An S-adenosylmethionine synthetase mutant SAM2-1 and its application

Abstract

The present invention discloses an S-adenosylmethionine synthetase mutant SAM2-1 and its application, belonging to the field of bioengineering technology. The amino acid sequence of the mutant is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The application of the mutant SAM2-1 in preparing the fermentation content of S-adenosylmethionine comprises the following steps: (1) constructing a recombinant vector containing the S-adenosylmethionine mutant SAM2-1; (2) constructing a recombinant strain containing the S-adenosylmethionine mutant SAM2-1; (3) fermenting and producing S-adenosylmethionine using L-methionine as a substrate. The present invention provides a new S-adenosylmethionine synthetase mutant, which has enhanced activity compared to the original S-adenosylmethionine synthetase and improves the efficiency of catalyzing the conversion of L-methionine to S-adenosylmethionine by 82.9%.

Description

Technical Field

[0001] The present invention relates to an S-adenosylmethionine synthetase mutant SAM2-1 and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] S-adenosylmethionine (SAM), discovered in 1951, is widely present in animals, plants, and microorganisms. It is also a physiologically active molecule present in all human tissues and body fluids. It is synthesized in vivo from L-methionine and ATP by S-adenosylmethionine synthetase. In this reaction, adenosine from ATP is transferred to L-methionine, forming a high-energy sulfur atom. This adenosine atom activates the attached carbon atom through nucleophilic attack, thus enabling transmethylation, transaminopropylation, and transsulfuration. S-adenosylmethionine participates in over 40 biochemical reactions and is a very important pharmaceutical molecule with broad application prospects in the treatment of conditions such as intrahepatic cholestasis and depression.

[0003] S-adenosylmethionine synthetase catalyzes the transfer of adenosine from ATP to L-methionine to produce S-adenosylmethionine. It is a key enzyme in S-adenosylmethionine synthesis. Researchers have constructed engineered strains by overexpressing or heterologously expressing S-adenosylmethionine synthetases from different sources in Escherichia coli and yeast, improving S-adenosylmethionine fermentation levels. However, production costs remain high. Developing new S-adenosylmethionine synthetases, improving S-adenosylmethionine synthesis efficiency, and reducing production costs have important practical implications for the application of S-adenosylmethionine. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide an S-adenosylmethionine synthetase mutant, which is used to construct a recombinant S-adenosylmethionine engineering strain, and the S-adenosylmethionine content obtained by fermentation is high.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides an S-adenosylmethionine synthetase mutant SAM2-1, the amino acid sequence of the mutant is shown in SEQ ID NO.1.

[0007] Furthermore, the encoding gene of the mutant is sam2-1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0008] Furthermore, the amino acid sequence shown in SEQ ID NO.1 is based on S-adenosylmethionine synthetase SAM2 in Saccharomyces cerevisiae S288C and has the following mutations: the polypeptide amino acid sequence corresponding to the nucleic acid sequence is replaced by C to S at amino acid position 23, the amino acid at amino acid position 48 is replaced by A to S, the amino acid at amino acid position 83 is replaced by D to H, the amino acid at amino acid position 139 is replaced by L to I, the amino acid at amino acid position 192 is replaced by V to L, the amino acid at amino acid position 269 is replaced by G to S, the amino acid at amino acid position 334 is replaced by I to V, and the amino acid at amino acid position 359 is replaced by I to S.

[0009] Furthermore, the amino acid sequence of the S-adenosylmethionine synthetase SAM2 is shown in SEQ ID NO.3, the encoding gene of the S-adenosylmethionine synthetase SAM2 is sam2, the nucleotide sequence of which is shown in SEQ ID NO.4, and the accession number in the NCBI database is: NP_010790.3.

[0010] The present invention also provides a use of an S-adenosylmethionine synthetase mutant SAM2-1 in synthesizing S-adenosylmethionine, comprising the following steps:

[0011] (1) Construction of a recombinant vector containing the S-adenosylmethionine mutant SAM2-1;

[0012] (2) Construction of a recombinant strain containing the S-adenosylmethionine mutant SAM2-1;

[0013] (3) Fermentation to produce S-adenosylmethionine using L-methionine as substrate.

