Recombinant escherichia coli for detecting s-adenosyl-l-methionine concentration, construction method and application thereof

Abstract

The application discloses recombinant escherichia coli for detecting S-adenosyl-L-methionine concentration and a construction method and application thereof, the construction method is (1) constructing recombinant plasmid pUC19-CAB; (2) introducing the recombinant plasmid pUC19-CAB into escherichia coli BL21 to obtain the recombinant escherichia coli BL21 / pUC19-CAB for detecting S-adenosyl-L-methionine concentration; the recombinant escherichia coli BL21 / pUC19-CAB of the application can be used for detecting S-adenosyl-L-methionine concentration, can be used for efficient screening of different yield SAM strains, and the recombinant escherichia coli for detecting S-adenosyl-L-methionine concentration of the application has the advantages that different SAM concentration fermentation liquor is screened with simple operation, short screening period and high detection efficiency.

Description

Technical Field

[0001] This invention relates to a recombinant Escherichia coli for detecting the concentration of S-adenosine-L-methionine, its construction method, and its application, belonging to the field of microbial technology. Background Technology

[0002] S-Adenosyl-L-methionine (SAM), synthesized from L-methionine and adenosine triphosphate via S-adenosylmethionine synthase, is present in all organisms and plays a crucial role in demethylation, desulfation, and amination. SAM has important applications in the pharmaceutical industry, commonly used in the treatment of arthritis, depression, liver disease, fibromyalgia, and Alzheimer's disease. SAM is also a major methyl donor in methylation reactions occurring primarily in living cells. However, the expensive nature of SAM significantly limits its applications. Therefore, improving its production process in a cost-effective manner to meet industry demands is essential.

[0003] SAM production can be achieved through chemical catalysis, enzymatic catalysis, or microbial catalysis. However, harsh reaction conditions, low productivity, environmental pollution, and purification difficulties limit the application of chemical catalysis. On the other hand, the high cost of the SAM precursor ATP also restricts enzymatic methods for preparing this compound. Therefore, inexpensive and efficient microbial catalysis is the preferred method for industrial applications. Furthermore, given the significant application value and huge market potential of SAM, finding SAM-producing strains with good fermentation performance is a key focus of research and development.

[0004] Currently, the commonly used method for screening SAM-producing strains mainly involves liquid fermentation of the strains, extraction of SAM samples from the fermentation broth, and finally detection of SAM content using HPLC (High Performance Liquid Chromatography). This method is time-consuming and cumbersome, and not suitable for screening large numbers of strains. Therefore, developing an effective high-throughput detection method is essential for high-throughput screening of SAM-producing strains. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant Escherichia coli for detecting the concentration of S-adenosine-L-methionine.

[0006] A second objective of this invention is to provide a method for constructing recombinant Escherichia coli for detecting S-adenosine-L-methionine concentration.

[0007] A third objective of this invention is to provide the application of recombinant Escherichia coli BL21 / pUC19-CAB in the detection of S-adenosine-L-methionine concentration.

[0008] The technical solution of this invention is summarized as follows:

[0009] The method for constructing recombinant Escherichia coli for detecting S-adenosine-L-methionine concentration includes the following steps:

[0010] (1) Construction of recombinant plasmid: The fusion sequence of MI37 promoter, SAM riboswitch CAB and marker gene GFP was synthesized into plasmid pUC19 to obtain recombinant plasmid pUC19-CAB;

[0011] The nucleotide sequence of the MI37 promoter is shown in SEQ ID NO.2;

[0012] The nucleotide sequence of the SAM riboswitch CAB is shown in SEQ ID NO.3;

[0013] The nucleotide sequence of the marker gene GFP is shown in SEQ ID NO.4;

[0014] The fusion sequence of the MI37 promoter, SAM riboswitch CAB, and marker gene GFP is shown in SEQ ID NO.5;

[0015] The nucleotide sequence of the plasmid pUC19 is shown in SEQ ID NO.1;

[0016] The nucleotide sequence of the recombinant plasmid pUC19-CAB is shown in SEQ ID NO.6;

[0017] (2) The recombinant plasmid pUC19-CAB was introduced into Escherichia coli BL21 to obtain recombinant Escherichia coli BL21 / pUC19-CAB for detecting the concentration of S-adenosine-L-methionine.

