METHIONINE LYASE, ENCODING GENE AND BIOSYNTHETIC METHOD THEREOF

Abstract

Embodiment of the present invention discloses a kind of methionine lyase and its encoding gene and biosynthetic method. According to the present invention, the gene encoding methionine lyase as shown in SEQ ID No. 1 is separated from the genome of C. rosea. Embodiment of the present invention further provides an efficient biosynthetic method of methionine lyase, comprising: (1) cloning gene (shown in SEQ ID No. 1) encoding methionine degradation enzyme into a yeast expression vector to construct recombinant yeast expression vector; (2) transforming the recombinant yeast expression vector into Saccharomyces cerevisiae to obtain expression strain; (3) inducing the expression strain to express the methionine lyase, collecting induced strains, purifying expressed recombinant methionine lyase. Purity of recombinant methionine lyase prepared according to the present invention is above 90%, and its efficiency of degradating methionine can reach 0.53±0.0030 μM MTL.Math.h.sup.−1.Math.mg protein.sup.−1.

Claims

1. A gene encoding methionine lyase from the fungus, cDNA sequence of which is (a), (b) or (c) as follows: (a) nucleotide as shown in SEQ ID No. 1; (b) nucleotide which is capable of being hybridized with complementary sequence of SEQ ID NO: 1 in strict hybridization condition; (c) nucleotide sequence with homology of at least 80% to the nucleotide sequence as shown in SEQ ID No. 1; preferably, nucleotide sequence with homology of at least 90% to the nucleotide sequence as shown in SEQ ID No. 1; more preferably, nucleotide sequence with homology of at least 95% to the nucleotide sequence as shown in SEQ ID No. 1; most preferably, nucleotide sequence with homology of at least 97% to the nucleotide sequence as shown in SEQ ID No. 1.

2. A recombinant expression vector containing the encoding gene of claim 1, wherein preferably, the recombinant expression vector is a recombinant yeast expression vector.

3. A recombinant host cell containing the recombinant expression vector of claim 2; wherein preferably, the recombinant host cell is a recombinant Saccharomyces cerevisiae cell.

4. A methionine degradation enzyme encoded by gene of claim 1, wherein the amino acid sequence of which is as shown in SEQ ID No. 2.

5. A biosynthetic method of protein of claim 4, comprising steps of: (1) cloning methionine degradation enzyme gene into a yeast expression vector to construct recombinant yeast expression vector expressing methionine degradation enzyme; (2) transforming the constructed recombinant yeast expression vector expressing the methionine degradation enzyme into Saccharomyces cerevisiae to obtain expression strain of methionine lyase; (3) inducing the expression strain of methionine lyase to express the methionine lyase, collecting induced expressing strain which is then broken, and purifying expressed recombinant methionine lyase.

6. The method of claim 5, wherein in the step (1), the nucleotide as shown in SEQ ID No. 1 was directionally cloned into a yeast expression vector pYES2 by double enzyme digest of HindIII and BamHI, to obtain recombinant yeast expression vector pYES2-STR3 for over-expressing the methionine degradation enzyme; wherein the Saccharomyces cerevisiae in step (2) is Saccharomyces cerevisiae INVSc1; wherein manner of inducing the expression strain of methionine lyase to express the methionine lyase in step (3) is to induce the expression methionine lyase by galactose;

7. The method of claim 6, wherein the recombinant yeast expression vector pYES2 is constructed by step of: taking genome of Clonostachys rosea as a template, taking SEQ ID No. 3 as a upstream primer and SEQ ID No. 4 as a downstream primer to amplify gene encoding methionine degradation enzyme, which is cloned into the expression vector pYES2, to obtain the recombinant yeast expression vector pYES2-STR3; wherein conditions of inducing in step (3) are that as follows: OD600 before induction is 0.4, concentration of the galactose was 2% based on w/v, inducing expression temperature is 30° C., inducting time is 16 hours.

8. The method of claim 5, wherein the purifying in the step (3) comprises steps of: cleaning and regenerating HisTrap FF Ni-column, column equilibration being conducted with column equilibration buffer; after the equilibration is completed, collected strain after induced expression being resuspended by the column equilibration buffer, then being sonicated, then being centrifuged to get supernatant for loading and binding; after loading is completed, washing away non-specific impurities on column by using elution buffer containing 20 mM of imidazole; collecting target protein with elution buffer containing 200 mM imidazole; purifying and collecting the target protein through desalting column to obtain purified methionine-degradating enzyme.

9. The method of claim 8, wherein components of the column equilibration buffer are: 20 mM Tris-HCl pH=8.5, 300 mM KCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF; components of elution buffer are: 20 mM Tris-HCl pH=8.5, 300 mM KCl, 20 mM imidazole, 10% glycerol, 1 mM PMSF.

10. Application of methionine degradation enzyme of claim 4 in degrading methionine.

Description

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

[0025] FIG. 1 shows a chemical structure of methionine.

[0026] FIG. 2 shows agarose gel electrophoresis of a PCR amplification for encoding methionine lyase gene; the M is DL5000.

