Isoleucine riboswitch and its applications

Abstract

The present invention discloses an isoleucine riboswitch and its application, belonging to the field of genetic engineering. The present invention provides a method for constructing a riboswitch mutant library, successfully constructs the mutant library, and screens from the library a gene regulatory element that can respond to isoleucine in the environment and is negatively regulated by isoleucine to achieve the inhibitory effect on a specific gene. By applying the riboswitch IleRS with different expression intensities screened by the present invention, in an environment containing isoleucine at 10-60 mM, the inhibition rate of egfp expression can reach more than 20%.

Description

Technical Field

[0001] This invention relates to isoleucine ribosome switches and their applications, belonging to the field of genetic engineering. Background Technology

[0002] Corynebacterium glutamicum, a recognized food safety production bacterium, is widely used in the industrial fermentation production of chemicals such as amino acids, organic acids, and alcohols. With in-depth research into dynamic metabolic engineering, an increasing number of regulatory tools are being used to optimize and modify the metabolic pathways of Corynebacterium glutamicum for the efficient production of high-value-added chemicals. Ribosomal switches, as non-coding RNA cis-regulatory elements, possess characteristics such as short response time, low metabolic burden, and high specificity, and have been used in bacterial metabolic engineering. However, the limited number of ligands that naturally occurring ribosomal switches can respond to restricts their application in the dynamic regulation of gene expression. Therefore, it is necessary to modify the ligand specificity of ribosomal switches to obtain ribosomal switches that can respond to target metabolites.

[0003] Ribosomal switches are RNA segments located in the 5'-UTR region of mRNA that regulate gene expression. They typically contain two domains: an aptamer domain that binds to a ligand and an expression platform that regulates gene expression. Upon binding to a ligand, the aptamer domain induces a conformational change in the expression platform, affecting transcription termination or translation initiation in downstream genes, thereby regulating gene expression. However, currently, relatively few naturally occurring amino acid-responsive ribosomal switches have been discovered in microorganisms, primarily including lysine ribosomal switches, glycine ribosomal switches, glutamine ribosomal switches, and S-adenosylmethionine ribosomal switches. Developing other amino acid-responsive ribosomal switches is crucial for regulating the metabolic pathways of amino acids and their derivatives to efficiently produce related chemicals.

[0004] (2S,3R,4S)-4-hydroxyisoleucine (4-HIL) is a natural non-protein amino acid with insulin-promoting biological activity, showing promising potential in the treatment of diabetes. Currently, methods for producing 4-HIL using microorganisms have been developed, primarily through the expression of an isoleucine dioxygenase (IDO) gene. Using either the bacteria's own isoleucine (Ile) or exogenously added Ile as precursors, the Ile undergoes a hydroxylation reaction at the C4 position to generate 4-HIL. Since Ile is the precursor for 4-HIL synthesis, a sufficient supply of Ile is necessary to increase 4-HIL production. However, IDO activity is inhibited by high concentrations of Ile. Static enhancement of the Ile biosynthesis pathway to increase the precursor supply for 4-HIL production can easily lead to Ile accumulation, inhibiting IDO activity and hindering 4-HIL synthesis, thus posing a technical challenge to 4-HIL production. Therefore, developing Ile-responsive dynamic regulatory tools to dynamically downregulate the Ile biosynthesis pathway is an important technical challenge. This dynamic regulation tool can achieve the following objectives: it activates the Ile synthesis pathway only when the intracellular Ile concentration is low, promoting the synthesis and supply of Ile; while it downregulates the Ile synthesis pathway when the intracellular Ile concentration is high, slowing down the synthesis and accumulation of Ile and avoiding inhibition of IDO. In this way, it dynamically regulates the supply level of Ile, maintains a stable and continuous supply of Ile, and solves the problem of Ile over-accumulation inhibiting IDO activity. Summary of the Invention

[0005] The present invention provides a ribosome switch that is negatively responsive to isoleucine, having a nucleotide sequence shown in any of SEQ ID NO.1 to 4.

[0006] The present invention also provides a recombinant plasmid containing the ribosomal switch.

[0007] In one implementation, pJYW-5 or pDTW107 is used as the starting plasmid.

