A method for synthesizing free selenocysteine using Bacillus subtilis

Abstract

The present invention discloses a method for synthesizing free selenocysteine by using Bacillus subtilis, which belongs to the field of bioengineering technology. grac The expression vector overexpressed serine acetyltransferase, realizing for the first time the intracellular accumulation and extracellular secretion of selenocysteine in recombinant Bacillus subtilis. It can increase the yield of selenocysteine produced by Bacillus subtilis fermentation from 0 to 348μg / g dry weight, and the extracellular production of selenocysteine reaches 373μg / L, which helps to achieve food-grade production of selenocysteine.

Description

Technical Field

[0001] The invention relates to a method for synthesizing free selenocysteine by utilizing Bacillus subtilis, and belongs to the technical field of bioengineering. Background Art

[0002] Selenium is an essential trace element for the human body, performing its physiological functions through the formation of selenoproteins with selenocysteine as the active center. The bioavailability of selenium varies with the source and nutritional status of the selenium. Compared to inorganic selenium, organic selenium has the advantages of low toxicity and ease of conversion and utilization. Selenocysteine is an important organic form of selenium. Selenocysteine is synthesized in the body in two ways: one is through a specialized protein translation system, which converts seryl-tRNA into selenocysteinyl-tRNA on the ribosome, which is then incorporated into proteins to form vital selenoproteins; the other is through the synthesis of free selenocysteine via the sulfur metabolism pathway. Selenoamino acids are used as selenium-based nutritional supplements, and enhancing their synthesis is an important way to enhance microbial accumulation of organic selenium. Currently, selenium-enriched yeast using selenomethionine as the primary form of selenium has been commercialized, but selenocysteine, a more potent form than selenomethionine, has not yet been synthesized in large quantities.

[0003] Selenocysteine accumulates at very low levels in microorganisms. Currently, only lactic acid bacteria have been found to accumulate free selenocysteine, but the accumulation is limited. Therefore, there is an urgent need to develop food-grade genetically engineered bacteria that can enhance selenocysteine synthesis, with both research value and potential for industrial application. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a recombinant Bacillus subtilis, which uses Bacillus subtilis as a host and expresses serine acetyltransferase.

[0005] In one embodiment, the serine acetyltransferase is derived from Arabidopsis thaliana or Escherichia coli.

[0006] In one embodiment, the serine acetyltransferase is serine acetyltransferase SATp or serine acetyltransferase SATm from Arabidopsis thaliana; the amino acid sequence of the serine acetyltransferase SATp is shown in SEQ ID NO.4; the amino acid sequence of the serine acetyltransferase SATm is shown in SEQ ID NO.5.

[0007] In one embodiment, the serine acetyltransferase is derived from Escherichia coli, and the amino acid sequence is shown as SEQ ID NO.6.

[0008] In one embodiment, the serine acetyltransferase is Arabidopsis thaliana-derived serine acetyltransferase SATp, Arabidopsis thaliana-derived serine acetyltransferase SATm, and Escherichia coli-derived serine acetyltransferase T197A and M256I double mutant EcCysE.

[0009] In one embodiment, the recombinant Bacillus subtilis uses Bacillus subtilis as a host to express a gene encoding serine acetyltransferase.

[0010] In one embodiment, the nucleotide sequence encoding serine acetyltransferase SATp is shown as SEQ ID NO.1, the nucleotide sequence encoding serine acetyltransferase SATm is shown as SEQ ID NO.2, and the nucleotide sequence encoding the Escherichia coli-derived serine acetyltransferase T197A and M256I double mutant EcCysE is shown as SEQ ID NO.3.

[0011] In one embodiment, the recombinant Bacillus subtilis uses Bacillus subtilis as a host and pHT01 plasmid as a vector to express a gene encoding a serine acetyltransferase protein.

[0012] The present invention also provides a method for constructing the above-mentioned recombinant Bacillus subtilis, characterized in that it comprises the following steps:

[0013] (1) The gene encoding the serine acetyltransferase protein and the vector are respectively subjected to enzyme digestion and ligation to obtain a recombinant vector carrying the serine acetyltransferase gene;

[0014] (2) The recombinant vector constructed in step (1) is transferred into Bacillus subtilis to obtain a recombinant bacterium.

[0015] The present invention also provides a method for preparing selenocysteine. The method uses the recombinant Bacillus subtilis as a production strain and produces selenocysteine by fermentation in a culture medium containing inorganic selenium.

