Heterologous expression system for synthesizing nisin and application of heterologous expression system
A nisin and nisin technology, applied in the field of bioengineering, can solve problems such as high production cost, low Nisin output, difficult gene synthesis and effective transformation of regulatory system, and achieve the effect of reducing the difficulty of modification
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Embodiment 1
[0050] Embodiment 1: Construction of the recombinant plasmid containing Nisin heterologous synthesis pathway
[0051] Specific steps are as follows:
[0052] (1) Through the NCBI database search, the Nisin synthesis gene cluster was determined, and the Lactococcus lactis genome (genebank accession number: CP069378.1) was used as a template to clone the precursor and modification enzyme gene nisA (nucleotide sequence such as SEQID NO .1), nisB (nucleotide sequence as shown in SEQ ID NO.2) and nisC (nucleotide sequence as shown in SEQ ID NO.3);
[0053] (2) Link the genes obtained above into the vector using the Gibson assembly method to prepare a recombinant vector; where nisA is linked to the first promoter of the vector pRSFDuet-1, and nisB is linked to the second promoter of the vector pRSFDuet-1 After the promoter, nisC was connected to the first promoter of the vector pACYCDuet-1, in which the homology arm was designed at the 5' end of the primer, which was bolded and lis...
Embodiment 2
[0062] Example 2: Nisin induced expression and Tris-Trince detection
[0063] Specific steps are as follows:
[0064] 1. Construction of recombinant Escherichia coli
[0065] Transform the recombinant plasmids pRS-nisA-nisB and pAC-nisC constructed in Example 1 into the same E.coliBL21(DE3), construct a dual-plasmid expression system, and prepare recombinant Escherichia coli E.coli BL21(DE3) / pRS -nisA-nisB / pAC-nisC.
[0066] 2. Preparation and expression of nisin
[0067] (1) Inoculate the recombinant Escherichia coli E. coli BL21(DE3) / pRS-nisA-nisB / pAC-nisC into 5 mL of 100 μg / mL of Kana antibiotics and 34 μg / mL of chloramphenicol LB liquid medium , cultured at 37°C, 200rpm, until the seed solution grows to OD 600 When it is 1.5 to 2.0, the first-grade seed liquid is obtained;
[0068] (2) Transfer the prepared first-grade seed solution to 50 mL of 2YT medium with 100 μg / mL of Kana antibiotics and 34 μg / mL of chloramphenicol at a ratio of 1% (v / v), at 37° C. at 200 rpm ...
Embodiment 3
[0081] Embodiment 3: Detection of Nisin activity by the inhibition zone
[0082] Specific steps are as follows:
[0083] (1) Preparation of trypsin solution: 0.25% (w / v) trypsin, 0.02% (v / v) EDTA, 0.01M PBS, pH 8.0.
[0084] (2) To the Nisin solution prepared according to the method of Example 2, add the trypsin solution prepared in step (1) at a ratio of 3:1 by volume, shake and react at 37° C. at 200 rpm for 1 hour, and cut the leader peptide. Make Nisin fully mature, and pass through a 0.22 μm filter membrane to sterilize after the reaction is used for activity detection.
[0085] (3) Adjust the indicator bacteria B.subtilis 168 and B.licheniformis cultivated overnight to OD with sterile water 600 1.7, inoculated into 0.75% agar medium containing 1% (v / v) Tween by 1% (v / v) inoculation amount, poured flat plate after mixing, the thickness was 4mm, and punched after the agar solidified.
[0086] (4) Add 80-100 μL of the mature Nisin product obtained in step (2) into the we...
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