[0008] In one embodiment, the plasmid further contains a constitutive promoter P. tacM The constitutive promoter P tacM It is located upstream of the ribosome switch.

[0009] In one embodiment, the recombinant plasmid uses pDTW107 as the starting plasmid and contains constitutive promoter P sequentially according to the direction of gene transcription. tacM The ribosome switch and the gene encoding the fluorescent protein.

[0010] In one embodiment, the gene encoding the fluorescent protein is egfp, and its nucleotide sequence is shown in SEQ ID NO.7.

[0011] The present invention also provides recombinant microbial cells containing the recombinant plasmid.

[0012] In one embodiment, the microorganism is Corynebacterium glutamicum, including but not limited to Corynebacterium glutamicum SN01 and Corynebacterium glutamicum ATCC 13032.

[0013] In one embodiment, the Corynebacterium glutamicum is a Corynebacterium glutamicum capable of synthesizing (2S,3R,4S)-4-hydroxyisoleucine, including but not limited to Corynebacterium glutamicum SN02 (disclosed in the paper "Improving the Synthesis of 4-hydroxyisoleucine in Recombinant Corynebacterium glutamicum using RBS Sequence") and Corynebacterium glutamicum HIL02 (disclosed in the paper "High production of 4-hydroxyisoleucine in Corynebacterium glutamicum by multistepmetabolic engineering").

[0014] In one embodiment, the Corynebacterium glutamicum expresses a target gene using the recombinant plasmid; the target gene includes, but is not limited to, the threonine dehydratase gene ilvA and the acetylhydroxy acid synthase gene ilvBN.

[0015] The present invention also provides a method for regulating the expression of a target gene, the method comprising:

[0016] (1) The target gene is ligated to the recombinant plasmid and the target gene is located downstream of the ribosome switch, and the recombinant plasmid carrying the target gene is transferred into the microbial cell;

[0017] (2) Cultivate the microbial cells and add isoleucine to the culture environment to inhibit the expression of the target gene.

[0018] In one embodiment, the final concentration of isoleucine in the culture environment is ≤60mM.

[0019] In one embodiment, the final concentration of isoleucine in the culture environment is 5–60 mM.

[0020] The present invention also provides the ribosome switch that negatively responds to isoleucine, the recombinant plasmid, the recombinant microbial cell, or the application of the method in regulating gene expression.

[0021] Beneficial effects: This invention provides a method for constructing a ribosome switch mutant library and successfully constructs the mutant library, from which IleRS are screened. After screening, four IleRS with different expression intensities were obtained. These IleRS are all ribosome switches negatively regulated by Ile, and the highest inhibition rate of egfp expression can reach more than 20%. Attached Figure Description

[0022] Figure 1 The starting plasmid pDTW107-P contains TPPRS. tacM -TPPRS-tetA.

[0023] Figure 2 For the detection plasmid p5-P containing IleRS tacM -IleRSN-egfp.

[0024] Figure 3 The inhibitory effects of IleRS1, IleRS2, IleRS3, and IleRS4 on eGFP expression levels were investigated. Detailed Implementation

[0025] Fluorescence intensity (FI) detection method: Cell culture was diluted to an appropriate concentration, sampled and added to a 96-well plate, and fluorescence intensity was measured using a microplate reader. Specific parameters were: excitation wavelength 479 nm (bandwidth 20 nm) and emission wavelength 520 nm (bandwidth 20 nm).

[0026] Method for calculating inhibition rate: Measure fluorescence intensity and calculate as follows: RFU = FI / OD 562 Inhibition rate (%) = (RFU - RFU0) / RFU0; where RFU0 represents the RFU value without adding Ile, and RFU represents the RFU value with adding Ile.

[0027] Selective culture medium: glucose 25 g / L, (NH4)2SO4 0.5 g / L, corn steep liquor 10 g / L, KH2PO4 1 g / L, MgSO4 0.75 g / L, FeSO4 1.5 g / L, pH 7.20.

[0028] LBB medium: yeast extract 2.5 g / L, sodium chloride 5 g / L, peptone 5 g / L, brain and heart extract 18.5 g / L.

