Recombinant bacillus subtilis and application thereof

Abstract

The present invention provides a recombinant Bacillus subtilis JY011802 that can produce sublancin in a high yield, which was deposited at the China General Microbiological Culture Collection Center on Oct. 31, 2018 with an accession number of CGMCC No. 16667, and an application thereof. The yield of the sublancin produced by the recombinant Bacillus subtilis can reach 3100 mg/L.

Claims

1. A recombinant Bacillus subtilis JY011802 which was deposited at the China General Microbiological Culture Collection Center on Oct. 31, 2018 with an accession number of CGMCC No. 16667.

2. A method for producing a recombinant Bacillus subtilis, the method comprises steps of: 1) ligating a target gene fragment expressing sublancin into a cloning vector to obtain a recombinant cloning vector, and then transforming the recombinant cloning vector into Escherichia coli for further cloning; 2) digesting the recombinant cloning vector cloned in step 1) with endonuclease, and then ligating the target gene into a pBS101 expression vector to obtain a recombinant plasmid; 3) transforming the obtained recombinant plasmid into a Bacillus subtilis 1A747 expression host by heat shock method, and collecting transformed Bacillus subtilis 1A747 cells; and 4) Spreading the transformed Bacillus subtilis 1A747 cells on a LB medium plate and culturing until a single colony appears, thereby producing the recombinant Bacillus subtilis.

3. A recombinant Bacillus subtilis obtained by the method of claim 2.

4. A method for producing sublancin, comprising cultivating the recombinant Bacillus subtilis JY011802 according to claim 1 in a fermentation medium in order to produce said sublancin.

5. A method for producing sublancin, comprising cultivating the recombinant Bacillus subtilis JY011802 according to claim 2 in a fermentation medium in order to produce said sublancin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the pJET1.2/Blunt cloning vector.

(2) FIG. 2 shows the pBS101 expression vector.

(3) FIG. 3 is a standard curve of the peak area and concentration of the sublancin standard.

(4) FIG. 4 shows the detection profile of the secondary structure of Sublancin detected by using circular dichroism spectrometer.

(5) FIG. 5 shows the 1H-15N HSQC of the Sublancin sample.

(6) FIG. 6 shows the sequence connection relationship and spin system determination of Sublancin.

(7) FIG. 7 is a schematic diagram of the three-dimensional structure of Sublancin.

(8) FIG. 8 shows the glycosyl moiety assignment and NOE information.

(9) FIG. 9 is a schematic diagram of the disulfide bond connection of Sublancin.

(10) FIG. 10 shows the disulfide bond connection pattern of Sublancin (SEQ ID. No:2).

DETAILED DESCRIPTION OF THE INVENTION

(11) The following embodiments merely exemplarily illustrate specific modes of the present invention, and do not limit the scope of the present invention. The scope of the present invention is limited only by the attached claims and the equivalents thereof.

Preparation Example

(12) Experimental Materials:

(13) Templates and Strains

(14) Bacillus subtilis 168 DNA template (NCBI reference sequence: NC_000964.3), Escherichia co/i BL21 competent cells (Item No.: CD901-03, supplier: Beijing TransGen Biotechnology Co., Ltd.). Bacillus subtilis 1A747 (purchased from Bacillus Genetic Stock Centre of the Ohio State University (Columbus, Ohio, USA))

(15) Vectors:

(16) The pJET1.2/Blunt cloning vector (FIG. 1) and pBS101 expression vector (FIG. 2) were products of Biovector.

(17) 1. PCR amplification of target gene fragment

(18) The full sequence name of the target gene fragment was: sunI and sunA-sunT-bdbA-sunS-bdbB (SEQ ID No. 1).

(19) Primer Premier software was used to design primers so that BamHI and SacII restriction sites were contained in both ends of the gene fragment product of interest, respectively.

