Construction method and application of genetically engineered bacteria expressing thanatin
Patent Information
- Application Number
- CN202611171084.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-04
- Publication Date
- 2026-09-15
Smart Images
Figure CN122750722A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and in particular to a method for constructing and applying a genetically engineered bacterium expressing the death factor thanatin. Background Technology
[0002] Antimicrobial peptides are an important component of the organism's innate immune system. They are a class of small-molecule, cationic, amphiphilic polypeptides with broad-spectrum antimicrobial activity, capable of rapidly and effectively inhibiting or killing Gram-negative bacteria, Gram-positive bacteria, fungi, parasites, and some enveloped viruses. They are considered one of the most promising candidates for addressing the antibiotic resistance crisis. Thanatin is an antimicrobial peptide with a monodisulfide β-hairpin structure. Naturally extracted thanatin has low content and complex processing. Currently, its preparation mainly employs two methods: chemical synthesis and heterologous expression. Chemical synthesis is costly, unsuitable for large-scale production, and has poor environmental friendliness. While heterologous expression of thanatin by microorganisms has great development potential, current technologies have not yet disclosed genetically engineered bacteria capable of achieving high levels of multi-copy expression of thanatin. Summary of the Invention
[0003] The purpose of this invention is to provide a method for constructing and applying a genetically engineered bacterium that expresses the death factor thanatin, thereby achieving stable and high expression of multiple copies of thanatin.
[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing a genetically engineered bacterium expressing the death factor thanatin, wherein recombinant plasmid pESCgp04-HIS-PiggyBac transposase and recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3 are co-transformed into competent yeast cells to obtain the genetically engineered bacterium; The nucleotide sequence of the recombinant plasmid pESCgp04-HIS-PiggyBac transposase is shown in SEQ ID NO. 1; The nucleotide sequence of the recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3 is shown in SEQ ID NO.2.
[0005] Preferably, the yeast competent cells are Saccharomyces cerevisiae CENPK2 competent cells.
[0006] Preferably, the co-conversion method is a chemical conversion.
[0007] The present invention also provides the application of the genetically engineered bacteria constructed by the above-described construction method in the preparation of drugs against drug-resistant bacteria.
[0008] The present invention also provides thanatin, a death-causing agent produced by genetically engineered bacteria constructed by the above-described construction method, wherein the amino acid sequence of thanatin is shown in SEQ ID NO.48.
[0009] Preferably, the genetically engineered bacteria described in claim 5 are fermented to obtain a fermentation broth, and the supernatant of the fermentation broth is collected to obtain the death factor Thanatin.
[0010] Preferably, the fermentation culture time is 24 to 96 hours.
[0011] The present invention also provides the application of the aforementioned thanatin and the thanatin produced by the aforementioned production method in the preparation of drugs against drug-resistant bacteria.
[0012] The present invention has the following technical effects and advantages: This invention successfully constructed a genetically engineered bacterium, CENPK2-Thanatin-PiggyBac, by integrating the PiggyBac transposon system into Saccharomyces cerevisiae CENPK2, which stably and highly expresses the death factor Thanatin. This bacterium exhibits good stability and high expression levels of the target protein, providing a new technical solution and application prospect for its large-scale preparation and anti-infective drug development. Attached Figure Description
[0013] Figure 1 The map shows the recombinant plasmid pESCgp04-HIS-PiggyBac transposase. Figure 2 The spectrum of the recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3; Figure 3 The colony PCR results are for the verification of the recombinant plasmid pESCgp04-HIS-PiggyBac transposase in genetically engineered bacteria. Figure 4 The colony PCR results are for the validation of the recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3 in genetically engineered bacteria. Figure 5The results of Tris-tricine-SDS-PAGE electrophoresis of Thanatin, the death factor expressed by genetically engineered bacteria, are shown in lane 1. Lane 1 shows the electrophoresis results of the CENPK2 expression product of Saccharomyces cerevisiae, and lane 2 shows the electrophoresis results of the CENPK2-Thanatin-PiggyBac expression product of genetically engineered bacteria. Figure 6 The results of secondary mass spectrometry for thanatin, a death-causing agent expressed by genetically engineered bacteria; Figure 7 Results of in vitro antibacterial activity assay for thanatin, a death-causing agent expressed by genetically engineered bacteria. Detailed Implementation
[0014] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0015] In this invention, the vectors pESCgp04-HIS, pUC57, pBluescript, pESC-LEU, and pESCtp01-Thanatin-EKx3 were obtained from the Laboratory of Biosynthesis of Green Storage and Feed Functional Compounds of the National Food and Strategic Reserves Administration. In this invention, the CENPK2 competent cells of Saccharomyces cerevisiae were obtained from the Laboratory of Biosynthesis of Green Storage and Feed Functional Compounds of the National Food and Strategic Reserves Administration, and the Uniclone One Step Seamless Cloning Kit was purchased from Beijing Jinsha Biotechnology Co., Ltd.