[0014] Furthermore, the specific operation of step (1) to construct a recombinant vector containing the S-adenosylmethionine mutant SAM2-1 is as follows:

[0015] (1-1) Design primers 306-sam2-1-F / R, whose sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. Use the high-fidelity polymerase Phanta HS and the sam2-1 gene as a template to amplify the S-adenosylmethionine synthetase gene of sam2-1 containing the homology arms of the pRS306 vector.

[0016] (1-2) Design primers pRS306-F / R, whose sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. Use the high-fidelity polymerase Phanta HS and the pRS306 plasmid as a template to amplify the linearized fragment of pRS306.

[0017] (1-3) The pRS306 linearized vector fragment and the S-adenosylmethionine synthetase gene of sam2-1 containing the vector homology arm were connected using homologous recombinase, transformed into Escherichia coli DH5α, and after resistance screening, the plasmid was extracted to obtain the recombinant expression vector pRS306-sam2-1.

[0018] Furthermore, the specific operation of step (2) to construct the recombinant strain containing the S-adenosylmethionine mutant SAM2-1 is as follows:

[0019] (2-1) Design primers Ura-F / R, whose sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively. Use high-fidelity polymerase Phanta HS and pRS306-sam2-1 plasmid as a template to amplify the linearized fragment of pRS306-sam2-1.

[0020] (2-2) The linearized fragment of pRS306-sam2-1 was integrated into the genome of Saccharomyces cerevisiae CEN.PK 2-1C by homologous recombination, and the engineered Saccharomyces cerevisiae strain SC-sam2-1 was obtained by screening.

[0021] Furthermore, the specific operation of step (3) to produce S-adenosylmethionine by fermentation using L-methionine as a substrate is as follows:

[0022] (3-1) Seed plate activation: Streak the recombinant expression strain on an eggplant-shaped flask containing solid YPD medium in a clean bench and culture at 30°C for 24 hours to obtain activated seeds.

[0023] (3-2) Seed solution culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at 30°C and 30-50% dissolved oxygen. The culture was continued until the OD value reached 10-15 to obtain the seed solution.

[0024] (3-3) Fermentation of expression strain: The seed liquid was proportionally added to a fermentation tank containing 8 L of fermentation medium, the temperature was 30°C, the dissolved oxygen was 30-50%, and the pH was adjusted to 5.5 with ammonia water. After the base sugar was consumed, a glucose solution with a concentration of 550 g / L was added, and the specific growth rate was adjusted to 0.1-0.3; when the OD grew to 50-150, L-methionine was added, and the concentration of L-methionine in the fermentation liquid was controlled to 0.5-2 g / L; after starting to add L-methionine, the sugar addition flow rate was adjusted to control the ethanol content in the fermentation liquid to 0.2-2 g / L, and the fermentation was completed after 60 h.

[0025] Furthermore, in step (3-2), the components and contents of the seed culture medium are as follows: 20.0 g / L glucose, 10.0 g / L peptone, 10.0 g / L yeast powder, 1.6 g / L potassium dihydrogen phosphate, 1.8 g / L dipotassium hydrogen phosphate, and 0.1 g / L GPE defoamer. Furthermore, in step (3-2), the components of the seed culture medium are as follows: 20.0 g / L glucose, 10.0 g / L peptone, 10.0 g / L yeast powder, 1.6 g / L potassium dihydrogen phosphate, 1.8 g / L dipotassium hydrogen phosphate, and 0.1 g / L GPE defoamer.

[0026] Furthermore, in step (3-3), the amount of the seed liquid added is 20% of the fermentation medium by volume.

[0027] Furthermore, in step (3-3), the fermentation medium components and contents are: glucose 10.0 g / L, peptone 2.0 g / L, yeast powder 3.0 g / L, ammonium sulfate 5.0 g / L, potassium dihydrogen phosphate 12 g / L, magnesium sulfate heptahydrate 2.0 g / L, calcium chloride 0.5 g / L, sodium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.5 g / L, zinc sulfate heptahydrate 0.5 g / L, manganese sulfate 0.2 g / L, sodium molybdate dihydrate 0.2 g / L, copper sulfate pentahydrate 5 mg / L, biotin 0.5 mg / L, GPE defoamer 0.05 g / L.

