S-adenosylmethionine synthetase mutant SAM2-1 and application thereof

Abstract

The invention discloses an S-adenosylmethionine synthetase mutant SAM2-1 and application thereof, and belongs to the technical field of biological engineering. The amino acid sequence of the mutant is as shown in SEQ ID NO.1, and the nucleotide sequence of the mutant is as shown in SEQ ID NO.2. The application of the mutant SAM2-1 in preparation of the S-adenosylmethionine fermentation content 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; and (3) producing S-adenosyl methionine by taking L-methionine as a substrate through fermentation. Compared with the original S-adenosylmethionine synthetase, the novel S-adenosylmethionine synthetase mutant provided by the invention has the advantages that the activity is enhanced, and the efficiency of catalyzing the conversion of L-methionine into S-adenosylmethionine is improved by 82.9%.

Description

Technical Field

[0001] The 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) was discovered in 1951. It is widely present in animals, plants and microorganisms. It is also a physiologically active molecule present in all tissues and body fluids of the human body. It is synthesized in the body from L-methionine and ATP by S-adenosylmethionine synthetase. In this reaction, adenosine in ATP is transferred to L-methionine to form a high-energy sulfur atom, which activates the carbon atom connected to it through nucleophilic attack, thus having the effects of transmethylation, transaminopropylation and transsulfuration. S-adenosylmethionine can participate in more than 40 biochemical reactions and is a very important pharmaceutical molecule. It has broad application prospects in the treatment of diseases such as intrahepatic cholestasis and mental depression.

[0003] S-adenosylmethionine synthetase catalyzes the transfer of adenosine from ATP to L-methionine to generate S-adenosylmethionine. It is a key enzyme in the synthesis of S-adenosylmethionine. Researchers have improved the fermentation level of S-adenosylmethionine by overexpressing or heterologously expressing S-adenosylmethionine synthetases from different sources in Escherichia coli and yeast, but the production cost is still high. Developing new S-adenosylmethionine synthetases, improving the efficiency of S-adenosylmethionine synthesis, and reducing production costs have important practical significance 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 a mutant of S-adenosylmethionine synthetase, 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 technical solutions provided by the present invention are as follows: 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.

[0006] Furthermore, the encoding gene of the mutant is sam2-1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0007] 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 has the 23rd amino acid replaced by C to S, the 48th amino acid replaced by A to S, the 83rd amino acid replaced by D to H, the 139th amino acid replaced by L to I, the 192nd amino acid replaced by V to L, the 269th amino acid replaced by G to S, the 334th amino acid replaced by I to V, and the 359th amino acid replaced by I to S.

[0008] Furthermore, the amino acid sequence of the S-adenosylmethionine synthetase SAM2 is shown in SEQ ID NO.3, the coding 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.

[0009] The present invention also provides an application of an S-adenosylmethionine synthetase mutant SAM2-1 in synthesizing S-adenosylmethionine, comprising the following steps: (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.

[0010] Furthermore, the specific operation of step (1) to construct a recombinant vector containing the S-adenosylmethionine mutant SAM2-1 is as follows: (1-1) Design primers 306-sam2-1-F / R, the sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, and use high-fidelity polymerase Phanta HS and sam2-1 gene as template to amplify the S-adenosylmethionine synthetase gene of sam2-1 containing homology arms of pBR306 vector; (1-2) Design primers pBR306-F / R, the sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and use high-fidelity polymerase Phanta HS to amplify the pBR306 linearized fragment using the pBR306 plasmid as a template; (1-3) The pRS306 linearized vector fragment and the S-adenosylmethionine synthetase gene of sam2-1 containing the vector homology arm were connected with homologous recombinase, transformed into Escherichia coli DH5α, and after resistance screening, the plasmid was extracted to obtain the recombinant expression vector pRS306-sam2-1.