[0018] The above-described method was used to construct recombinant Escherichia coli BL21 / pUC19-CAB for detecting S-adenosine-L-methionine concentration.

[0019] The application of the above-mentioned recombinant Escherichia coli BL21 / pUC19-CAB in the detection of S-adenosyl-L-methionine concentration includes the following steps: The recombinant Escherichia coli BL21 / pUC19-CAB is inoculated into M9 medium and cultured to obtain a seed culture; the seed culture is then inoculated into M9 medium containing SAM at concentrations increasing from 0 μM to 2000 μM, and cultured at 37°C for 24 hours. 200 μL of each culture is then taken, and the fluorescence intensity and OD of the fermentation broth are detected using a microplate reader. 600 Value; calculate relative fluorescence intensity / OD 600 The recombinant Escherichia coli BL21 / pUC19-CAB showed a negative correlation with the SAM concentration in the experiment.

[0020] Advantages of this invention:

[0021] The recombinant Escherichia coli BL21 / pUC19-CAB of the present invention for detecting S-adenosine-L-methionine concentration can be used for efficient screening of SAM strains with different yields. Using the recombinant Escherichia coli of the present invention for detecting S-adenosine-L-methionine concentration to screen fermentation broths with different SAM concentrations has the advantages of simple operation, short screening cycle and high detection efficiency. Attached Figure Description

[0022] Figure 1 This is a map of plasmid pUC19.

[0023] Figure 2 This is a plasmid map of the recombinant plasmid pUC19-CAB.

[0024] Figure 3 The effect of different concentrations of SAM on the fluorescence intensity in the fermentation broth obtained from the fermentation of recombinant Escherichia coli BL21 / pUC19-CAB. Detailed Implementation

[0025] The present invention will be further described below through specific embodiments.

[0026] The Escherichia coli BL21 used in the following examples was purchased from TransGen Biotech; the pUC19 plasmid used in the following examples was purchased from Addgene.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1

[0030] The method for constructing recombinant Escherichia coli for detecting S-adenosine-L-methionine concentration includes the following steps:

[0031] (1) Construction of recombinant plasmid: The fusion sequence of MI37 promoter, SAM riboswitch CAB and marker gene GFP was synthesized into plasmid pUC19 to obtain recombinant plasmid pUC19-CAB;

[0032] The nucleotide sequence of the MI37 promoter is shown in SEQ ID NO.2;

[0033] The nucleotide sequence of the SAM riboswitch CAB is shown in SEQ ID NO.3;

[0034] The nucleotide sequence of the marker gene GFP is shown in SEQ ID NO.4;

[0035] The fusion sequence of the MI37 promoter, SAM riboswitch CAB, and marker gene GFP is shown in SEQ ID NO. 5; it was directly synthesized by Jinkairui Company into the vector pUC19. The plasmid map of plasmid pUC19 is shown in [link to plasmid map]. Figure 1 The plasmid map of recombinant plasmid pUC19-CAB is shown below. Figure 2 .

[0036] The nucleotide sequence of the plasmid pUC19 is shown in SEQ ID NO.1;

[0037] The nucleotide sequence of the recombinant plasmid pUC19-CAB is shown in SEQ ID NO.6;

[0038] Alternatively, the pUC19 vector sequence in this embodiment can be replaced with the pUC18 vector sequence or other vectors, and the rest is the same as in this embodiment, to obtain similar pUC18-CAB plasmids, etc.

[0039] (2) The recombinant plasmid pUC19-CAB was introduced into Escherichia coli BL21; the transformed E. coli were plated on LB solid medium and incubated in a 37°C incubator for 10 hours; the transformants were picked for colony PCR verification, and the correct verification was the recombinant E. coli BL21 / pUC19-CAB used to detect the concentration of S-adenosine-L-methionine.

[0040] Example 2

[0041] The application of recombinant Escherichia coli BL21 / pUC19-CAB in the detection of S-adenosine-L-methionine concentration includes the following steps:

[0042] Recombinant Escherichia coli BL21 / pUC19-CAB was inoculated into M9 medium and cultured at 37°C for 10 hours to obtain seed culture.