(20) 2. Ligation of the target fragment into pJET1.2/Blunt vector and plasmid transformation

(21) 2.1 Ligation of the target fragment into pJET1.2/Blunt vector

(22) TABLE-US-00001 TABLE 1.1 Enzyme-linked reaction system ddH.sub.2O 13.5 μl 10*T4 DNA Ligase Buffer 2 μl pJET1.2/Blunt vector 0.5 μl DNA 3 μl T4 DNA Ligase 1 μl Total 20 μl

(23) The PCR product was well mixed with the above system, then the mixture was centrifuged briefly and left to stand at 16° C. overnight. The ligated system was stored in a refrigerator at 4° C. for later use.

(24) 2.2 Plasmid Transformation by Heat-Shock Method

(25) a. The prepared 100 μl of Escherichia coli BL21 competent cells were taken out from the freezer at −80° C. and were left on ice for 10 min to make them enter the 0° C. reception state. In a super clean bench, 10 μl of the corresponding enzyme-linked product was added to competent cells, the mixture was gently rotated and the contents were mixed well, and the mixture was placed on ice for 30 min (a control without plasmid DNA can be set in the test);

(26) b. Heat shock: A thermometer was used to accurately adjust the temperature of the water bath to 42° C. The sample was taken out and immediately placed into a water bath at 42° C. for 90 s for accurate heat shock;

(27) c. Icing: The EP tube was quickly removed and put into ice to cool the cells for 2 minutes;

(28) d. Resuscitation: 400 μl of LB medium that has been pre-heated in an incubator at 37° C. was added to the EP tube and the EP tube was incubated with shaking in a shaker at 37° C. at 180 r/min for 1.5 h to recover the bacteria;

(29) e. Spread plate: In the super clean bench, 300 μl and 150 μl of transformed competent cells were taken and transferred to a petri dish, respectively, and the transformed cells were spread uniformly on the surface of an agar plate with a sterile elbow glass rod;

(30) f. Cultivation: The petri dishes were placed right side up in an incubator at 37° C. for cultivation until the liquid was absorbed, and then the petri dishes were inverted for cultivation. 12-16 hours later, colonies may appear.

(31) g. MasterPlate: The petri dishes that have been cultured overnight were taken out. In the super clean bench, 10 single colonies from each petri dish were picked to 2 solid LB Petri dishes with a diameter of 15 cm and streaked for monoclonal expansion and further screening. The picked colonies were numbered 1-10 and cultured in an incubator at 37° C. for 12 hours.

(32) 3. Double digestion (BamHI and SacII) and ligation of plasmid and expression vector pBS101.

(33) 3.1 First the Mixture of the digestion system without the plasmid and the expression vector was prepared, and then each of the aliquots of Mixture was charged into 0.6 ml EP tube.

(34) TABLE-US-00002 TABLE 1.2 Endonuclease Digestion System ddH.sub.2O 30 μl 10*Proteinase buffer 4 μl BamH I 1 μl SacII 1 μl plasmid 4 μl Total 40 μl

(35) The endonuclease digestion was performed in an incubator at 37° C. for not more than 1.5 hours. 2 μl of 10× Buffer can be added to terminate the digestion. It was shown from the results that the digestion effect was good. The plasmid digested in the same step was used to recover the target fragment. The digested expression vector was first stored in a refrigerator at 4° C. for use.

(36) 3.2 Ligation of the recovered target fragment into the pBS101 expression vector

(37) TABLE-US-00003 TABLE 1.3 Enzyme-linked reaction system ddH.sub.2O 13.5 μl 10*T4 DNA Ligase Buffer 2 μl pBS101 0.5 μl DNA 3 μl T4 DNA Ligase 1 μl Total 20 μl

(38) The components of reaction system were well mixed and centrifuged briefly. The prepared system was left to stand at 16° C. overnight. The ligated system was stored in a refrigerator at 4° C. for later use.

(39) 3.3 Plasmid transformation by heat shock method

(40) The recombinant plasmid was transformed into a Bacillus subtilis 1A747 (purchased from Bacillus Genetic Stock Centre of the Ohio State University (Columbus, Ohio, USA)) expression host by a heat shock method, and the transformed Bacillus subtilis 1A747 cells were collected and spread on LB medium plate (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 15 g of agar were dissolved in purified water and made up to 1 L with purified water). The cells were cultured at 37° C. until single colonies appear. After the transformants were correctly verified by plasmid extraction and PCR, the next cultivation and fermentation studies were performed.