[0011] Furthermore, 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, the sequences of which are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively, and use high-fidelity polymerase Phanta HS to amplify the pBR306-sam2-1 plasmid as a template to obtain the linearized fragment of pBR306-sam2-1; (2-2) The linearized fragment of pBR306-sam2-1 was integrated into the genome of Saccharomyces cerevisiae CEN .PK 2-1C through homologous recombination, and the Saccharomyces cerevisiae engineered strain SC-sam2-1 was obtained by screening.

[0012] Furthermore, the specific operation of fermenting S-adenosylmethionine using L-methionine as a substrate in step (3) is as follows: (3-1) Seed plate activation: Streak the recombinant expression strain on an eggplant-shaped bottle filled with solid YPD medium in a clean bench and culture at 30°C for 24 h to obtain activated seeds.

[0013] (3-2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at a temperature of 30°C and a dissolved oxygen content of 30-50%. The culture was continued until the OD value was 10-15 to obtain seed liquid. (3-3) Fermentation of expression strain: The seed liquid was added in proportion to a fermentation tank containing 8 L of fermentation medium, the temperature was 30°C, the dissolved oxygen was 30-50%, the pH was adjusted to 5.5 with ammonia water, and after the base sugar was consumed, a glucose solution with a concentration of 550 g / L was added to adjust the specific growth rate to 0.1-0.3; when the OD grew to 50-150, L-methionine was added to control the concentration of L-methionine in the fermentation liquid 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 terminated after 60 h.

[0014] Further, in step (3-2), the components and contents of the seed culture medium are: 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. Further, in step (3-2), the components of the seed culture medium are: 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.

[0015] Furthermore, in step (3-3), the amount of the seed liquid added is 20% of the fermentation medium by volume.

[0016] 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.

[0017] Furthermore, in step (3-3), the seed liquid is proportionally added to a fermentation tank containing 8L 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 550g / L is added to adjust the specific growth rate to 0.1-0.2; when the OD grows to 120-140, L-methionine is added to control the concentration of L-methionine in the fermentation liquid to 0.5-1g / L; after starting to add L-methionine, the sugar addition flow rate is adjusted to control the ethanol content in the fermentation liquid to 0.3-0.8g / L, and the fermentation is terminated after 60h of fermentation.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a novel S-adenosylmethionine synthetase mutant, which has enhanced activity compared with the original S-adenosylmethionine synthetase and improves the efficiency of catalyzing the conversion of L-methionine to S-adenosylmethionine by 82.9%.

[0019] (2) The present invention uses an S-adenosylmethionine synthetase mutant to construct a recombinant expression strain and ferment 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

[0020] Example 1 According to the tertiary predicted structure of Saccharomyces cerevisiae S288C S-adenosylmethionine synthetase SAM2 (the amino acid sequence is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.4) and the chemical structures of the substrates L-methionine and adenine nucleoside triphosphate, after simulated mutation using bioinformatics software, an S-adenosylmethionine synthetase mutant SAM2-1 with improved binding ability to the substrate ectoine and cosubstrate and good stability was obtained, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2.

[0021] Example 2 Construction of S-adenosylmethionine synthetase mutant SAM2-1 (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 NO.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. (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; (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 using Ni-NTA resin to obtain the S-adenosylmethionine synthetase mutant SAM2-1.

[0022] Example 3 Construction of a recombinant vector containing the S-adenosylmethionine mutant SAM2-1 (1) Design primers 306-sam2-1-F / R, the sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, and use high-fidelity polymerase Phanta HS and sam2-1 gene as template to amplify the S-adenosylmethionine synthetase gene of sam2-1 containing homology arms of pBR306 vector; (2) Design primers pBR306-F / R, the sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and use high-fidelity polymerase Phanta HS to amplify the pBR306 linearized fragment using the pBR306 plasmid as a template; (3) The pRS306 linearized vector fragment and the S-adenosylmethionine synthetase gene of sam2-1 containing the vector homology arm were connected with homologous recombinase, transformed into Escherichia coli DH5α, and after resistance screening, the plasmid was extracted to obtain the recombinant expression vector pRS306-sam2-1.