[0043] Seed culture was inoculated at 10% (v / v) into SAM M9 medium with concentrations increasing from 0 μM to 2000 μM (concentrations of 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM, 1200 μM, 1300 μM, 1400 μM, 1500 μM, 1600 μM, 1700 μM, 1800 μM, 1900 μM, and 2000 μM). The cultures were incubated at 37°C for 24 hours. 200 μL of each culture was then collected, and the fluorescence intensity and OD of the fermentation broth were measured using a microplate reader. 600 Value; calculate relative fluorescence intensity / OD 600 The result is as follows Figure 3As shown, the recombinant Escherichia coli BL21 / pUC19-CAB showed a negative correlation with the SAM concentration in the experiment.

[0044] In summary, recombinant Escherichia coli BL21 / pUC19-CAB can respond to SAM concentrations in the range of 0-2000 μM. Therefore, it can be used to screen for SAM-producing strains.

[0045] Example 3

[0046] Application of recombinant Escherichia coli BL21 / pUC19-CAB in detecting SAM concentration in Saccharomyces cerevisiae fermentation broth

[0047] (1) Construction of a SAM-producing Saccharomyces cerevisiae library

[0048] The SAM2 gene was randomly mutated using error-prone PCR to establish a SAM-producing Saccharomyces cerevisiae library.

[0049] The SAM2 gene is derived from Saccharomyces cerevisiae, and its nucleotide sequence is shown in SEQ ID NO.7.

[0050] The specific steps are as follows:

[0051] As shown in Table 1, using the genome of Saccharomyces cerevisiae ATCC4040002 as a template, the PGK1p fragment was amplified by PCR using Prs-PGK1p-F (SEQ ID NO. 8) and PGK1p-R-PGK1t (SEQ ID NO. 9);

[0052] Using the genome of Saccharomyces cerevisiae ATCC4040002 as a template, the PGK1t fragment was amplified by PCR using PGK1p-PGK1t-F (SEQ ID NO.10) and PGK1t-R-Prs (SEQ ID NO.11);

[0053] Using the plasmid vector Prs426 (ATCC, USA) with the nucleotide sequence shown in SEQ ID NO.12 as a template, the linear plasmid backbone fragment of Prs426 was amplified by PCR using PGK1t-Prs-F (SEQ ID NO.13) and Prs-R-PGK1p (SEQ ID NO.14).

[0054] Using the Seamless Cloning Kit (Minerva Super Fusion Cloning Kit, purchased from [website name])

[0055] (http: / / www.uebio.com / index.html) The PGK1p and PGK1t fragments obtained in the above steps were ligated to the Prs426 linear plasmid backbone to obtain the recombinant plasmid named Prs426-PGK (SEQ ID No. 15).

[0056] Using recombinant plasmid Prs426-PGK as a template, the linear backbone of recombinant plasmid Prs426-PGK was amplified by PCR using Err-Prs-F (SEQ ID NO.16) and Err-Prs-R (SEQ ID NO.17);

[0057] Using the Saccharomyces cerevisiae ATCC4040002 genome as a template, the SAM2 gene was amplified by error-prone PCR using Err-SAM2-F (SEQ ID NO.18) and Err-SAM2-R (SEQ ID NO.19) to obtain the SAM2m fragment. Using the seamless cloning kit described above, the SAM2m fragment was ligated to the linear backbone of the recombinant plasmid Prs426-PGK to obtain the SAM2 mutant plasmid library, named Prs426-SAM2m.

[0058] Table 1 Primers and templates for constructing the SAM2 mutant library.