[0029] Table 1. Ribosome switch mutant sequences

[0030]

[0031]

[0032] Note: The underline indicates the ligand-binding region of the aptamer domain; the gray background indicates the truncated 5' end (87 bp).

[0033] Example 1: Construction of a starting plasmid containing a TPP ribosomal switch

[0034] Using genomic DNA from *E. coli* MG1655 as a template, the TPP ribosome switch TPPRS (nucleotide sequence shown in SEQ ID NO. 5) was amplified using primers thiM-F and thiM-R. Using plasmid pIT (disclosed in patent publication number CN111440797A) as a template, the tetA gene (nucleotide sequence shown in SEQ ID NO. 6) was amplified using primers tetA-F and tetA-R. The amplified fragment was digested with NotI and SalI enzymes and inserted between the NotI and SalI sites of plasmid pJYW-5 (disclosed in patent publication number CN103834679B) to obtain plasmid p5-P. tacM -tetA. The amplified TPPRS sequence was ligated into the NotI-digested plasmid p5-P using a one-step cloning process. tacM In -tetA, we obtain p5-P tacM -TPPRS-tetA. Then with p5-P tacM Using -TPPRS-tetA as a template, P was amplified with primers RS-F and RS-R. tacM -TPPRS-tetA. Using pDTW109 (disclosed in patent application CN103409446A) as a template, the linear pDTW107 plasmid was amplified using primers pDTW108-F and pDTW108-R, and then ligated to P via one-step cloning. tacM -TPPRS-tetA and pDTW107 were used to obtain the plasmid pDTW107-P containing TPPRS. tacM -TPPRS-tetA, such as Figure 1 As shown.

[0035] thiM-F: 5'-GTGAGCGGATAACAATTTTTATTAACGCGATTGTA-3';

[0036] thiM-R: 5'-CATCCTATAACTCCTTCTGCCATAACGTGAAGAAGCAATG-3';

[0037] tetA-F: 5'-ATAGCGGCCGCAGAAGGAGTTATAGGATGAAATCTAACAATG-3';

[0038] tetA-R: 5'-AGCTGTCGACTCCTTCAGGTCGAGGTGGCCCGG-3';

[0039] RS-F: 5'-CTAGCGAGCTCGTATACTGAGCTGTTGACAATTAATC-3';

[0040] RS-R: 5'-AGACCCGCGATGAGCTGTCTAGAGAGCTCGAATTC-3';

[0041] pDTW108-F: 5'-CAGCTCATCGCGGGTCTACG-3';

[0042] pDTW108-R: 5'-GTATACGAGCTCGCTAG-3'.

[0043] Example 2 Construction of TPPRS mutant library

[0044] 1) The plasmid pDTW107-P constructed in Example 1 tacM Using TPPRS-tetA as a template, and primers TPP-M-F1019 and TPP-M-R1019, linear plasmids containing random mutant bases were amplified. The mutant library containing the mutant region is shown in SEQ ID NO. 8.

[0045] TPP-M-F1019: 5'-NNNNCCAGCGTAGNGAAGTCACGGACCACCAGG-3'

[0046] pDTW108-R: 5'-NNCCAGNNCAGNNNNNNCGGGTATTTCTCAGCCTTCACG-3'

[0047] 2) The linear plasmid containing random mutant bases constructed in Example 1 was recovered and purified. After being treated with DpnI, it was phosphorylated and then treated with T4 DNA ligase for 16 hours to obtain the plasmid ligation solution.

[0048] 3) The plasmid ligation solution obtained in step 2) was introduced into E. coli JM109 competent cells by chemical transformation and cultured for 12 h;

[0049] 4) Extract the plasmid from the cell culture medium obtained in step 3) to obtain the library plasmid pDTW107-P tacM -TPPRS lib -tetA;

[0050] 5) Electroporate the library plasmid obtained in step 4) into Corynebacterium glutamicum SN01 (accession number: CCTCCNO:M2014410, recorded and published in Appl Microbiol Biotechnol, 2015, 99(9):3851-3863) competent cells to obtain a TPPRS mutant library.

[0051] Example 3: Screening of TPPRS mutant libraries

[0052] 1) The *Corynebacterium glutamicum* containing the TPPRS mutant library constructed in Example 2 was inoculated into LBB medium, and 30 mg / L kanamycin sulfate was added. The medium was then incubated at 200 r / min and 30 °C for 8 h.

