A gene of a deep archaea transmethylase i, protein sequence and preparation method thereof

Abstract

The application provides a gene and a protein sequence of a deep archaea transmethylase I and a preparation method thereof, belongs to the technical field of genetic engineering, and the transmethylase I is derived from a deep archaea genome and has transmethyl activity, so that the methyl in a methoxyl aromatic compound is transferred to a corrin ring protein. The deep archaea transmethylase I is cloned into a pCold-TF expression vector, is induced to express in Escherichia coli through a recombination expression vector, is purified through immobilized nickel ion affinity, and the transmethylase is prepared. The transmethylase I can be used for transmethylation of the methoxyl aromatic compound.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the gene and protein sequence of a deep archaea transmethylase I and its preparation method. Background Technology

[0002] The transmethylation process using methoxy aromatic compounds as substrates requires the combined action of multiple enzymes, including transmethylase I, a corrinoid protein, AE activator, and transmethylase II. Transmethylase I is responsible for the crucial substrate recognition and catalytic process, catalyzing the transfer of methyl groups from methoxy aromatic compounds to corrinoid proteins. Transmethylase II catalyzes the transfer of methyl groups from corrinoid proteins to tetrahydrofolate. AE activator can catalyze the conversion of inactive divalent cobalt to active monovalent cobalt.

[0003] Transmethylase complexes are mostly found in acetic acid-producing bacteria. Recently, it has been found that this type of transmethylase complex also exists in methanogenic archaea and other archaea. However, the transmethylases reported so far are all of bacterial origin and have high affinity. Summary of the Invention

[0004] The purpose of this invention is to provide a gene and protein sequence of a deep archaea transmethylase I and its preparation method. The deep archaea transmethylase I has the reactivity of transmethylation using guaiacol as a substrate, transferring methyl groups to a cycloporin protein, and can be used for the transmethylation of methoxy aromatic compounds.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a protein sequence of a deep archaea transmethylase I, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] The present invention further protects the gene fragment encoding the above-mentioned deep archaea transmethylase I protein sequence, characterized in that its nucleotide sequence is as shown in SEQ ID NO: 2.

[0008] The present invention further protects a recombinant expression vector comprising the above-mentioned gene fragment.

[0009] The present invention further protects a recombinant bacterium, characterized in that it comprises the above-mentioned recombinant expression vector.

[0010] As a further improvement of the present invention, the recombinant bacteria is Escherichia coli.

[0011] This invention further protects a method for preparing transmethylase I from deep archaea, comprising the following steps:

[0012] S1. Construct the above-mentioned recombinant expression vector;

[0013] S2. The recombinant expression vector obtained in step S1 is transferred into host cells, and the host cells are cultured and induced to express.

[0014] S3. Collect the host cells after induced culture, break them up, heat them, and centrifuge them to obtain the supernatant;

[0015] S4. Purify the supernatant to obtain the transmethylase I.

[0016] The present invention further protects a deep archaea transmethylase I prepared by the above-described preparation method.

[0017] The present invention further protects a transmethylation reaction buffer for the above-mentioned archaea transmethylase I, characterized in that the buffer reaction system contains 35 mmol / L Tris-HCl, 70 mmol / L KCl, 12 mmol / L MgCl2, 0.5 mmol / L Ti(III) citrate, 2.3 mmol / L ATP, 0.08 mg / mL activator enzyme, 1.2 mg / mL Co(II)-corrin cyclic protein, 2.3 mmol / L guaiacol and 0.015 mg / mL Trigger Factor tagging archaea methyltransferase I.

[0018] The present invention has the following advantages: The transmethylase I sequence in this invention is not homologous to currently reported transmethylase I sequences. The enzyme size also differs; currently reported transmethylase I sequences are around 47 kDa, while the transmethylase I of this invention is around 32 kDa. Furthermore, currently discovered transmethylase I sequences are widely found in various types of microorganisms, while the transmethylase I of this invention is only found in deep archaea.