[0016] Example 1: Construction of recombinant plasmid pESCgp04-HIS-PiggyBac transposase The nucleotide sequence of PiggyBac transposase (NCBI ID: ABS12112.1) was optimized using the codons of Saccharomyces cerevisiae and used as a template. The PiggyBac transposase fragment was amplified using primers tpos1 / tpos50 and inserted into the vector pESCgp04-HIS to obtain the recombinant plasmid pESCgp04-HIS-PiggyBac transposase, the nucleotide sequence of which is shown in SEQ ID NO.1. The primer sequences are shown in Table 1. The results are as follows: Figure 1 As shown.
[0017] SEQ ID NO.1: Example 2: Construction of recombinant plasmid pUC57-LEU-CL1-pFull Using plasmid pESC-LEU as a template, the amplification products were obtained by using primers LEU2-CL1-F(Gib) / LEU2-CL1-R(Gib) and LEU2-CL1-F1(Gib) / LEU2-CL1-R1(Gib). The amplification products were then assembled with the vector pUC57 using the Uniclone OneStep Seamless Cloning Kit to obtain the recombinant plasmid pUC57-LEU-CL1-pFull. The primer sequences are shown in Table 1.
[0018] Example 3: Construction of recombinant plasmid pUC57-3'piggyBac ITR+insulator Fragment A was obtained by sequentially ligating primers 3'-1 to 3'-8 using KOD high-fidelity enzyme, and fragment B was obtained by sequentially ligating primers 3'-9 to 3'-16 using Q5 high-fidelity enzyme. Fragment A and fragment B were then ligated to obtain the ligation fragment, which was then assembled with the vector pUC57 using the Uniclone One Step Seamless Cloning Kit via Gibson assembly to obtain the recombinant plasmid pUC57-3'piggyBac ITR+insulator. The primer sequences are shown in Table 1.
[0019] Example 4: Construction of recombinant plasmid pUC57-5'piggyBac ITR+insulator Using the recombinant plasmid pUC57-3'piggyBac ITR+insulator as a template, the insulator fragment was amplified with primer insulator-F / 3'-16. Primers 5'-1 to 5'-10 were sequentially ligated to obtain fragment C. Fragment C, the insulator fragment, and the vector pUC57 were assembled using the Uniclone One Step Seamless Cloning Kit via Gibson assembly to obtain the recombinant plasmid pUC57-5'piggyBac ITR+insulator. The primer sequences are shown in Table 1.