[0016] In one embodiment, the inorganic selenium includes but is not limited to selenite.

[0017] In one embodiment, the seed liquid of the recombinant Bacillus subtilis is first inoculated into a fermentation medium and cultured at a temperature of 30-38° C. and a rotation speed of 150-300 rpm for at least 10 hours.

[0018] In one embodiment, the fermentation is to culture the recombinant Bacillus subtilis at 33°C and 200 r / min until the OD 600 When the OD value reached 0.4, IPTG was added to induce gene expression at a final concentration of 1 mM. 600When the concentration reaches 0.7, sodium selenite is added to a final concentration of ≤12 mg / L to continue fermentation.

[0019] In one embodiment, the fermentation is to culture the recombinant Bacillus subtilis at 37°C and 200 r / min until the OD 600 When the OD value reached 0.4, IPTG was added to induce gene expression at a final concentration of 1 mM. 600 When the concentration reached 0.7, sodium selenite was added to a final concentration of 50 mg / L to continue fermentation.

[0020] In one embodiment, the seed solution concentration of the recombinant Bacillus subtilis is not less than OD 600 1.0.

[0021] In one embodiment, the method further employs IPTG for induction.

[0022] In one embodiment, the final concentration of IPTG is 1 mM.

[0023] The present invention also provides the recombinant Bacillus subtilis or the recombinant Bacillus subtilis obtained by the above construction method, or the use of the above method in preparing selenocysteine or a selenocysteine-containing product.

[0024] Beneficial effects:

[0025] (1) The present invention utilizes a strong inducible promoter P grac The expression vector overexpresses the gene encoding serine acetyltransferase, thereby enhancing the synthesis of intracellular selenocysteine and achieving the secretion of selenocysteine. The present invention can increase the yield of selenocysteine produced by Bacillus subtilis fermentation from 0 to 348 μg / g dry weight. The strain with the highest selenocysteine yield reported so far is the lactic acid bacterium Fructobacillus tropaeoli, with a total selenium-rich content of 580 μg Se / g dry weight, of which the selenocysteine content is 25%, that is, the selenocysteine yield is 145 μg / g dry weight. Therefore, the selenocysteine yield of the present invention is significantly improved.

[0026] (2) This invention achieves, for the first time, the intracellular accumulation and extracellular secretion of selenocysteine in recombinant Bacillus subtilis, resulting in a selenocysteine yield of 145 μg / g dry weight and an extracellular selenocysteine yield of 373 μg / L. This method for preparing selenocysteine can significantly reduce production costs.

[0027] (3) Since Bacillus subtilis is a food safety grade strain, the selenocysteine produced by recombinant Bacillus subtilis obtained using Bacillus subtilis as a host can reach food grade. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Reconstructing the selenocysteine biosynthesis pathway in Bacillus subtilis.

[0029] Figure 2 This is the effect of sodium selenite addition on the growth of HTSATp. DETAILED DESCRIPTION

[0030] The pHT01 plasmid involved in the following examples was purchased from Protin Biotechnology (Beijing) Co., Ltd.

[0031] The culture medium involved in the following examples is as follows:

[0032] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride. LB medium can be used as a seed medium and fermentation medium.

[0033] Synthetic medium: 2% glycerol (v / v), 1mM (NH4)2SO4, 150mM NH4Cl, 5mM potassium phosphate, 4mM sodium citrate, 2mM MgCl2, 0.7mM CaCl2, 50μM MnCl2, 5μM FeCl3, 1μM ZnCl2, 2μM CuCl2, 3μM CoCl2, 2.5μM Na2MoO4 and 0.25mM of L-tryptophan, pH 7.0; synthetic medium can be used as seed medium and fermentation medium.

[0034] The detection methods involved in the following embodiments are as follows:

[0035] Selenocysteine content detection: UPLC-MS detection conditions are as follows:

[0036] Instrument: Waters XevoTQS-micro ultra-performance liquid chromatography-triple quadrupole mass spectrometer; Chromatographic conditions: ACQUITY UPLC HSS T3 C18 Column, 2.1 × 100 mm, 1.8 μm; column temperature, 40°C; sample chamber temperature, 10°C; injection volume, 5 μL; flow rate, 0.2 mL / min; run time, 7 min; mobile phase A, 0.1% formic acid in water; mobile phase B, acetonitrile; mobile phase concentration gradient, see Table 1.