[0019] This invention relates to transmethylase I, derived from the genome of a deep archaea, which possesses transmethylation activity. Using methoxylated aromatic compounds as substrates, it transfers the methyl group to a corrin ring protein. The deep archaea transmethylase I was cloned into the pCold-TF expression vector, induced to express in *E. coli* using the recombinant expression vector, and purified via immobilized nickel ion affinity polymerization to prepare the transmethylase. This transmethylase I can be used for the transmethylation of methoxylated aromatic compounds. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1The results of agarose gel electrophoresis of the two gene fragments amplified by PCR in Example 1 are shown.

[0022] Figure 2 The results of SDS-PAGE electrophoresis analysis of the protein in Example 1 are shown.

[0023] Figure 3 This is a graph showing the transmethylation activity test results of the archaea transmethylase I in Example 1;

[0024] Figure 4 This is a comparison chart showing the results of liquid chromatography detection of catechol, a product of guaiacol transmethylation, in Example 1. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Preparation and application of deep archaea transmethylase I and corrin cyclic protein fragments

[0027] 1. Construction of prokaryotic recombinant expression vectors

[0028] Using the metagenomic sequence of the deep archaea obtained by sequencing, the gene fragments encoded by both were determined by bioinformatics analysis. The gene sequence of the deep archaea methyltransferase I is shown in SEQ ID NO: 2, the gene fragment of the clinoporin ring is shown in SEQ ID NO: 3, and the protein fragment is shown in SEQ ID NO: 4.

[0029] First, using *Archaeopteryx* genomic DNA as a template, the *Archaeopteryx* methyltransferase I fragment and the *Crocynium cyclic protein* fragment were amplified using primer pairs F1 / R1 and F2 / R2, respectively. The agarose gel electrophoresis results of the two amplified gene fragments are shown below. Figure 1 As shown, (A) is the PCR result of the *Archaeopteryx* transmethyltransferase I gene fragment, and (B) is the PCR result of the *Cruciferone* cyclic protein gene fragment. The sizes of the *Archaeopteryx* transmethyltransferase I and *Cruciferone* cyclic protein gene fragments are 903 bp and 429 bp, respectively. The primer sequences are as follows:

[0030] Methyltransferase I primers:

[0031] F1:5'-GGTACCCTCGAGGGATCCATGAAGTTTGGAATGTTCATTTATG-3'

[0032] R1:5'-CAGGTCGACAAGCTTTTAGTCTAGTATACTTTCCCACTTGTC-3'

[0033] Gurin cyclic protein primers:

[0034] F2:5'-GCCGCGCGGCAGCCATATGATGTCTTGGTTAAAATCTATGATG-3'

[0035] R2:5'-CGAGTGCGGCCGCAAGCTTTTATTTGGATGCCATTGCTTTTTT-3'

[0036] The pCold-TF vector was digested with BamHI and HindIII, and the pET28a vector was digested with NdeI and HindIII. The digested products were then recovered using a DNA product purification kit. The *Archaeopteryx* methyltransferase I fragment gene was recombined with the pCold-TF vector digested with BamHI / HindIII, and the clinocyclic cyclic protein gene was recombined with the pET28a vector digested with NdeI / HindIII. After successfully constructing these two vectors, they were transformed into DH5α competent cells. Single cells were picked the following day, and positive recombinant clones were verified by colony PCR. The positive clones were then subjected to DNA sequencing to verify the correctness of the *Archaeopteryx* methyltransferase I fragment gene and clinocyclic cyclic protein gene sequences.

[0037] 2. Prokaryotic expression of recombinant archaea methyltransferase I fragment protein and corrugated ring protein

[0038] The prokaryotic recombinant expression vectors of archaea methyltransferase I and cyclophosphamide were transformed into *E. coli* BL21(DE3) competent cells. The transformed bacterial cultures were evenly spread onto solid LB agar plates containing 100 μg / mL ampicillin and 50 μg / mL kanamycin, respectively, and incubated upside down at 37°C for 16 hours. Single colonies were picked and transferred to 20 mL of LB liquid medium containing the corresponding antibiotics and incubated overnight at 37°C × 200 rpm. The 20 mL bacterial culture was then transferred to a 500 mL culture system and cultured at 37°C × 200 rpm until OD (digestive activity) was observed. 600 When the measured value reached 0.7-1.0, IPTG was added to a final concentration of 0.5 mmol / L, and the mixture was cultured for another 18 h at 20℃ × 200 r / min to induce the expression of Trigger Factor tag / deep archaea methyltransferase I fusion protein and gurnolin ring protein, respectively.