[0059]

[0060] A single colony of *Saccharomyces cerevisiae* BY4741 (ATCC4040002, purchased June 2016, website: https: / / www.atcc.org / ) was inoculated into 3 ml of YPD liquid medium and cultured at 30°C and 220 rpm for 12 h. The culture was then transferred to a fresh 3 ml YPD liquid medium and cultured at 30°C and 220 rpm for 5 h. 1 ml of the bacterial culture was transferred to a pre-sterilized 1.5 ml centrifuge tube and centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the bacterial cells were collected. The cells were washed once with 1 ml of sterile water and centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the bacterial cells were collected. Then, 1 ml of 100 mM LiAc aqueous solution was added to the bacterial cells and mixed well. The mixture was allowed to stand at room temperature for 5 min. After standing, the cells were centrifuged at 4000 rpm for 3 min, and the LiAc liquid was removed using a pipette. Boil salmon DNA (Solarbio, 10 mg / ml) in boiling water for 5 minutes, then quickly place it on prepared ice to cool.

[0061] Add 120 μl of PEG3350 (50 g PEG3350 / 100 ml water), 18 μl of 1.0 M LiAc aqueous solution, 5 μl of salmon sperm DNA (boiled in boiling water for 5 min and cooled), and 800 ng of Prs426-SAM2m to the bacterial pellet centrifuge tube in sequence. The final volume is made up to 180 μl with sterile water. Then, gently pipette the mixture for 1 minute to mix it thoroughly. Place the mixed centrifuge tube in a 42°C water bath for 30 minutes, then centrifuge at 4000 rpm for 3 minutes and remove the supernatant. Add 1 ml of clean YPD liquid medium and incubate at 30°C and 220 rpm for 2 hours. Centrifuge the incubated centrifuge tube at 4000 rpm for 3 minutes, discard the upper medium, and wash twice with 1 ml of sterile water. Finally, add 200 μl of sterile water to the centrifuge tube containing the cells and mix well. Spread the mixture on uridine-deficient SC medium plates and incubate at 30°C for 2 days. All the transformants obtained are the SAM-producing Saccharomyces cerevisiae library.

[0062] (2) Screening for high-SAM-producing Saccharomyces cerevisiae strains using recombinant Escherichia coli BL21 / pUC19-CAB.

[0063] SAM-producing *Saccharomyces cerevisiae* strains were inoculated into 1 mL of YPD medium supplemented with 2 g / L L-Met and cultured in 96-well plates at 30°C for 48 hours using a shaker. 200 μL of the fermentation broth was then used to determine the OD of each yeast strain. 600 The fermentation broth was then allowed to stand for 4 hours. 600 μL of the fermentation supernatant was added to each new sterile 96-well plate, followed by 100 μL of recombinant E. coli BL21 / pUC19-CAB cultured in M9 medium for 10 hours. The mixed culture was then incubated at 37°C in a shaker for 12-16 hours. 200 μL of the culture was then used to detect the OD of the recombinant E. coli using a microplate reader. 600 Value and fluorescence intensity. Use fluorescence intensity / OD 600 (E. coli) / OD 600 The relative fluorescence intensity (RFI) is represented by the yeast strain. A lower RFI value indicates a higher SAM content. High-SAM-producing strains were screened by comparing the RFI with a control strain. This screening process identified 1140 SAM2 mutant yeast strains within one week, resulting in a mutant strain M20 with approximately twice the SAM yield compared to the starting strain. Its SAM yield was determined to be 221.95 mg / L by HPLC.

[0064] (3) HPLC detection method for SAM

[0065] The fermentation broth was mixed with a 10% (v / v) perchloric acid aqueous solution in equal proportion and incubated on a shaker at 30°C for 1 hour. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was collected and filtered through a 0.22 μm microporous membrane into a liquid chromatography vial for UV-Vis detection of SAM. The mobile phase was 0.01 M ammonium formate solution, with pH adjusted to 3.5 by glacial acetic acid; the flow rate was 0.5 mL / min; the column was Elite C18 (4.6 mm × 250 mm); and the detection wavelength was 260 nm.

[0066] Culture medium used in the examples

[0067] YPD liquid medium: final glucose concentration 25 g / L, final yeast extract concentration 10 g / L, final peptone concentration 20 g / L, prepared with distilled water. YPD solid medium is YPD liquid medium with 20 g / L agar powder added.

[0068] LB liquid medium: peptone concentration of 10 g / L, yeast extract concentration of 5 g / L, sodium chloride concentration of 10 g / L, prepared with distilled water.

[0069] LB solid medium: peptone concentration of 10 g / L, yeast extract concentration of 5 g / L, sodium chloride concentration of 10 g / L, agar powder concentration of 20 g / L, prepared with distilled water.