Example 1 Cultivation of Recombinant Bacillus Subtilis Transformants

(41) 4 transformant single colonies and 1 Bacillus subtilis 1A747 single colony were picked and inoculated into 25 ml of liquid medium (30 g of corn flour, 18 g of soybean meal, 12.5 g of peptone, 15 g of glucose, 3 g of KH.sub.2PO.sub.4 and 1.25 g of ammonium sulfate were dissolved in purified water and made up to 1 L with purified water), respectively. They were cultured with shaking at 200 rpm for 12-18 hours at 37° C., and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected.

(42) The concentration of the target polypeptide Sublancin in the supernatant was detected by high performance liquid chromatography.

(43) The chromatographic conditions of high performance liquid chromatography were as follows:

(44) Octylsilane-bonded silica gel was used as filler (High Performance Liquid Chromatograph: Agilent 1260, C8 chromatographic column: ZORBAX 300SB-C8, 5 m, 4.6×150 mm), and trifluoroacetic acid-water (1:1000) was used as mobile phase A, and trifluoroacetic acid-water-acetonitrile (0.85:200:800) was used as mobile phase B; column temperature was 25° C.; detection wavelength was 280 nm; flow rate was 1 ml/min; injection volume was 20 L; gradient elution was carried out according to the following Table 5. The number of theoretical plates was calculated to be not less than 2000 according to the Sublancin peak.

(45) TABLE-US-00004 TABLE 2.1 Elution gradients for HPLC Time (minutes) A (%) B (%) 0 70 30 1 70 30 15 57 43 25 57 43 25.01 0 100 30 0 100 30.01 70 30 35 70 30

(46) The peak position of the Sublancin standard was 8.5 min-10 min. The standard curve of the peak area and Sublancin concentration was shown in FIG. 3. The standard curve equation was y=1833.5x-20.405 (x was the standard concentration, the unit was mg/ml, and y was average peak area).

(47) It was shown from the results that the concentrations of Sublancin in the supernatants obtained by the four transformed strains were 2980 mg/L, 2920 mg/L, 3010 mg/L, and 3100 mg/L, respectively, while the concentration of Sublancin in the supernatant obtained by Bacillus subtilis 1A747 was 100 mg/L. According to references.sup.[6,7], the current yields of Sublancin produced by Bacillus subtilis were 129 mg/L and 642 mg/L, respectively.

(48) The strain with the highest yield of Sublancin (that is, the strain with a Sublancin concentration of 3100 mg/L in the supernatant) was named JY011802. Bacillus subtilis JY011802 has been deposited on Oct. 31, 2018 at the China General Microbiological Culture Collection Center (CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences, Building 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Postcode 100101) with an accession number of CGMCC No. 16667.

Verification Examples

(49) 1. Mass Spectrometry

(50) The Sublancin in the supernatant of strain JY011802 obtained in Example 1 was qualitatively analyzed by mass spectrometry, and the specific steps were as follows:

(51) Name and model of test instrument: electrospray tandem mass spectrometer micrOTOF-Q II (Bruker), Agilent 1100 Series High Performance Liquid Chromatograph (HPLC, Agilent) Test sample: Sublancin

(52) The test results were shown in Table 3.1 below.