[0020] Example 5: Construction of recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3 Using recombinant plasmid pUC57-3'piggyBac ITR+insulator as a template, the 3'piggyBac ITR+insulator fragment was amplified using primers 3'piggy-F(Gib) / 3'piggy-R(Gib); using plasmid pESCtp01-Thanatin-EKx3 as a template, primers Than-pig1-F(Gib) / Than-pig1-R(Gib), Than-pig2-F(Gib) / Than-pig2-R(Gib), and Than-pig3-F(Gib) / Than-pig3-R were used to amplify the fragment. Thanatin1, Thanatin2, and Thanatin3 fragments containing α-factor secretion signals were amplified using (Gib) respectively; LEU2-CL1 fragment was amplified using primers LEU2-pFull-F(Gib) / LEU2-pFull-R(Gib) as a template; Thanatin1, LEU2-CL1, and 3'piggyBac ITR+insulator fragments were inserted into the vector pBluescript to obtain the recombinant plasmid pBluescript-3'piggyITR-Thanatinx1. Using the recombinant plasmid pUC57-5'piggyBac ITR+insulator as a template, the 5'piggyBac ITR+insulator fragment was amplified using primers 5'piggy-F(Gib) / 5'piggy-R(Gib). This fragment, along with Thanatin1 and Thanatin3 fragments, was then inserted into the recombinant plasmid pBluescript-3'piggyITR-Thanatinx1, resulting in the recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3, whose nucleotide sequence is shown in SEQ ID NO.2. The primer sequences are shown in Table 1. The results are as follows: Figure 2 As shown.
[0021] SEQ ID NO.2: Table 1. Nucleotide sequences of each primer
[0022] Example 6: Construction of genetically engineered bacteria Preparation of SC-HL solid medium: 6.7g YNB, 0.68g DO / -His / -Leu and 20g agar powder were diluted to 800mL of distilled water, sterilized at 121℃ for 15min, cooled to below 55℃, and then 200mL of 20% glucose was added and the mixture was poured into plates. Preparation of SC-UHL solid medium: 6.7g YNB, 0.68g DO / -Ura / -His / -Leu and 20g agar powder were diluted to 800mL of distilled water, sterilized at 121℃ for 15min, cooled to below 55℃, and then 200mL of 20% glucose was added and the mixture was poured into plates. The recombinant plasmids pESCgp04-HIS-PiggyBac transposase and pBluescript-3'5'piggyITR-Thanatinx3 were co-transformed into competent *Saccharomyces cerevisiae* CENPK2 cells using a chemical transformation method. After transformation, 5 mL of sterile physiological saline was added, and the cells were plated on SC-HL and SC-UHL solid culture bases and cultured at 29°C for 3 days to obtain the genetically engineered strain CENPK2-Thanatin-PiggyBac expressing the thanatin. The results are as follows: Figures 3-4 As shown.
[0023] Example 7: Fermentation culture of genetically engineered bacteria and expression of the death factor Thanatin The genetically engineered strain CENPK2-Thanatin-PiggyBac was inoculated into YPD liquid medium and cultured overnight to obtain a seed culture. The seed culture was then inoculated into 100 mL of YPD medium at a 1% inoculum and cultured at 29°C and 220 rpm with shaking for 5 days to obtain the fermentation broth. The fermentation broth was centrifuged at 4°C and 12000 rpm for 10 min to obtain the supernatant. After being concentrated 5-fold, it was analyzed by Tris-tricine-SDS-PAGE electrophoresis. The stacking gel concentration was set to 4%, the interlayer gel concentration to 15.5%, and the separating gel concentration to 15.5%. The results are as follows: Figure 5 As shown.
[0024] The results showed that the amino acid sequence of Thanatin obtained by fermentation and expression was as shown in SEQ ID NO.48, and its band size was 2.9 kDa, which was as expected.
[0025] SEQ ID NO.48: GSKKPVPIIYCNRRTGKCQRMDDDKGSKKPVPIIYCNRRTGKCQRM Experimental Example 1: Secondary Mass Spectrometry Identification Cut the target protein band from the Tris-tricine-SDS-PAGE gel described in Example 7, wash three times with ddH2O, add destaining solution for destaining and wash four times with water, wash the gel block sequentially with 300 μL 25 mmol / L ammonium bicarbonate, 50% acetonitrile, and 100% acetonitrile until dehydrated and white, add 50 μL 10 mmol / L DTT and reduce in a water bath at 37°C for 2 h, cool and add an equal volume of 50 mmol / L IAA for alkylation in the dark for 30 min; repeat the above operation, add 40 μL 0.01 μg / μL trypsin and swell on ice until transparent, then add 40 μL of 50 mmol / L NH4HCO3 containing 10% acetonitrile, digest overnight at 37°C and collect the supernatant, add 100 μL of extraction solution containing 67% acetonitrile and 2% formic acid to the gel block, incubate at 37°C for 30 min, sonicate for 15 min, centrifuge and combine the supernatants, concentrate and dry to obtain Thanatin.