[0037] Table 1 Mobile phase gradient

[0038]

[0039]

[0040] Example 1: Construction of Serine Acetyltransferase Expression Vector

[0041] The gene encoding SATp (nucleotide sequence shown in SEQ ID NO. 1) was chemically synthesized and digested with Bam HI and Sal I to recover the fragments. The gene encoding SATm (nucleotide sequence shown in SEQ ID NO. 2) was chemically synthesized and digested with Bam HI and Sal I to recover the fragments. The gene encoding EcCysE (nucleotide sequence shown in SEQ ID NO. 3) was chemically synthesized and digested with Bam HI and Sal I to recover the fragments. pHT01 was digested with Bam HI and Sal I to recover the fragments. The SATp fragment, SATm fragment, and EcCysE fragment were inserted into the pHT01 vector P, respectively. grac The transformation product was obtained between the Bam HI and Sal I restriction sites downstream of the promoter; the transformation product was spread in LB culture to obtain transformants, and the transformants were picked and inoculated into LB culture medium for culture. After that, the plasmid was extracted for enzyme digestion verification and sequencing verification. If the verification was correct, the recombinant plasmids pHT01-SATp, pHT01-SATm and pHT01-EcCysE were obtained.

[0042] Example 2: Construction of recombinant Bacillus subtilis

[0043] According to the Spizizen transformation method, Bacillus subtilis 168 competent cells were prepared and transformed; the expression vectors pHT01-SATp, pHT01-SATm, pHT01-EcCysE and pHT01 empty vector constructed in Example 1 were respectively transformed into Bacillus subtilis 168 competent cells, and the transformation products were cultured on chloramphenicol-resistant plates and inverted at 37°C for 12 hours until colonies appeared. Single colonies were picked and cultured for preservation, and the genome was extracted for PCR identification to obtain genetically engineered bacteria HTSATp, HTSATm, HTEcCysE and HT.

[0044] Example 3: Verification of Fermentation Performance of Genetically Engineered Bacillus subtilis in LB Medium

[0045] Bacillus subtilis 168, HT, HTSATp, HTSATm and HTEcCysE constructed in Example 2 were inoculated onto LB seed medium, respectively, and cultured overnight at 37° C. to obtain seed liquid.

[0046] The seed liquid of each strain was inoculated into LB fermentation medium at a 5% inoculum volume, and the OD 600 To 0.4, add IPTG to a final concentration of 1 mM to induce gene expression, OD 600 Sodium selenite was added to a final concentration of 6 mg / L at 0.7°C. Fermentation was continued for 9 h (total fermentation time 12 h) to obtain a fermentation broth. As a control, another group was prepared without IPTG addition, with all other fermentation conditions being identical.

[0047] The fermentation broths obtained above were centrifuged. The supernatant was filtered through a microporous membrane and analyzed for extracellular selenocysteine production using UPLC-MS. The centrifuged cells were lysed with hydrochloric acid (pH 2.5), transferred to lysis medium B (MP Biomedicals), and disrupted using a FastPrep-24 instrument. The supernatant was then centrifuged and filtered through a microporous membrane, followed by ultrafiltration. The filtrate was then analyzed for intracellular selenocysteine content using UPLC-MS.

[0048] The results are shown in Table 2. Without the addition of IPTG, none of the strains synthesized selenocysteine. Even with the addition of IPTG, no intracellular or extracellular selenocysteine could be detected in wild-type Bacillus subtilis strains 168 and HT. However, all genetically engineered bacteria carrying serine acetyltransferase constructed in Example 2 showed intracellular selenocysteine synthesis, with the HTSATp strain producing the highest yield. However, no extracellular selenocysteine was detected in the fermentation broth of the genetically engineered bacteria cultured in LB medium.

[0049] Table 2 The yield of selenocysteine from genetically engineered bacteria in LB medium

[0050]

[0051] Example 4: Verification of Fermentation Performance of Genetically Engineered Bacillus subtilis in Synthetic Medium

[0052] Bacillus subtilis 168, HT, and HTSATp, HTSATm, and HTEcCysE constructed in Example 2 were inoculated into a synthetic medium as a seed culture medium, and cultured overnight at 37° C. to obtain a seed solution;

[0053] The above seed solution was inoculated into the synthetic culture medium at a 5% inoculum volume, and cultured at 37°C and 200 r / min to obtain OD 600 When the OD value reached 0.4, IPTG at a final concentration of 1 mM was added to induce gene expression. 600 Sodium selenite was added to a final concentration of 50 mg / L at 0.7°C. Fermentation was continued for 9 h (total fermentation time 12 h) to obtain a fermentation broth. As a control, another group was prepared without IPTG addition, with all other fermentation conditions being identical.