(53) TABLE-US-00005 TABLE 3.1 Sublancin Comparison before and after reduction Molecular Molecular weight weight Molecular Relative before after weight area reduction reduction increment increment Sample (Da) (Da) (Da) (%) Remarks Sublancin 3875.7292 3879.7669 4.0377 10.0 Two pairs of disulfide bonds of component 1 were reduced 3713.6663 3717.6932 4.0269 −9.0 Two pairs of disulfide bonds of component 2 were reduced 7751.4231 7759.5087 8.0856 −1.0 Four pairs of disulfide bonds of component 3 were reduced

(54) Conclusion: LC-MTQ-MS was used to determine the exact molecular weight of Sublancin before and after reduction. It was shown from the results that the main component was the target molecule, whose complete molecular weight is 3875.7249D, and the relative deviation from the theoretical molecular weight (containing 2 pairs of disulfide bonds and 1 Hex modification) is less than 0.0004%. The measured molecular weight was consistent with the theoretical molecular weight. The molecular weight of the sample shown in the test result was basically the same as 3878.78 Da and 3875.75 Da of the molecular weights of Sublancin as reported in the References.sup.[1, 2, 3]. It can be seen that this sample had the structural information corresponding to Sublancin.

(55) 2. Amino Acid Sequence Determination

(56) Name and model of detection instrument: Applied Biosystems 491 Protein Sequence Analyzer

(57) Test sample: Sublancin

(58) Test results:

(59) TABLE-US-00006 (SEQ ID. No: 2) H-Gly-Leu-Gly-Lys-Ala-Gln-Cys-Ala-Ala-Leu-Trp-Leu- Gln-Cys-Ala-Ser-Gly-Gly-Thr-Ile-Gly-Cys-Gly-Gly- Gly-Ala-Val-Ala-Cys-Gln-Asn-Tyr-Arg-Gln-Phe-Cys- Arg-OH

(60) The amino acid sequence of the sample shown in this test result was completely consistent with the amino acid sequence of Sublancin reported in References.sup.[1, 2, 3]. It can be seen that this sample had structural information corresponding to Sublancin.

(61) 3. Secondary Structure Detection

(62) Name and model of detection instrument: circular dichroism spectrometer (model: JASCO J-810), Cell length=1 mm.

(63) Test sample: Sublancin

(64) Test results: The test profile was shown in FIG. 4.

(65) The analysis results are shown in Table 3.2 below.

(66) TABLE-US-00007 TABLE 3.2 Structure English Chinese fraction ratio Helix α-helix 0.1 35.1% Beta β-sheet 0.1 40.5% Turn β-turn angle 0.0  0.0% Random Random coil 0.1 24.4% Total 0.3  100%
4. Nuclear Magnetic Resonance (Tertiary Structure)

(67) Name and model of test instrument: NMR spectrometer Agilent DD2 600 MHz

(68) Test sample: Sublancin

(69) Test results: see FIG. 5, FIG. 6 and Table 3.3.

(70) TABLE-US-00008 TABLE 3.3 Sublancin structure calculation statistics table Total number of structural calculation constraints 562 Total NOE Constraints 508 Within residue 218 Interresidue 103 Medium range 75 Remote 68 Multi-homing NOE 44 Dihedral angle constraint Φ angle 30 Ψ angle 30 Disulfide bonds C7—C36, C14—C29 2 Hydrogen bond 0 Experimental constraint rmsd Bond length ({acute over (Å)}) 0.003 ± 0.000 Bond angle (degrees) 0.473 ± 0.016 Discomfort torsion angle (degrees) 1.626 ± 0.095 Average pairwise rmsd Main chain: secondary structure region ({acute over (Å)}) 0.15 Main chain: 1-37 residues ({acute over (Å)}) 0.86 Heavy atom: secondary structure region ({acute over (Å)}) 0.63 Heavy atom: 1-37 residues ({acute over (Å)}) 1.05 Procheck analysis Optimum area (%) 91.9 Additional permitted area (%) 8.1 General allowed area (%) 0 Not allowed area (%) 0

(71) Analysis: The calculated three-dimensional structure of the Sublancin sample was two nearly parallel α-helix structures connected by a central loop region (residues 16-24), where Helix A included residues 4-15 and Helix B included residues 25-35, the two helix structures were pulled closer to each other by two pairs of disulfide bonds (C7-C36, C14-C29) at the ends (FIGS. 7A and 7B). The 562 constraints were used for structure calculation (see Table 3.3), with an average of more than 15 constraints per residue. Without using hydrogen bonding constraints, a clear secondary structure region (residues 4-15 and residues 25-35) was obtained. Due to the constraints of the disulfide bond (C7-C36) at the end of the helix structure, a large number of NOE between L2-F35, and the non-secondary structure regions at the N and C terminals can be seen, and the structural calculation also converges well (FIG. 7A). There are fewer constraints in the central loop region, indicating greater flexibility in this region (FIG. 7A).