[0039] 3. Affinity purification of archaea methyltransferase I and corrin cyclic protein (under anaerobic conditions)

[0040] Induced *E. coli* cells were centrifuged at 8000 rpm for 3 min to collect the cells. The cells were resuspended in 40 mL of lysis buffer (20 mmol / L Tris-HCl, pH 7.6, 150 mmol / L NaCl, 5% glycerol) and subjected to sonication. The sonication conditions were 600 W for 4 seconds, followed by a 4-second pause, for a total of 20 min. The cells were then centrifuged at 10000 rpm for 30 min at 4 °C, and the supernatant was collected to obtain the crude recombinant protein solution.

[0041] The supernatant was added to a chromatographic column packed with 2 mL of Ni-NTA purification resin, and buffer was allowed to flow through the column to allow the 8-consecutive-histidine tag of the archaea methyltransferase I and the 6-consecutive-histidine tag of the clinocyclic cyclic protein to specifically bind to the nickel ions immobilized on the Ni-NTA resin, respectively. The resin was then washed with lysis buffer containing 20 mmol / L imidazole to remove non-specifically bound proteins. Next, the Ni-NTA resin was eluted with 20 mL of elution buffer (containing 250 mmol / L imidazole), and the eluent was collected. The eluent contained the Trigger Factor tag / archaea methyltransferase I fusion protein and the clinocyclic cyclic protein, respectively.

[0042] The above protein was transferred to a storage solution [20 mmol / L Tris-HCl (pH 7.6), 1 mmol / L DTT, 150 mmol / L NaCl, 5% glycerol] and stored at -20°C. The protein was identified by SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown, (A) is the SDS-PAGE electrophoresis result of the Trigger Factor tag / deep archaea methyltransferase I fusion protein, and (B) is the SDS-PAGE electrophoresis result of the clinocyclic protein. The Trigger Factor tag / deep archaea methyltransferase I fusion protein is approximately 100 kDa, and the clinocyclic protein is approximately 17 kDa.

[0043] 4. Assay of Methyltransferase I Fragment Protease Activity in Deep Archaea

[0044] The Trigger Factor tag purified by affinity (Ni-NTA resin) in step 3 was subjected to a transmethylation test with the fusion protein of the deep archaea methyltransferase I fragment.

[0045] (1) Preparation of Co(II)-corrin cyclic protein (under anaerobic conditions)

[0046] The corin cyclic protein also originates from the archaea *Benchenia*, and its gene sequence is shown in the sequence listing as SEQ ID NO: 3. 1.5 mL of corin cyclic protein (30 mg) solution was added to 8.5 mL of refolded solution, followed by the addition of DTT to a final concentration of 1 mmol / L. The refolded solution contained 50 mmol / L Tris, 300 mmol / L betaine, and 1 mmol / L hydroxycobalamin, and the pH was adjusted to 7.6. This mixture was then incubated at 4°C in the dark for 16 hours. The next day, the Co(II)-corin cyclic protein was prepared by exchanging the solution several times with a Tris HCl solution containing 1 mmol / L DTT at pH 7.6 using a 10 kDa ultrafiltration tube until the filtrate containing hydroxycobalamin appeared distinctly clear instead of red.