[0028] Furthermore, in step (3-3), the seed liquid is proportionally added to a fermentation tank containing 8 L of fermentation medium, the temperature is 30°C, the dissolved oxygen is 30-50%, the pH is regulated to 5.5 with ammonia water, and after the base sugar is consumed, a glucose solution with a concentration of 550 g / L is added, and the specific growth rate is regulated to 0.1-0.2; when the OD grows to 120-140, L-methionine is added, and the concentration of L-methionine in the fermentation broth is controlled to 0.5-1 g / L; after the addition of L-methionine begins, the sugar addition flow rate is adjusted to control the ethanol content in the fermentation broth to 0.3-0.8 g / L, and the fermentation is completed after 60 hours.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention provides a new S-adenosylmethionine synthetase mutant, which has enhanced activity compared to the original S-adenosylmethionine synthetase and improves the efficiency of catalyzing the conversion of L-methionine to S-adenosylmethionine by 82.9%.

[0031] (2) The present invention applies a mutant of S-adenosylmethionine synthetase to construct a recombinant expression strain and ferments it to synthesize S-adenosylmethionine, thereby improving the synthesis efficiency of S-adenosylmethionine. The preparation cost of S-adenosylmethionine is low, which is conducive to the promotion and application of S-adenosylmethionine. DETAILED DESCRIPTION

[0032] Example 1

[0033] Based on the predicted tertiary structure of Saccharomyces cerevisiae S288C S-adenosylmethionine synthetase SAM2 (amino acid sequence shown in SEQ ID NO. 3, nucleic acid sequence shown in SEQ ID NO. 4) and the chemical structures of its substrates L-methionine and adenosine triphosphate, a mutant S-adenosylmethionine synthetase SAM2-1 with improved binding affinity to the substrate ectoine and cosubstrate and superior stability was obtained after simulated mutagenesis using bioinformatics software. The amino acid sequence of the mutant SAM2-1 is shown in SEQ ID NO. 1, and the nucleic acid sequence is shown in SEQ ID NO. 2.

[0034] Example 2 Construction of S-adenosylmethionine synthetase mutant SAM2-1

[0035] (1) Based on the sequences of sam2-1 and pET32a(+), primers 32a-sam2-1-F / R and pET32a(+)-F / R were designed. The nucleotide sequences were as shown in SEQ ID NOs. 11-14. The linear fragments were connected using homologous recombinase, transformed into DH5α, and screened for ampicillin resistance to obtain the recombinant vector pET32a(+)-sam2-1-6His.

[0036] (2) The recombinant expression vector pET32a(+)-sam2-1-6His was transformed into Escherichia coli BL21(DE3) to construct the recombinant expression strain BL21(DE3) / pET32a(+)-sam2-1-6His;

[0037] (3) The recombinant expression strain BL21 (DE3) / pET32a (+)-sam2-1-6His was cultured in LB medium, expression was induced, the cells were collected at low temperature, resuspended in PBS, and ultrasonically disrupted. The supernatant was purified with Ni-NTA resin to obtain the S-adenosylmethionine synthetase mutant SAM2-1.

[0038] Example 3 Construction of a recombinant vector containing the S-adenosylmethionine mutant SAM2-1

[0039] (1) Design primers 306-sam2-1-F / R, whose sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. Use the high-fidelity polymerase Phanta HS and the sam2-1 gene as a template to amplify the S-adenosylmethionine synthetase gene of sam2-1 containing the homology arms of the pRS306 vector;

[0040] (2) Design primers pRS306-F / R, whose sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. Use high-fidelity polymerase Phanta HS and pRS306 plasmid as template to amplify the linearized fragment of pRS306.

[0041] (3) Use homologous recombinase to connect the pRS306 linearized vector fragment and the S-adenosylmethionine synthetase gene of sam2-1 containing the vector homologous arm, transform it into Escherichia coli DH5α, perform resistance screening, extract the plasmid, and obtain the recombinant expression vector pRS306-sam2-1.

[0042] Example 4 Construction of recombinant strain containing S-adenosylmethionine mutant SAM2-1

[0043] (1) Design primers Ura-F / R, whose sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. Use high-fidelity polymerase Phanta HS and pRS306-sam2-1 plasmid as template to amplify the linearized fragment of pRS306-sam2-1.

[0044] (2) The linearized fragment of pRS306-sam2-1 was integrated into the genome of Saccharomyces cerevisiae CEN.PK 2-1C through homologous recombination, and the engineered Saccharomyces cerevisiae strain SC-sam2-1 was obtained by screening.