(72) Glycan part only H1, Hβa, b of H1 and C22

(73) For the glycosyl moiety, only the NOE signals between H.sub.1, C22 H.sub.α, H.sub.βa,b were shown (FIG. 8), indicating the position and the R configuration of the glycosyl linkage (if it was a configuration, there should also be a NOE signal between H.sub.2 and C22 H.sub.α, Hp.sub.α,β). No other glycosyl signals and NOE information of the peptide other than the C22 residue were observed, indicating that the glycosyl moiety was a region with great flexibility, which had no definite relative positional relationship with the polypeptide moiety.

(74) Conclusion: The NMR detection and analysis results of this sample were basically consistent with the Sublancin NMR detection and analysis results reported in References.sup.[4,5], and this sample had the structural information corresponding to Sublancin.

(75) 5. Infrared Absorption Spectrum

(76) Name and model of detection instrument: VERTEX 70 Fourier transform infrared

(77) spectrometer (Bruker, German)

(78) Test sample: Sublancin

(79) Test results: see Table 3.4.

(80) TABLE-US-00009 TABLE 3.4 Sublancin FT-IR characteristic peak analysis Peak Wave Type of Number Number Absorption Chemical Bond # (cm−1) Intensity Vibration Characteristics 1 3300.1 s ν.sub.O—H, ν.sub.N—H Hydroxyl, peptide bond, amino 2 3063.6 m ν.sub.Ar—H, δ.sub.N—H octave benzene ring, peptide peak bond 3 2960.0 m ν.sub.—CH3 alkyl 4 2935.4 m ν.sub.—CH2 alkyl 5 2873.9 m ν.sub.—CH3 alkyl 6 1657.8 s ν.sub.C═O, amide I peptide bond, α-helix 7 1541.4 s δ.sub.N—H, ν.sub.C—N, ν.sub.C═C peptide bond, benzene ring 8 1454.8 m δ.sub.—CH3   δ.sub.—CH2 alkyl 9 1410.4 m δ.sub.—CH3   δ.sub.—CH2 alkyl 10 1334.6 m γ.sub.—CH2 alkyl 11 1299.4 m amide III α-helix 12 1242.9 m amide III β-sheet 13 1170.4 m ν.sub.C—O side chain hydroxyl 14 1104.5 w ν.sub.C—O sugar, side chain hydroxyl 15 1071.6 w γ.sub.—CH, ν.sub.C—O alkyl, sugar 16 1032.0 w δ.sub.—C—O silk amino acid side chain, sugar 17 891.9 w δ.sub.—CH alkyl, sugar 18 743.3 w δ.sub.═CH benzene ring 19 612.9 m ν.sub.C═O, ν.sub.—SH carboxylic acid, amide conjugated system, disulfide bond 20 551.4 m ν.sub.C═O, ν.sub.—SH carboxylic acid, amide conjugated system, disulfide bond Note: ν-stretching vibration, δ-bending vibration, γ-out-of-plane bending vibration, s-strong peak, m-medium strong peak, w-weak peak. Analysis: Sublancin as the sample to be tested was tableted by using potassium bromide powder and then detected by using Fourier transform infrared spectroscopy. The wave number correlation coefficient was 1.00 (equivalent to 100% similarity). The molecular structure of the sample contained main structures such as amide I, amide III, amino, peptide bond, methyl, methylene, phenyl, glycosyl, disulfide bond, α-helix, β-sheet and the like. Conclusion: In the infrared absorption spectrum of this sample, its absorption peak had the structural information corresponding to Sublancin, including main structural features such as amide I, amide III, amino, peptide bond, methyl, methylene, phenyl, glycosyl, disulfide bond, α-helix, ,β-sheet and the like, which were more obvious.