[0070] M9 medium: per 1L contains: 20g glucose, 6.8g disodium hydrogen phosphate, 3g potassium dihydrogen phosphate, 0.5g sodium chloride, 1.0g ammonium chloride, 0.24g magnesium sulfate, 0.11g calcium chloride, 10mL trace element stock solution, 20mg ferric ammonium citrate, 2.8mg ferrous sulfate, and 0.5g glycine, prepared with distilled water.

[0071] Trace element stock solution: 10.2 g / L ZnSO4·7H2O, 15 g / L EDTANa2·2H2O, 5.12 g / L FeSO4·7H2O, 0.5 g / L anhydrous CuSO4, 0.5 g / L MnCl2·4H2O, 0.86 g / L CoCl2·6H2O, 3.84 g / L CaCl2·2H2O, 0.56 g / L Na2MoO4·2H2O, prepared with distilled water.

[0072] SC medium: final glucose concentration of 20 g / L, final concentration of amino-free yeast nitrogen source (YNB) of 6.7 g / L, final concentration of amino acid mixture of 0.2 g / L, 20 g / L agar powder, prepared with distilled water. The missing amino acid mixture refers to the amino acid mixture with the corresponding components removed.

[0073] Amino acid mixture: glycine, 2.0g; alanine, 2.0g; methionine, 2.0g; lysine, 2.0g; arginine, 2.0g; serine, 2.0g; asparagine, 2.0g; aspartic acid, 2.0g; phenylalanine, 2.0g; cysteine, 2.0g; proline, 2.0g; tyrosine, 2.0g; glutamic acid, 2.0g; valine, 2.0g; threonine, 2.0g; serine, 2.0g; isoleucine, 2.0g; inositol, 2.0g; glutamine, 2.0g; para-aminobenzoic acid, 0.2g; adenine, 0.5g; leucine, 10g; methionine, 2g; tryptophan, 2g; histidine, 2g; uracil, 2g.

Claims

1. A method for constructing a recombinant Escherichia coli for detecting the concentration of S-adenosyl-L-methionine, characterized by The method comprises the following steps: (1) constructing a recombinant plasmid: synthesizing a fusion sequence of a MI37 promoter, a SAM riboswitch CAB and a marker gene GFP on a plasmid pUC19 to obtain a recombinant plasmid pUC19-CAB; The nucleotide sequence of the MI37 promoter is shown as SEQ ID NO. 2; The nucleotide sequence of the SAM riboswitch CAB is shown as SEQ ID NO. 3; The nucleotide sequence of the marker gene GFP is shown as SEQ ID NO. 4; The fusion sequence of the MI37 promoter, the SAM riboswitch CAB and the marker gene GFP is shown as SEQ ID NO. 5; The nucleotide sequence of the plasmid pUC19 is shown as SEQ ID NO. 1; The nucleotide sequence of the recombinant plasmid pUC19-CAB is shown as SEQ ID NO. 6; (2) introducing the recombinant plasmid pUC19-CAB into Escherichia coli BL21 to obtain a recombinant Escherichia coli BL21 / pUC19-CAB for detecting the concentration of S-adenosyl-L-methionine.

2. The recombinant Escherichia coli BL21 / pUC19-CAB for detecting the concentration of S-adenosyl-L-methionine constructed by the construction method of claim 1.

3. Use of recombinant E. coli BL21 / pUC19-CAB according to claim 2 for detecting the concentration of S-adenosyl-L-methionine, characterized in that The method comprises the following steps: inoculating the recombinant E. coli BL21 / pUC19-CAB into M9 medium, culturing to obtain a seed liquid; inoculating the seed liquid into M9 medium with SAM at concentrations from 0 μM to 2000 μM, respectively, culturing at 37°C for 24 hours, taking 200 μL of each culture liquid, detecting the fluorescence intensity and the OD of the fermentation liquid by means of an enzyme label instrument 600 ; calculating the relative fluorescence intensity / OD 600 ; the recombinant E. coli BL21 / pUC19-CAB is negatively correlated with the concentration of SAM in the experiment.

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