[0045] Example 5 Fermentation production of S-adenosylmethionine using L-methionine as substrate

[0046] (1) Seed plate activation: streak the recombinant expression strain SC-sam2-1 on an eggplant-shaped flask filled with solid YPD medium in a clean bench and culture at 30°C for 24 h to obtain activated seeds;

[0047] (2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at 30°C and 30-50% dissolved oxygen. The culture was continued until the OD value reached 13 to obtain the seed liquid.

[0048] The components and contents of the seed culture medium are as follows: glucose 20.0 g / L, peptone 10.0 g / L, yeast powder 10.0 g / L, potassium dihydrogen phosphate 1.6 g / L, dipotassium hydrogen phosphate 1.8 g / L, GPE defoamer 0.1 g / L;

[0049] (3) Fermentation of expression strain: The seed liquid was proportionally added to a fermentation tank containing 8 L of fermentation medium, the temperature was 30 ° C, the dissolved oxygen was 30-50%, and the pH was adjusted to 5.5 with ammonia water. After the base sugar was consumed, a glucose solution with a concentration of 550 g / L was added, and the specific growth rate was adjusted to 0.15; when the OD grew to 130, L-methionine was added, and the L-methionine concentration in the fermentation liquid was controlled to 0.8 g / L; after starting to add L-methionine, the sugar addition flow rate was adjusted to control the ethanol content in the fermentation liquid to 0.5 g / L, and the fermentation was terminated after 60 h;

[0050] The amount of the seed liquid added is 20% of the volume of the fermentation medium; the components and content of the fermentation medium are: glucose 10.0 g / L, peptone 2.0 g / L, yeast powder 3.0 g / L, ammonium sulfate 5.0 g / L, potassium dihydrogen phosphate 12 g / L, magnesium sulfate heptahydrate 2.0 g / L, calcium chloride 0.5 g / L, sodium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.5 g / L, zinc sulfate heptahydrate 0.5 g / L, manganese sulfate 0.2 g / L, sodium molybdate dihydrate 0.2 g / L, copper sulfate pentahydrate 5 mg / L, biotin 0.5 mg / L, and GPE defoamer 0.05 g / L.

[0051] Comparative Example 1

[0052] (1) Based on the sequences of sam2 and pET32a(+), primers 32a-sam2-F / R and pET32a(+)-F / R were designed. The nucleotide sequences of primers 32a-sam2-F / R are shown in SEQ ID NO. 15-16. The linear fragments were connected using homologous recombinase, transformed into DH5α, and screened for ampicillin resistance to obtain the recombinant vector pET32a(+)-sam2-6His.

[0053] (2) The recombinant expression vector pET32a(+)-sam2-6His was transformed into Escherichia coli BL21(DE3) to construct the recombinant expression strain BL21(DE3) / pET32a(+)-sam2-6His.

[0054] (3) The recombinant expression strain BL21 (DE3) / pET32a (+)-sam2-6His was cultured in LB medium, expression was induced, the cells were collected at low temperature, resuspended in PBS, and ultrasonically disrupted. The supernatant was purified with Ni-NTA resin to obtain S-adenosylmethionine synthetase SAM2.

[0055] Comparative Example 2

[0056] (1) Primers 306-sam2-F / R were designed, with the sequences shown in SEQ ID NO.17 and SEQ ID NO.18, respectively. The high-fidelity polymerase Phanta HS was used to amplify the S-adenosylmethionine synthetase gene of sam2 containing the homologous arms of the pRS306 vector using the sam2 gene as a template.

[0057] (2) Primers pRS306-F / R were designed, with the sequences shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The pRS306 linearized fragment was amplified using the high-fidelity polymerase Phanta HS and the pRS306 plasmid as a template.

[0058] (3) Use homologous recombinase to connect the pRS306 linearized vector fragment and the S-adenosylmethionine synthetase gene of sam2 containing the vector homologous arm, transform it into Escherichia coli DH5α, perform resistance screening, extract the plasmid, and obtain the recombinant expression vector pRS306-sam2.

[0059] Comparative Example 3 Construction of SAM2 recombinant strain containing S-adenosylmethionine

[0060] (1) Design primers Ura-F / R, whose sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. Use high-fidelity polymerase Phanta HS and pRS306-sam2 plasmid as template to amplify the linearized fragment of pRS306-sam2.