[0053] 2) Collect the cells obtained in step 1), wash the cells twice with sterile physiological saline, and then transfer all the cells to selective medium. Add 20 mM Ile and 0.5 mM NiCl2 to perform negative selection and culture at 200 r / min and 30 ℃ for 24 h.

[0054] 3) Inoculate the cells obtained in step 2) into fresh selective medium at a seeding rate of 5% (v / v), add tetracycline to a final concentration of 0.9 mg / L for positive selection, and culture at 200 r / min and 30℃ for 24 h.

[0055] 4) Inoculate the cells obtained in step 3) into fresh selective medium at a seeding rate of 5% (v / v), add 20 mM Ile and 0.6 mM NiCl2 to perform negative selection, and culture at 200 r / min and 30 ℃ for 24 h; repeat step 3) to complete another round of negative and positive selection;

[0056] 5) Repeat steps 3) and 4) to complete the next round of negative and positive selection. During negative selection, the final concentration of Ile is 20 mM, and the final concentration of NiCl2 increases by 0.1 mM in each round; during positive selection, the final concentration of tetracycline is 0.9 mg / L. A total of four rounds of screening are performed (each round includes one negative selection and one positive selection) until the final concentration of NiCl2 in the negative selection increases to 0.8 mM.

[0057] 6) Spread the bacterial cultures from the last round of negative selection and the last round of positive selection onto LBB plates containing 30 mg / L kanamycin sulfate, and randomly select 20 single bacteria from each plate for sequencing.

[0058] The ribosomal switch mutant TPPRS obtained through screening M 1. TPPRS M 2. As shown in Table 1.

[0059] Example 4: Truncation of the Ribosome Switch TPPRS

[0060] The ribosomal switch TPPRS screened in Example 3 M 1. TPPRS M Using 2 as a template, primers TPPRSP-F and TPPRSN-R were used to amplify ribosomal switches IleRS1 and IleRS2, which were truncated by 87 bp.

[0061] TPPRS M 1. The truncated IleRS1 (nucleotide sequence as shown in SEQ ID NO.1); TPPRS M 2. The truncated IleRS2 (nucleotide sequence as shown in SEQ ID NO.2).

[0062] TPPRSP-F: 5'-TGTGAGCGGATAACAATTGCAACCAAACGACTCGGGGTG-3';

[0063] TPPRSN-R: 5'-CCTATAACTCCTTCTGCCATAACGTGAAGAAGCAATG-3'.

[0064] Example 5 TPPRS M N lib Construction of mutant libraries

[0065] 1) Using the ribosomal switch mutant TPPRS M plasmid pDTW107-P tacM -TPPRS M Using 1-tetA as a template, linear plasmids containing random mutant bases were amplified using primers Tlib2-F and M1Tlib2-R. The mutant library containing the mutant region is shown in SEQ ID NO.9. The plasmid was then used to carry the ribosome switch mutant TPPRS. M plasmid pDTW107-P 2 tacM -TPPRS M Using 2-tetA as a template, linear plasmids containing random mutant bases were amplified using primers Tlib2-F and M2Tlib2-R. The mutant library containing the mutant region is shown in SEQ ID NO.10.

[0066] Tlib2-F: 5'-NNNNNCCAGCGTAGGGAAGTCACGGACCAC-3';

[0067] M1Tlib2-R: 5'-NNNNNCCAGATCAGTAGGTACGGGTATTTCTCAGCC-3';

[0068] M2Tlib2-R: 5'-NNNNNCCAGATCAGAGTGGACGGGTATTTCTCAGCC-3';

[0069] 2) Recovery and purification: The linear plasmid containing the mutant library constructed in step 1) was treated with DpnI, then phosphorylated, and then treated with T4 DNA ligase for 16 hours to obtain the plasmid ligation solution.

[0070] 3) The plasmid ligation solution from step 2) above was chemically transformed and introduced into E. coli JM109 competent cells and cultured for 12 h.