[0026] Thanatin was analyzed by mass spectrometry using an UltiMate 3000 RSLC nano-liquid chromatography system coupled with a ThermoFisher Q Exactive plus mass spectrometry system. The spray voltage was set to 1.9 kV, the ion transfer tube temperature to 320 °C, the primary mass spectrometry resolution to 70,000 m / s, and the scan range to 350–1500 m / s. Secondary spectra were acquired in DDA mode. The raw data were retrieved and analyzed using Proteome Discoverer 2.5 software. The results are as follows: Figure 6 As shown.
[0027] Secondary mass spectrometry results showed that the peptide GSKKPVPIIYCNR was detected and matched the amino acid sequence of the death factor Thanatin, confirming the correct expression of the target protein.
[0028] Experimental Example 2: Determination of Antibacterial Activity Indicator strains of *Escherichia coli* O157:H7, *Salmonella typhimurium*, and *Aeromonas vesiculosus* JL-2 were inoculated into LB broth, while indicator strain *Acinetobacter baumannii* was inoculated into TSB broth. All cultures were incubated overnight at 37°C and 220 rpm until OD (out of control) was reached. 600 =0.01, and 100 μL was inoculated into each well of a 96-well plate; 40 mL of the fermentation broth prepared in Example 7 was concentrated 10 times and then added to each well with 100 μL. After incubation at 37°C for 12 h, the OD was measured.600 The antibacterial activity was calculated, and the results are as follows: Figure 7 As shown.
[0029] The results showed that the fermentation broth of the genetically engineered strain CENPK2-Thanatin-PiggyBac of the present invention exhibited significant antibacterial activity against all indicator strains.
[0030] As can be seen from the above embodiments, the present invention provides a method for constructing and applying a genetically engineered bacterium expressing the death factor Thanatin. The present invention integrates the PiggyBac transposon system into Saccharomyces cerevisiae CENPK2, successfully constructing a genetically engineered bacterium that stably and highly expresses multiple copies of the death factor Thanatin. This bacterium exhibits good stability and high expression levels of the target protein, providing a new technical solution and application prospect for its large-scale preparation and anti-infective drug development.
[0031] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for constructing a genetically engineered bacterium expressing the death factor thanatin, characterized in that, The recombinant plasmid pESCgp04-HIS-PiggyBac transposase and the recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3 were co-transformed into competent yeast cells to obtain genetically engineered bacteria; The nucleotide sequence of the recombinant plasmid pESCgp04-HIS-PiggyBac transposase is shown in SEQ ID NO.1; The nucleotide sequence of the recombinant plasmid pBluescript-3'5'piggyITR-Thanatinx3 is shown in SEQ ID NO.
2.
2. The construction method according to claim 1, characterized in that, The yeast competent cells were Saccharomyces cerevisiae CENPK2 competent cells.
3. The construction method according to claim 2, characterized in that, The co-transformation method is a chemical transformation.
4. The use of the genetically engineered bacteria constructed by the construction method according to any one of claims 1 to 3 in the preparation of drugs against drug-resistant bacteria.
5. Thanatin, a death-causing agent produced by the genetically engineered bacteria constructed according to any one of claims 1 to 3, is characterized in that... The amino acid sequence of the death-causing substance Thanatin is shown in SEQ ID NO.
48.
6. The method for producing the death-causing agent thanatin according to claim 5, characterized in that, The genetically engineered bacteria described in claim 5 are fermented to obtain a fermentation broth, and the supernatant of the fermentation broth is collected to obtain the death factor Thanatin.
7. The production method according to claim 6, characterized in that, The fermentation culture time is 24 to 96 hours.
8. The use of Thanatin as described in claim 5, or Thanatin produced by the production method described in claim 6 or 7, in the preparation of drugs against drug-resistant bacteria.