(81) 6. Confirmation of Sugar Structure

(82) Name and model of detection instrument: Agilent 1100 Series High Performance

(83) Liquid Chromatograph (HPLC, Agilent), micrOTOF-Q II Mass Spectrometer (Brook)

(84) Test sample: Sublancin

(85) Test results: see Table 3.5 and Table 3.6.

(86) TABLE-US-00010 TABLE 3.5 Identification results of glycation modification sites Measured theoretical Molecular Molecular Weight Weight Ion Sample (Da) (Da) Characteristic sequence Modification Score Sublancin 2204.8798 2204.9136 LQCASGGTIGCGGGA C14, 484 VACQNY C29: Carbamidomethyl (SEQ ID. No: 3) (C); C22: Hex (C)* C22: Hex (C) Note*: Hex was hexose (glucose), Reference.sup.[8].

(87) TABLE-US-00011 TABLE 3.6 Results of saccharification rate at the SublancinCys22 site Characteristic Relative Category Lot Number Ionic Strength* Strength (%) Glycosylation Sublancin 3671596 99.1  Modification Non-glycosylation Sublancin  34420 0.9 Modification Note*: “.sup.12LQCASGGTIGCGGGAVACQNY.sup.32” (SEQ ID. No: 3) was selected as a reliable characteristic peptide based on the ion score and detection times. CONCLUSION: The Sublancin as the sample to be tested was subjected to enzymatic hydrolysis and then reductive alkylation. It was detected by HPLC-MS/MS. It was shown from the results that all the glycation modification sites of the samples were at Cys22, the glycation rate was 99.1%, and the non-glycosylation modification rate was 0.9%.

(88) The glycosylation modification sites shown in this test result were consistent with the conclusion that Sublancin had a glucose linked to the cysteine at position 22 reported in References.sup.[2, 4]. It can be seen that this sample had structural information corresponding to Sublancin.

(89) 7. Three-Dimensional Configuration of Disulfide Bonds

(90) Name and model of detection instrument: Agilent 1110 HPLC, electrospray quadrupole time-of-flight tandem mass spectrometer (Q-TOF) micrOTOF-Q II

(91) Test sample: Sublancin

(92) Test results: see Table 3.7 and Table 3.8.

(93) TABLE-US-00012 TABLE 3.7 Measured disulfide bond connection and detection frequency of Sublancin Disulfide Bond Connection Sublancin Notes (C1)—(C5) 17 Expected disulfide bond (C2/C3/C4) 29 Expected disulfide bond, including 1 Hex (C2/C3/C4)—(C1)—(C5) 3 Unexpected disulfide bond, including 1 Hex

(94) TABLE-US-00013 TABLE 3.8 Relative strength and ratio of sublancin measured disulfide bond connection Disulfide Bond Connection 2016111601 Notes Strength (C1)—(C5) 560306 Expected disulfide bond (C2/C3/C4) 4096141 Expected disulfide bond, including 1 Hex (C2/C3/C4)—(C1)—(C5) 11091 Unexpected disulfide bond, including 1 Hex relative Expected connection 99.8 proportion (%) Unexpected connection 0.2 Conclusion: The samples were subjected to enzymolysis by using Chymotrypsin, and the comparison of samples was performed before and after reduction, and the samples were detected by using HPLC-MS/MS mass spectrometry. The main disulfide bond connection modes of Sublancin were obtained through analysis strategies such as “software comparison differences .fwdarw. data optimization and filtering .fwdarw. mother-child peptide verification .fwdarw. mass spectrum verification” . The main disulfide bond connection modes were: (C1)—(C5), (C2)—(C4). Very few unexpected connections were also detected: (C2/C3/C4)—(C1)—(C5). See FIG. 10 for the connection mode. The disulfide bond connection sites shown in this test result were completely consistent with the Sublancin disulfide bond connection sites reported in the literatures .sup.[1, 2, 3, 4]. It can be seen that this sample had structural information corresponding to Sublancin.

REFERENCES

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