[0047] (2) Activity assay reaction

[0048] Enzyme activity assays were performed in 350 μl quartz cuvettes sealed with rubber stoppers and aerated with N2, with all measurements in triplicate. The activity of *Archaeopteryx* methyltransferase I was determined in a 300 μl volume buffer (pH 7.6) containing 35 mmol / L Tris-HCl and 70 mmol / L KCl. First, recombinant Co(II)-corrin cyclic protein was converted to Co(I)-corrin cyclic protein at a final concentration of 1.2 mg / mL by adding 12 mmol / L MgCl2, 0.5 mmol / L Ti(III) citrate (freshly prepared), 2.3 mmol / L ATP, and 0.08 mg / mL activator enzyme (GenBank accession no. ACJ01666.1). Second, methyltransferase I was added to a final concentration of 2.3 mmol / L guaiacol and reacted overnight at 25 °C. Three aliquots of the samples were taken before the addition of methyltransferase I and after the activity assay for HPLC analysis of methoxy compounds. CH3 is transferred from the substrate guaiacol to Co(I)-corrin cyclic protein to form CH3-Co(III)-corrin cyclic protein, resulting in increased UV absorption at around 530 nm. This phenomenon can be observed using a UV-Vis spectrophotometer. The above results are shown in […]. Figure 3 , Figure 3 The deep archaea methyltransferase I transfers the methyl group of guaiacol to Co(I)-corrin cyclic protein, producing methylated Co(III)-corrin cyclic protein (red); Figure 4 The formation of catechol, a product of guaiacol transmethylation, was detected by liquid chromatography.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

