombinant vector or a recombinant bacterium containing the nucleic acid molecule in catalyzing production of ethylene from α-ketoglutarate and arginine.

3. A method for producing ethylene, comprising the step of catalyzing production of ethylene from α-ketoglutarate and arginine with the protein as claimed in claim 1.

Images