[0071] 4) Extract plasmids from the cell culture medium prepared in step 3) to obtain plasmid pDTW107-P containing the mutant library. tacM -TPPRS M 1 lib -tetA and pDTW107-P tacM -TPPRS M 2 lib -tetA;

[0072] 5) Electroporate the plasmids containing the mutant library obtained in step 4) into Corynebacterium glutamicum SN01 competent cells to obtain TPPRS. M 1 lib and TPPRS M 2 lib The mutant library.

[0073] Example 6 TPPRS M N lib Screening of mutant libraries

[0074] 1) The TPPRS-containing sample prepared in Example 5 M 1 lib or TPPRS M 2 lib The mutant libraries of Corynebacterium glutamicum were inoculated into LBB medium, and 30 mg / L kanamycin sulfate was added. The cultures were then incubated at 200 r / min and 30 ℃ for 8 h.

[0075] 2) Collect the cells cultured in step 1), wash the cells twice with sterile physiological saline, and then transfer all the cells to their respective selective culture media. Add 20 mM Ile and 0.6 mM NiCl2 to perform negative selection and culture at 200 r / min and 30 ℃ for 24 h.

[0076] 3) Inoculate the cells obtained in step 2) into fresh selective medium at a seeding rate of 5% (v / v), add tetracycline to a final concentration of 0.9 mg / L for positive selection, and culture at 200 r / min and 30℃ for 24 h;

[0077] 4) Inoculate the cells obtained in step 3) into fresh selective medium at a seeding rate of 5% (v / v), add 20 mM Ile and 0.7 mM NiCl2 to perform negative selection, and culture at 200 r / min and 30 ℃ for 24 h; repeat step 3) to complete another round of negative and positive selection;

[0078] 5) Repeat steps 3) and 4) to complete the next round of negative and positive selection. During negative selection, the final concentration of Ile is 20 mM, and the final concentration of NiCl2 increases by 0.1 mM in each round; during positive selection, the final concentration of tetracycline is 0.9 mg / L. A total of three rounds of screening are performed (each round includes one negative selection and one positive selection) until the final concentration of NiCl2 in the negative selection increases to 0.8 mM.

[0079] 6) Spread the bacterial cultures from the last round of negative selection and the last round of positive selection onto LBB plates containing 30 mg / L kanamycin sulfate, and randomly select 20 single bacteria from each plate for sequencing.

[0080] The ribosomal switch mutant TPPRS obtained through screening M 3. TPPRS M 4. As shown in Table 1. TPPRS M 3. The truncated IleRS3 (nucleotide sequence shown in SEQ ID NO.3); TPPRS M 4. The truncated IleRS4 (nucleotide sequence shown in SEQ ID NO.4).

[0081] Example 7: Construction of a probe plasmid containing IleRS

[0082] The IleRSN sequences shown in SEQ ID NO. 1–4 (N being 1, 2, 3, and 4, respectively) were synthesized using a total chemical synthesis method. The fluorescent reporter gene egfp (nucleotide sequence shown in SEQ ID NO. 7) was synthesized using a total chemical synthesis method. The IleRSN-egfp fragment was obtained by overlap PCR using primers TPPRSP-F and egfp-R.

[0083] TPPRSP-F: 5'-TGTGAGCGGATAACAATTGCAACCAAACGACTCGGGGTG-3';

[0084] egfp-R: 5'-TCGAATTCGTCGACGGATCCTTACTTGTACAGCTCGTCC-3';

[0085] An IleRSN-egfp fragment was inserted between the NotI and BamHI sites in plasmid pJYW-5 to obtain the probe plasmid p5-P containing IleRS. tacM -IleRSN-egfp, such as Figure 2 As shown.

[0086] Example 8 Activity detection of IleRS

[0087] The detector plasmid p5-P constructed in Example 7 was used. tacM -IleRSN-egfp was electroporated into Corynebacterium glutamicum ATCC13032 to obtain strain ATCC 13032 / p5-P tacM -IleRSN-egfp. This will enable ATCC 13032 / p5-P. tacM -IleRSN-egfp was cultured in LBB medium at 200 rpm and 30°C for 10 h, and the final OD was determined. 562 0.02 μL of the culture was transferred to fresh LBB medium, which was then supplemented with Ile at concentrations ranging from 0 to 60 mM (0, 5, 10, 20, 40, 60 mM). After culturing for 12 h, 1 mL of bacterial suspension was collected every 4 h. The cells were washed twice with physiological saline, resuspended in physiological saline, diluted to an appropriate concentration, added to 96-well plates, and the fluorescence intensity was measured using a microplate reader.