[0027] FIG. 3 shows PCR screening pYES2-STR3 positive monoclonal agarose gel electrophoresis; M is DL2000, N is the negative control with water, the others are positive clone screening results.

[0028] FIG. 4 shows mass spectrometry result of methionine lyase.

[0029] FIG. 5 is a SDS-PAGE diagram of expressing and purifying methionine degradation enzyme STR3 under optimized conditions; 1: Marker, 2: supernatant obtained by cell disruption and centrifugation, 3: fluid wear (a protein mixture liquid flows through uncombined part of combining column), 4: STR3 after Nickel column purified.

EMBODIMENTS

[0030] Combined with the specific examples to further describe the invention, the advantages and characteristics of the invention will be more clear with the description. But these examples are only exemplary, and do not limit the scope of the present invention. Technicians in this field should be understood, in, without departing from the spirit and scope of the invention can to modify or replace the details and the form of the technical scheme of the invention, but these modifications, and alternatives are falling into the scope of protection of the invention.

1. Experimental Materials

[0031] Clonostachys rosea (isolated and stored in the inventors' laboratory, GeneBank accession number KT007105);

[0032] Saccharomyces cerevisiae INVSc1 and expression plasmid pYES2 are purchased from Invitrogen Corporation (Improved method for high efficiency transformation of intact yeast cells Nucleic Acids Res 1992, 20: . . . 1425).

Embodiment 1: Cloning and Expression Vector Construction of Methionine Lyase Gene STR3

[0033] (1) Based on the amino acid methionine lyase sequence conservation and codon degeneracy, homologous cloning the conserved sequences of methionine lyase in black truffle spore, amplifying 5′ and 3′ ends of the conserved sequences by RACE technology, conducting NCBI Blast after sequence splicing, then finding its consistency in amino acid level with homologous protein in the model strain is 78%. The nucleotide sequence thereof is for example the nucleotide sequence shown in SEQ ID NO. 1, and its deduced amino acid sequence is as shown in SEQ ID NO. 2.

[0034] (2) Extraction of the expression plasmid pYES2

Taking a test tube containing culture medium of 10 mL LB (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, Ampicillin 100 mg/L) to inoculate E. coli Top10 containing pYES2 plasmid in the tube, culture at 37° C. and 180 rpm overnight, extracting plasmid according to a method in the plasmid kit; after plasmid extraction, marking and placing it in refrigerator at 4° C. to wait for use in next step.

[0035] (3) PCR amplification of STR3 with sequence encoding His tag.

Reaction system of PCR amplification: black truffle spore genome 0.5 μL, 10×buffer 5 μL, dNTPs 4 μL, STR3-5′ upstream primer 1 μL, STR3-3′ downstream primer 1 μL, pfu polymerase 0.4 μL, plus ddH2O to 50 μL.

TABLE-US-00001 STR3-5′ upstream primer is 5′-CCCAAGCTTAACACAATGTCTGCCCCGCCTCCGCCAAATG-3′, STR3-3′ downstream primer is 5′-CGCGGATCCTTAGTGGTGGTGGTGGTGGTGTTTGGCTGTGCGTG GAGTTCGTC-3′.

[0036] Underlined italics therein mark the enzyme cleavage site HindIII and BamHI, bold parts mark encoding His tag sequence.

[0037] PCR amplification conditions: 94° C. initial denaturation 10 min; 94° C. 30 s, 55° C. 30 s, 72° C. 2 min, 30 cycles; final extension to 72° C. 5 min.

[0038] After the PCR product STR3-His is detected correctly by agarose gel electrophoresis (FIG. 2), the PCR product was recovered by the kit.

[0039] (4) Construction and transformation of expression vector

using BamHI and HindIII double enzyme to digest the expression plasmids and PCR products, then recovering the enzyme digestion products by PCR product recovery kit, establishing enzyme-linkded system: pYES2 and STR3 mixed enzyme-digestion product in 8 μL, 10×T.sub.4 DNA ligase buffer 1 μL, T.sub.4 DNA ligase 1 μL; putting enzyme-linked system into constant temperature drying bath at 16° C.; after the treatment for 8 h, the enzyme-linked product being added to the E. coli Top10 competent cells; EP tube being reset in ice for 30 min, then put into 42° C. water bath for heat shock for 30 s, then put into ice statically for 2 min; 500 μL LB culture medium was added into each tube for cultivation in shaker for 60 min at 37° C. and 180 rpm; centrifuging at 4000 rpm for 4 min, taking 400 μL supernatant and gently mixing precipitate, plating it on LB plate containing 100 mg/L ampicillin, and cultivating in 37° C. thermostat chamber for 10-12 hours.

[0040] (5) Screening of positive clones

single colonies growing on the plate being inoculated on the LB plate containing 100 mg/L ampicillin, and cultured for 6 h at 37° C.; the colonies being selected for PCR analysis (FIG. 3); taking ddH.sub.2O as negative control, the PCR product being detected by agarose gel electrophoresis. If the molecular weight of the target band amplified being about 1.4 kb, it was preliminarily proved the vector was successfully constructed.