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[0057] sequence list <110> Shanghai Jiao Tong University <120> Gene, protein sequence and preparation method of transmethylase I from deep archaea <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 300 <212> PRT <213> Methanogenic archaea (absent) <400> 1 Met Lys Phe Gly Met Phe Ile Tyr Glu Pro Thr Pro Val Glu Gly Phe 1 5 10 15 Asp Leu Glu Val Tyr Arg Leu Lys Ser Glu Arg Gly Ile Val Gly Asn 20 25 30 Pro Asn Pro Asp Met Thr Thr Asn Ile Ala Cys Phe Gly Asp Asn Asn 35 40 45 Met Ala Glu Gln His Pro Asp Trp Val Ala Gln Ser Ala His Gly Pro 50 55 60 Ala Leu Arg Thr Asn Lys Tyr Tyr Asn Leu Arg Trp Asp Ile Val Cys 65 70 75 80 Asn Gln Glu Lys Gln Met Ile Asp Tyr Met Leu Asp Leu Ile Glu Lys 85 90 95 Thr Ala Asp Tyr Thr Lys Gly Ile Thr Val Ser Ser Met His Val Ala 100 105 110 Asp His Gly Phe Cys Thr Cys Pro Arg Cys Lys Ala Ala His Ala Lys 115 120 125 Ser Gly Met Asp Leu Leu Asp Trp Arg Ala Gln Thr Val Thr Asp Phe 130 135 140 Ile Gly Ala Ala Lys Glu Arg Val Lys Asn Lys Pro Phe Tyr Val Gly 145 150 155 160 Leu Leu Pro Asp Pro Val Asn Cys Arg Glu Arg Phe Gly Leu Asp Phe 165 170 175 Asp Ala Leu Ala Glu Phe Ala Thr Ala Phe Val Val Pro His Trp Ser 180 185 190 Lys Thr Tyr Ala Thr Pro Trp Tyr Phe Glu Thr Met Ala Arg Ser Phe 195 200 205 Lys Lys Leu Leu Lys Lys Pro Val Tyr Pro Gly Leu Tyr Ile Gln Gly 210 215 220 Pro Gly Asp Asp Pro Asn Glu Leu Glu Asn His Thr Gln Leu Leu Lys 225 230 235 240 Thr Ala Cys Arg Ile Ala Arg Thr Gly Val Asp Gly Leu Ile Phe Leu 245 250 255 Ala Ala Asn Ala Gln Ile Met Lys Asp Phe Gln Arg Thr Cys Val Glu 260 265 270 Asp Arg Glu Leu Arg Glu Phe Leu Asp Gly Tyr Gly Gly Lys Pro Val 275 280 285 Leu Asp Leu Val Asp Lys Trp Glu Ser Ile Leu Asp 290 295 300 <210> 2 <211> 903 <212> DNA <213> Methanogenic archaea (none) <400> 2 ttgaagtttg gaatgttcat ttatgaacct actcctgttg aaggattcga cctagaagta 60 taccgtctaa aatcagaacg aggaattgtt ggaaatccaa atcctgatat gacaactaac 120 attgcctgct tcggcgacaa caacatggca gaacaacacc ccgactgggt tgcacaatct 180 gcccatggac ctgcactgcg aacaaacaaa tactataacc tacgctggga tatcgtttgt 240 aatcaagaaa aacaaatgat tgattacatg cttgacttaa tcgaaaaaac cgcagattac 300 accaaaggaa tcactgttag cagcatgcac gtagcagatc acggattttg cacctgccca 360 agatgtaaag cagcccacgc aaaaagtgga atggacttgc tagattggag ggcacaaaca 420 gtaacagatt tcatcggtgc agcaaaagaa cgcgtaaaaa acaaaccatt ctatgttggt 480 ttgcttcctg acccagttaa ctgccgtgaa cgattcggct tggactttga cgctctagca 540 gaattcgcaa ccgcctttgt tgtaccccac tggtcaaaga catatgcaac tccatggtac 600 tttgaaacaa tggcccgttc atttaagaaa ctactcaaaa agccagtgta tccaggtctt 660 tacattcaag gaccaggaga tgaccccaac gaactagaaa accacactca actgctcaaa 720 acagcatgtc gtatcgcccg aactggtgta gatggactta ttttcctagc cgcaaacgct 780 caaatcatga aagacttcca aagaacatgt gtcgaagaca gagagcttcg cgaatttttg 840 gatggatatg gcggcaaacc agtcttagat ctggttgaca agtgggaaag tatactagac 900 taa 903 <210> 3 <211> 429 <212> DNA <213> Artificial sequence (without origin) <400> 3 atgtcttggt taaaatctat gatggaaaaa gaacccgaag aatctgacaa cccaattgga 60 attgtagtaa tcggaacact tgaccccgac gtacacttaa cccctaaaga aatggtaaga 120 aaatcactca ccaaagccga attcaaatgc tacgacgtag gcaaaaaagc accagcagca 180 gactttgcta acaaagcaaa agaagttaat gcagacatca tcgcagtttc aatcaacact 240 gctccagcaa aaaacaacat ccctgcacta atgcaagaaa ttgaagccgc aggcctcaaa 300 ggcaaagtcg taatcatgat cggcggcgca gcagtcgacg aagacgacgc agaagaaatc 360 ggtgcactat tcggcgaaac tcgagaagaa gcagtagcca tcgccaaaaa agcaatggca 420 tccaaataa 429 <210> 4 <211> 142 <212> PRT1] <213> Artificial Sequence (unnamed) <400> 4 Met Ser Trp Leu Lys Ser Met Met Glu Lys Glu Pro Glu Glu Ser Asp 1 5 10 15<00002(27)>Asn Pro Ile Gly Ile Val Val Ile Gly Thr Leu Asp Pro Asp Val His 20 25 30 Leu Thr Pro Lys Glu Met Val Arg Lys Ser Leu Thr Lys Ala Glu Phe 35 40 45[[ID=]37] Lys Cys Tyr Asp Val Gly Lys Lys Ala Pro Ala Ala Asp Phe Ala Asn 50 55 60 Lys Ala Lys Glu Val Asn Ala Asp Ile Ile Ala Val Ser Ile Asn Thr 65 70 75 80 Ala Pro Ala Lys Asn Asn Ile Pro Ala Leu Met Gln Glu Ile Glu Ala 85 90 95 Ala Gly Leu Lys Gly Lys Val Val Ile Met Ile Gly Gly Ala Ala Val 100 105 110 Asp Glu Asp Asp Ala Glu Glu Ile Gly Ala Leu Phe Gly Glu Thr Arg 115 120 125 Glu Glu Ala Val Ala Ile Ala Lys Lys Ala Met Ala Ser Lys 130 135 140

Claims

1. A deep archaeal transmethylase I, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding the archaea transmethylase I as described in claim 1.

3. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 2.

4. A recombinant bacterium, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. The recombinant bacteria of claim 4, wherein, The recombinant bacteria is Escherichia coli.

6. A method for the production of a deep archaeal methyltransferase I, characterized in that, Includes the following steps: S1. Construct the recombinant expression vector as described in claim 3; S2. The recombinant expression vector obtained in step S1 is transferred into host cells, and the host cells are cultured and induced to express. S3. Collect the host cells after induced culture, break them up, heat them, and centrifuge them to obtain the supernatant; S4. The supernatant was purified to obtain the deep archaea transmethylase I.

7. An archaea transmethylase I prepared by the method described in claim 6.

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