[0061] (2) The linearized fragment of pRS306-sam2 was integrated into the genome of Saccharomyces cerevisiae CEN.PK 2-1C through homologous recombination, and the engineered Saccharomyces cerevisiae strain SC-sam2 was obtained.

[0062] Comparative Example 4

[0063] The only difference from Example 5 is that the recombinant expression strain in step (1) is SC-sam2.

[0064] Comparative Example 5

[0065] The only difference from Example 5 is that in step (3), the specific growth rate is regulated to 0.35.

[0066] Comparative Example 6

[0067] The only difference from Example 5 is that in step (3), the specific growth rate is regulated to 0.05.

[0068] Comparative Example 7

[0069] The only difference from Example 5 is that in step (3), L-methionine was added when the OD grew to 40.

[0070] Comparative Example 8

[0071] The only difference from Example 5 is that in step (3), L-methionine was added when the OD grew to 170.

[0072] Comparative Example 9

[0073] The only difference from Example 5 is that the L-methionine concentration in the fermentation broth in step (3) is controlled to be 3 g / L.

[0074] Comparative Example 10

[0075] The only difference from Example 5 is that the L-methionine concentration in the fermentation broth in step (3) is controlled to be 0.2 g / L.

[0076] Comparative Example 11

[0077] The only difference from Example 5 is that in step (3), the ethanol content in the fermentation liquid is controlled to be 0.1 g / L.

[0078] Comparative Example 12

[0079] The only difference from Example 5 is that in step (3), the ethanol content in the fermentation liquid is controlled to be 3 g / L.

[0080] Table 1. Primer sequence list

[0081]

[0082] Experimental Example 1 Enzyme activity determination

[0083] Enzyme activity assay reaction system: ATP 33 g / L, L-methionine 10 g / L, S-adenosylmethionine synthetase (mutant) 0.5 g / L, magnesium sulfate 3.0 g / L, potassium dihydrogen phosphate 5.0 g / L. Adjust the pH to 7.0 with potassium dihydrogen phosphate. After vortexing and mixing, react at 30°C for 2 h, then add perchloric acid solution to stop the reaction. The reaction liquid was collected and the S-adenosylmethionine content was determined by HPLC according to the USP standard S-Adenosyl-l-methionine Disulfate Tosylate.

[0084] S-adenosylmethionine specific enzyme activity (U / g) = amount of S-adenosylmethionine generated / (amount of S-adenosylmethionine synthetase × reaction time)

[0085] Wherein U represents the amount of S-adenosylmethionine synthetase that catalyzes the production of 1 mg of S-adenosylmethionine per minute in this reaction system.

[0086] Table 2. Specific enzyme activity of S-adenosylmethionine synthetase

[0087]

[0088] As can be seen from the data in Table 2, the specific enzyme activity of the S-adenosylmethionine synthetase mutant SAM2-1 is 52.3 U / g, and the specific enzyme activity of the original S-adenosylmethionine synthetase SAM2 is 28.6 U / g. Compared with the original S-adenosylmethionine synthetase SAM2, the specific enzyme activity of the S-adenosylmethionine synthetase mutant SAM2-1 is increased by 82.9%, indicating that compared with the original S-adenosylmethionine SAM2 synthetase, the efficiency of the S-adenosylmethionine synthetase mutant SAM2-1 provided by the present invention in catalyzing the conversion of L-methionine to S-adenosylmethionine is increased by 82.9%, and the enzyme activity is greatly improved.

[0089] Experimental Example 2 Comparison of fermentation content

[0090] After fermentation in Example 5 and Comparative Examples 4-12, the fermentation broth was centrifuged to collect the cells, an equal volume of purified water was added, and the cells were incubated at 70°C for 10 minutes. The supernatant was centrifuged and appropriately diluted, and the intracellular S-adenosylmethionine concentration was determined by HPLC. Three measurements were performed, and the average value was calculated. The results are listed in Table 3.

[0091] Table 3. S-adenosylmethionine fermentation content

[0092]

[0093] As can be seen from the data in Table 3, the fermentation content of S-adenosylmethionine in Comparative Example 4 is 5.4 g / L, and the fermentation content of S-adenosylmethionine in Example 5 is 16.7 g / L. The fermentation content in Example 5 is 11.3 g / L higher than that in Comparative Example 4, with an increase of 209.3%. It can be seen that the efficiency of fermentation production of S-adenosylmethionine by the strain SC-sam2-1 in Example 5 is significantly higher than that of the strain SC-sam2 in Comparative Example 4, indicating that the efficiency of the S-adenosylmethionine synthetase mutant SAM2-1 in catalyzing the conversion of L-methionine to S-adenosylmethionine is greatly improved compared with the original S-adenosylmethionine synthetase SAM2.