[0023] Example 4 Construction of the recombinant strain containing the S-adenosylmethionine mutant SAM2-1 (1) Design primers Ura-F / R, the sequences of which are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively, and use high-fidelity polymerase Phanta HS to amplify the pBR306-sam2-1 plasmid as a template to obtain the linearized fragment of pBR306-sam2-1; (2) The linearized fragment of pBR306-sam2-1 was integrated into the genome of Saccharomyces cerevisiae CEN .PK 2-1C through homologous recombination, and the Saccharomyces cerevisiae engineered strain SC-sam2-1 was obtained by screening.

[0024] Example 5 Fermentation to produce S-adenosylmethionine using L-methionine as substrate (1) Seed plate activation: streak the recombinant expression strain SC-sam2-1 on an eggplant-shaped bottle filled with solid YPD medium in a clean bench and culture at 30°C for 24 h to obtain activated seeds; (2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at a temperature of 30°C and a dissolved oxygen content of 30-50%. The culture was continued until the OD value reached 13 to obtain the seed liquid. 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 potassium hydrogen phosphate, and 0.1 g / L GPE defoamer; (3) Fermentation of expression strains: The seed liquid was inoculated into a fermentation tank containing 8 L of fermentation medium in proportion, the temperature was 30 °C, the dissolved oxygen was 30-50%, the pH was adjusted to 5.5 with ammonia water, and after the base sugar was consumed, a glucose solution with a concentration of 550 g / L was added to adjust the specific growth rate 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 the addition of 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 of fermentation; The amount of the seed liquid added is 20% of the volume of the fermentation medium; the components and contents of the fermentation medium are: 10.0 g / L glucose, 2.0 g / L peptone, 3.0 g / L yeast powder, 5.0 g / L ammonium sulfate, 12 g / L potassium dihydrogen phosphate, 2.0 g / L magnesium sulfate heptahydrate, 0.5 g / L calcium chloride, 0.5 g / L sodium chloride, 0.5 g / L ferrous sulfate heptahydrate, 0.5 g / L zinc sulfate heptahydrate, 0.2 g / L manganese sulfate, 0.2 g / L sodium molybdate dihydrate, 5 mg / L copper sulfate pentahydrate, 0.5 mg / L biotin, and 0.05 g / L GPE defoamer.

[0025] Comparative Example 1 (1) Based on the sequences of sam2 and pET32a(+), primers 32a-sam2-F / R and pET32a(+)-F / R were designed. The nucleotide sequence of primer 32a-sam2-F / R is 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.

[0026] (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.

[0027] (3) The recombinant expression strain BL21 (DE3) / pET32a (+) -sam2-6His was cultured in LB medium, the expression was induced, the cells were collected at low temperature, resuspended in PBS, ultrasonically disrupted, and the supernatant was purified using Ni-NTA resin to obtain S-adenosylmethionine synthetase SAM2.

[0028] Comparative Example 2 (1) Primers 306-sam2-F / R were designed, and the sequences were 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 pBR306 vector using the sam2 gene as a template.

[0029] (2) Primers pBR306-F / R were designed, and their sequences were shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. High-fidelity polymerase Phanta HS was used to amplify the pBR306 linearized fragment using the pBR306 plasmid as a template.

[0030] (3) The linearized vector fragment of pRS306 and the S-adenosylmethionine synthetase gene of sam2 containing the vector homology arm were connected with homologous recombinase, transformed into Escherichia coli DH5α, and after resistance screening, the plasmid was extracted to obtain the recombinant expression vector pRS306-sam2.

[0031] Comparative Example 3 Construction of SAM2 recombinant strain containing S-adenosylmethionine (1) Design primers Ura-F / R, the sequences of which are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively, and use high-fidelity polymerase Phanta HS to amplify the pBR306-sam2 plasmid as a template to obtain the linearized fragment of pBR306-sam2; (2) The linearized fragment of pBR306-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 by screening.

[0032] Comparative Example 4 The only difference from Example 5 is that the recombinant expression strain in step (1) is SC-sam2.