[0088] The results show ( Figure 3 In Ile-containing medium, the eGFP expression levels of IleRS1, IleRS2, IleRS3, and IleRS4 were significantly lower than those without Ile. In the presence of 10–60 mM Ile, the relative fluorescence intensity of eGFP decreased from 101664 RFU to 80231–92168 RFU, with an inhibition rate of 9.3–21.1%, reaching 14.7% at an Ile concentration of 20 mM. In the presence of 5–40 mM Ile, the relative fluorescence intensity of eGFP decreased from 4208 RFU to 3176–3902 RFU, with an inhibition rate of 7.3–24.5%, reaching 24.5% at an Ile concentration of 20 mM. In the presence of 5–60 mM Ile, the relative fluorescence intensity of eGFP decreased from 13884 RFU to 9776–13161 RFU, with an inhibition rate of 5.2–29.6%, reaching 26.9% at an Ile concentration of 40 mM. In the presence of 5–40 mM Ile, the relative fluorescence intensity of eGFP decreased from 101664 RFU to 80231–92168 RFU, with an inhibition rate of 9.3–21.1%, reaching 14.7% at an Ile concentration of 20 mM. In the presence of Ile, the relative fluorescence intensity of eGFP decreased from 4261 RFU to 3879–4242 RFU, with an inhibition rate of 0.4–9.0%. At an Ile concentration of 40 mM, the inhibition rate was 9.0%.

[0089] Example 9: Application of the ribosome switch mutant IleRS in repressor genes

[0090] The specific implementation method is the same as in Example 7, except that the egfp gene is replaced with the Ile synthesis gene threonine dehydratase gene ilvA. This plasmid was transformed into strain SN02, which has the ability to synthesize 4-HIL, to verify the inhibitory effect of IleRS on the ilvA gene. The results showed that the ilvA gene was inhibited under high concentrations of Ile. Optionally, this ribosome switch can also be used to inhibit gene expression on chromosomes, achieving a level of inhibition comparable to that on plasmids.

[0091] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A riboswitch responsive to negative isoleucine, with a nucleotide sequence as shown in any one of SEQ ID NO.1 to 4.

2. A recombinant plasmid containing the riboswitch described in claim 1.

3. The recombinant plasmid according to claim 2, wherein The recombinant plasmid also contains a constitutive promoter P tacM ; The constitutive promoter P tacM is located upstream of the riboswitch.

4. The recombinant plasmid according to claim 2, wherein Using pDTW107 as the starting plasmid, it successively contains the constitutive promoter P tacM , the riboswitch and the gene encoding the fluorescent protein.

5. A recombinant microbial cell containing the recombinant plasmid described in any one of claims 2 to 4.

6. A recombinant Corynebacterium glutamicum, characterized in that, Express the target gene using the recombinant plasmid according to any one of claims 2 to 4; the target gene includes the threonine dehydratase gene ilvA or the acetohydroxy acid synthase gene ilvBN .

7. A method for regulating the expression of a target gene, characterized in that, The method comprises: (1) ligating a target gene to the recombinant plasmid described in any one of claims 2 to 4, and positioning the target gene downstream of the riboswitch described in claim 1, and transferring the recombinant plasmid carrying the target gene into a microbial cell; (2) culturing the microbial cell, and adding isoleucine to the culture environment to inhibit the expression of the target gene.

8. The method according to claim 7, characterized in that, The microorganism includes Corynebacterium glutamicum.

9. The method according to claim 7 or 8, characterized in that The final concentration of the isoleucine in the culture environment is ≤ 60 mM.

10. Use of the riboswitch described in claim 1, or the recombinant plasmid described in any one of claims 2 to 4, or the recombinant microbial cell described in claim 5, or the recombinant Corynebacterium glutamicum described in claim 6, or the method described in any one of claims 7 to 9 in inhibiting the expression of a gene in a culture environment.

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