[0041] The bacterial colonies corresponding to correct band are activated, and the bacteria are sequenced. The resulted sequence of sequencing showed that loaded STR3-His fragment length is 1386 bp, indicating that the expression vector pYES2-STR3 is constructed successfully.

Embodiment 2: Expression and Purification of Methionine Degradation Enzyme

[0042] (1) Preparation of yeast competent cell

saved Saccharomyces cerevisiae INVSc1 being activated on solid YPD culture medium, transferred for three times, and inoculated into YPD liquid culture medium for shaking culture at 30° C. and 200 rpm until OD.sub.600=1.0; cells being collected and washed by precooled ultrapure water, and resuspended in 200 μL 1M sorbitol solution for the preparation of yeast competent cells.

[0043] YPD culture medium formula: Yeast Extract 10 g, Trypton 20 g, 121° C. high pressure sterilization for 20 min, adding 2% (w/v) glucose.

[0044] (2) Transformation of STR3 plasmid over-expression in Saccharomyces cerevisiae

PYES2-STR3 plasmid in E. coli TOP10 being extracted; the extracted plasmid being transformed into Saccharomyces cerevisiae in competent state and gently mixed, then added to precooled 0.2 cm electrode cup; gently tapping on the super clean bench to make the mixture flow to the bottom of the electrode cup; putting in an ice bath for 5 min; the electrode cup being placed in an electric conversion instrument at 1.5 kV, 5 mS, for exponential decay pulse conversion; quickly adding 1 mL precooled 1M sorbitol to the electrode cup; transferring the contents respectively to sterilized 1.5 mL EP tube; taking 300 uL thereof to be deposited to SC cultivate medium absent of uracil, inversion cultured for 2-3 d at 30° C., the selecting transformants growing on the plate for expanding culture.

[0045] Ingredients of SC culture medium: 0.67% yeast nitrogen base (without amino acid), 2% (glucose or cottonseed sugar).

[0046] (3) Induction of methionine degradation enzyme

inoculating all activated yeast suspension in 50 ml SC liquid culture medium absent of uracil for 24 h continuous activation, taking all of bacterial liquid to inoculate in 200 mL Sc—U for 24 h continuous activation; after 4000 rpm centrifuge for 5 min, collecting the yeast to inoculate in 1 L SC induced culture medium, and adding 2% galactose to induce culture at 30° C., 200 rpm for 16 h induction; the inducted yeast being collected after centrifuged for 15 min by a high speed refrigerated centrifuge (J-26XP, Beckman) at 4° C., 4000 rpm.

[0047] (4) Purification of methionine degradation enzyme

collected yeast being quickly frozen in liquid nitrogen, then fully grinded, then added to 25 mL of lysis buffer for resuspending; ultrasound breaking (running for 3 seconds, stopping for 3 seconds, with 99 cycles) is conducted until the solution is in translucent state; after the end of the ultrasonic, centrifuging at 4° C. and 12000 rpm for 1 h to collect supernatant obtained after cells breaking and centrifuging.

[0048] Ni-NTE column being prepared, washed by ultra pure water for 3 times, washed with lysisi buffer for 3 times; the supernatant flowing through Ni-NTE column twice, collecting protein mixture liquid and going through uncombined part of combining column; nickel column is washed with washing buffer, removing impurity protein on a nickel column, with 3 mL each time and washing three times; target bands is eluted with eluntion buffer, to obtain purified STR3, then removing imidazole in elution buffer via desalting column.

[0049] Buffer for protein purification:

lysisi buffer, components of which are: 20 mM Tris HCl (pH=8.5), 300 mM KCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF; washing buffer group components of which are: 20 mM Tris-HCl (pH=8.5), 300 mM KCl, 20 mM imidazole, 10% glycerol, 1 mM PMSF;
elution buffer, components of which are: 20 mM Tris-HCl (pH=8.5), 300 mM KCl, 200 mM imidazole, 10% glycerol, 1 mM PMSF;

[0050] (5) Yield of methionine degradation enzyme

using BCA method to measure STR3 eluent concentration purified by nickel column, the concentration is 0.67 mg/L, collecting total of 9 mL protein eluent, and final quality of protein is 6.03 g; because volume of starting bacteria liquid is 1 L, calculated yield of methionine degradation enzyme being 6.03 g/L.

[0051] (6) Analysis of enzymatic properties of methionine degradation enzyme

With a methionine as a substrate to detect effect of removing thiol of SRT3, 5 mL enzymatic reaction system includes: 1 μg/mL STR3, 50 mM Tris-HCl (pH8.0), 5 μm PLP and 20 mM methionine. The enzyme catalyzed reaction system was placed under the temperature of 25° C. for 1 h, and the product of Methyl thiol was detected by gas chromatography with reference to Liu RS, et al. Metabolism of L-methionine linked to the biosynthesis of volatile organic sulfur-containing compounds during the submerged fermentation of Tuber melanosporum. Appl Microbiol Biotechnol 2013, 97: 9981-9992. Calculation result shows degradation efficiency of methionine prepared by embodiment of the present invention was 0.53±0.0030 μM MTL.Math.h.sup.−1.Math.mg protein.sup.−1.