[0033] Comparative Example 5 The only difference from Example 5 is that in step (3), the specific growth rate is regulated to 0.35.

[0034] Comparative Example 6 The only difference from Example 5 is that in step (3), the specific growth rate is regulated to 0.05.

[0035] Comparative Example 7 The only difference from Example 5 is that in step (3), L-methionine is added when the OD grows to 40.

[0036] Comparative Example 8 The only difference from Example 5 is that in step (3), L-methionine is added when the OD grows to 170.

[0037] Comparative Example 9 The only difference from Example 5 is that in step (3), the L-methionine concentration in the fermentation broth is controlled to be 3 g / L.

[0038] Comparative Example 10 The only difference from Example 5 is that in step (3), the L-methionine concentration in the fermentation broth is controlled to be 0.2 g / L.

[0039] Comparative Example 11 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.

[0040] Comparative Example 12 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.

[0041] Table 1. Primer sequence list Experimental Example 1 Enzyme Activity Determination Enzyme activity detection reaction system: ATP 33g / L, L-methionine 10g / L, S-adenosylmethionine synthetase (mutant) 0.5g / L, magnesium sulfate 3.0g / L, potassium dihydrogen phosphate 5.0g / L, adjust pH 7.0 with potassium dihydrogen phosphate, shake and mix, react at 30℃ for 2h, then add perchloric acid solution to stop the reaction, take the reaction liquid and determine the content of S-adenosylmethionine by HPLC, and the determination method refers to USP standard S-Adenosyl-l-methionineDisulfate Tosylate.

[0042] S-adenosylmethionine specific enzyme activity (U / g) = amount of S-adenosylmethionine generated / (amount of S-adenosylmethionine synthetase × reaction time) Wherein U represents the amount of S-adenosylmethionine synthetase that catalyzes the production of 1 mg S-adenosylmethionine per minute in the reaction system.

[0043] Table 2. Specific enzyme activity of S-adenosylmethionine synthetase From the data in Table 2, it can be seen that 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 into S-adenosylmethionine is increased by 82.9%, and the enzyme activity is greatly improved.

[0044] Experimental Example 2 Comparison of fermentation content After the fermentation of Example 5 and Comparative Examples 4-12 was completed, the fermentation broth was centrifuged to collect the bacterial cells, an equal volume of purified water was added, and the mixture was water-bathed at 70°C for 10 min. The supernatant was centrifuged and appropriately diluted, and the concentration of intracellular S-adenosylmethionine was determined by HPLC, and the determination was performed three times, and the average value was taken. The results are listed in Table 3.

[0045] Table 3. S-adenosylmethionine fermentation content From the data in Table 3, it can be seen that 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 strain SC-sam2-1 in Example 5 is significantly higher than that of strain SC-sam2 in Comparative Example 4, indicating that the efficiency of S-adenosylmethionine synthetase mutant SAM2-1 in catalyzing the conversion of L-methionine into S-adenosylmethionine is greatly improved compared with the original S-adenosylmethionine synthetase SAM2.

[0046] 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 of Example 5 is 13.5 g / L higher than that of Comparative Example 5, with an increase of 421.9%, and 10.3 g / L higher than that of 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.

[0047] 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 when the fermentation OD grows to 50-150, and the efficiency of fermentation production of S-adenosylmethionine is high, indicating that the present invention controls 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.

[0048] 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 L-methionine concentration in the fermentation broth is controlled to be 0.5-2 g / L, and the efficiency of fermentation production of S-adenosylmethionine is high, indicating that the present invention can significantly improve the efficiency of fermentation production of S-adenosylmethionine by regulating the concentration of L-methionine in the fermentation broth.

[0049] 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 of 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 liquid is controlled to be 0.2-2 g / L, and the efficiency of fermentation production of S-adenosylmethionine is high. It shows that the present invention can significantly improve the efficiency of fermentation production of S-adenosylmethionine by regulating the ethanol content in the fermentation liquid.