[0094] The fermentation content of S-adenosylmethionine in Comparative Example 5 is 3.2 g / L, the fermentation content of S-adenosylmethionine in Comparative Example 6 is 6.4 g / L, and the fermentation content of S-adenosylmethionine in Example 5 is 16.7 g / L. The fermentation content in Example 5 is 13.5 g / L higher than that in Comparative Example 5, with an increase of 421.9%, and 10.3 g / L higher than that in Comparative Example 6, with an increase of 160.9%. It can be seen that under the same conditions, the fermentation efficiency of the fermentation strain Example 5 is high when the fermentation control specific growth rate is 0.1-0.3, indicating that the regulation of the specific growth rate of the present invention can significantly improve the efficiency of the fermentation production of S-adenosylmethionine.

[0095] The fermentation content of S-adenosylmethionine in Comparative Example 7 is 8.9 g / L, the fermentation content of S-adenosylmethionine in Comparative Example 8 is 12.8 g / L, and the fermentation content of S-adenosylmethionine in Example 5 is 16.7 g / L. The fermentation content in Example 5 is 7.8 g / L higher than that in Comparative Example 7, with an increase of 87.6%, and 3.9 g / L higher than that in Comparative Example 8, with an increase of 30.5%. It can be seen that under the same conditions, the fermentation strain starts to add L-methionine after the fermentation is regulated to grow to 50-150, and the efficiency of fermentation production of S-adenosylmethionine is high, indicating that the present invention regulates the OD growth to a certain range before starting to add L-methionine, which can significantly improve the efficiency of fermentation production of S-adenosylmethionine.

[0096] The fermentation content of S-adenosylmethionine in Comparative Example 9 is 10.5 g / L, the fermentation content of S-adenosylmethionine in Comparative Example 10 is 13.2 g / L, and the fermentation content of S-adenosylmethionine in Example 5 is 16.7 g / L. The fermentation content in Example 5 is 6.2 g / L higher than that in Comparative Example 9, with an increase of 59.0%, and 3.5 g / L higher than that in Comparative Example 10, with an increase of 26.5%. It can be seen that the efficiency of fermentation production of S-adenosylmethionine is high when the L-methionine concentration in the fermentation broth is controlled to be 0.5-2 g / L, indicating that the present invention can significantly improve the efficiency of fermentation production of S-adenosylmethionine by regulating the L-methionine concentration in the fermentation broth.

[0097] The fermentation content of S-adenosylmethionine in Comparative Example 11 is 14.2 g / L, the fermentation content of S-adenosylmethionine in Comparative Example 12 is 7.7 g / L, and the fermentation content of S-adenosylmethionine in Example 5 is 16.7 g / L. The fermentation content in Example 5 is 2.5 g / L higher than that in Comparative Example 11, with an increase of 17.6%, and 9.0 g / L higher than that in Comparative Example 12, with an increase of 116.9%. It can be seen that after the present invention starts to add L-methionine, the ethanol content in the fermentation broth is controlled to be 0.2-2 g / L, and the efficiency of fermentation production of S-adenosylmethionine is high. This shows that the present invention can significantly improve the efficiency of fermentation production of S-adenosylmethionine by regulating the ethanol content in the fermentation broth.

[0098] In summary, the S-adenosylmethionine synthetase mutant Sam2-1 and its application provided by the present invention solve the problem of S-adenosylmethionine production, are conducive to the large-scale production of S-adenosylmethionine, can effectively reduce the production cost of S-adenosylmethionine, and are of great significance to the promotion and application of S-adenosylmethionine.

Claims

1. An S-adenosylmethionine synthetase mutant SAM2-1, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

2. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 1 in synthesizing S-adenosylmethionine, characterized in that: The following steps are involved: (1) Construction of a recombinant vector containing the S-adenosylmethionine mutant SAM2-1; (2) Construction of a recombinant strain containing the S-adenosylmethionine mutant SAM2-1; (3) Fermentation to produce S-adenosylmethionine using L-methionine as substrate.

3. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 2 in synthesizing S-adenosylmethionine, characterized in that: The specific operation of step (1) constructing a recombinant vector containing the S-adenosylmethionine mutant SAM2-1 is as follows: (1-1) Design primers 306-sam2-1-F / R, whose sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. Use the high-fidelity polymerase Phanta HS and the sam2-1 gene as a template to amplify the S-adenosylmethionine synthetase gene of sam2-1 containing the homology arms of the pRS306 vector. (1-2) Design primers pRS306-F / R, whose sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. Use the high-fidelity polymerase Phanta HS and the pRS306 plasmid as a template to amplify the linearized fragment of pRS306. (1-3) The pRS306 linearized vector fragment and the S-adenosylmethionine synthetase gene of sam2-1 containing the vector homology arm were connected using homologous recombinase, transformed into Escherichia coli DH5α, and after resistance screening, the plasmid was extracted to obtain the recombinant expression vector pRS306-sam2-1.

4. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 2 in synthesizing S-adenosylmethionine, characterized in that: The specific operation of step (2) to construct the recombinant strain containing the S-adenosylmethionine mutant SAM2-1 is as follows: (2-1) Design primers Ura-F / R, whose sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively. Use high-fidelity polymerase Phanta HS and pRS306-sam2-1 plasmid as a template to amplify the linearized fragment of pRS306-sam2-1. (2-2) The linearized fragment of pRS306-sam2-1 was integrated into the genome of Saccharomyces cerevisiae CEN.PK 2-1C by homologous recombination, and the engineered Saccharomyces cerevisiae strain SC-sam2-1 was obtained by screening.

5. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 2 in synthesizing S-adenosylmethionine, characterized in that: The specific operation of step (3) to produce S-adenosylmethionine by fermentation using L-methionine as substrate is as follows: (3-1) Seed plate activation: Streak the recombinant expression strain on an eggplant-shaped flask filled with solid YPD medium in a clean bench and incubate at 30°C for 24 hours to obtain activated seeds; (3-2) Seed solution culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at 30°C and 30-50% dissolved oxygen. The culture was continued until the OD value reached 10-15 to obtain the seed solution. (3-3) Fermentation of expression strain: The seed liquid was proportionally added to a fermentation tank containing 8 L of fermentation medium, the temperature was 30°C, the dissolved oxygen was 30-50%, and the pH was adjusted to 5.5 with ammonia water. After the base sugar was consumed, a glucose solution with a concentration of 550 g / L was added, and the specific growth rate was adjusted to 0.1-0.3; when the OD grew to 50-150, L-methionine was added, and the concentration of L-methionine in the fermentation liquid was controlled to 0.5-2 g / L; after starting to add L-methionine, the sugar addition flow rate was adjusted to control the ethanol content in the fermentation liquid to 0.2-2 g / L, and the fermentation was completed after 60 h.

6. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 5 in synthesizing S-adenosylmethionine, characterized in that: In step (3-3), the fermentation medium components and contents are as follows: glucose 10.0 g / L, peptone 2.0 g / L, yeast powder 3.0 g / L, ammonium sulfate 5.0 g / L, potassium dihydrogen phosphate 12 g / L, magnesium sulfate heptahydrate 2.0 g / L, calcium chloride 0.5 g / L, sodium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.5 g / L, zinc sulfate heptahydrate 0.5 g / L, manganese sulfate 0.2 g / L, sodium molybdate dihydrate 0.2 g / L, copper sulfate pentahydrate 5 mg / L, biotin 0.5 mg / L, and GPE defoamer 0.05 g / L.

7. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 5 in synthesizing S-adenosylmethionine, characterized in that: In step (3-3), the seed liquid is proportionally added to a fermentation tank containing 8 L of fermentation medium, the temperature is 30° C., the dissolved oxygen is 30-50%, the pH is regulated to 5.5 with ammonia water, and after the base sugar is consumed, a glucose solution with a concentration of 550 g / L is added, and the specific growth rate is regulated to 0.1-0.2; when the OD grows to 120-140, L-methionine is added, and the concentration of L-methionine in the fermentation broth is controlled to 0.5-1 g / L; after the addition of L-methionine begins, the sugar addition flow rate is adjusted to control the ethanol content in the fermentation broth to 0.3-0.8 g / L, and the fermentation is completed after 60 hours.

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