[0054] The fermentation broths obtained above were centrifuged separately. The supernatant after centrifugation was filtered through a microporous membrane and then analyzed for extracellular selenocysteine production using UPLC-MS. The centrifuged cells were dissolved in hydrochloric acid (pH 2.5), transferred to lysis medium B, and disrupted on a FastPrep-24 instrument. The supernatant was then centrifuged and filtered through a microporous membrane, followed by ultrafiltration. The filtrate was then analyzed for intracellular selenocysteine content using UPLC-MS. The results are shown in Table 3.

[0055] The results showed that without the addition of IPTG, none of the strains synthesized selenocysteine. Even with the addition of IPTG, no selenocysteine could be detected in wild-type Bacillus subtilis strains 168 and HT. However, all genetically engineered strains carrying serine acetyltransferases of the present invention showed detectable selenocysteine synthesis both intracellularly and extracellularly. The genetically engineered Bacillus subtilis strains constructed in the present invention were able to secrete selenocysteine in synthetic culture medium. The HTSATp strain exhibited the highest production of both extracellular and intracellular selenocysteine.

[0056] Table 3 Production of selenocysteine from genetically engineered bacteria in synthetic culture medium

[0057]

[0058]

[0059] Comparative Example 1

[0060] The specific implementation method is the same as Example 3 and Example 4, except that Bacillus subtilis serine acetyltransferase BsCysE (nucleotide sequence shown in SEQ ID NO.7, amino acid sequence shown in SEQ ID NO.8) was used to construct the Bacillus subtilis genetically engineered bacteria HTBsCysE; the results showed that regardless of whether LB or synthetic medium was used as the seed medium and fermentation medium, with or without the addition of IPTG, no selenocysteine was detected in the extracellular and cytosolic compartments.

[0061] Comparative Example 2

[0062] The specific implementation is the same as that of Example 3, except that OD 600 When the temperature reaches 0.7, 3 mg / L, 6 mg / L, 12 mg / L and 20 mg / L sodium selenite are added respectively. The results are as follows Figure 2 It shows that when the sodium selenite exceeds 12 mg / L, the growth of bacteria is inhibited, and when the sodium selenite reaches 20 mg / L, the bacteria hardly grow.

[0063] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Bacillus subtilis, characterized in that Serine acetyltransferase is expressed in Bacillus subtilis 168 using an expression vector containing a strong inducible promoter Pgrac; the amino acid sequence of the serine acetyltransferase is shown in any one of SEQ ID NOs. 4 to 6.

2. The recombinant Bacillus subtilis according to claim 1, characterized in that The pHT01 plasmid was used as a vector to express the gene encoding serine acetyltransferase protein.

3. A method for constructing the recombinant Bacillus subtilis according to claim 1 or 2, characterized in that: The following steps are involved: (1) Connecting the gene encoding serine acetyltransferase to an expression vector to obtain a recombinant vector carrying the serine acetyltransferase gene; (2) The recombinant vector constructed in step (1) was transferred into Bacillus subtilis 168 to obtain a recombinant bacterium.

4. The method according to claim 3, characterized in that The nucleotide sequence of the gene encoding serine acetyltransferase is shown in any one of SEQ ID NOs. 1 to 3.

5. A method for preparing selenocysteine, characterized in that: The recombinant Bacillus subtilis according to claim 1 or 2 is used as a production strain to ferment and produce selenocysteine in a culture medium containing sodium selenite.

6. The method according to claim 5, characterized in that The recombinant Bacillus subtilis can also express serine acetyltransferase by inducing IPTG.

7. The method according to claim 5, characterized in that The fermentation is to culture the recombinant Bacillus subtilis at 33°C until OD 600 When the OD reaches 0.4, IPTG was added to induce gene expression. 600 When the concentration reaches 0.7, sodium selenite is added to a final concentration of ≤12 mg / L to continue fermentation.

8. The method according to claim 5, characterized in that The fermentation is to culture the recombinant Bacillus subtilis at 37°C until OD 600 When the OD value reaches 0.4, IPTG was added to induce gene expression. 600 When the concentration reached 0.7, sodium selenite was added to a final concentration of 50 mg / L to continue fermentation.

9. Use of the recombinant Bacillus subtilis according to claim 1 or 2, or the method according to any one of claims 5 to 8, in the preparation of selenocysteine or a selenocysteine-containing product.

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