[0050] 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. A S-adenosylmethionine synthetase mutant SAM2-1, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

2. The S-adenosylmethionine synthetase mutant SAM2-1 according to claim 1, characterized in that: The coding gene of the mutant is sam2-1, and its nucleotide sequence is shown in SEQ ID NO.

2.

3. The S-adenosylmethionine synthetase mutant SAM2-1 according to claim 1, characterized in that: 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 has the 23rd amino acid replaced by C to S, the 48th amino acid replaced by A to S, the 83rd amino acid replaced by D to H, the 139th amino acid replaced by L to I, the 192nd amino acid replaced by V to L, the 269th amino acid replaced by G to S, the 334th amino acid replaced by I to V, and the 359th amino acid replaced by I to S.

4. The S-adenosylmethionine synthetase mutant SAM2-1 according to claim 3, characterized in that: The amino acid sequence of the S-adenosylmethionine synthetase SAM2 is shown in SEQ ID NO.3, the coding 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.

5. 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.

6. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 5 in synthesizing S-adenosylmethionine, characterized in that: The specific operation of step (1) to construct a recombinant vector containing the S-adenosylmethionine mutant SAM2-1 is as follows: (1-1) Design primers 306-sam2-1-F / R, the sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, and use high-fidelity polymerase Phanta HS and sam2-1 gene as template to amplify the S-adenosylmethionine synthetase gene of sam2-1 containing homology arms of pBR306 vector; (1-2) Design primers pBR306-F / R, the sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and use high-fidelity polymerase Phanta HS to amplify the pBR306 linearized fragment using the pBR306 plasmid as a template; (1-3) The pRS306 linearized vector fragment and the S-adenosylmethionine synthetase gene of sam2-1 containing the vector homology arm were connected with homologous recombinase, transformed into Escherichia coli DH5α, and after resistance screening, the plasmid was extracted to obtain the recombinant expression vector pRS306-sam2-1.

7. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 5 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, the sequences of which are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively, and use high-fidelity polymerase Phanta HS to amplify the pBR306-sam2-1 plasmid as a template to obtain the linearized fragment of pBR306-sam2-1; (2-2) The linearized fragment of pBR306-sam2-1 was integrated into the genome of Saccharomyces cerevisiae CEN .PK 2-1C through homologous recombination, and the Saccharomyces cerevisiae engineered strain SC-sam2-1 was obtained by screening.

8. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 5 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 bottle filled with solid YPD medium in a clean bench and culture at 30°C for 24 h to obtain activated seeds; (3-2) Seed liquid culture: The activated seeds were inoculated into a fermentation tank containing 10 L of seed culture medium at a temperature of 30°C and a dissolved oxygen content of 30-50%. The culture was continued until the OD value was 10-15 to obtain seed liquid. (3-3) Fermentation of expression strain: The seed liquid was added in proportion to a fermentation tank containing 8 L of fermentation medium, the temperature was 30°C, the dissolved oxygen was 30-50%, the pH was adjusted to 5.5 with ammonia water, and after the base sugar was consumed, a glucose solution with a concentration of 550 g / L was added to adjust the specific growth rate to 0.1-0.3; when the OD grew to 50-150, L-methionine was added to control the concentration of L-methionine in the fermentation liquid 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 terminated after 60 h.

9. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 8 in synthesizing S-adenosylmethionine, characterized in that: 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.

10. Use of the S-adenosylmethionine synthetase mutant SAM2-1 according to claim 8 in synthesizing S-adenosylmethionine, characterized in that: In step (3-3), the seed liquid is proportionally added to a fermentation tank containing 8L 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 550g / L is added to adjust the specific growth rate to 0.1-0.2; when the OD grows to 120-140, L-methionine is added to control the concentration of L-methionine in the fermentation liquid to 0.5-1g / L; after starting to add L-methionine, the sugar addition flow rate is adjusted to control the ethanol content in the fermentation liquid to 0.3-0.8g / L, and the fermentation is terminated after 60h of fermentation.

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